THE BIOLOGICAL BULLETIN PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY Editorial Board HAROLD C. BOLD, University of Texas ARTHUR W. POLLISTER, Columbia University FRANK A. BROWN, JR., Northwestern University c L PROSSER) University of Illinois JOHN B. BUCK, National Institutes of Health T. H. BULLOCK, University of California, Los Angeles LIBBIE H. HYMAN, American Museum of Natural History V. L. LOOSANOFF, U. S. Fish and Wildlife Service CARROLL M. WILLIAMS, Harvard University MARY E. RAWLES, Carnegie Institution of Washington FRANZ SCHRADER, Columbia University WM. RANDOLPH TAYLOR, University of Michigan DONALD P. COSTELLO, University of North Carolina Managing Editor VOLUME 116 FEBRUARY TO JUNE, 1959 Printed and Issued by LANCASTER PRESS, Inc. PRINCE & LEMON STS. LANCASTER, PA. 11 THE BIOLOGICAL BULLETIN is issued six times a year at the Lancaster Press, Inc., Prince and Lemon Streets, Lancaster, Penn- sylvania. Subscriptions and similar matter should be addressed to The Biological Bulletin, Marine Biological Laboratory, Woods Hole, Massachusetts. Agent for Great Britain : Wheldon and Wesley, Limited, 2, 3 and 4 Arthur Street, New Oxford Street, London, W. C. 2. Single numbers $2.50. Subscription per volume (three issues), $6.00. Communications relative to manuscripts should be sent to the Managing Editor, Marine Biological Laboratory, Woods Hole, Massachusetts, between June 1 and September 1, and to Dr. Donald P. Costello, P.O. Box 429, Chapel Hill, North Carolina, during the remainder of the year. Second-class postage paid at Lancaster, Pa. LANCASTER PRESS, INC., LANCASTER, PA. CONTENTS No. 1. FEBRUARY, 1959 PAGE CHANG, JOSEPH JIN, AND FRANK H. JOHNSON The influence of pressure, temperature and urethane on the luminescent flash of Mnemiopsis leidyi 1 DAVIS, CHARLES C. Osmotic hatching in the eggs of some fresh-water copepods 15 FlNGERMAN, MlLTON, MlLDRED E. LOWE AND BANGALORE I. SUNDARARAJ Dark-adapting and light-adapting hormones controlling the distal retinal pigment of the prawn Palaemonetes vulgaris 30 FLEMISTER, SARAH C. Histophysiology of gill and kidney of crab Ocypode albicans 37 GIESE, A. C., L. GREENFIELD, H. HUANG, A. FARMANFARMAIAN AND R. LASKER Organic productivity in the reproductive cycle of the purple sea urchin . . 49 GOREAU, THOMAS F. The physiology of skeleton formation in corals. I. A method for meas- uring the rate of calcium deposition by corals under different conditions 59 GREEN, JAMES W., MARY HARSCH, LLOYD BARR AND C. LADD PROSSER The regulation of water and salt by the fiddler crabs, Uca pugnax and Uca pugilator 76 ICHIKAWA, M., AND J. NlSHIITSUTSUJI-U\VO Studies on the role of the corpus allatum in the Eri-silkworm, Philosamia cynthia ricini 88 ITO, TOSHIO, AND Moxozo TANAKA Beta-glucosidase of the midgut of the silkworm Bombyx mori 95 JENKINS, MARIE M. The effects of thiourea and some related compounds on regeneration in planarians 106 JONES, N. S., AND W. D. BURHANCK Almyracuma proximoculi gen. et sp. nov. (Crustacea, Cumacea) from brackish water of Cape Cod, Massachusetts 115 KLEINHOLZ, L. H. Purines and pteridines from the reflecting pigment of the arthropod retina 125 KURLAND, CHARLES G., AND HOWARD A. SCHNEIDERMAN The respiratory enzymes of cliapausing silkworm pupae: A new interpre- tation of carbon monoxide-insensitive respiration 136 LEEDALE, GORDON F. Periodicity of mitosis and cell division in the Euglenineae 162 in iv CONTENTS SCHEER, BRADLEY T. The hormonal control of metabolism in crustaceans. IX. Carbohydrate metabolism in the transition from intermoult to premoult in Carcinides maenas 175 STUNKARD, HORACE W., AND JOSEPH R. UZMANN The life-cycle of the digenetic trematode, Proctoeces maculatus (Looss, 1901) Odhner, 1911 [syn. P. subtenuis (Linton, 1907) Hanson, 1950], and description of Cercaria adranocerca n. sp 184 No. 2. APRIL, 1959 BALECH, ENRIQUE Two new genera of dinoflagellates from California 195 BEETON, ALFRED M. Photoreception in the opossum shrimp, Mysis relicta Loven 204 BROOKBANK, JOHN W. The respiration of unfertilized sea urchin eggs in the presence of antisera against fertilizin 217 CHACE, FENNER A., JR., AND GEORGE M. MOORE A bicolored gynandromorph of the lobster, Homarus americanus 226 FULTON, CHANDLER Re-examination of an inhibitor of regeneration in Tubularia 232 GEORGE, J. C., AND R. M. NAIK Studies on the structure and physiology of the flight muscles of birds. 4. Observations on the fiber architecture of the pectoralis major muscle of the pigeon 239 GROSS, WARREN J. The effect of osmotic stress on the ionic exchanges of a shore crab 248 KANWISHER, JOHN Histology and metabolism of frozen intertidal animals 258 KRIVANEK, JEROME O., AND ROBIN C. KRIVANEK Chromatographic analyses of amino acids in the developing slime mold, Dictyostelium discoideum Raper 265 RIEGEL, J. A. Some aspects of osmoregulation in two species of sphaeromid isopod Crustacea 272 RONKIN, R. R. Motility and power dissipation in flagellated cells, especially Chlamy- domonas 285 RUSTAD, RONALD C. Consequences of unilateral ultraviolet irradiation of sea urchin eggs . . . 294 SUSSMAN, MAURICE, AND HERBERT L. ENNIS The role of the initiator cell in slime mold aggregation 304 TUCKER, JOHN S., AND ARTHUR C. GIESE Shell repair in chitons 318 WILLIAMS, CARROLL M. The juvenile hormone. I. Endocrine activity of the corpora allata of the adult Cecropia silkworm 323 CONTENTS v No. 3. JUNE, 1959 ALLEN, M. JEAN Embryological development of the polychaetous annelid, Diopatra cuprea (Bosc) 339 BOOLOOTIAN, R. A., A. C. GIESE, J. S. TUCKER AND A. FARMANFARMAIAN A contribution to the biology of a deep sea echinoid, Allocentrotus fragilis (Jackson) 362 COSTLOW, JOHN D., JR., AND C. G. BOOKHOUT The larval development of Callinectes sapidus Rathbun reared in the laboratory 373 DA vi SON, JOHN Studies on the form of the amphibian red blood cell 397 ENGELMANN, FRANZ The control of reproduction in Diploptera punctata (Blattaria) 406 ERASER, RONALD C. Somite genesis in the chick. II. Analysis of nutrients from yolk 420 GRANT, WILLIAM C., JR., AND GRACE E. PICKFORD Presence of the red eft water-drive factor prolactin in the pituitaries of teleosts 429 HARRISON, JOHN R. Developmental characteristics of low temperature chick blastoderms. I. Influence of the hypoblast on development in vitro 436 HUMPHRIES, A. A., JR., AND W. X. HUGHES A study of the polysaccharide histochemistry of the oviduct of the newt, Triturus viridescens 446 LENHOFF, HOWARD M., AND HOWARD A. SCHNEIDERMAN The chemical control of feeding in the Portuguese man-of-war, Physalia physalis L. and its bearing on the evolution of the Cnidaria 452 MCLACHLAN, JACK, AND CHARLES S. YENTSCH Observations on the growth of Dunaliella euchlora in culture 461 METZ, CHARLES B. Inhibition of fertilizhi agglutination of sperm by the dermal secretion from Arbacia 472 MORRISON, PETER Body temperatures in some Australian mammals. I. Chiroptera 484 YOST, HENRY T., JR., AND HOPE H. ROBSON Studies on the effects of irradiation of cellular participates. III. The effect of combined radiation treatments on phosphorylation 498 AUTHOR'S ERRATUM Reference : Scheer, Bradley T. The hormonal control of metabolism in crus- taceans. IX. Biol Bull., 116: 175-183 (February, 1959). Table III on page 179 should be Table IV ; Table III was inadvertently omitted. The mean total carbohydrate of the blood for 15 animals was 10.3 mg. per 100 ml., with a range of 5.8 to 15.9. There were no differences between normal and eye- stalkless animals in Stage C 4 or D^ Vol. 116, No. 1 February, 1959 THE BIOLOGICAL BULLETIN PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY THE INFLUENCE OF PRESSURE, TEMPERATURE AND URETHANE ON THE LUMINESCENT FLASH OF MNEMIOPSIS LEIDYI 1 JOSEPH JIN CHANG 2 AND FRANK H. JOHNSON .lltirinc Biological Laboratory, Woods Hole, Mass., Department of Biology, Bronw University, Providence, R. I., and Department of Biology, Princeton University, Princeton, N. J. The luminescent flash response, induced by electrical stimulation of small seg- ments of excised meridional canals of Mneniiopsis leidyi, has been recently analyzed in relation to temperature and certain other factors (Chang, 1954). This flash, as well as that of the firefly (Chang, 1956), has been found to resemble, in important respects, the contraction response of directly stimulated muscle fibers. Temperature relations of muscular contraction and various other biological processes, including specific enzyme action, bacterial luminescence, cell division, nerve activity, etc., are subject to modification by increased hydrostatic pressure. Moreover, temperature- pressure relations may be influenced by the presence of narcotics such as alcohol or urethane as well as other chemical agents (cf., Johnson, Eyring and Polissar, 1954; Johnson, 1957; Brown, 1957; Marsland, 1957; Tasaki and Spyropoulos, 1957; Spyropoulos, 1957a, 1957b). Since studies of pressure-temperature-inhibitor relations have proved a useful approach to understanding certain aspects of the chemical and physiological con- trol of biological processes, and since studies incorporating all three variables are yet available with respect to relatively few processes, the present investigation of the Mneniiopsis flash was undertaken. Unfortunately, no separate biochemical com- ponents of the luminescent system have been obtained thus far from this organism, and it does not secrete a luminous slime, so the pressure-temperature relations could not be studied in regard to the luminescence of both whole organs and the reaction system in vitro, as was recently done w r ith Chaetopterus (Sie, Chang and Johnson, 1958). More than 8000 individual flashes of the excised Mneniiopsis organs, however, have been accurately measured and carefully analyzed to constitute the basis of this study. 1 This study was aided in part by contract Nonr 1353 (00), Project NR 165-233, between the Office of Naval Research and Princeton University, and in part by the Eugene Higgens Fund Allocated to Princeton University. Reproduction in whole or in part is permitted for any purpose of the United States Government. - Present address : National Institutes of Health, Bethesda, Maryland. 1 Copyright 1959, by the Marine Biological Laboratory JOSEPH JIN CHANG AND FRANK H. JOHNSON MATERIALS AND METHODS Mnemiopsis leidyi collected around Woods Hole. Massachusetts, and kept in large aquaria with very slowly running sea water for not more than two days, were used for this study. As previously shown (Chang, 1954), reproducible responses to electrical stimulation are obtained only with small portions of the photogenic organs, which are closely associated with the meridional canals. For experiments, the canals, with their closely adjacent tissues, were carefully excised. A small piece, measuring from 1.5 to 4 mm. in length, and including from one to four paddle plates, was cut out for the test material. This piece was then placed in a Incite chamber which in turn was sealed in a pressure bomb with a glass window as previously described (Sie, Chang and Johnson, 1958) for the purpose of stimulation at normal or under increased hydrostatic pressures. A pair of Ag-AgCl electrodes in the specimen chamber was connected to an electronic stimulator which had controllable parameters of pulse amplitude, dura- tion, repetition frequency and synchronization delay. The flash response was re- corded by means of a stabilized photomultiplier-amplifier unit described by Chang (1954). The two beams of a dual-beam cathode-ray oscillograph were fed respec- tively by the output of the light detection unit and by the stimulus signal, and were photographed on a continuously moving film or with a single-frame camera. Increased pressure was applied by means of an oil-filled hydraulic pump operated by hand. Pressures up to 10,000 pounds per square inch (psi) could be applied within approximately one second. RESULTS The time course of the flash response The time course of luminescent intensity in the Mnemiopsis flash has been shown to remain unaltered with increasing flash maxima due to increasing strength of stimulation (Chang, 1954). Results obtained in the present study show that, with a given strength of stimulation, increased pressure reduces the flash maxima but the time course of intensity again remains essentially the same. Figure 1 illustrates superimposed tracings of oscilloscope records from a single specimen under dif- ferent pressures up to 1000 psi at room temperature. With this specimen, higher pressures diminished the flash intensity so much that the form of the response was hardly analyzable. Temperature has a marked effect on the time course of the responding flash, which becomes progressively prolonged as the temperature is lowered (Chang, 1954). At a given constant temperature, between 35 and 15.5 C., however, the time course was found to remain unaltered by increased pressure. Latent period According to a limited amount of data obtained in the present study with re- spect to the latent period between the time of stimulation and the onset of luminescent response, no significant variation was induced by pressure. While a critical study of this relationship would require additional experiments specifically designed for this purpose, it appears likely that the differences in the latent period under normal and increased pressures w r ould be quite small, if any. PRESSURE AND MNEMIOPSIS FLASH Pressure versus flash height at constant temperature The initial effect of increased pressure was always to reduce the intensity of the flash, and remarkably small amounts of pressure were required to produce a detectable decrease in flash height, so small in fact that they could not be read accurately on the hydraulic pump's gauge, which was not calibrated for pressures less than 200 psi. Moreover, when applied suddenly, as little as 1000 to 1500 psi T 60 90 120 150 MILL I SECONDS 180 FIGURE 1. Superimposed tracings of oscillograph records of luminescent responses of a single specimen at 22 C., under normal and various increased pressures, applied in a step-wise series. The time was measured from the front edge of the square pulse used for stimulation. often caused a virtually complete inhibition of the luminescent response (Fig. 2, A and C). Frequently, though not invariably, however, a process of adaptation under a sustained pressure took place, whereby during continued stimulation at a given frequency the flash reappeared and facilitated to successively higher maxima, some- times reaching intensities several times greater than the highest intensity observed with identical stimuli prior to compression (Fig. 2, C). Apparently, this same 4 JOSEPH JIN CHANG AND FRANK H. JOHNSON process of adaptation occurred to various extents during step-wise application of pressure in small increments, inasmuch as such step-wise increases up to a given pressure were considerably less inhibitory than a sudden increase to that pressure (Fig. 2). 5- 3t /5.5 H4- | A O ^v ^^ UJ I J O 3 I U ll J d2- 4, 4, j, ^ 1 _ ^ S: U: o o ll. Q ll,. L. ] Illllllllll i \ " 2 3 4 5 6 MINUTES 7- /5.5 /5.5 T 3 6- B C k o 5 5 ~ - u O I i 4 - I 5 i M | u3- * ^ o o M. *O -. o 2- *l 1 1 1 i W/ W ^ i t 1- III 1, II Illll , Illllllll k 11 Illll mil llllllllll II ..,., nil 1 2 C ) 1 2 3 F 4 MINUTES FIGURE 2. Intensities of flashes responding to repeated, identical rectangular pulses at the rate of one every 3 seconds. B and C were taken from the same specimen, and A from another. The downward arrows represent the time of application of the various pressures indicated in psi, and upward arrows represent decompression to normal pressure. The flashes that went off scale in C reached a height of 16 or above on the relative scale of the figure when measured at a lower sensitivity of the phototube. The initial effect of decompression was essentially always an increase in flash maxima over those occurring while under pressure, or in some instances those occurring prior to compression (Fig. 2, A, B, C). Such increases sometimes at- tained dramatic proportions, especially in those instances wherein adaptation under pressure had taken place to a very marked extent, as indicated in Figure 2, C and PRESSURE AND MNEMIOPSIS FLASH LJ H; z LJ h- z r in 20 "^ | LJ P LJ cr .2 \ \ \ x . II ! /55 * \ \ N. \ \ i i i 1,000 2DOO PRESSURE CPS I) 3,000 FIGURE 3. Relation between pressure and logarithm of flash intensity, in a step-wise series of pressure increases during repetitive responses to identical stimuli. Dashed lines were drawn by inspection, the lower two lines pertaining to a specimen in sea water, and the uppermost line to a specimen in sea water containing 0.1 M urethane. illustrated more clearly in later figures. The only exceptions to increasing flash maxima following decompression occurred when, for unknown reasons, the speci- men deteriorated under pressure with complete loss of excitability (Fig. 6, A). Because of a wide variability in the quantitative effects of pressure on different specimens, and the phenomenon of adaptation that occurred to various extents at unpredictable rates, reliable data concerning the relation between amount of pres- sure and of effect produced are obviously difficult to achieve. The physiological state of the specimen at the moment of the experiment was evidently an important factor in the results obtained. The most feasible approach to investigating the quantitative relation between amount of pressure and effect produced appeared to 6 JOSEPH JIN CHANG AND FRANK H. JOHNSON be through a series of rapid, step-wise pressure increases, that would permit a minimum of adaptation in a given specimen, under repetitive stimulation by square pulses of identical voltage and duration fired at a constant frequency. The results of such a series at 15.5 C. are shown in Figure 2, A and 2, B. Although analysis of these results is subject to the complicating factors referred to above, data from Figure 2 and from two other experiments are plotted in an analytical manner (cf., Johnson, Eyring and Polissar, 1954) in Figure 3, where each point represents the height of an individual flash in a series of three to eight flashes immediately before or after a change in pressure, at a constant stimulation frequency of one every three seconds throughout. Despite the numerous factors that potentially influence the observed results, the relationship between the logarithm of relative flash height and the amount of pres- sure under which the response occurred appears to be roughly linear. The slopes of the dashed lines drawn by inspection in Figure 3 indicate a molecular volume change of about 170 cc. per mole for the over-all process. Pressure effects at different temperatures At a temperature as low as 5 C., strong stimuli elicited only a weak response at normal pressure. Under 1000 psi the response was abolished and it failed to return after decompression, so further experiments at temperatures this low were abandoned. A large number of experiments were done within the range 15 to 36 C., however, and representative results are illustrated in Figures 4, 5, and 6, in addition to Figure 2. Qualitatively, no pronounced differences in the effects of pressure at the different temperatures were found. The same phenomena, and same sort of variability as described above for experiments at 15.5, were encountered at all the higher tem- peratures studied. Quantitative differences are difficult to make certain of, for the reasons already indicated. Certain generalizations, however, may be adduced from the data, as follows. First, at all temperatures the initial effect of pressure was to reduce the intensity of the flash. Second, at all temperatures a sudden compression was more effective in reducing the flash intensity than was a more gradual or step-wise increase in pressure. Third, adaptation and facilitation under pressure varied unaccountably. Out of the total number of experiments performed, they failed to occur in a larger number of instances than they did occur. In some instances they failed to occur during reasonably long periods of sustained pressure (Figs. 2, A ; 4. A; 5, B ; 6, A) even though excitability was not destroyed, as shown by recovery after decompression. In other instances, they occurred readily, sometimes resulting in flash intensities greatly exceeding those at normal pressure as already noted (Fig. 2, C, 1000 psi), or at pressures as high as 3000 psi (Fig. 5, A). 4000 psi (Fig. 6, B) and 5000 psi (not illustrated). Moreover, adaptation and facilitation sometimes occurred promptly on raising the pressure from a given high pressure, where they had not appreciably occurred, to a still higher pressure, e.g., after raising from 2000 psi to 3000 psi (Figs. 4, A and 6, B). Fourth, although sudden decompression always led to an increase in flash in- tensity, the pattern of changing maxima in successive flashes varied considerably. PRESSURE AND MNEMIOPSIS FLASH 5- ^Z O 21 4- | A 1- I 03- Q UJ X i 1 ^ ^. W2- i "i . < Til [7 b. O 1 ill III ^ l.in.ihiinilll 1 lillllllll,ll,.ll ( III 10 20 30 40 50 60 70 SECONDS 5- o 21 4 ^ B 1- I ^ O . u 3- T I < 2- u. t 1- o r 1 1 1 ,, ||||| III) , 1 5 10 15 20 25 30 SECONDS 8- i 4 X ^jl 21.6' ^ 5 7- ki C 6- : 1 ^ 5- k i O 1^ 5- * ^ O ui O ' _ o C) ^ _ _ o o X 3 ~" ^^j- ^5 fy ^ j Q ^ L_ 2- y <*i Ci (^ | $ * 1- 1 1 * iillllllllll Lin, ,,n, llllniMilMMi 1 Itiii*- 2 3 4 5 6 7 MINUTES FIGURE 4. Intensities of flash response to repeated identical stimuli, at the rate of one every 3 seconds (A and C) and of 3 per second (B). Arrows represent the time of applying or of releasing, pressure indicated as psi. Room temperature. 8 JOSEPH JIN CHANG AND FRANK H. JOHNSON In some instances there was a relatively large "overshoot" in the first one or two flashes after decompression, followed by a fairly rapid decline (Figs. 2, A; 4, C; 5, A; 5, B; 6, A), whereas in other instances decompression was followed by a more or less gradual facilitation (Fig. 2, C and Fig. 4, B, after 1000 psi; Fig. 4, A, 7- 6- 5- 4- 3- 2- I 0- 25' O CJ minutes for the controls (the nauplii from the experimental eggs became turgid and weak in their movements after hatching, and died by bursting in 15 to 20 minutes). A 0.1 M sucrose solution was used for another set of eggs. Some of the eggs already had hatched, one was in the process of hatching, and a group of five in the egg mass had not yet begun to hatch. The inner membrane of the hatching egg quickly shrank back against the larva. None of the larvae was obviously distorted from the osmotic effects of the solution, and they continued to twitch in a normal fashion. No sign of hatching was observed in any of the eggs. The eggs were maintained in the 0.1 M sucrose for 4 hours, at which time they were transferred to lake water. Immediately all of them began to swell. Spheres of normal size formed, but they were not entirely freed from the outer membranes. The nauplii were very weak. All of them hatched, but they died soon. It is thought that these deaths may have been the result of some other factor than osmotic effects, for example from anoxia. This is suggested by subsequent experiments and by the fact that some of the already hatched siblings of the experimental nauplii were placed in 0.1 M sucrose for 24 hours, then transferred to lake water, with no ill effects. A set of eggs of D. siciloides, some of which were hatching, was placed in 0.05 M HATCHING IN COPEPOD EGGS 21 sucrose solution. Some larvae began the hatching process. There was some swell- ing, but apparently insufficient pressure was built up to burst the outer membrane. The eggs were placed back in lake water after 30 minutes in the sugar solution. Hatching began immediately, the first larva being freed 8 minutes later. Another set of eggs of the same species at hatching time was placed in 0.04 M sucrose solution. Those that had already formed spheres hatched. Those still in the outer membranes (including those that had started to hatch) failed to hatch or change in any way during 17 minutes. The eggs were then placed in 0.03 M sucrose. Swelling was immediate (in 3 out of 4 eggs). One of these hatched in about three minutes. Another swelled considerably but failed to squeeze out of the outer membrane. There was no further change for 15 minutes. The remaining O.I MM FIGURE 7. Two hatching eggs of Diaptomus siciloides after immersion in 1 M sucrose solu- tion. The nauplii and the inner membranes have collapsed through the osmotic loss of water. The outer membranes are unshrunken, though somewhat contorted. eggs were then placed in 0.02 M sucrose. In 2V 2 minutes one larva had hatched, but when the membrane broke, the larva was not thrown out. In another egg a sphere was formed (136 p. in diameter considerably smaller than the average). When the membrane broke, it did so slowly, taking a full two seconds to collapse. The larva was not thrown out in the usual manner, but temporarily remained en- tangled in the collapsed membrane. The results described above are summarized, along with additional information, in Table I. 22 CHARLES C. DAVIS The resistance of the outer membrane of eggs in the early stages of hatching interfered with efforts to ascertain the approximate osmotic pressure of the fluid within the expanded inner membrane. It was necessary to experiment with spheres that already had been extruded (thus very shortly before completion of hatching). Successful observations were completed in 18 instances, results being similar both for D. ashlandi and D. siciloides. The results obtained with isolated extruded spheres are condensed in Table I. The following are representative experiments: 1) A nauplius of D. ashlandi in the twitching stage in a sphere was placed in 0.05 M sucrose. The diameter of the sphere decreased very gradually over a period of 12 seconds (plus an unknown portion of the duration of time needed to find it under the microscope). When all the space within the membrane had disappeared, the larva moved, puncturing the membrane with one of its antennae, after which it escaped. 2) A nauplius of D. ashlandi in a sphere was placed in 0.04 M sucrose. The sphere shrank. It was then placed back in lake water where it swelled up again. It was placed in 0.04 M sucrose a second time, and shrank again. The nauplius then moved, punctured the membrane, and escaped. 3 ) A larva of D. ashlandi in a sphere was placed in TABLE I Summary of the effect of various concentrations of sucrose on the swelling and hatching of eggs. (-j- = occurring, = sometimes occurring, and sometimes not, -- = not occurring.) Sucrose cone. (M) Swelling of intact egg Bursting of outer membrane Extrusion of inner membrane Swelling of inner membrane Hatching from extruded sphere Shrinking of inner membrane Lake water + + + + + 0.01 + + + + 0.02 + + + 0.03 + 0.04 + 0.05 + 0.10 + 0.03 M sucrose. The membrane shrank somewhat, but a considerable space re- mained between the nauplius and the membrane. The membrane no longer formed a perfect sphere, but was distorted to an ovoid shape with dimensions of 119 p. X 146 fji. The larva hatched successfully. From the results of such experiments, it appears that the osmotic pressure of the fluid within the extruded inner membrane was approximately equivalent to that of 0.03 M to 0.04 M sucrose. Such solutions have osmotic pressures of 0.672 to 0.896 atmosphere. The A of the fluid (A f ) would be 0.056 to 0.074. The freezing point of the Lake Erie water used was 0.03 C. The A of the internal fluids of nauplii has never been measured, but Aj for fresh-water Crustacea, as summarized by Krogh (1939) and Harnisch (1951), lies between 0.30 (Daphnia magna) and 0.81 (Potamobius). Przylecki (1921), reported by Krogh (1939), observed the A of older eggs (50-80 hours) of Daphnia magnet to be 0.74. It would therefore appear that A 4 is greater than A f , which in turn is greater than A . The above reported results indicate very clearly that the hatching of the copepod eggs studied here was by osmotic means. HATCHING IN COPEPOD EGGS 3. Test of the permeability of the egg membranes Ziegelmayer (1927) concluded that a change in the permeability of the (outer) membrane initiated the process of hatching. In contrast, Marshall and Orr ( 1954 ) suggested the possibility that there was a sudden increase of the osmotic pressure of the fluid within the inner membrane, after which hatching proceeded. To the present author this latter hypothesis seemed reasonable, for the alternate hypothesis apparently would be that the non-living inner membrane would have to change its permeability suddenly. Such a sudden change certainly would not be unexpected in a living membrane, but would not be as likely for a non-living membrane. There- fore the results reported below were unexpected. As a preliminary experiment, to test the effect of some of the higher osmotic concentrations on the nauplius itself, larvae of Diaptonms siciloides which had just hatched were placed in a series of sucrose solutions as follows: 1 M, 0.5 M, 0.4 M, 0.3 M, 0.2 M, and 0.1 M, with other nauplii remaining in lake water as controls. Larvae became contorted and succumbed instantly in 1 M sucrose. In a 0.5 M solution they died within a few seconds, and likewise showed distinct evidence of the osmotic removal of water from their tissues. Results were the same in 0.4 M sucrose, though the larvae lived somewhat longer. In 0.3 M sucrose they lived over ten minutes, but the end result was similar. In the 0.2 M solution, at the end of 10 minutes they appeared normal, but moved seldom and weakly. Subsequently they died. In the 0.1 M solution they lived normally for many hours, but moved somewhat less vigorously than did the controls. Thus the larvae can withstand a solution with an osmotic pressure as great as that of 0.1 M sucrose, but not as great as that of a 0.2 M solution. In a subsequent experiment, an egg sac of D. ashlandi containing hatching eggs was placed consecutively in 0.1 M, 0.2 M, 0.3 M, 0.4 M, and 0.5 M sucrose, and observed for shrinkage of the inner membranes and of the enclosed nauplii. No shrinkage was apparent in the 0.1 M or 0.2 M solutions. A slight shrinkage could be seen in the 0.3 M solution, but it was somewhat obscure. In 0.4 M and 0.5 M solutions, however, shrinkage was considerable. The nauplii within the shrunken inner membranes appeared to be destroyed. However, in three out of the ten eggs in the egg case, no shrinkage occurred, even in the 0.5 M solution, and the enclosed nauplii continued to twitch. A few minutes after being transferred to 0.5 M sucrose, one of the three suddenly began to shrink (not timed) but the other two remained as they were. Approximately one-half hour later the second one rather suddenly shrank. The third, on the other hand, still maintained life, and was intact, at the end of 2 1 /^> hours, although by this time (in the conditions of the experiment) considerable evaporation had occurred, and therefore the sugar concentration was higher than 0.5 M. A second egg case of D. siciloides was placed in 0.5 M sucrose. Again, three out of ten eggs failed to shrink in the 0.5 M solution, but the remainder clearly showed the effects of the hypertonic external medium. Two of the three started to shrink 19 minutes after the egg case was placed in the sucrose, and the other began 2 minutes later. The process of shrinking took approximately 3 to 4 minutes (the time at which shrinking was completed was difficult to judge exactly). One-half hour after the egg case was placed in the sucrose, it was returned to lake water. Hatching was successful in nine of the ten eggs, though the nauplii were not normal (see below in section 4 for a more detailed description of this hatching). 24 CHARLES C. DAVIS These results suggest that in those eggs where hatching had begun, or was ready to begin, the inner membrane rather suddenly became permeable to water, whereas in those eggs not yet ready to hatch the inner membrane was impermeable. To test the hypothesis that a permeability change takes place when the nauplius is ready to hatch, some eggs definitely not yet to the hatching point were tested by placing the egg cases in 0.5 M sucrose solution. The eggs of Diaptomus ashlandi, laid less than two hours previously by a gravid female, failed to shrink although they remained in the solution for an hour. Two egg cases from D. siciloides were tested. Both contained eggs with embryos that were twitching and with eyes that were fully developed. In neither did shrink- ing take place at first. In one egg case there still was no shrinking after 17 y hours, at which time it was replaced in lake water. No hatching took place, though the embryos were alive, as shown by the fact that they continued to twitch. After 8 hours the eggs were again placed in sucrose. Again no shrinkage occurred. When removed to lake water 16 hours later, the eggs appeared normal except that there was no twitching, but before long the embryos disintegrated. In the second egg case containing twitching nauplii there was no shrinkage in 0.5 M sucrose at the end of an hour. In 21A hours, however, 3 of the 16 eggs were shrunken. This egg case was thereupon return to lake water. By the time the solution was changed and the eggs located under the microscope, the shrunken eggs had swollen again, and one was beginning the process of hatching. Hatching then continued in egg after egg, and was perfectly normal in all instances except one, where the inner membrane after extrusion must have been perforated only slightly and lost its internal pressure slowly, collapsing completely around the larva, which struggled for a few seconds before it broke out. These observations confirm that there is a change of permeability of the inner egg membrane at the time of hatching. 4. Observations on the bursting of the inner egg membrane As discussed above, the nauplius always began its characteristic movements in normal hatching about a minute before the inner egg membrane burst and liberated it. It appeared as though the nauplius ruptured the membrane in some way by its activities, although it was not clear how this was done inasmuch as the rupture almost always occurred at the head end of the nauplius. Marshall and Orr (1954) said of this final act of hatching: "Quite suddenly it [nauplius] tears the membrane and swims away" (p. 393). Similarly, Ziegelmayer (1927) stated that the larva ruptured the inner membrane by the movements of its second antenna, but he thought the inner membrane was closely appressed around the nauplius after it was liberated from the outer membrane passively by an explosion-like bursting of the latter. In the observational section (section 1 above) of the present paper, the rupture of the membrane was implied to be the result of the struggling of the nauplius, because this was the way it appeared. However, one of the experiments unexpectedly gave very revealing results. As reported in section 3 (above), an egg case in which the eggs w r ere in the process of hatching was placed in 0.5 M sucrose for half an hour, then replaced in lake water. All the eggs but one hatched, but the nauplii were very weak. As many of the hatchings as possible were watched carefully and continuously until hatching was HATCHING IN COPEPOD EGGS 25 completed. If the nauplii twitched or moved at all during hatching, they did so only by very slight and slow movements of the appendages. Three hatchings were followed where the entire process took place with no evidence of any muscular movement whatsoever on the part of the nauplius. Two other cases were similar, but there were some slight movements. These, however, were by no means sufficient to burst the membrane. The remaining hatchings could not be followed throughout (and one egg did not hatch) . In spite of lack of naupliar movements, at the proper time the inner egg membrane burst and the nauplii were liberated. A second egg case (D. siciloidcs} in which hatching was taking place was treated in the same manner. Of the 9 eggs in the egg sac, 8 shrank at once or very soon after immersion in the sucrose solution. One however, shrank only just before the case was returned to lake water, one-half hour later. In the lake water all of the 9 eggs hatched, although three of them had been "hatched" artificially by the inevitable rough treatment of rapidly changing solutions (these three, although appearing normal, never moved after liberation). Of the remaining six eggs, four hatched without any movements, and after hatching, three of these never moved (the fourth moved its appendages slightly during the process of dying, immediately after hatching). One of the nauplii twitched regularly, though weakly, before hatching, but during the period of the final bursting of the membrane there was no further movement, and the larva never moved after hatching. Only one of the nauplii (presumably from the egg that shrank at the last minute) hatched normally and lived indefinitely after hatching. It is not believed that the bursting took place through the continued swelling of the sphere. Both before and after the above observations were made, numerous attempts were undertaken by measuring extruded spheres in normal eggs, to as- certain whether the swelling of the sphere continued until the time of breaking. No evidence of such growth after extrusion was obtained. 5. Attempts to demonstrate the existence of a hatching enzyme In the observations and experiments described above, the hatching eggs were immersed in less than 0.5 cc. of lake water during hatching. There never was any evidence that the liberation of a hatching enzyme by the bursting of hatching eggs speeded up the hatching of those eggs in the cluster that still remained unhatched. In the eggs of Diaptomus, as reported above, the volume of the fluid within the inner egg membrane just before the nauplius was freed averaged 1,875,400 p?. This is less than 1/300,000 the volume of 0.5 cc. ( = 5 X 10 11 /A 3 ). With such a dilution of any hatching enzyme that might be present, one would hardly expect an effect. Therefore, the volume of water involved was reduced (three experiments on D. siciloides) by drawing detached egg sacs in which the eggs were actively hatching into capillary tubes (i.d. = 1 mm.), along with half of an egg sac in which no hatching was occurring. The other half of the non-hatching batch of eggs was kept as a control. In one of the three experiments the experimental eggs were in a rather early developmental stage. There were 10 experimental eggs and 15 hatching eggs en- closed in the capillary tube, with 12.7 X 10 /A 3 of water. Hence the ratio of fluid from the bursting membranes to the amount of diluting water was approximately 1 : 450. Neither the experimental eggs nor the controls hatched. 26 CHARLES C. DAVIS In the other two similar experiments the experimental eggs were in a very late stage of development. During the experimental period, hatching occurred in the experimental eggs some time after the other eggs had hatched. However, hatching took place almost simultaneously in the controls (in both instances hatching began first, and was completed first, in the experimental eggs, but the difference is not thought to be significant, inasmuch as some of the control eggs hatched before the last of the experimental ones). Thus, the existence of a hatching enzyme was not clearly demonstrated, in the conditions of these experiments. DISCUSSION The results reported in the present paper fully confirm the osmotic nature of the hatching process in the eggs of copepods. The observations of Marshall and Orr (1954) on the events of hatching are supported and supplemented. No evidence was obtained in support of Ziegelmayer's (1927) contention that the outer membrane expanded osmotically while the inner membrane remained closely appressed around the enclosed nauplius. Furthermore, the observation reported by Ziegelmayer that the membrane began swelling 6 to 12 hours before hatching was not confirmed.. Repeated attempts to detect an increase in volume of the egg previous to the few 7 minutes before the hatching process was completed gave negative results. Marshall and Orr (1954) stated concerning the discrepancy between their observations and those of Ziegelmayer (p. 400) : "It is difficult to decide whether Ziegelmayer was unable to see the bulging out of the inner membrane or whether the specimens he examined behaved in a different way." Ziegelmayer studied 17 (unlisted) species of Cyclops. Inasmuch as Marshall and Orr observed hatching in Cyclops agilis and C. viridis, and I observed it in C. biciispidatus and Mesocyclops edax, and the behavior of all these was unlike that reported by Ziegelmayer, it would appear that his observations were faulty or deficient, either through the use of too little magnifica- tion, or through failure to follow through hatching in individual eggs. On the other hand, the results reported above support Ziegelmayer's belief that hatching is initiated by a change of the permeability of the membrane. The lack of a similar conclusion by Marshall and Orr undoubtedly is associated with their lack of extensive experimentation. From the above, two unsolved questions arise : 1 ) what is the origin and the nature of the dissolved material within the inner egg membrane that gives rise to a A f of this fluid greater than the A of the external medium, and 2) what is the cause of the sudden change in the permeability of the inner membrane? In fresh- water copepods, such as those reported here, the osmotic pressure of the fluid within the inner membrane conceivably could have its origin simply in the attainment of an equilibrium between A f and A s . However, the hatching of marine copepods, as reported by Marshall and Orr (1954), occurs in the same manner as that of the fresh-water forms. In most marine invertebrates the osmotic pressure of the internal medium is in equilibium with that of sea water, and there is no reason to believe that such species as Calanus finmarchicus, Metridia longa, and Euchaeta norvegica, which are stenohaline, are an exception. Therefore, in marine species. A f must be greater than A , and greater than A, at the time the egg was laid. No information exists at present bearing on the relation of A s of the nauplius to A f in these marine species just before hatching. A 4 might either equal A fi , or it HATCHING IN COPEPOD EGGS 27 might be less than A r . A A,- that is greater than A,, can be attained only by the action of the embryo or larva enclosed in the egg. It could result, as suggested by Marshall and Orr (1954), through the excretion of metabolic wastes by the embryo and/or nauplius, or there could be an active secretion of substances with osmotic value by special glands or gland cells (or both of these processes could be involved simultaneously). In view of the sudden change of permeability of the inner egg membrane described above, if excretory products are involved they need not be excreted suddenly as postulated by Marshall and Orr, but could accumulate grad- ually, and become osmotically effective suddenly through the rapid alteration of membrane permeability that initiates hatching. These matters can be settled only through further experimentation, particularly on stenohaline marine species of copepods. It appears unlikely that a non-living membrane, such as the inner egg membrane of copepods. would be so constituted that its chemical or colloidal nature would suddenly be altered spontaneously at the proper time for hatching. If there is no spontaneous change, the influence for the alteration must come either from outside, or from the larva inside. Conceivably, the chemical nature of the membrane could be such that bacterial action from without would alter it in a definite course of time, but such an adaptation in evolution (especially considering that the bacterial population of waters is far from constant) seems far less likely than the evolution of a special hatching enzyme whose function is the chemical alteration of the mem- brane. Such a chemical alteration could change the membrane into a semi-permeable membrane (permeable to water) from its initial impermeable condition. It is true that the preliminary experiments described in section 5 above failed to detect the presence of such a hatching enzyme, but these experiments need repetition and refinement, and furthermore, from the nature of the experiment, negative results are not conclusive (although positive results would have been). The presence or absence of glands or gland cells producing a hatching enzyme has not been as- certained, but should be demonstrable histologically. Hatching enzymes, such as are postulated above for the hatching of copepod eggs, have been proven to exist in certain fish eggs. Here, however, hatching apparently does not involve osmotic phenomena. The hatching enzyme, which is produced by special embryonic glands, digests the egg membranes, and the fry emerges more or less without the benefit of its own muscular movements (e.g., see Bourdin, 1926 and Privolnev. 1943). Similar enzymes have been proven to exist in eggs of other aquatic animals, including Rana pipiens (Cooper, 1936). On the other hand, Wilson (1958) obtained results very similar to my own in the hatching of the eggs of the nematode, Trichostrongylus retortaejormis. Despite his negative results, he concluded from qualitative observations that some ''hatching factor" is secreted which weakens the protein membrane before hatching. The hatching process itself in T. rctortaejormis he thought to be osmotically determined. Both Ziegelmayer and Marshall and Orr described the final rupture of the inner membrane as due to the active movements of the nauplius. The present results contradict this, and show that the hatching process can proceed to completion with- out any movements on the part of the enclosed nauplius. Although there apparently was no further increase of the volume of the fluid enclosed by the inner membrane during the final period of the hatching act, the possibility is not eliminated that there 28 CHARLES C. DAVIS was a continuation of the entry of osmotic water. With the membrane already stretched to its physical capacity, such a further entry would build up the internal pressure to the bursting point of the membrane. A further hypothesis suggests itself, however, namely that the membrane is destroyed chemically by a secretion from the anterior end of the larva. This would account for the fact the membrane almost invariably burst at the head end of the nauplius. These hypotheses also can be tested only by further experimentation. Pyatakov (1926) studied hatching in the arguloid, Argulus foliaceus. Although his paper dealt primarily with the formation of the seam in one of the egg membranes along which splitting occurred during hatching, it is clearly implied that the hatching process itself is similar to that occurring in the Eucopepoda. Ziegelmayer (1927) reported, but did not describe, osmotic hatching in the eggs of an isopod (Asellus) and in an anostracan (Branchipus) . Hall (1953) described hatching in the an- ostran Chirac cphalus, and suggested that osmotic factors were involved. Przylecki (1921) and Ramult (1925) have presented results, summarized by Krogh (1939) and by Needham (1931), showing that in certain Cladocera, hatching is by osmotic means. In these forms, however, hatching differs considerably from that of the Copepoda, for it is the embryo itself that swells osmotically, and its increase in vol- iime stretches the egg membrane until it bursts. A similar method of hatching was reported by Manton (1928) for Hemimysis latnornae. In some unpublished ob- servations, the present author determined that hatching in the fresh-water decapod, Palacmonctcs kadiakensis, occurs in part through osmosis. For a discussion of and references concerning osmotic hatching in other invertebrates see Needham (1931). On the other hand, all Crustacea do not hatch osmotically. Le Roux (1933) described hatching in the amphipod Gammarus, where the young emerges from the egg by the active use of special egg teeth on the telson. This method was cor- roborated by the present author in the examination of hatching in Gammarus jasciatns in western Lake Erie. SUMMARY 1. The hatching process is described for the fresh-water copepods Diaptomus ashlandi, D. siciloides, D. oregoncnsis, Cyclops bicuspidatus, and Mesocyclops cda.v. In all of these species the inner membrane expands by the osmotic entry of water. The internal pressure thus produced ruptures the outer membrane, and the inner membrane containing the nauplius is extruded, forming a sphere whose volume is more than 2^/- 2 times that of the original egg. Subsequently the inner membrane bursts and the nauplius is thrown out. 2. It is shown that the osmotic pressure of the fluid within the expanded inner membrane is equivalent to that of a 0.03 to 0.04 M sucrose solution. 3. The inner membrane remains impermeable to water until the egg is ready to hatch. Thereupon the membrane changes its permeability within a short period of time. Hatching can be prevented indefinitely in eggs that are ready to hatch by immersing them in sufficiently concentrated sucrose solution. 4. Although during normal hatching the nauplius is active for a period of ap- proximately a minute before hatching, this activity is not necessary for the com- pletion of the hatching act. Nauplii hatched, even though they had been completely immobilized. HATCHING IN COPEPOD EGGS 29 5. Attempts to demonstrate the presence of a hatching enzyme were unsuccessful. 6. It is suggested that the pre-hatching change in permeability of the membrane is caused by the action of chemicals produced by the larva. It is further suggested that the greater osmotic pressure of the fluid within the inner membrane is caused by external metabolites of the larva either excretory or secretory. LITERATURE CITED (References marked with an asterisk (*) have not been seen, but are summarized by Krogh and by Needham.) BOURDIN, JEANNE, 1926. Le mecanisme de 1'eclosion chez les teleosteens. I. fitude biologique et anatomique. C. R. Soc. Biol, 95 (32) : 1149-1151. COOPER, KENNETH W., 1936. Demonstration of a hatching secretion in Rana pipicns Schreber. Proc. Nat. Acad. Sci., 22 : 433-434. HALL, R. E., 1953. Observations on the hatching of eggs of Chirocephalus diaphanous Prevost. Proc. Zoo/. Soc. Land.. 123: 95-109. HARNISCH, OTTO, 1951. Hydrophysiologie der Tiere. Die Binnengewasser, 19: i-vii, 1-299. KROGH, AUGUST, 1939. Osmotic Regulation in Aquatic Animals. Cambridge Univ. Press, pp. 1-242. LE Roux, M. L., 1933. Recherches sur la sexualite des Gammariens. Croissance, reproduc- tion, determinisme des caracteres sexuels secondaires. Bull. Biol. France Bclqe, Suppl.. 16: 1-138. MANTON, S. M., 1928. On the embryology of a mysid crustacean, Hciniinysis lamornac. Phil. Trans. Roy. Soc. London, Scr. B.\ 216: 363-463. MARSHALL, S. M., AND A. P. ORR, 1954. Hatching in Calanus finmarchicits and some other copepods. /. Mar. Biol. Assoc., 33: 393-401. MARSHALL, S. M., AND A. P. ORR, 1955. Biology of a Marine Copepod, Calanus fimnarchicus (Gunnerus). Oliver and Boyd, Edinburgh, pp. i-vii, 1-188. NEEDHAM, JOSEPH, 1931. Chemical Embryology. Cambridge Univ. Press, Vol. 2, pp. i-xvi, 615-1253; Vol 3, pp. i-xvi, 1255-2021. PRIVOLNEV, T. I., 1943. The mechanism of hatching in fish embryos. Zoo/. Zhunial, 22 (3) : 170-173. (In Russian, English summary.) *PRZYLECKI, ST., 1921. Recherches sur la pression osmotique chez les embryons de Cladoceres, provenants des oeufs parthenogenetiques. Trav. Inst. Nencki, I. (In Polish, French summary.) PYATAKOV, M. L., 1926. The dorsal organs of Arguhts and their relation to the hatching of the larva. Quart. J. Micr. Sci., 70: 159-171. *RAMULT, M., 1925. Development and resisting power of Cladocera embryos in the solutions of certain inorganic salts. Bull. Inst. Acad. Sci. Cracovie, 1925 : 135-194. WILSON, P. A. G., 1958. The effect of weak electrolyte solutions on the hatching rate of the eggs of Trichostrongylus retortaeformis (Zeder) and its interpretation in terms of a proposed hatching mechanism. /. Exp. Biol., 35 (3) : 584-601. ZIEGELMAYER, W., 1927. Untersuchungen zum Quellungsmechanismus von Eizellen. Zeitschr. f. Zcllforschnng, 4(1): 73-124. DARK-ADAPTING AND LIGHT-ADAPTING HORMONES CONTROL- LING THE DISTAL RETINAL PIGMENT OF THE PRAWN PALAEMONETES VULGARIS 1 MILTON FINGERMAN, MILDRED E. LOWE AND BANGALORE I. SUNDARARAJ Department of Zoology, Newcomb College, Tulane University, New Orleans 18, Louisiana, and Marine Biological Laboratory, Woods Hole, Massachusetts The first direct evidence that a light-adapting hormone is involved in the regu- lation of the distal retinal pigment of crustaceans was provided by Kleinholz (1936). He found that when extracts of the eyestalks of the prawn Palaemonetes vulgaris were injected into dark-adapted specimens kept in darkness, the distal retinal pig- ment approached the fully light-adapted condition. That this hormone is normally involved in retinal pigment migration was indicated by the fact that the eyestalks of dark-adapted specimens did not contain as much light-adapting hormone as those of light-adapted individuals. Brown, Hines and Fingerman (1952) found a distal retinal pigment light- adapting hormone in the supraesophageal ganglia, circumesophageal connectives, and ventral nerve cord of Palaemonetes vulgaris. In addition, these investigators re- ported the presence in Palaemonetes of a distal retinal pigment dark-adapting hor- mone. Their evidence was indirect, having been based on differences in rates of dark-adaptation between control prawns and those injected with extracts of various organs, followed by transfer to darkness. No one has supplied direct evidence for such a hormone by causing the distal retinal pigment of a specimen of Palaemonetes, or of any crustacean, to approach the fully dark-adapted state while the specimens were kept under constant illumination (Knowles and Carlisle, 1956). The aim of the present investigation was to provide direct evidence for a distal retinal pigment dark-adapting hormone in Palaemonetes. MATERIALS AND METHODS The prawns, Palaemonetes vulgaris, used in the experiments described below were collected in the Eel Pond at Woods Hole, Massachusetts. In the laboratory the stock supply of animals was kept in aquaria with running sea water. The method used to determine the effects of tissue extracts on the distal retinal pigment was that devised by Sandeen and Brown ( 1952) . The technique involves di- rect measurement of the position of this pigment in the living animal. The prawns were placed, one at a time, ventral surface down on the stage of a stereoscopic dis- secting microscope. With the aid of an ocular micrometer and transmitted light (1) the width of the translucent portion of the compound eye in a plane parallel to the long axis of the eyestalk and (2) the length of the eye from the corneal surface to the dorsal pigmented spot at the base of the eye proper were measured. 1 This investigation was supported by Grant No. B-838 from the National Institutes of Health. 30 HORMONES AND RETINAL PIGMENT 31 To render the distal clear portion of the eye more translucent and the proximal edge of this area more definite, the prawns were submerged in a dish of sea water on the stage of the microscope. The ratio of width of clear area (measurement 1) to total length (measurement 2) will be referred to as the distal retinal pigment index. Use of this ratio minimized the effect of size differences. In the fully dark- adapted eye the distal pigment abutted against the cornea ; the distal pigment index was 0.00. In the fully lighted-adapted eye the distal pigment index was about 0.25. A typical ratio for a fully light-adapted eye was 10/40. A magnification of 60 X was used in the measurements. Each unit of the ocular micrometer at this magnification was equivalent to 24.4 p.. The distal pigment index of 10 prawns could be determined with ease in three minutes. For all experiments the specimens were placed into black enameled pans con- taining sea water approximately 2.5 cm. deep. The pans were then exposed to an illumination of 20 ft. c. At this intensity the distal retinal pigment was about one- third of the distance toward the fully light-adapted position from the fully dark- LJ OX 0.15 Q.IO cO Q O.O5 FIGURE 1. 3 HOURS Responses of the distal retinal pigment to an extract of tritocerebral commissures, circles. Control, dots. adapted one. Under these conditions the distal pigment could respond to either light-adapting or dark-adapting hormone. Specimens with one eyestalk removed received the injections. Removal of one eyestalk resulted in the loss of an important source of retinal pigment light-adapting hormone ( Brown, Hines and Fingerman, 1952). Presumably, therefore, one-eyed prawns would not be as readily able to antagonize any injected dark-adapting hormone as would intact specimens. Extracts of eyestalks and of supraesophageal ganglia plus the circumesophageal connectives were prepared as follows. The organs to be assayed were extirpated and placed in sea water. When the desired number of each organ was available, the organs were transferred with a minimum of sea water to a glass mortar, trit- urated. and suspended in a sufficient volume of sea water to yield the desired con- centration. When the extracts of sinus glands and optic ganglia were prepared, these tissues, because of their small size, were placed directly into mortars rather than into sea water. Every extract was assayed on 10 specimens. Control speci- mens were injected with 0.02 ml. sea water. All experiments unless otherwise 32 FINGERMAN, LOWE AND SUNDARARAJ stated were performed three times. Student's t test was used to determine the level of significance. The 95 % level was taken as the minimal value for a significant difference between two means. EXPERIMENTS AND RESULTS Influence of the tritocerebral commissure on the distal retinal pigment Brown, Hines and Fingerman (1952) postulated that the tritocerebral com- missure that runs posterior to the esophagus from one circumesophageal connective to the other contains little or no light-adapting hormone but does possess dark- 0.25 XQ.20 o z z u 5 O 0.15 0.10 0.05 0.00 I FIGURE 2. I 2 3 4 J 6 HOURS Responses of the distal retinal pigment to eyestalk extract, circles. Sea water control, dots. adapting hormone. The first experiment was designed to test this hypothesis and to determine if the distal retinal pigment of specimens kept under constant illumination could be made to approach the fully dark-adapted state. A sufficient volume of an extract containing three tritocerebral commissures in each 0.02 ml. sea water was prepared. In Figure 1 are shown the results obtained when 10 prawns were each injected with 0.02 ml. of this extract. A transitory light-adaptation was produced that was followed by a dark-adaptation that lasted for several hours. This dark- adapting effect was highly significant statistically but the light-adaptation was not. The same experiment was also performed with one and two tritocerebral com- missures per dose. In both of these experiments the distal retinal pigment became HORMONES AND RETINAL PIGMENT slightly more dark-adapted than the controls but the differences were not statistically significant. The data of these experiments are, therefore, not included herein. Distal retinal pigment dark-adapting hormone in the cyestalk of Palaenionetes The aim of this series of experiments was to ascertain whether a dark-adapting hormone is present in the eyestalks of Palaenionetes. For the first experiment of this group, eyestalks were extracted in a sufficient volume of sea water to yield a x Ld Q < c/) 0.25 0.20 - 0.15 h- Z Ld o al Q_ 0.05 0.00 HOURS FIGURE 3. Responses of the distal retinal pigment to extracts of sinus glands (dots) and optic ganglia (circles). Sea water control, half-filled circles. final concentration of one-third of a pair per 0.02 ml. This extract was injected into 10 specimens and its effect determined over a period of seven hours. Control speci- mens were also used. A strong light-adaptational response was observed. This was followed by a large dark-adaptational response. Because of the importance of this experiment it was done five more times. The data for the six experiments were averaged. The results are presented in Figure 2 where each point represents the mean of 60 individuals. These results are statistically significant. 34 FINGERMAN, LOWE AND SUNDARARAJ The sinus gland in the eyestalk of crustaceans is thought to be merely a storage and release center for neurosecretory products produced elsewhere, e.g., in the optic ganglia (Knowles and Carlisle, 1956). The aim of the next experiment, therefore, was to determine whether the two retinal pigment hormones are found in the sinus glands and in the optic ganglia. These structures were dissected out, triturated, and suspended in sufficient sea water such that the final concentration was one-third of a complement per 0.02 ml. The experiment was performed three times with the 0.25 XQ.20 Q Z z 0.15 LJ o Q_ 0.10 0.05 0.00 I 01 2345 HOURS FIGURE 4. Responses of the distal retinal pigment to extracts of supraesophageal ganglia with the circumesophageal connectives attached from which the tritocerebral commissures had been removed (circles). Sea water control, dots. same results. A light-adaptational response occurred that was followed by a dark- adaptational one (Fig. 3) just as \vas found with extracts of whole eyestalks (Fig. 2). The amplitudes of the responses show r n in Figure 3 were slightly less than in Figure 2, presumably because of the decreased quantity of hormonal material in the extracts when the components of the eyestalks were separated from one another. The responses of the prawns to the extracts of sinus glands and optic ganglia were strikingly similar. Since the volume of the sinus gland is about one per cent HORMONES AND RETINAL PIGMENT 35 that of the tissue in one eyestalk, the concentration of the hormones must be much greater in the sinus glands than in the optic ganglia. An objection may be raised to the interpretation that the dark-adaptational re- sponse is due to a dark-adapting hormone, namely that the response is merely over- compensation on the part of the organism when removing the injected light-adapting hormone from the blood. To offset such an objection the final experiment was performed. Supraesophageal ganglia plus the circumesophageal connectives were dissected out. The tritocerebral commissures were then removed from these organs. These supraesophageal ganglia with the circumesophageal connectives attached were then extracted in sufficient sea water so that the final concentration was one- third of a complement per 0.02 ml. Such an extract would contain considerable light-adapting hormone in the virtual absence of a dark-adapting substance. The extract was then injected into 10 specimens. This experiment was also done three times. The averaged results (Fig. 4) revealed a statistically significant light- adaptational response and no dark-adaptation. If the dark-adaptational response shown in Figures 2 and 3 had been merely overcompensation then it would have occurred here also. DISCUSSION The results presented herein provide direct unequivocal evidence for a distal retinal pigment dark-adapting hormone in Palaemonetes. The indirect evidence for this endocrine factor presented by Brown, Hines and Fingerman (1952) finds support in these experiments. The results represent the first time that dark- adaptation has been induced in light-adapted specimens kept under constant illumination. The dark-adapting hormone appears to be subordinated to the light-adapting one, being able to function only after the latter hormone has run its course. However, the effect of the dark-adapting hormone persists much longer than that of the light- adapting substance (Figs. 2 and 3). The presence of these antagonistically functioning hormones probably provides Palaemonetes with more precise control of the position of its distal retinal pigment than it would have if these prawns produced light-adapting hormone alone. The prawns can secrete an antagonist when the pigment must be moved rapidly to the dark-adapted state rather than be forced to wait for the light-adapting hormone to be eliminated from the circulation. As information is being gathered about en- docrines in crustaceans, we find more instances where processes are controlled by oppositely functioning substances. Such was also the case with the red chroma- tophores of Palacuwnctcs. Brown, Webb and Sancleen (1952) demonstrated red pigment concentrating and dispersing hormones in this prawn where only the con- centrator had been found previously. SUMMARY AND CONCLUSIONS 1. The distal retinal pigment of the prawn Palaemonetes vidgaris is regulated by light-adapting and dark-adapting hormones. 2. These hormones are found in the sinus glands and central nervous organs. 3. The dark-adapting hormone was demonstrated by inducing with tissue ex- tracts dark-adaptation of the distal retinal pigment of light-adapted specimens 36 FINGERMAN, LOWE AND SUNDARARAJ maintained under constant illumination, the first time this has been accomplished in any crustacean. LITERATURE CITED BROWN, F. A., JR., M. N. HINES AND M. FINGERMAN, 1952. Hormonal regulation of the distal retinal pigment of Palaemonctes. Biol. Bull., 102 : 212-225. BROWN, F. A., JR., H. M. WEBB AND M. I. SANDEEN, 1952. The action of two hormones regulating the red chromatophores of Palacnioiietcs. J. Exp. ZooL, 120:391-420. KLEINHOLZ, L. H., 1936. Crustacean eye-stalk hormone and retinal pigment migration. Biol. Bull,, 70: 159-184. KNOWLES, F. G. W., AND D. B. CARLISLE, 1956. Endocrine control in the Crustacea. Biol. Rev., 31 : 396-473. SANDEEN, M. I., AND F. A. BROWN, JR., 1952. Responses of the distal retinal pigment of Palae- monetcs to illumination. Physiol. ZooL, 25 : 223-230. HISTOPHYSIOLOGY OF GILL AND KIDNEY OF CRAB OCYPODE ALBICANS SARAH C. FLEMISTER Edzi'ard Martin Biological Laboratories, Szvarthnwre College, Sivarthmorc, Pennsylvania, and Bermuda Biological Station, 1 St. George's ll'sst, Bermuda Brachyuran crabs possess the ability to regulate the internal level of the chloride ion against shifts in the external level of this ion. Webb (1940) and Jones (1941) showed that this ability varies in different species of crabs, and becomes functional over an increasingly wider range as the animals move away from the sea into an estuarine or shore habitat. Data presented by Flemister and Flemister (1951) indi- cated that the ghost crab, Ocypode albicans (Bosq), is able to regulate the internal chloride ion against a hypotonic environment of 200 millimoles of chloride per liter, and a hypertonic environment of 600 millimoles per liter. That is, within this range of environmental chloride ion, the internal chloride ion of the crab is main- tained at 375 millimoles per liter. Such regulation is accomplished by reciprocal mechanisms for uptake and loss or absorption and secretion of the chloride ion, these mechanisms being located in cells which occupy appropriate sites in reference to the external and internal environments. The principal site of chloride ion uptake by decapod Crustacea is thought to be the gills. The uptake of ions from the environment by fresh water animals was reported by Krogh (1937) as a probable function of the gill. Webb (1940) sug- gested that the histology of the gill of Carcinus inaenas was compatible with the process of salt and water transfer. Isolated gills of the crab Eriocheir sinensis were shown to absorb ions from the environment by Koch, Evans and Schicks (1954). Chloride ions are excreted from the body by the kidney or antennal gland. It has been established that under conditions of excess chloride ion in the environment, the urine secreted by the kidney contains a higher proportion of chloride ion than it does under conditions of low chloride ion in the environment. An investigation of the relation of oxygen consumption to chloride ion regulation reported by Flemister and Flemister ( 1951 ) led to the conclusion that chloride ion regulation by the kidney was supplemented by the activity of some other tissue or tissues. Excretory cells are found in the gill and in the hepato-pancreas, in addition to the kidney. Early accounts of crustacean anatomy by Cuenot (1895) and Pearson (1908) describe these cells and attribute an excretory function to them. A more recent study by Lison (1942) emphasizes their possible excretory role. These findings suggest that gill tissue may be active in chloride ion loss, as well as uptake. The hepato- pancreas is likewise a site at which chloride ion may be absorbed or excreted, al- though its role in regulation is doubtful. Travis (1955) has described the functional histology of this structure in detail. Similarly, the tufts of branchial epithelium 1 Contribution No. 250 from the Bermuda Biological Station. Assisted by a Grant-in-Aid from the National Science Foundation through the Bermuda Biological Station. 37 38 SARAH C. FLEMISTER which partially line the gill chamber of Ocypodc alhicaus occupy a position in which they might contribute to the transfer of ions and water. An investigation of the histophysiology of gill, kidney and branchial epithelium of Ocypodc albicans was undertaken with the special objective of examining their role in the transfer of the chloride ion. METHODS Ocypodc albicans was collected in the summer months on the beaches at Reho- both, Delaware, and at Bermuda in March and April. The animals were brought into the laboratory where they were maintained on damp sand and offered food until they were subjected to experimental treatment. The entire period of their stay in the laboratory was not longer than ten clays or two weeks. No animals showed indications of impending molting and none molted in the laboratory. Animals were acclimatized in sea water containing, respectively, 200, 400 and 600 millimoles of chloride per liter. The chloride ion level of the blood of Ocypode albicans is maintained in the range of 375 to 400 millimoles of chloride per liter ; thus the range of salinities was hypotonic, isotonic and hypertonic in reference to the internal chloride ion concentration. Animals remained in the experimental tanks for seventy-two hours. Blood and urine were collected and analyzed for chloride ion content before and after acclimatization, using methods described in a previous paper (Flemister and Flemister, 1951). At the end of the period of acclimatization, tissues were removed for study. Tissues taken included gill and antennal gland, which were fixed without further dissection, and the branchial epithelium. This latter tissue in Ocypodc albicans is the tufted lining of the inner face of the branchial chamber. It was removed, cut into two portions and these fixed flattened out. Fixation in formalin or Bourn's fixative, followed by hematoxylin and eosin or Mallory's connective tissue stain, was used for general histological examination. Regaud's fixative followed by post- chroming resulted in fixation of mitochondria which were then stained by iron hema- toxylin or Altman's acid aniline fuchsin. The Leschke method for the detection of chlorides was used according to the sequence described by Copeland (1948). Tissues were removed from the animal and fixed, without washing, in one per cent silver nitrate made acid with nitric acid. This fixation was accomplished in the dark, as was the development in Eastman D-ll (diluted 1:4) and final fixing in Eastman F-5 (diluted 1:5). The tissues were then washed, dehydrated, embedded in paraffin and sectioned. Sections were gold toned and sometimes counter-stained with eosin. The picture of silver de- position which resulted, as will be discussed below, led to testing for a clue as to the nature of the material reacting with the silver. Polyphenols, urates and fats might possibly be expected to react with the silver in the procedure described. These were individually tested for by the following procedures. The Hollande method for the detection of urates was used as described by Click (1948). This involved fixation in one per cent silver nitrate solution in neutral formalin, in the dark. Polyphenols were tested for by treating sections of formalin- fixed material in ammoniacal silver nitrate in the dark by Masson's method (Lison, 1936). Reduced silver deposits in each of these indicates presence of material tested for. Some formalin-fixed tissues were washed and imbedded in Carbowax CRAB GILL AND KIDNEY 3 C ) after the method of Blank and McCarthy (1950) and the sections stained with Sudan III for the detection of fats. RESULTS The gill Descriptions of the histology of decapod crustacean gills are to be found in the paper of Cuenot (1895) and the monograph on Cancer by Pearson (1908). The gill of Oc\f>odc olbicans, as that of other brachyurans, is formed of a number of lamellae, or broad flattened plates arranged serially in pairs along a central gill stem (Fig. 1). The gill stem provides support for the lamellae and is the pathway for the afferent and efferent branchial vessels. The entire outer surface of the gill is covered by a thin layer of chitin which is about 1 /x in thickness. The individual leaflet or lamella may be likened to a flattened thin-walled sac. Underlying the chitin is a continuous lining of epithelial cells (Fig. 2). At ir- regular intervals the faces of the lamella are joined by large cells whose cytoplasm contains distinct fibrils, and which constitute pillar cells. The distal border of the lamella is expanded, being free of pillar cells and traversed by an occasional con- nective tissue fiber. The irregular cavity within the lamella resulting from this arrangement of pillar cells and fibers is filled with blood in life and an occasional blood cell is seen in sections. The lamellar blood space communicates with the afferent and efferent branchial vessels. The epithelial cells of the lamellae are continued as the lining of the gill stem. Collagen fibers of some thickness are found in the stem itself. Large connective tissue cells conforming to the classifica- tion of Leydig cells of the first order, according to Kiikenthal (1926-1927), com- pose the chief support of the gill stem. Smaller spindle-shaped Leydig cells of the third order may also be found. Blood cells are commonly seen lying in the in- terstitial spaces. In addition to the cells described, there is yet another type which appears to be unique to the gill. It was termed a branchial excretory cell by Cuenot (1895) and a branchial athrocyte by Lison (1942). These cells are large, oval in shape, with the cytoplasm arranged in a peripheral layer surrounding a vacuole. The nucleus is displaced to one side, close to the cell membrane. The vacuole contains material which in fixed sections appears as an aggregate of granules incompletely filling the space. The cells are arranged in irregular rows or aggregates which protrude into and are bathed by the blood which passes through the stem. Lison (1942) and earlier investigators have observed that certain classes of dyes injected into the blood stream may be accumulated by these cells and from this function is derived their classification as branchial excretory cells. In mitochondrial preparations it is immediately apparent that the lamellar epithelium is rich in mitochondria, while the branchial excretory cells show a sparse or absent population (Fig. 3). It is also apparent that although the lamellar epithe- lium is continuous with the lining of the gill stem, there is a marked decrease in the mitochondrial count in the cells lining the gill stem. The mitochondria may be filamentous or arranged in minute rows of granules ; they occupy the cytoplasm of the cell on either side of the nucleus, and do not appear to be constantly located either toward the base or toward the distal surface (Fig. 4 ). In preparations made 40 SARAH C. FLEMISTER FIGURE 1. Portion of gill. 17 X- Several lamellae attached to central stem. Afferent, ventral and efferent, dorsal, vessels appear as light, roughly circular areas. Darker part of stem is area of branchial athrocytes. FIGURE 2. Longitudinal section of stem of gill with several lamellae. 200 X. Note loosely packed athrocytes, blood spaces and occasional connective tissue fibers in stem. Lamellae are lined by epithelium below a thin chitin cover. Pillar cells appear to join faces of lamellae. FIGURE 3. Portion of stem and lamella stained with iron hematoxylin. 380 X. Mito- chondria show as dark clumps in lamellar epithelium. Athrocyte in extreme left center field shows enclosed material lightly stained. Very dark cells in stem are blood cells. FIGURE 4. Section of lamella, iron hematoxylin. 860 X. Mitochondria appear as dark clumps and threads. Chitin covering heavily stained. CRAB GILL AND KIDNEY 41 from crabs acclimatized to 200 millimoles of chloride per liter the lamellar cells show some vacuolation ; in such instances the mitochondria line up at the borders of the vacuoles. Vacuolation is less in lamellar cells of material taken from crabs which had been living in sea water containing 400 and 600 millimoles of chloride per liter, but otherwise the picture is the same. Mitochondria appear as scattered granules in the peripheral cytoplasm of the branchial excretory cells. However, the material present in the vacuoles of these cells often stained with the mitochondrial stain. The Leschke test for the detection of chlorides gave clear results on one point: there is never, under any circumstances, any evidence of silver deposition in the lamellar epithelium, the epithelium of the stem, any of the connective tissue of the stem, or in any of the blood cells. There is blackening of the covering chitin, as would be expected since the tissues were not washed before fixing. There is occasional outlining of the nucleus, and connective tissue fibers may show blackening. In all of the tissues prepared from Rehoboth Beach crabs there is blackening of the material contained within the vacuole of the branchial excretory cells. This is not uniformly true of the crabs taken in Bermuda. Careful examination of material taken from crabs from the three classes of environmental situations shows no obvious quantitative differences which might reflect activity in the regulatory mechanism. The interpretation of the occurrence of deposits of reduced silver as evidence of the localization of chloride is open to dispute. The Leschke method depends upon the formation of silver chloride after treatment with acid silver nitrate and sub- sequent reduction of silver by means of a quinone-containing solution. Such a method was meticulously explored by MacCallum (1905) using a variety of biolog- ical materials. It was his conclusion that only halides of all substances in biological materials would give this particular reaction. It is to be noted that in his experi- ments, proteins and other substances used were purified by repeated treatment to rid them of all free chloride. His conclusions have been used by a number of workers to justify their results with the Leschke method. Keys and ^'illmer (1932) used this method in determining the location of the "salt cells" of fish. There are two lines of objection to a blanket acceptance of silver deposits as evidence of the localization of chlorides. One of these is based on the highly diffusi- ble nature of the chloride ion which tends to move freely in biological fluids and across membranes. Silver is a heavy metal and tends to be adsorbed upon mem- branes so that penetration of the solution may be uneven. Thus, any reduced silver found after the treatment is complete may, or may not, represent a true localization of chloride present in 1'k'o. The second objection, which in some respects is more serious, is that certain substances found in tissues are capable of immediately re- ducing acid silver nitrate in the dark. The best known of these is Vitamin C, as- corbic acid, and this property is the basis of the method of Giroud (1938) for de- termining the location of Vitamin C in tissues. Polyphenols give a silver precipitate after treatment with ammoniacal silver nitrate, and urates yield a silver precipitate after treatment in neutral silver nitrate. Tissues from Ocypode albicans subjected to these tests showed no evidence of reduced silver in the locations observed after the Leschke test or in any other locations. Vitamin C is regularly found in the cytoplasm of the cell, occupying a position in the neighborhood of the Golgi appara- tus. Thus the nature of the material contained within the vacuoles of the branchial 42 SARAH C. FLEMISTER IP Iro^rtr^fcta^^ By^p FIGURE 5. Section of kidney, hematoxylin and eosin-stained. 100 X. Typical view of labyrinth, showing renal tubule cells surrounding saccule cells. The latter are very pale. Occasional very dark cells between the two layers are blood cells in the blood spaces. FIGURE 6. Labyrinth, hematoxylin and eosin. 380 X. The lower left hand portion of the picture shows renal tubule cells. Note brush border, and the secretion bleb being extruded into the lumen. In the upper right hand corner are a few saccule cells, very pale and appearing almost empty. FIGURE 7. Labyrinth, iron hematoxylin. 380 X. At top renal tubule cells surround saccule cells which have not been stained by iron hematoxylin. Section of renal tubule at bottom shows mitochondria situated towards the hemocoele, in the basal portion of the cells. CRAB GILL AND KIDNEY 43 excretory cells is still undetermined. It seems reasonable to speculate, however, that under some circumstances this might he a complex molecule which is capable of combining loosely with chloride. An alternate interpretation would be that it is a molecule which under some circumstances carries a reducing radical of yet un- determined nature. The material is not fat : it persists after normal dehydration and clearing procedures ; it stains with cytoplasmic stains generally ; it does not stain with Sudan III after Carbowax embedding and sectioning. What it is re- mains undetermined ; that the reduced silver indicates the location of a chloride re- mains to be positively substantiated. The lei el n cv The histology of decapod crustacean kidneys is amply treated in the descriptive works of Marchal (1892) and Pearson (1908). The kidney of Ocypode albicans conforms to these descriptions. The labyrinthine structure is the result of the growth in close proximity of two sac-like portions of the excretory tubule. The floor < if the more dorsal end sac pushes into the roof of the more ventral renal tubule with a consequent close interdigitation of the layers. The lumen of the end sac communicates with the lumen of the renal tubule which in turn empties through a bladder to the outside. There is no direct connection between the hemocoele and the lumen of the excretory apparatus ; all materials eliminated must pass through the cells either of the end sac or renal tubule. Typical sections through the kidney show portions of the end sac, renal tubule, and areas of interdigitation (Fig. 5). Comparisons of end sac epithelium and renal tubule epithelium can be made easily in the areas of interdigitation, at which locations the end sac epithelium always constitutes the inner layer of cells, surrounded by an outer layer of renal tubule epithelium. The appearance and staining capacities are sufficiently different so that renal tubule cells may always be distinguished from end sac cells. Renal tubule cells are cuboidal in shape, stain deeply and have a well de- nned brush border on the surface of the cell facing the lumen (Fig. 6). In contrast the cells of the end sac are large oval or cuboidal cells generally arranged in one layer although they may occasionally form two indistinct layers. A considerable portion of the cell is occupied by a vacuole which may contain granular material, and the nucleus is consequently displaced to one side. Staining is invariably light or pale in contrast to the deeper staining renal tubule cells. The end sac cells resemble closely the branchial excretory cells described for the gill, and they have been termed kidney athrocytes by Lison (1942). Mitochondria are found in renal tubule cells, usually as filaments occupying the area of the cell towards the hemocoele (Fig. 7). There are generally few or no mitochondria observed in the end sac cells, and the material of the vacuole does not stain with the mitochondria! stain. Kidney tissue treated by the Leschke method shows silver deposition as follows. The contents of the vacuoles of the end sac cells are blackened. The degree of blackening is not uniform, and shows no correlation with the observed regulatory FIGURE 8. Labyrinth, silver fixation. 860 X. Portion of renal tubule from crab acclima- tized to hypertonic environment. Heavy deposition of silver apparently in brush border area of cell, and outlining secretion blebs. Note that silver also accumulates within cell in discrete particles. They are not nuclei, and significance of their occurrence is not known. 44 SARAH C. FLEMISTER activity of the animal. Tests similar to those made on gill tissues for polyphenols, urates and fats gave negative results. The nature of this material is undetermined. In kidney tissue removed from crabs acclimatized to 600 millimoles of chloride per liter there is a pronounced deposit on the lumen side of the renal tubule cell (Fig. 8). This blackening appears to involve the brush border as well as the immediately ad- jacent lumen edge of the cell. The blebs of secretion characteristic of this type of cell are also outlined with faint depositions of silver. The cytological picture here coincides with the known physiological activity of the kidney, which is excretion of excess chloride under the conditions of regulation to the hypertonic environment. There seems to be little doubt that the site of excretion is through the renal tubule cells. Silver deposition in kidneys taken from animals acclimatized to 200 milli- moles of chloride per liter shows along the lumen border of cells, but not out- lining the secretion blebs; the silver always seems to be within the cell. In several specimens from Bermuda, the sections show no silver except at the blood side of the cell. This was not observed uniformly in the tissues of crabs acclimatized to a hypotonic environment. Under such conditions the kidney is excreting a dilute urine with reference to the chloride content and it is perhaps impossible to detect accumulation or reabsorption of chloride which might be occurring. The branchial epithelium The lining of the inner surface of the gill chamber is the branchial epithelium, which in Ocypodc albieans is developed into numerous tufts which presumably offer increased surface for respiratory exchange. The membrane is composed chiefly of a large blood space surrounded by the stellate and spindle-shaped connective tissue cells characteristic of Crustacea. The main blood channel protrudes into finger-like projections which follow poorly denned ridges. Within the network of connective tissue are scattered large cells corresponding to the reserve cells of Cuenot (1895). These cells have the property of accumulation and storage of proteinaceous materials. The outer covering is composed of a single layer of flattened epithelium covered by a very thin layer of chitin. None of the cell types show any evidence of possible active absorption or secre- tion. The mitochondria! picture does not indicate such activity on the part of any cells ; there are no athrocytes ; and there is no evidence of tegmental glands as- sociated with this epithelium. It seems probable that respiratory exchange occurs across this membrane, although the contiguity of external and internal environments is not as close as in the gill lamellae. Although salt transfer, and that of water, could be accomplished by direct osmotic forces it does not seem possible that this membrane takes an active part in salt and water regulation. DISCUSSION The microscopic anatomy of the gill and kidney of Ocypode albicaiis can be re- lated to the function of transfer of chloride ion between the blood of the animal and the environment. The hepato-pancreas and the branchial epithelium are likewise areas at which transfer may occur, but these sites do not appear to be concerned with regulation. Gill and kidney on the other hand are not only areas of passive transfer, but are also elements of the regulatory mechanism. The regulatory mechanism has CRAB GILL AND KIDNEY 45 been demonstrated by Flemister and Flemister (1951) to l)e effective in Ocypodc albicans over a hypotonic and hypertonic range of environmental chloride ion con- tent, and it has been further demonstrated that this regulation requires the expendi- ture of energy. The chief portal of entry of the chloride ion into the body of the crab is at the gill surface. The lamellar cells which constitute the cellular surface of the gill are closely associated with the blood stream, which bathes one surface, and the external environment which bathes the other surface. The cells give evidence of being active in some secretion or absorption process by their rich population of mitochondria, which is a generally accepted sign of a metabolically active cell. The lamellar cells under no circumstances showed any evidence of accumulation of chloride ion, and it is assumed that the absorption process is a continuing one and does not involve even temporary accumulation within the cell. These same cells are the final route of BLOOD cr LAMELLAR CELL H 2 O + CO 2 C.A. HC0 3 ~ H+ t H 2 C0 3 ENVIRONMENT HCo: cr FIC.URE 9. carbon dioxide as it leaves the body. It is known from the work of Ferguson, Lewis and Smith (1937) that the gills of crustaceans contain a large amount of the enzyme carbonic anhydrase as compared with the blood or other tissues taken from the animals. The diagram (Fig. 9) shows how the excretion of carbon dioxide may be related to the function of chloride ion uptake by the gill of the crab. Carbon dioxide from the body tissues arrives at the lamellar cell in the form of bicarbonate, and is transferred across the cell to the environment. As bicarbonate leaves the cell, chloride ion enters, maintaining the ionic balance ; similar exchange occurs at the blood surface of the cell, with the net result that chloride ion enters the blood stream as carbon dioxide is lost from the body. This uptake is augmented by some specific cellular activity which results in the production of carbon dioxide which is removed from the cell as bicarbonate produced by the activity of carbonic anhydrase. It is believed that this absorption mechanism is working under all conditions of tonicity of the environment, and is independent of the ion content of the environment. It 46 SARAH C. FLEMISTER is effective in supplying enough chloride ion to maintain the internal level until the external level falls below about twenty-five per cent of the internal level (Flemister and Flemister, 1951). Below this level it is still working as can be observed from the uptake experiments cited above. When the crab is subjected to a hypertonic environment, the mechanism is still at work, and supplements the osmotic force tending to drive chloride ion into the body. The internal chloride ion level then is maintained by excretion of excess chloride ion by the kidney, and perhaps at other sites. It is interesting to note that in Gecarcinus lateralis as reported by Flemister (1958) the blood chloride ion level tends to rise above the normal level when the crab is living in hypertonic environment. It appears that the excretory mechanism cannot keep up with the intake in this particular situation. The renal tubule cells of the kidney are the principal sites of chloride ion ex- cretion. The cells lie between the blood stream and the lumen of the kidney which communicates with the exterior ; they show the brush border and mitochondria characteristically associated with absorbing or secreting cells ; it is reported by Krug- ler and Burkner (1948) that alkaline phosphatase is found in these cells; and the cytological picture resulting from the Leschke test adds evidence that chloride ion may be excreted from the body at this site. Analysis of the urine of crabs reported by Flemister and Flemister (1951) showed that there is always chloride ion in the urine, and the amount increases directly with increasing chloride ion in the environ- ment. The mechanism of secretion is not known, but it appears to be a function of the renal tubule cells. There is no evidence here that these cells can reabsorb chloride ion from the urine. Hence the renal tubule cells are the route of chloride ion out of the body. The athrocytes of the end-sac and the branchial athrocytes are concerned with the removal of large, poorly diffusible molecules from the blood stream. According to Lison (1942) this is accomplished by a process of accumulation, the exact nature of which is not understood. Final removal from the body results from the breaking away of the end-sac athrocytes so that they float freely in the lumen of the kidney and are lost from the bodv with the urine. There is no such obvious final route j for the gill-stem athrocytes, and they may perhaps be looked upon as analogous to the fixed macrophages of the vertebrate reticulo-endothelial system. However, it should be noted that specific evidence for a phagocytic action is lacking. It is dif- ficult to imagine how the athrocytes could be involved in the mechanism resulting in chloride ion regulation, unless the ion is somehow attached to a large poorly diffusible molecule within the cell, and thus removed from the blood stream. If the Leschke test is assumed valid as an indicator of the presence of halides, then the athrocytes contain halides in quantity far greater than other cells. There is no quantitative difference in the amount of halicle bound by the athrocytes of crabs taken from hypotonic or hypertonic environments. Perhaps the only function of these cells is accumulation, and there can be no eventual release back into the blood stream. These cells would then act antagonistically to the lamellar cells which are continually absorbing chloride ion from the environment, and they would supplement the function of the renal tubule cells. It is to be noted that this speculation is based on an assumed validity of the Leschke test. There is some indication that the kidney is not the only site of chloride ion loss from the body, according to Flemister (1958). Granting that the role of the CRAB GILL AND KIDNEY 47 athrocytes is hypothetical, one other source of leaking of chloride ion could be at the branchial epithelial surface. Since this surface is relatively thin and lies between blood stream and environment, there is ample opportunity here for exchange of ions as a result of osmotic differentials. Thus the branchial epithelium might serve as a portal of entry of chloride ion in a hypertonic medium, a site of chloride ion loss in a hypotonic medium. The regulation of the blood chloride ion level must depend on those cells which are active in absorbing or secreting chloride ions, those of the lamella of the gill and the renal tubule of the kidney. SUMMARY 1. The fine structure of the gill and kidney of Ocypode albicans was examined for evidence of participation in the transfer of water and the chloride ion. 2. The epithelium of the gill lamellae was found to have the characteristics of a secreting epithelium. No other cells associated with the gill structure had either the position or morphology to be considered important in this function. The athro- cytes of the gill stem probably do not participate in salt-water regulation, but are concerned principally with the removal of poorly diffusible ions from the blood stream. No mechanism of such removal can be interpreted from the present study. 3. The cells of the renal tubule of the kidney were found to have the charac- teristic brush border and mitochondrial picture associated with actively secreting or reabsorbing epithelia. Silver deposition following the Leschke test gave evidence that these cells are involved in the excretion of chloride, and may possibly also act to reabsorb chloride from the urine. The athrocytes of the kidney end-sac, like those of the gill stem, probably do not function in salt or water regulation. Materi- als accumulated within these cells are lost to the body when the cells break away and float free in the urine. 4. Absorption of salt from the environmental medium is accomplished by an energy-using mechanism in the lamellar cells. This is a constant function and is associated with the carbonic anhydrase mechanism working in the excretion of carbon dioxide. Excretion of salt by the renal tubule is probably also a constant function, although no evidence as to its possible mechanism is available. Reabsorp- tion of salt at this location is a possibility ; the triggering mechanism is probably a falling chloride concentration in the blood. LITERATURE CITED BLANK, H., AND P. L. MCCARTHY, 1950. General method for preparing histologic sections with a water soluble wax. /. Lab. and Clin. Med., 36: 776-781. COPELAND, D. E., 1948. The cytological basis of chloride transfer in the gills of Fundulus heteroclitus. J. Morph., 82: 201-227. CUENOT, L., 1895. fitudes physiologiques sur les Crustaces decapodes. Arch, de Biol., 13 : 245-303. FERGUSON, J. K. W., L. LEWIS AND J. SMITH, 1937. The distribution of carbonic anhydrase in certain marine invertebrates. /. Cell. Com p. Physiol., 10 : 395-400. FLEMISTER, L. J., 1958. Salt and water anatomy, constancy and regulation in related crabs from marine and terrestrial habitats. Biol. Bull., 115: 180-200. FLEMISTER, L. J., AND S. C. FLEMISTER, 1951. Chloride ion regulation and oxygen consumption in the crab Ocypode albicans (Bosq). Biol. Bull., 101 : 259-273. GIROUD, A., 1938. L'acide ascorbique dans la cellule et les tissus. Protoplasma Monographs, Berlin. 48 SARAH C. FLEMISTER CLICK, D., 1948. Techniques of Histo- and Cytochemistry. Interscience Publishers, Inc., New York. JONES, L. L., 1941. Osmotic regulation in crabs. /. Cell. Com p. Physio!., 18: 79-92. KEYS, A., AND E. N. WILLMER, 1932. Chloride secreting cells in the gills of fish with special reference to the common eel. /. Physio!., 76 : 368-378. KOCH, H. J., J. EVANS AND E. SCHICKS, 1954. The active absorption of ions by the isolated gills of the crab Eriochcir sinensis. Med. Kon. Acad. Weten., 16:1-16. KROGH, A., 1937. Osmotic regulation in fresh water fishes by active absorption of chloride ions. Zeitschr. f. vergl. PhysioL, 24 : 656-666. KRUGLER, O. E., AND M. L. BURKNER, 1948. Histochemical observations of alkaline phos- phatase in the integument, gastrolith sac, digestive gland and nephridium of the cray- fish. PhysioL Zool, 21:105-110. KUKENTHAL, W., 1926-1927. Handbuch der Zoologie, 3 : 845-849. LISON, L., 1936. Histochimie Animale. Gauthiers-Villars, Paris. LISON, L., 1942. Recherches sur 1'histophysiologie comparee de 1'excretion chez les arthropodes. Mem. Acad. Roy. Belgian (Classc dc Sciences), 19: Part 5, 1-107. MACCALLUM, A. B., 1905. On the nature of the silver reaction in animal and vegetable tissues. Proc. Roy. Soc. London, Ser. B, 76: 217-229. MARCHAL, P., 1892. Recherches anatomique et physiologique sur 1'appareil excreteur des Crustaces decapodes. Arch. Zool. Exper. et Gen., Ser. 2, 10 : 57-275. PEARSON, J., 1908. Cancer. Liverpool Marine Biological Committee Memoires, London. TRAVIS, D., 1955. The molting cycle of the spiny lobster, Panulirus argns (Latreille). II. Pre-ecdysial histological and histochemical changes in the hepato-pancreas and in- tegumental tissues. Biol Bull, 108: 88-113. WEBB, D. A., 1940. Ionic regulation in Carcinits ntacnas. Proc. Rov. Soc. London, Ser. B, 129: 107-136. ORGANIC PRODUCTIVITY IN THE REPRODUCTIVE CYCLE OF THE PURPLE SEA URCHIN * A. C. GIESE, L. GREENFIELD, H. HUANG, A. FARMANFARMAIAN, R. BOOLOOTIAN 2 AND R. LASKER 3 Hopkins Marine Station of Stanford University, California The gonads of a gravid purple sea urchin (Strongylocentrotus purpuratus) may contribute as much as one-fifth to the total wet weight of the animal. On the other hand, the shrunken gonad of an immature animal or one which has recently spawned may be only one-eighteenth as large. The development of the gonad represents a remarkable synthesis of organic material, since the larger part of the protoplasm of a sea urchin is gonadal during the breeding season, the only other organ of any bulk being the intestine. The intestine in turn owes part of its bulk to its food contents, the epithelium itself being quite delicate. The volume of perivisceral fluid bears an inverse relation to the gonads, being present in larger amounts when the gonad is less well developed. The perivisceral fluid, however, contains little organic material (Lasker and Giese, 1954). Furthermore, its organic constituents do not vary in any striking or systematic way during the year (Bennett and Giese, 1955). A fairly good measure of organic productivity in the sea urchin might therefore be gained by a study of the increase in organic constituents in the gonads during their growth from immature (or spent) to gravid condition. The results of such study are reported in this paper. METHODS For most of the experiments reported here, sea urchins were collected at the monthly low tide at Yankee Point, near Carmel, California. In a few instances speci- mens were obtained near Moss Beach, California. The gonad index was deter- mined for each of ten specimens, the index being the ratio of the volume of gonad to wet weight of animal, times 100. The total nitrogen (TN), non-protein nitrogen (NPN), lipid, and glycogen contents of samples of gonad were determined. For one male and one female, water and ash content of the sample were also determined monthly. From samples at the height of the season, and also after the spawn-out, determinations were made of the desoxyribonucleic acid (DNA) and ribonucleic acid (RNA), as well as lipid, total nitrogen and non-protein nitrogen at the same time, and in a few samples reducing sugar (RS) content was determined. These data give a biochemical picture of the constituents of the gonads correlated with the gonadal cycle over an entire year. For the biochemical determinations on the gonads of each animal, several samples 1 Supported by funds made available by National Science Foundation Grant GS 482, U. S. Public Health Grant 4578, and the Rockefeller Foundation. We are indebted to Mr. F. Fal- coner, head librarian of the Biological Libraries, for verification of the literature cited. - Now at the University of California at Los Angeles. 3 Now at Scripps Institution of Oceanography, La Jolla, California. 49 50 GIESE, ET AL. of various wet weights (several grams) were placed in a vacuum desiccator over concentrated sulfuric acid and dried for about 12 hours. When tissues were to be used for glycogen analysis, a few drops of 10 per cent trichloracetic acid were in- jected before drying to prevent glycolysis by enzymes during the drying process. All analyses were done in duplicate ; the duplicates varied by only a few per cent. For determination of total nitrogen, a given sample was digested in sulfuric acid with selenium catalyst over electric heat or gas flame, and from an aliquot of the digest the ammonia was distilled with a Markham still (Markham, 1942), or in a Conway diffusion cell (Conway, 1947), into borate buffer containing brom-cresol green and methyl red as indicators. The borate was then titrated to the original color with 0.01 N sulfuric acid. Usually several weights of samples were tested and to one of them a known weight of a nitrogen-containing compound (glycine) was added to serve as a check on the accuracy of the method. For determining non-protein nitrogen 1 ml. of 10 per cent trichloracetic acid (TCA) was added to a 10-30 mg. sample of gonad and the tissue was macerated with a glass rod. It was heated to 80-100 C. in an oven for 15 minutes, allowed to cool, centrifuged, and the supernatant plus two washings of the precipitate were added to the flask which was then placed on the digestion rack and the nitrogen con- tent determined as described above. The non-protein nitrogen subtracted from the total nitrogen is taken to give the protein nitrogen (PN) 3 . This is multiplied by the factor 6.25 to convert to protein. Total lipids in gonadal tissue were determined by extracting 100-mg. samples with 10 ml. ethyl ether in a micro-Soxhlet apparatus, refluxing being continued for two hours. Many samples were extracted at the same time on a sand bath. Glycogen was determined in the following way (Good ct al., 1933; Meyer, 1943). The ground dry sample was treated with an equal volume of 10 per cent TCA, cooled, and the supernatant was transferred to a lusteroid tube to which was added 1 ml. of distilled water wash of the precipitate. After addition of 2.5 ml. of 95 per cent ethanol and mixing, the sample was centrifuged and the supernatant fluid was discarded and the tube allowed to drain for several minutes. To it was added enough warm water to give about 70 //,gm glycogen per ml. and the content of glycogen was determined by the anthrone method (Seifter et al., 1950). Re- ducing sugar was determined in the supernatant fluid of a homogenized gonad by the Somogyi method (1945; 1952) which involves first the precipitation of the protein by TCA, centrifuging the sample, and testing of the supernatant solution. Water content was determined by weighing minced tissue before and after drying in the desiccator over sulfuric acid. Ash content was determined on a known dry weight of gonad (about 100 mg.) heated to 450-500 C. in a porcelain crucible for three to eight hours. Nucleic acids were extracted using the Hershey, Dixon and Chase (1953) adap- tation of the Schmidt-Thannhauser (1945) and Schneider (1945) procedures, acid- soluble phosphorus being removed by cold 10 per cent TCA, phospholipid being re- 3 Although it is classical procedure, some question exists whether this is entirely justified here, because when a direct test for protein nitrogen is made on the residue remaining after extracting acid-soluble phosphates, phospholipids and nucleic acids from the tissue mash, only about a half to a third as much is obtained as by the difference between total nitrogen and non-protein nitrogen. It is possible that some of the proteins are dissolved by the extraction procedures, but additional studies are desirable. ORGANIC PRODUCTIVITY OF SEA URCHINS 51 moved with ethanol and a mixture of ethyl ether and ethanol (60 C.). RNA was removed with KOH, DNA being precipitated with 5 per cent TCA (Leslie, 1955) . The indole reaction of Ceriotti ( 1952) was used for DNA and the orcinol re- action of Ogur and Rosen (1950) was used for RNA; the details of the method as used here have been described elsewhere (Iverson and Giese, 1957). Some studies were made determining the nucleic acids by the phosphorus method (Fiske and Sub- barow, 1925) but they were considered less reliable and are not reported here. RESULTS The average values for some chemical contituents of gonads of male and female sea urchins taken each month of the year 1956 are given in Table I. Certain trends TABLE I Chemical constituents of gonads of the purple sea urchin (Jan. to Dec. 1956) (Water in % wet weight, all others in % dry weight) Date Av. GI* Lipid NPN Protein Glycogen Water Ash cf 9 cf 9 cf 9 d" 9 2 1000 u ^oo ~> 800 ? 700 % 60 Z 500 O O 400 >- 300 o O O UJ CL 200 100 10 20 30 40 50 60 70 60 90 100 TEMPERATURE IN C FIGURE 1. Calcium-45 exchange of small pieces of corallum from Manicina areolata with sea water at 4 C., 28 C, 58 C. and 100 C. The coral was carefully cleaned to remove all organic matter, and the experiments ran for twenty-four hours. The ordinate is the specific activity plotted on a logarithmic scale. 62 THOMAS F. GOREAU CURVE A, LIVING CORAL CURVE B, SKELETON CONTROL B 10 15 20 25 30 TIME IN HOURS FIGURE 2. SKELETON FORMATION IN CORALS 63 rather slowly, but in most species tested, the process was sixty to eighty per cent complete at the end of twenty-four hours. As expected, the rate of exchange with sea water was strongly temperature-dependent. This is demonstrated in Figure 1, which shows the specific activity of small pieces of Manicina areolata which have been allowed to equilibrate at different temperatures in sea water containing calcium- 45. In most species tested, the rate of calcium-45 deposition in the living coral was much faster than in the skeleton controls. This is shown in Figure 2 for Acropora prolijera, in which the specific activity of the dead corallum is about five per cent that of the living coral at the end of twenty-nine hours. In water of a given specific activity the equilibration rate appears to be much slower in imperforate corals such as Oculina or Phyllangia than in perforate species such as Acropora or Porites. The effect of the total skeletal surface on the exchange rate is being studied. There is some evidence that the living coenosarc forms a barrier which re- stricts calcium exchange of the skeleton with the sea water. In a number of experi- ments in which the calcium rate of the experimental colonies was very low, it was noted that the specific activity of the skeleton controls was higher than that of the living coral. It has been previously demonstrated by Goreau and Bowen (1955) that the exchangeable calcium in the tissues of the cold water coral Astrangia danae is maintained at only about eighty-eight per cent of the calcium concentration in the sea, i.e., calcium tends to be excluded from the tissues of coral. Until more evidence is available, it is difficult to state precisely the extent to which coral tissues can restrict the calcium exchange of the underlying skeleton with sea water. This problem is now under investigation. THE EFFECT OF LIGHT ON CALCIUM DEPOSITION IN CORALS AND OTHER HERMATYPES Light has long been recognized as an essential environmental factor in the growth of tropical reef building corals (Vaughan, 1919; Edmondson, 1928; Verwey, 1930; Kawaguti, 1937a, 1937b; Yonge, 1940; Vaughan and Wells, 1943) and other hermatypes such as Lithothanmion and Millepora. Yonge and Nicholls (1931a), Yonge (1940) and Kawaguti (1944) stated that this was due to photosynthesis by unicellular zooxanthellae contained within the cells of the gastrodermis. Kawaguti and Sakumoto (1948) claimed that in five species of reef corals the uptake of cal- cium was greater in light than in darkness. Their observations were based on changes in the calcium content of small volumes of sea water when corals were put in, the results being expressed in terms of milligrams of calcium taken up per hour per gram of coral. In our experiments, the effect of illumination on deposition of calcium-45 was determined by exposing one series of coral colonies to a standard light source while keeping a control series in darkness under otherwise equal conditions. The light source was a twin bank of 20-watt fluorescent tubes in a reflector housing located about one foot above the experimental vessels. FIGURE 2. Comparison of the calcium-45 deposition and exchange in living and dead colonies of Acropora prolijera. The results from the living coral have been re-calculated in terms of the specific activity to permit direct comparison with the exchange controls which were devoid of organic matter. Both controls and experimentals were run under identical conditions at the same time. The specific activity is plotted on a logarithmic ordinate. 64 THOMAS F. GOREAU The results of our preliminary experiments are given in Table I which shows calcium uptake in nine species of coral, and a coralline alga (Porolithon}. In two of these species, dark experiments were not run ; only the results of light experiments are shown. In most species, there was a significant increase in the calcification rate on exposure of the coral to a light. The course of a typical experiment is seen in Figure 3 which shows the progressive incorporation of calcium-45 into the skele- ton of the Caribbean staghorn coral Acropora prolifera in light and darkness. The pH in both light and dark vessels was measured every six hours with a Beckman Model G pH meter. This showed that the observed differences in the calcification rate in the light and dark experiments were not due to a decrease in the pH of the water of the dark experiments, as such changes were prevented by continuous aeration with a stream of air. It is probable that the negative calcium balance found by Kawaguti and Sakumoto (1948) in some corals in darkness was caused by a lowering of the pH, due to the failure of these workers to aerate or stir the water in their experimental vessels. TABLE I Calcification rates in the apical polyps of branching coral species, in Number of samples in brackets calcium mg. N~ l hr.~ l Species Calcification in light Calcification in dark P Cladocora arbuscula Porites divaricata Porites compressa Acropora prolifera 6.31.58 (9) 9.80.54 (10) 7.81.70 (11) 12.46.50 (12) 80 _i_ 2 if. M r\\* 6.10.20* (10) 5.01.00 (8) 7.42.10 (7) 7.25.00* (11) 0.7 0.01 <0.7 HC.XV, hence the enzyme cannot be a primary factor in calcification as was previously as- sumed by Stolkowsky (1950) for mollusk shells. An interesting problem arises from our data on calcification rates of reef corals from which the zooxanthellae had been removed. The second part of Table II shows that in darkness normal corals calcify from two to three times faster than corals which have lost their zooxanthellae. This suggests that the presence of these algal symbionts, even when not photosynthesizing, may have a potentiating effect on the calcification rate of the coral host. It is thus considered possible that the zooxanthellae can exert a general stimulant effect on the host's metabolism, mediated through a vitamin or hormone-like factor. This function of the zooxanthellae would to some extent be independent of the photosynthetically controlled "janitorial" activ- ities of these algae which result in the assimilation of the animal host's metabolic waste products. It is hoped that work now in progress will provide more evidence for this interesting possibility. This work \vas in part supported by grants from the New York Zoological Society and the National Science Foundation (Grant Number G-4017), and by in- stitutional funds from the University College of the West Indies. Studies on Pacific corals were made at the Eniwetok and Hawaii Marine Laboratories with the aid of AEC contract AT (29-2) -226 with the University of Hawaii. The nitro- gens were determined by N. I. Goreau. Boats and other facilities of the University SKELETON FORMATION IN CORALS 73 College Marine Biological Station at Port Royal, Jamaica were made available through the kindness of Professor D. M. Steven. Grateful acknowledgment is hereby made to all the persons and institutions whose generous assistance made this work possible. SUMMARY 1. A method is described for the accurate measurement of calcification rates in reef-building corals under various controlled conditions, using calcium-45 as tracer. 2. At the temperatures of the experiments, there was a slow but appreciable isotopic exchange between the coral skeleton and sea water. There are indications that this is considerably less in living coral where the tissue forms a barrier against such exchange. 3. In many of the reef-building corals tested so far, the calcification rate was significantly lowered by the exclusion of light. 4. The calcification rate of reef corals grown in darkness for prolonged periods of time to remove the zooxanthellae is considerably reduced and seems independent of the light intensity. 5. Variations in the growth rates of different parts of coral colonies were meas- ured. The existence of growth gradients was demonstrated in a number of species. 6. Calcium uptake was greatly reduced on the addition of Diamox, a specific carbonic anhydrase inhibitor. In those species tested, the effect of carbonic anhy- drase inhibition and exclusion of light was in the same direction. In the presence of complete inhibition of carbonic anhydrase there was still an uptake, even in darkness. 7. It was concluded that the effect of light on reef coral growth is in part mediated through the zooxanthellae. The decreased calcification rates of reef corals in darkness, in the absence of zooxanthellae or in the presence of a carbonic anhy- drase inhibitor suggest that the rapid calcification of these corals may be dependent on efficient removal of H 2 CO 3 . LITERATURE CITED ABE, N., 1940. Growth of Fungia acthtifonnis var. palawensis (Doderlein) and its environ- mental conditions. Palac Trap. Biol. Stat. Rep., 2 : 105-145. AGASSIZ, A., 1890. On the rate of growth of corals. Bull. Mus. Comp. Zool. Harvard, 20: 61-64. . BOSCHMA, H., 1924. On the food of madreporaria. Proc. Acad. Sci. Amsterdam, 27: 13-23. BOSCH MA, H., 1925a. The nature of the association between Anthozoa and zooxanthellae. Proc. Nat. Acad. Sci., 11 : 65-67. BOSCHMA, H., 1925b. On the symbiosis of certain Bermuda coelenterates and zooxanthellae. Proc. Amer. Acad. Arts Sci., 60: 451-461. BOSCHMA, H., 1925c. 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Barrier Reef Exped. Sci. Rep., 1 : 353-391. VONGE, C. M., AND A. G. NICHOLLS, 1930. Studies on the physiology of corals. II. Digestive enzymes with notes on the speed of digestion. Gt. Barrier Reef Exped. Sci. Rep., 1 : 59-81. ONGE, C. M., AND A. G. XICHOLLS, 1931a. Studies on the physiology of corals. IV. The structure, distribution and physiology of zooxanthellae. Gt. Barrier Reef Exped. Sci. Rep., 1 : 135-176. ONGE, C. M., AND A. G. NICHOLLS, 1931b. Studies on the physiology of corals. V. The effect of starvation in light and darkness on the relationship between corals and zooxanthellae. Gt. Barrier Reef Exped. Sci. Rep., 1 : 179-211. THE REGULATION OF WATER AND SALT BY THE FIDDLER CRABS, UCA PUGNAX AND UCA PUGILATOR JAMES W. GREEN, MARY HARSCH, LLOYD BARR AND C. LADD PROSSER Department of Physiology and Biochemistry, Rutgers University, New Brunsivick, New Jersey; Department of Physiology, University of Illinois, Urbana, Illinois; and the Marine Biological Laboratory, Woods Hole, Massachusetts Ionic and osmotic regulation in decapod Crustacea are the result of selective ionic absorption and excretion through several routes (Prosser et al., 1950; Robert- son, 1953). The gills have been implicated as the primary site of absorption (Huf, 1936; Krogh, 1938; Webb, 1940; Koch, 1954; Gross, 1957) but the alimentary tract may also be important (Burger, 1957). The antennary glands are considered the chief organs of selective excretion (Nagel, 1934; Webb, 1940; Robertson, 1949; Prosser et al., 1955; Burger, 1957). The cellular mechanisms of ionic absorption and excretion in crustaceans are poorly understood and hypo-osmotic regulation has been less extensively studied than hyper-osmotic regulation. Recently (Prosser et al., 1955) it has been shown that Pachygrapsus crassipcs, when maintained in 170% sea water (S.W.) excretes a urine higher in Mg but significantly lower in Na than animals in normal sea water. The Na which is not excreted in the urine may be stored in tissues for short periods (Gross, 1958) or may be excreted by extra-antennary gland routes, as suggested by the finding (Gross, 1957) that salt exchanges, as measured by electroconductivity methods, occur in the gill chamber of Pachygrapsus. Since Jones (1941) had shown that Uca crenulata is a stronger hypo-osmotic regulator than P. crassipes, studies were undertaken on several species of Atlantic coast Uca to determine their ability to excrete Na by extra-antennary gland routes. In a preliminary survey it was found that Uca mina.v, U. pugilator and U. pugnax all show hypo-osmotic regulation and reduced urine Na in concentrated sea water. These properties were not found in Callinectes and Carcinus. The object of the present paper is to report a detailed study of the response of the body fluids and tissues of Uca pugnax and U. pugilator to prolonged exposure to concentrated sea water. MATERIALS AND METHODS Uca pugnax and U. pugilator were acclimated to 175% sea water during three- day periods by increasing the concentration of the sea water 25% per day. The crabs were held at 175% sea water in large finger bowls containing a small amount of the bathing medium for 2 to 4 days after reaching this concentration. They were not fed in the laboratory but the sea water in the bowls was changed daily. Usually crabs were used within 7 to 14 days after collection. Some of the variability in the experiments may be attributed to the starvation of the animals and their varied nutritional states upon collection. Greater experimental variability is found between different batches of crabs than between the sexes of the two species. 76 IONIC REGULATION IN FIDDLER CRABS 77 Urine was collected from single animals by mounting the crab, caudal end down, on a microscope stage, attaching one wire from a Harvard inductorium to the mouth and stimulating the opercular region at the base of the antenna with the other. A small capillary drawn out at the end was simultaneously placed near the opercular covering. Usually moderate shocks resulted in the expulsion of urine, as much as 10-20 microliters from a single crab. Urine from three crabs was generally pooled on a piece of Parafilm which was kept in a high humidity chamber. Blood was collected in the manner described for Pachygrapsus (Prosser et al., 1955). Gill fluid was collected through small openings made in the gill plate prior to the experiment. Care was taken to prevent bleeding at the time the openings were made. After exposing crabs to isotopic solutions for a 12-hour period, the animals were exposed to non-isotopic sea water for 15 minutes and transferred to dry finger bowls for 30 minutes before removing gill fluid by fine capillaries through the gill plate openings. Stomach fluid was collected in capillaries from excised stomachs. Some studies were performed with isotopic Na 24 . This ion was obtained with Na,CO 3 as the carrier and was initially made up in a small amount of distilled water. Ten-mi, aliquots of this highly active sample, containing 0.1-0.2 mc./mg., were placed in finger bowls containing 490 ml. of the appropriate sea water. Crabs were exposed to these isotope solutions for 12-18 hours before sampling. Exploratory experiments had shown that the relative specific activity of the serum of crabs re- mained nearly constant after 12 hours during the period of sampling. Routinely, samples of blood, urine, gill fluid and stomach fluid were pooled from three animals for analysis. Twenty-five microliters of such a sample were added to 10 ml. of glass-distilled water in small Pyrex tubes. From this solution Na 24 counts were made with a well counter and scintillation tube. Sodium. K, Ca and Mg were analyzed by flame photometry in a conventional manner using the Beckman flame attachment with a photomultiplier tube. Chloride was analyzed by the method of Schales and Schales (1941), SO 4 by the method of Nalefski and Takano (1950) and NH 4 by the method of Russell (1944). Osmotic determinations were made on all fluid samples, using the Jones method (1941) as modified by Gross (1954). In those studies where Na 24 counts of tissue were made, tissues were removed to Parafilm, weighed, placed in test tubes with the Parafilm and counted in the same manner as were the fluids. Counts were expressed per 25 mg. of wet tissue. RESULTS Several preliminary experiments were performed to test our methods and to establish optimum levels for Na 24 use. Table I presents the results from our two most extensive experiments for osmotic and ionic analyses of several fluids from crabs in 100% and 175% sea water. The measure of variability is the standard error. Significant differences between 100% and 175% groups were found for all components of serum except Mg, K, Ca and NH 4 ; for urine components except Ca, NH 4 and Cl ; for gill fluid components except NH 4 , and for components of stomach fluid except K and osmotic concentration. A statistical evaluation of the difference between the analytical values (from 78 GREEN, HARSCH, BARR AND PROSSER TABLE I Osmotic and electrolyte concentrations in Uca expressed as niM/L Fluid No. crabs Osmotic cone.* Na Mg K Ca NH 4 Total mEq.+ Cl SO 4 Total mEq.~ For Crabs in 100% S.W. Serum 28 .497 328 46 11 16 20 483 537 42 621 .012 4.40 2.55 0.32 1.35 1.28 7.75 1.26 Urine 23 .583 276 108 16 17 75 617 622 47 716 .014 17.4 11.2 1.10 0.89 7.2 25.8 1.90 Gill fluid 28 .506 314 64 10 12 18 494 569 36 641 .011 9.73 4.63 0.50 0.41 2.04 6.99 1.96 Stomach fluid 13 .758 335 101** 17 31 63 679 542 143 828 .036 21.1 21.2 0.88 3.19 3.84 17.7 8.52 For Crabs in 175% S.W. Serum 33 .587 375 55 15 14 21 549 574 49 672 .011 9.1 3.64 0.48 0.61 1.88 6.96 1.11 Urine 33 .683 218 255 20 20 116 904 704 120 944 .012 18.18 12.9 0.71 1.77 7.7 14.1 4.89 Gill fluid 33 .860 503 123 15 19 18 820 855 60 975 .023 6.56 6.69 0.39 0.31 2.19 9.40 3.13 Stomach fluid 18 .828 393 167 16 22 43 830 704 111 926 .015 7.87 12.6 0.48 0.65 3.34 37.0 5.33 Composition of S.W. used for experiments 100% S.W. .560 .750 397 579 88 139 9 17 12 20 606 914 576 941 22 29 620 999, * Equivalent moles of NaCl; stomach average of 10 crabs. ** Average of 8 crabs. Table I) of each of the fluids from crabs within the same medium is given in Table IV. The different fluids from animals within the same medium are as quantitatively distinct as are the same kinds of fluids from crabs in the two different media. For example, serum and urine from crabs in 100% sea water are as different as sera from TABLE II Analysis of Na 2 * counts in Uca tissues. Counts per 25 mg. wet weight 100% S.W. Aver. cts. 175% S.W. Aver. cts. % change 175%/100% P values 100 vs. 175 P values 100 vs. sera P values 175 vs. sera Serum 3891.9 4365.1 112 <.01 Muscle 1243.43 2035.06 166 <.005 <.005 <.005 Mid-gut gland Stomach 2020.95 2663.85 1507.44 3508.3 75 132 <.005 <.005 <.005 >.050 <.005 <.005 Gill 5000.96 3576.9 72 <.005 <.01 <.005 Heart 1819.1 2122.36 117 >.100 <.005 <.005 Intestine 948.62 1132.15 119 >.050 <.005 <.005 IONIC REGULATION IN FIDDLER CRABS 79 crabs in normal and concentrated sea water. This finding emphasizes the existence of homeostatic mechanisms in this group of crabs. The ability of these crabs to maintain their sera hypo-osmotic to the medium in both normal and concentrated sea water as shown by the osmotic concentration, appears to be shared with other mem- bers of the grapsoid group (Robertson, 1953). More striking is the finding that crabs in both types of media produce a urine which is hypertonic to the serum. The data from Tables I and IV show that the crabs regulate all serum ions in concen- trated sea water and all but Ca in normal sea water. With the exception of Na all other electrolytes occur in higher concentrations in urine than in serum. Since the degrees to which these ions are concentrated in the urine varies in crabs from the same medium and between the two media and also varies for the different ions, it is probable that their concentration is a result of secretion or selective ion reabsorption. Table I indicates that the gill fluid from crabs in 175% sea water is hyper-osmotic to the medium, the serum and the urine. That this hypertonicity results from water and solute absorption as well as solute secretion will be apparent later. Directly related to the gill fluid hypertonicity is the urine Na concentration which is signif- TABLE III Relative specific activities for crabs in normal and hypertonic sea water 100% S.W. 175% S.W. P Values Fluid No. crabs Na mEq./L. Counts per min. RSA CPM/ Na23 No. crabs Na mEq./L. Counts per min. RSA CPM/ Na*! RSA 100 vs. RSAns Serum 13 349 2335 6.7 18 403 2955 7.8 0.01 Urine 13 258 2144 6.6 18 210 1599 7.6 0.05 Gill fluid 13 320 1099 3.7 18 486 2487 4.8 0.01 Stomach fluid 13 346 1816 5.8 18 393 2111 5.3 0.10 icantly lower (Table IV) in crabs in concentrated than in normal sea water. And while this result is not unexpected (Prosser ct al., 1955) it indicates the extra- antennary gland excretion of this ion. possibly through the gills, and hence its as- sociation with gill fluid hypertonicity. The stomach fluid of crabs in 100% sea water (Table I) is marked by its significant hypertonicity to serum, urine and gill fluid. Its ion content is different from serum except for Na and Cl ; from urine except for Na, Mg and K and from gill fluid except for Na, Mg and Cl . The stomach fluid from crabs in 175% sea water is hypertonic to serum and urine but not to gill fluid. Its ion content is greater than that of serum for all ions except Na and K; stomach fluid is more concen- trated than urine except for Ca, Cl and SO 4 and more concentrated than gill fluid except for K. Both water and solute absorption probably occur from the stomach and the distribution of electrolytes in the stomach fluid makes some secretion into the gut probable. Fluid/serum ratios have been summarized in Figure 1 for osmotic concentration and the electrolyte values. The extent to which the urine/serum ratio (U/S) de- parts from unity has been used as a measure of antennary gland regulation (Prosser 80 GREEN, HARSCH, BARR AND PROSSER TABLE IV Probability values of analyses Fluid Osmotic cone. Na Mg K Ca MH, CI SO 4 A. Comparison of fluids of crabs in 100% and 175% S.W. Serum Urine Gill fluid Stomach fluid <.02 = .05 <.02 >:?o >.05 >.50 >.50 >.50 i 100% S.W. B. Comparison of fluids from crabs in the same medium Serum vs. urine <.01 <.01 <.01 <.01 >.50 <.01 <.01 <.02 Serum vs. gill fluid >.50 >.10 <.01 >.10 <.01 >.10 <.01 >.02 Serum vs. stomach fluid <.01 >.50 <.02 <.01 <.01 <.01 >.50 <.01 Urine vs. gill fluid <.01 >.05 <.01 <.01 <.01 <.02 >.05 <.01 Stomach fluid vs. urine <.01 = .05 >.50 >.10 <.01 >.10 <.02 <.01 Gill fluid vs. stomach fluid <.01 >.10 >.05 <.01 <.01 <.01 >.10 <.01 175% S.W. Serum vs. urine Serum vs. gill fluid Serum vs. stomach fluid Urine vs. gill fluid Stomach fluid vs. urine Gill fluid vs. stomach fluid <.01 H Si >.02 >.50 <:jjj >.50 i 100%, S.W. C. *Comparisons of ratios of fluids from crabs in the same medium U/S vs. one SW/S vs. GF/S SW/S vs. SF/S <.02 <.02 i >.02 >.50 <:o! = .01 <:2! 175% S.W. U/S vs. one SW/S vs. GF/S SW/S vs. SF/S <:'! .os <.02 = .01 >.50 >.05 <:o! X5 1 <:S! * See Figure 1 for meaning of ratios. ct al., 1955). When the U/S ratios for crabs in 100% sea water are compared with those from 175% sea water only the ratios for osmotic concentration, K and Cl are found to be alike; the 175% sea water U/S ratio for Na is lower and all others higher than the corresponding 100% sea water values. The considerable regulation exhibited by the antennary glands of these crabs in normal sea water (Tables I and IV) is increased under the stress of concentrated sea water, partic- ularly for Mg, NH 4 and SO 4 . IONIC REGULATION IN FIDDLER CRABS 81 Because both gills and stomach have a direct contact with the external medium and appear to be the most likely sites of exchange of water and salts with the medium, it is reasoned that the extent to which the gill fluid/serum (GF/S) and the stomach fluid/serum (SF/S) ratios deviate from the sea water/serum ratio should provide a measure of the absorptive and secretory capacities of gill and stomach tissues. These ratios are presented in Figure 1, and the statistical signif- icances of a variety of internal comparisons (for example, GF/S with SF/S ratios from crabs in 100% sea water) are given in Table IV. o I U 3. 2 j F ^ : X 1. u ll ._tx ftf I S s xl Y/////// i 1 S! A B C D A BCD A B C D Chloride Sulfate Osmotic Cone. 8. \ Legend ' 7_ A Urine/Serum Ratio (U/S) X B Gil Fluid/Serum Ratio (GF/S) X 6. C Stomach Fluid/Serum Ratio (SF/S) t -, - D Sea Water/Serum Ratio (SW/S) ^ N , U 100% SW \ \ x Fx^ 4- bJ 175% SW . 3. S Paired Ratios Differ Significantly 1 , x x , V XI X X X \ X X 2- p s __ X s X jf -1 x x x X 1- rl r| 1 ~L R r Iri 1 J X 1 X 1 x \ ral fel4 ~|s| 1 s i ts FN sKjsx s li s sx s s i 1 s s \ v II BCD Sodium A B C D Ftotassium BCD Calcium B C Magnesium BCD Ammonium FIGURE 1. Ratios of the osmotic and electrolyte concentrations of fluids from crabs in 100 and 175% sea water. Statistical significance was attributed to P values of 0.02 or less. In a few experiments crabs were exposed to sea water containing Na 24 . The same quantity of the isotope was added to equal volumes of 100% and 175% sea water. Since the Na 23 concentration of the 175% sea water was greater than that of the 100% sea water, a factor was used to correct the counts obtained from fluids and tissues of crabs in the concentrated sea water to make them comparable to those from normal sea water. This correction factor was obtained by dividing the Na 24 / Na 23 ratio in 100% sea water by the Na 21 /Na 23 ratio in 175% sea water. Multiply- ing the counts from the fluids and tissues from crabs in 175% sea water by this factor gave the corrected counts. Approximate isotopic equilibrium was attained in blood and urine in both groups of crabs after 12 hours. Isotopic analyses of GREEN, HARSCH, BARR AND PROSSER tissues were made in a number of experiments and the assumption was made that these too had attained isotopic equilibrium. The results of the tissue studies are summarized in Table II. The high Na 24 count in the gills from crabs in 100% sea water implies that these are the primary means of Na entrance or else that Na can- not be excreted as rapidly as it enters and dams up in this tissue. Since Na 24 counts increased more in muscle (67%) and stomach tissues (32%) relative to the increase in sera in going from 100% sea water to 175% sea water, these tissues may serve for Na storage during the stress of high serum Na. Only gill and mid-gut gland tissues from crabs in 175% sea water had lower Na 24 counts than their 100% sea water counterparts; serum, muscle and stomach all had higher counts while heart and intestine were not significantly different. With the exception of the gill tissue from the crabs in 100% sea water the tissues of both groups had lower counts than their sera, which is in part an indication that the Na'- 4 is restricted primarily to the extracellular space. The fact that in tissues from the 175% sea water animals, the counts from mid-gut gland and gills were propor- tionately lower while those of muscle and stomach were proportionately higher with respect to their sera than the same tissues from the 100%> sea water crabs, in- dicates a differential tissue response to the Na stress. A study of the rate at which Na 2 * can penetrate these crabs in 100% and 175% sea water has shown (Green and Harsch, 1958) that the isotope enters the crabs in the concentrated sea w r ater more readily. This finding, coupled with the low Na 24 counts in gills and mid-gut gland shown in Table II, affords evidence that these tissues are concerned with the excretion of Na under the conditions of these experiments. When a comparison is made of the relative specific activities (RSA) (counts per minute/meq of Na 23 /L ) for serum, urine, gill fluid and stomach fluid for the two groups of crabs, as summarized in Table III, only serum and gill fluid values are found to be significantly different. The high RSA value of serum for the crabs in 175% sea water indicates a greater exchange rate of Na 24 for Na 23 as compared with normal sea water. The higher RSA value in the gill fluid is interpreted to mean that the crabs in hypertonic sea water excrete more Na by the gills than do crabs in normal sea water. DISCUSSION The osmotic concentration data of Table I indicate that in both normal and concentrated sea water Uca is a hypo-osmotic regulator; sera of 100% sea water crabs were 12% lower in osmotic concentration and of 175% sea water crabs 22% lower than their respective media. Hypo-osmotic regulation occurs in crabs which spend much time out of water (Jones, 1941) and in shrimps and prawns (Parry, 1954). Ionic regulation in Uca is quantitatively different in the two media. In 100% sea water the serum concentrations as per cent of medium concentrations are : Na, 83; Mg, 52; K, 122; Ca, 133; Cl, 93; SO 4 , 191; while in 175% sea water they are : Na, 65 ; Mg, 40 ; K, 88 ; Ca, 70 ; Cl. 61 ; SO 4 , 169. In the concentrated sea water each ion is proportionately less concentrated in serum relative to the me- dium than in normal sea water ; however, the extent to which the ions are regulated, as measured by the per cent increase in serum concentrations in 175% sea water relative to the sera concentrations in 100% sea water, is, in order of decreasing order of regulation: Cl, 7; Na, 14; Ca, 14; SO 4 , 17; Mg, 20; K, 36. Uca differs from IONIC REGULATION IN FIDDLER CRABS 83 Pachygrapsus crassipes (Prosser et al., 1955) under similar conditions, especially in the greater ability of the fiddler crab to regulate Na. The osmotic concentration of the 100% sea water crabs approximates that of Pachygrapsus marmoratus as measured by Robertson (1953), as do the K, Ca, and SO 4 values relative to Cl, while Mg values in Uca are relatively higher. A cation deficit of 12% exists in the serum of crabs in 100% sea water ; a deficit of 10% in crabs in 175% sea water. The urine cation deficit is smaller in both groups. The serum deficits are attributed to organic cations. The lower cation deficit in urine than in serum is associated with the higher urine concentration of ammonia ; however, if the NH 4 excreted by the antennary glands is subtracted from the total cation deficit, the cation deficit is still smaller than that in serum. Tentative conclusions can be drawn concerning the formation of urine, gill fluid and stomach fluid in Uca. The urine in both normal and concentrated sea water has a higher osmotic concentration and a higher total electrolyte concentration than serum. This ability to produce a blood hyper-osmotic urine is one means of keep- ing the blood hypo-osmotic to the medium. Pachygrapsus crassipes failed to show a hyper-osmotic urine (Prosser ct al., 1955). Uca appears to spend more time out TABLE V pH of Uca urine Treatment of crabs Crabs in 100% S.W. Crabs in 175% S.W. Equilibrated to medium for 3 days Equilibrated to medium for 4 weeks 6.92.15* 6.38.ll 7.16.10 6.42.ll * Standard error. The pH was measured with the Beckmau micro-glass electrode. Urine of crabs equilibrated at the same time was not significantly different. Differences in the urine pH of crabs in the same media for different lengths of time were real. of water than Pachygrapsus and may be a better hypo-osmotic regulator, partly be- cause of its ability to produce hyper-osmotic urine. It was not feasible to obtain true urine volumes, and excretion of solutes which are unlikely to be transported actively was not studied. The urine/serum ratios (U/S) differ for different ions and are maximal for NH 4 (4 and 8 in 100% and 175% sea water). If NH 4 were excreted by simple filtration, marked reabsorption of all other ions would be required to give such high NH 4 values ; hence it is prob- able that NH 4 is either secreted or its excretion is accelerated by acidification of the urine. This latter alternative appears unlikely from the pH data presented in Table V. The U/S ratio is next highest for Mg, increases proportionately in the concentrated sea water. The high U/S ratios for Mg and SO 4 in (175% sea water) could result from marked reabsorption of water and other ions (except NH 4 ) ; they could indicate secretion of Mg and, at least in 175% sea water, also of S0 4 . The treatment of Na by the antennary glands is unique. Its U/S ratio is less than one in normal sea water and is decreased in 175% sea water. This reduction in urine Na was found in Pachygrapsus (Prosser et al., 1955) and has been seen in another semi-terrestrial genus, Ocypode (Gifford, unpublished data). Reduced urine Na in concentrated sea water is not necessary for hypo-osmotic regulation, 84 GREEN, HARSCH, BARR AND PROSSER however, since it does not occur in hypo-osmotic shrimps and prawns (Parry, 1954) . The decreased urine Na in 175% sea water could result from reduced secretion or increased reabsorption. In Pachygrapsus urine Na was not reduced in 175% sea water which lacked Mg (Prosser et ctl., 1955) ; injection of extra Mg into land crabs in 100% sea water reduces urine Na (Gifford, unpublished data). In Uca in 175% sea water Mg excretion increases more than Na excretion decreases w r hen both are compared with responses in 100% sea water. It seems probable from these ob- servations that Mg secretion interferes in some way with Na secretion. Filtration and reabsorption of Na might serve a useful function in causing water absorption. However, one would expect such Na reabsorption to be associated with some Cl or SO 4 absorption ; this does not appear to be the case. If the Na were reabsorbecl by exchange with Mg one would expect two Na ions to be exchanged for one of Mg ; the finding for 175% sea water was 1.3 ions of Mg for each ion of Na. Hence on a quantitative basis it is difficult to attribute an increased Na reabsorption to an increased Mg excretion. On an energetic basis Na reabsorption seems improbable. If the crab needs to remove Na in 175% sea water and is able to filter it in the kidney, why would this Na be reabsorbed against a Na gradient, using energy for this purpose, only to be secreted at another site using energy a second time ? It is possible that Na is exchanged for NH 4 or for hydrogen ions, in which case its absorption would serve a useful function. By exclusion, active secretion of Na is indicated, a process which is reduced under a high Mg load. The U/S ratios for K, Cl and Ca are slightly above one and increase slightly in 175% sea water. Since the ionic gradients of these elements are from urine to plasma (presumably because of water reabsorption) the differences among them could result from differences in back permeability among these ions. Rather than postulate secretion of all ions, it seems more reasonable that there is filtration coupled with reabsorption of Na and water and some secretion of NH 4 and Mg (possibly also SO 4 ). The composition of fluid from the gill chamber indicates a combination of dif- fusion and secretion and a mixing with sea water. The osmotic concentration is intermediate between serum and 100% sea water; it may be as high or higher than 175% sea water. This could mean outward secretion of some ions or absorp- tion of water. Ammonia in gill fluid is intermediate between serum and the me- dium, hence NH 4 appears to be lost from the gills only by diffusion. Sodium concentration in gill fluid is similar to Na in serum in 100% sea water, but is much higher in 175% sea water. In both concentrations it is lower than in the medium. The gill fluid specific activity is significantly higher in 175% than 100% sea water while the gill tissue has significantly fewer Na 24 counts in 175% than 100% sea water. It is concluded that active secretion of Na occurs in the gills, at least in 175% sea water. The diffusion gradient for SO 4 is outward in 100% sea water but in 175% sea water the SO 4 in gill fluid is higher than in either serum or medium ; hence there might be some SO 4 secretion along with Na. Magnesium and Cl in gill fluid resemble Na in being close to serum levels in 100% sea water and higher than serum but lower than 175% sea water. These gradients could result from diffusion, secretion (in 175% sea water) or from water absorption. Potassium is similar in serum, gill fluid and medium ; Ca in gill fluid is similar to both media, lower than serum in 100% and slightly higher in 175% IONIC REGULATION IN FIDDLER CRABS 85 sea water. It is difficult to see how these concentrations could be so similar if there were much absorption of water. The relative importance of differences in perme- ability, in secretion and of water uptake by the gills cannot be evaluated from the present data. However it appears that the gills are important in ionic regulation in Uca and that the univalent ions do not separate from the divalent ions in route of excretion as they do in marine teleosts. It is probable that, like the lobster (Burger, 1957), Uca swallows some sea water, hence stomach fluid is a modification of sea water. Since NH 4 is absent from sea water and is higher in stomach fluid than in serum, gastric secretion of NH 4 is probable ; however the concentration of NH 4 in stomach fluid is less than in urine, where more secretion is indicated, and greater than in gill fluid where NH 4 may be lost only by diffusion. Sodium in stomach fluid is similar in concentration to Na in serum in both me- dia but lower than sea water, especially 175% sea water, hence absorption of Na in the stomach is indicated. With the Na, water is also probably absorbed, as in- dicated by the higher osmotic concentration in stomach fluid than in sea water. Concentration of the other ions might then be established by different inward perme- abilities. A less likely alternative would be absorption of sea water and then active secretion of the different ions. Sulfate in stomach fluid is so high that it may well be secreted. In any case there must be some absorption of ions other than Na along with water ; presumably this is the source of the Mg which is excreted in such large amounts by the antennary glands (kidneys). The significantly higher Na 24 levels found in stomach and muscle tissues of crabs in concentrated sea water indicate that during Na stress these tissues may serve as repositories for Na, as indicated by Gross (1958). It is unlikely that this storage mechanism is confined to a single kind of ion or that it can account for ionic regulation in a concentrated medium for long periods of time. In the absence of data on fluid volumes and kidney clearances, a tentative quali- tative summary is as follows : Ammonia diffuses from the gills, is actively excreted in the stomach and very much concentrated in the urine. Sea water is swallowed, especially in 175% sea water, Na and water are absorbed, other ions to a less extent. Filtration occurs in the kidney although Mg and Na may be actively excreted ; Na and water may be reabsorbed. In 175% sea water the heavy load of Mg excretion is coupled with decreased secretion or increased reabsorption of Na. Sodium (also probably SO 4 ) appears to be actively secreted by the gills, more in concentrated than in normal sea water. The various fluids which have been measured represent steady-state concen- trations resulting from diffusion and selective permeabilities combined with active transport, and fluxes can only be inferred. SUMMARY 1. Analyses were made of the serum, urine, gill and stomach fluids for total osmotic concentration and the electrolytes Na. Mg, K, Ca, NH 4 , Cl and SO 4 in Uca pngna.v and U. pugilator when these two species were kept in 100% and 175% sea water. 2. For crabs in 100% sea water the serum electrolyte values for Na, Mg and Cl are lower and those for K, Ca, NH 4 and SO 4 higher than in the medium ; for crabs 86 GREEN, HARSCH, BARR AND PROSSER in 175% sea water the serum electrolyte values of Na, Mg, K, Ca, NH 4 and Cl are lower and only SO 4 higher than the values in the medium. The sera of crabs from both media are hypotonic to their saline environment. 3. The electrolyte values of sera from crabs in normal sea water differ signifi- cantly from the gill fluid electrolytes for Mg, Ca and Cl only ; while similar sera values from crabs in concentrated sea water differ significantly for Na, Mg, Ca, Cl and SO 4 . In all cases except for Ca from crabs in normal sea water the significant gill fluid electrolyte concentrations are greater than the corresponding sera values. 4. Crabs in normal and concentrated sea water maintain their stomach fluids more concentrated than the external medium. Sera electrolyte concentrations from crabs in 100% sea water are significantly lower than stomach fluid concentration for Mg, K, Ca, NH 4 and SO 4 . In crabs from 175% sea water corresponding serum electrolyte significance is found for Mg, Ca, NH 4 , Cl and SO 4 . 5. All electrolytes are regulated by the antennary gland by crabs in the high salinity medium and all except Ca in the normal sea water ; Mg and NH 4 are espe- cially controlled by the antennary gland. In concentrated media the antennary gland excretion of Na is significantly lower than in normal sea water while the Mg ex- cretion is markedly elevated. 6. Ammonia appears to be secreted by both the antennary gland and the stomach but its appearance in the gill fluid is attributed to diffusion. 7. Urine osmotic and electrolyte concentrations are significantly higher than the corresponding serum concentrations for animals in both media. 8. For crabs in 100% sea water the average fluid osmotic concentrations are equivalent to the following moles of NaCl : serum, 0.497 ; urine, 0.583 ; gill fluid, 0.506 and stomach fluid, 0.758 ; for crabs in 175% sea water the corresponding values are : serum, 0.587 ; urine, 0.683 ; gill fluid, 0.860 and for stomach fluid, 0.828. 9. By the use of Na 24 , the relative specific activities of serum and gill fluid from crabs in 175% sea water are shown to be significantly higher than the corresponding serum and gill fluid values from crabs in 100% sea water while the RSA values of the urines are not significantly different. Na 24 counts in gill tissue from the 175% sea water crabs are significantly lower than in the 100% sea water crabs. Active excretion of Na by the gills is indicated. 10. The low isotopic concentration of the mid-gut gland from crabs in concen- trated sea water, comparable to that of gill tissue, suggests a Na secretory mechanism for this organ. The high isotopic Na concentrations found in muscle and stomach tissues of crabs in 175% sea water indicate that these tissues may be serving as storage depots during periods of serum Na stress. 11. The data show that the chief sites of entrance of water and electrolytes into these fiddler crabs are the stomach and the gills. 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Saunders Co., Philadelphia. PROSSER, C. L., J. W. GREEN AND T. J. CHOW, 1955. Ionic and osmotic concentrations in blood and urine of Pachygrapsus crassipes acclimated to different salinities. Biol. Bull., 109 : 99-107. ROBERTSON, J. D., 1949. Ionic regulation in some marine invertebrates. J. Exp. Biol, 26 : 182-200. ROBERTSON, J. D., 1953. Further studies on ionic regulation in marine invertebrates. /. Exp. Biol, 30 : 277-296. RUSSELL, J. A., 1944. Colorimetric estimation of small amounts of ammonia by the phenol- hypochlorite reaction. /. Biol. Chem., 156: 457-461. SCHALES, O., AND S. S. SCHALES, 1941. A simple and accurate method for the determination of chloride in biological fluids. /. Biol. Chem., 140 : 879-884. WEBB, D. A., 1940. Ionic regulation in Carcinus maenas. Proc. Roy. Soc. London, Series B , 129: 107-136. STUDIES ON THE ROLE OF THE CORPUS ALLATUM IN THE ERI-SILKWORM. PHILOSAMIA CYNTHIA RICINI 1 M. ICHIKAWA AND J. NISHIITSUTSUJI-UWO Zoological Institute, University of Kyoto, Japan The corpus allatum of insects has two known functions. In the developing in- sect, it furnishes a hormone which, in collaboration with the growth and differentia- tion hormone of the prothoracic glands (or their homologues), brings about larval molts. In the adult female, presumably the same corpus allatum hormone stimulates gonadal development, especially yolk deposition in the eggs. The latter effect has been demonstrated in a variety of species representing, among others, Orthoptera (Pfeiffer, 1939; Scharrer, 1946), Hemiptera (Wigglesworth, 1936), and Diptera (Thomsen, 1940, 1942; Vogt, 1941, 1943; Day, 1943). On the other hand, the adult ovaries of several representatives of Lepidoptera tested proved independent of the corpus allatum hormone (Bounhiol, 1942; Fukuda, 1944; Williams, 1946). In another lepidopteran, the Eri-silkworm, Philosamia cynthia ricini, the corpus allatum of the newly emerged moth is 20 times larger than that of the last instar larva, an observation which suggests that this gland is functionally active in the adult of this species. In the course of experiments designed to demonstrate this physiological activity in adult Philosamia, a new role of the corpus allatum was. discovered. MATERIAL AND METHODS Larvae of Philosamia were reared at around 25 C. Pupae from which the brain had been removed not later than 22 hours after pupation (artificially induced diapause) were used as test animals. Four to 6 corpora allata from donors of different stages were implanted into these diapausing pupae through a small hole in the dorsal integument of the second or third abdominal segment. The hole was then covered with a piece of integument and the wound was coated with melted paraffin. In some additional experiments, brains were implanted together with corpora allata; in others, corpora cardiaca were added, since they are known to store neurosecretory material originating in the brain. Following the implantation, the specimens were kept again at about 25 C. and were examined at appropriate intervals. RESULTS 1. Implantation of corpora allata from adult donors Implants of corpora allata from male or female donors whose adult age was 1-2 days, into diapausing pupae that had been deprived of their brains for two months, 1 This work was supported by a research grant from the Ministry of Education, Japan. A part of this paper was presented at the 28th Annual Meeting of the Zoological Society of Japan, held at Sapporo, 1957. We wish to thank Dr. Berta Scharrer, Albert Einstein College of Medicine, New York, for her assistance in the preparation of this manuscript. 88 CORPUS ALLATUM IN PHILOSAMIA 89 were effective in 9 out of 10 cases (Table I). Within 22-32 days after implanta- tion the hosts underwent an additional pupal molt. These animals were unable to shed the old pupal cuticle by themselves, but molting fluid was present so abund- antly that the old cuticle could be easily removed by forceps. The new pupal skin thus exposed was of normal color in the posterior half of the animal, but it ap- peared yellowish white in the anterior part. The imaginal discs of wings, antennae, and legs showed a very slight development toward the adult form while other organs displayed no sign of adult differentiation. This result reveals two important effects of the corpus allatum of Philosamia: (1) the implants must have furnished juvenile hormone since the molt following their implantation was pupal rather than adult. This effect is in keeping with the known role of the corpora allata in a variety of insect species. (2) The implants, in addition to the juvenile hormone, must have furnished a principle which initiated molting in a diapausing host deprived of its brain. It was concluded that this molt- inducing hormone originated in the neurosecretory cells of the brain of the donor and was stored in its corpus allatum. An axonal transport of neurosecretory ma- TABLK I Implantation of endocrine organs isolated from adults Endocrine organ Number of implanted organs Number of experimental specimens Number of deaths or undeveloped cases Number of adults Number of second pupal instars Corpus allatum 4-6 10 1 9 Corpus rardiarum 4-6 10 1* (10%) Brain-cardiaca-allata complex 3 15 3 12 (80%) * The interval needed for its development was abnormally long. terial produced in the insect protocerebrum has already been demonstrated in earlier investigations (Scharrer and Scharrer, 1944; Scharrer, 1952; M. Thomsen, 1954, and others). In many species, the neurosecretory material can be traced only as far as the corpora cardiaca which in these forms are considered as the main storage and release center of neurosecretory hormones. Therefore, corpus cardiacum im- plants and brain implants, either alone or in combination with corpus allatum im- plants, also were tested. When four to six corpora cardiaca were implanted into each of ten diapausing pupae, only one of the recipients emerged 47 days later, an interval much longer than that normally required for adult development. The other nine hosts remained unchanged. This result demonstrates at best only a minor role of the corpus card- iacum of Philosamia as a storage center for neurosecretory material. Each of 15 diapausing pupae (417 days after their brain was extirpated) re- ceived three complexes of brain-corpora cardiaca-allata plus subesophageal gang- lion. Three animals died. Twelve of the hosts pupated again within three weeks after implantation ; none proceeded to become an adult moth. These results do not differ from those after the implantation of corpora allata alone. 90 M. ICHIKAWA AND J. NISHIITSUTSUJI-UWO 2. Implantation of corpora allata jrotn pupal donors The pupae which furnished the corpora allata in this series had passed from 11 to 13 days in the pupal state. Again each of the diapausing hosts received six corpora allata. Seventeen out of 22 pupae thus operated upon differentiated quite normally into moths within 25 days after the implantation (Table II). The re- maining five hosts remained pupae or died before showing any positive result. It is of interest that none in this group underwent a second pupal molt. Thus the result differs from that of the previous experiment in which adult corpus allatum implants had been used. One must conclude that pupal corpora allata contain only the hormone which stimulates the prothoracic glands, but are devoid of appreciable amounts of juvenile hormone. The addition of pupal brains and corpora cardiaca to corpus allatum implants did not alter the outcome of the results. Twenty-one out of 22 diapausing animals TABLE II Implantation of corpora allata isolated from pupae and larvae Endocrine organ Number of implanted organs Number of experimental specimens Number of deaths or undeveloped cases Number of adults Number of second pupal instars Pupal donors: Corpus allatum 6 22 5 17 (77.3%) Brain-cardiaca -allata 3 22 1 21 complex (95.4%) Brain 3 23 4 19 (82.6%) Larval donors: Corpus allatum (5th instar) 6 24 5* 19 (20.8%) (79.2%) Corpus allatum (4th instar) 6 27 6 21 (77.8%) One specimen required an abnormally long interval. Thus, receiving these grafts emerged after about 25 days ; the remaining one died, none of these animals underwent an additional pupal molt. In another group of test animals each of which received three pupal brains, emergence occurred after the same period of time in 19 out of 23 specimens. These results show that (a) implants of either pupal brains or pupal corpora allata furnish the hormone necessary for the initiation of adult differentiation, and (b) pupal corpora allata do not contain appreciable amounts of juvenile hormone. 3. Implantation of corpora allata from larval donors Among 27 test animals which received corpora allata removed from fourth in- star caterpillars two days before the next molt, 21 underwent a second pupal molt within 11 to 14 days. None showed adult differentiation. The result was some- what different when the donors were fifth instars which had just entered the spin- CORPUS ALLATUM IN PHILOSAMIA 91 ning stage. In this group 19 out of 24 test animals had another pupal molt while four became adult moths after a normal, and one after a prolonged, interval of time. It seems that in the last mentioned five cases the corpora allata had already ceased to secrete juvenile hormone. 4. Extirpation of corpora allata from pupae Since the preceding experiments had demonstrated the presence of juvenile hor- mone in the corpora allata not only of larval but also of adult Philosamia, the question arose which role is played by these glands in the imago. A possible control over gonadal activity was tested by removing the corpora allata from pupae not older than 40 hours which were then allowed to complete their adult development. Twelve allatectomized specimens did not differ essentially from 20 sham operated controls. In each group about the same number of eggs became mature (Table III) . In other TABLE III Comparison of egg development in allatectomized and control females Number of specimens examined Average number of eggs Mature Immature Total Allateetomized 12 128 150 278 Control 20 154 131 285 words, in Philosamia ovarian function seems to be independent of the corpora allata. Future tests with biochemical methods will be needed to show whether or not the corpora allata in this species have a metabolic function. DISCUSSION The present experiments have revealed that in Philosamia brainless pupae can be induced to molt by the implantation of corpora allata. Depending on the stage of the donor, the molt caused may or may not be coupled with adult differentiation. Larval and adult corpora allata furnish enough juvenile hormone to render the en- suing molt of the test animal a second pupal molt. By contrast, pupal corpora allata lack effective doses of juvenile hormone. The type of molt occurring is, how- ever, of less interest than the fact that molts can be induced at all by corpus allatum implants in cases where they would otherwise not occur. While it has been known for some time that corpora allata from larval and adult donors can furnish juvenile hormone, the present study offers the first evidence that corpus allatum implants can induce molting. Theoretically, the molt-inducing hormone present in the corpus allatum implants used in our experiments either could have originated in the corpora allata themselves, or it could merely have been stored there. The first possibility seems less likely. The reasons for assuming the second mode of action are as fol- lows. In Philosamia as well as other forms of insects, neurosecretory cells of the brain are known to furnish a hormone which stimulates the prothoracic glands into releasing a molt-promoting hormone. It is also known that this neurosecretory 92 M. ICHIKAWA AND J. NISHIITSUTSUJI-UWO material is transported along axons and stored at some distance from the site of origin. In a variety of species the storage and release center is the corpus cardiacum. In some species, including Philosamia cynthia, neurosecretory material has been ob- served to enter also the corpus allatum. However, the possibility that this gland stores neurosecretory material in appreciable amounts has never been tested ex- perimentally with positive results. So far, the presence of neurosecretory material within the corpus allatum tissue has been interpreted as a possible morphological indication for the existence of an allatotropic action on the part of neurosecretory cells (E. Thomsen, 1954). The present study neither contradicts nor supports this view. However, judging from the result with pupal donors of Philosamia, juvenile hormone can be absent in corpora allata in which brain hormone is known to be stored. Therefore, one would have to assume that corpus allatum cells do not necessarily respond under all circumstances to stimulation by an "allatotropic hormone." Fur- thermore this factor may or may not be identical with the molt-inducing hormone. The present study offers evidence that implants of corpora allata in Philosamia furnish brainless pupae with a sufficient amount of neurosecretory material to in- duce them to molt. It does not prove that in the intact animal the corpus allatum tissue serves as the main storage and release center of a hormone produced by the brain. The possibility exists that neurosecretory material which reaches the organ via the nervi corporis allati accumulates within the corpus allatum in gradually in- creasing amounts without being given off into the circulation. This situation would perhaps be comparable to the accumulation of juvenile hormone in the abdomen of adult males of Platysamia (Williams, 1956). Further experiments will be needed to determine whether in species such as Philosamia with inconspicuous corpora cardiaca the corpora allata indeed take over the main storage and release function. The experimental demonstration of the presence of molt-promoting hormone in the corpora allata of Philosamia is paralleled by morphological data showing the existence of a corresponding neurosecretory pathway. The presence of neuro- secretory material in the nervi corporis allati has been observed in Bonibyx (Bounhiol, Gabe and Arvy, 1953, 1954; Kobayashi, 1957) as well as Philosamia (unpublished observations of the authors). Whatever the mechanism of release of neurosecretory hormones under normal physiological conditions, the fact remains that, with the exception of the pupal stage, the corpora allata of Philosamia contain two hormones controlling post-embryonic development, the "prothoracotropic hormone" of neurosecretory origin and the "juvenile hormone" produced by the corpus allatum cells themselves. SUMMARY 1. Pupae of Philosamia cynthia ricini in which diapause had been artificially in- duced by the removal of the brain, served as test animals for the effects of corpus allatum implants. Four to six corpora allata from donors in different stages in- duced molting in hosts which otherwise would have remained pupae. It was con- cluded that in Philosamia the corpus allatum, in addition to producing juvenile hor- mone, contains an appreciable amount of molt-inducing hormone furnished by neuro- secretory cells of the brain. The interpretation is supported by the existence, in Philosamia as well as other insect species, of a neurosecretory pathway which links CORPUS ALLATUM IN PHILOSAMIA the secretory part of the brain with the corpora cardiaca-allata and which permits the storage of hormones produced in the brain at some distance from the cells of origin. While in most species studied so far the main storage center is the corpus cardiacum, this role may have been taken over by the corpus allatum in Philosamia. 2. As might be expected, the molt induced may or may not be coupled with adult differentiation depending on the stage of the donor. Implants of corpora allata from adult or fourth instar larval donors caused an additional pupal molt because, in addition to molt-inducing hormone, they also supplied juvenile hormone to the host. By contrast, implants from pupal donors contained no appreciable amount of juvenile hormone with the result that they brought about an imaginal molt. Some of the fifth instar implants had the same effect as those from pupae, while others acted like tissues from fourth instars. It seems that during the fifth larval stage the change from activity to temporary inactivity of the corpus allatum cells occurs gradually. Thus implants of larval and adult corpora allata furnish two hormones controlling post-embryonic development, while pupal corpora allata contain only one, namely, the neurosecretory material derived from the protocerebrum. 3. Even though the presence of corpus allatum hormone has been demonstrated in glands from adult donors in the present experiments, the role normally played by this hormone in the adult moth is still unknown. Extirpation of corpora allata from female pupae of Philosamia did not prevent egg maturation in the resulting moths. LITERATURE CITED BOUNHIOL, J. J., 1942. L'ablation des corps allates au dernier age larvaire n'affecte pas, ulterieurement, la reproduction chez Bomby.r mori. C. R. Acad. Sci., 215: 334-336. BOUNHIOL, J. J., M. GABE AND L. ARVY, 1953. Donnees histophysiologiques sur la neuro- secretion chez Bombvx mori L., et sur ses rapports avec les glandes endocrines. Bull. Biol. France Bclg., 87 : 323-333. BOUNHIOL, J. J., M. GABE AND L. ARVY, 1954. Donnees histophysiologiques sur la neurosecre- tion chez Botnbyx mori L. et sur ses rapports avec les glandes endocrines. Pubbl. Stas. Zool. Napoli, Suppl, 24: 52-53. DAY, M. F., 1943. The function of the corpus allatum in muscoid Diptera. Biol. Bull., 84 : 127-140. FUKUDA. S., 1944. The hormonal mechanism of larval moulting and metamorphosis in the silkworm. /. Fac. Sci. Tokyo (Imp.} Univ., Sect. IV, 6: 477-532. KOBAYASHI, M., 1957. Studies on the neurosecretion in the silkworm, Bombyx mori L. Bull. Scricult. Exp. Station, 15: 181-273. PFEIFFER, I. W., 1939. Experimental study of the function of the corpora allata in the grass- hopper, Mclanoplus diffcrentialis. J. E.rp. Zool., 82: 439-461. SCHARRER, B., 1946. The relationship between corpora allata and reproductive organs in adult Leucophaca madcrae (Orthoptera). EndocrinoL, 38: 46-55. SCHARRER, B., 1952. Neurosecretion. IX. The effects of nerve section on the intercerebralis- cardiacum-allatum system of the insect Leucophaca madcrae. Biol. Bull., 102 : 261-272. SCHARRER, B., AND E. SCHARRER, 1944. Neurosecretion. VI. A comparison between the inter- cerebralis-cardiacum-allatum system of the insects and the hypothalamo-hypophyseal system of the vertebrates. Biol. Bull., 87 : 242-251. THOMSEN, E., 1940. Relation between corpus allatum and ovaries in adult flies (Muscidae). Nature, 145: 28-29. THOMSEN, E., 1942. An experimental and anatomical study of the corpus allatum in the blow-fly, Calliphora er\throcephala Meig. Vid. Medd. Dansk Naturh. Forcn., 106 : 319^05. 94 M. ICHIKAWA AND J. NISHIITSUTSUJI-UWO THOMSEN, E., 1954. Experimental evidence for the transport of secretory material in the axons of the neurosecretory cells of Calliphora erythrocephala Meig. Pubbl. Stas. Zool. Napoli, Suppl., 24: 48-49. THOMSEN, M., 1954. Neurosecretion in some Hymenoptera. Dan. Biol. Skr., 7, no. 5 : 1-24. VOGT, M., 1941. Bemerkung zum Corpus allatum von Drosophila. Naturwiss., 29 : 80-81. VOGT, M., 1943. Zur Produktion gonadotropen Hormones durch Ringdriisen des ersten Lar- venstadiums bei Drosophila. Biol. Zentralbl., 63 : 467^470. WIGGLESWORTH, V. B., 1936. The function of the corpus allatum in the growth and reproduc- tion of Rhodnius prolixiis (Hemiptera). Quart. J. Micr. Sci., 79: 91-121. WILLIAMS, C. M., 1946. Physiology of insect diapause. The role of the brain in the pro- duction and termination of pupal dormancy in the giant silkworm, Platvsamia cecropia. Biol. Bull., 90: 234-243. WILLIAMS, C. M., 1956. The juvenile hormone of insects. Nature, 178: 212-213. BETA-GLUCOSIDASE OF THE MIDGUT OF THE SILKWORM BOMBYX MORI TOSH1O 1TO AND MOTOZO TANAKA Scriciilhinil Experiment Station, Sitf/iiinmi-kit, Tokyo, Japan Contrary to a wide distribution of /y-glucosidase in plants, the occurrence of this enzyme in insects seems to be rare, since neither utilization of /3-glucosides nor presence of the enzyme activity has been often recognized in insects. Until re- cently, the demonstration of this enzyme in insects has been discussed on the basis of the utilization of /^-glucosides in growth experiments (see the review by Lipke and Fraenkel. 1956). Studies of this enzyme from the enzymic points of view, however, have been lately carried out with the wood louse Pored! io ( Newcomer, 1952, 1956) and the cockroach Periplaneta unierieuna (Newcomer, 1954). Ap- plying a highly sensitive fluorimetric method for /?-glucosidase assay, Robinson (1956) has demonstrated the occurrence of the enzyme in the locust Locitsta uii- gratorla, the mealworm Tenebrio niulitor, the water-boatman Notonecta, the cock- roach Periplaneta ainerieana, and the black aphis Aphis jubae. The occurrence of this enzyme has also been reported for the bean weevil Callosobruchus ehinensis, the bean blister beetle Epieanta (/or/mini, the silkworm Boiuby.v inori, and the wild silk- worm Dictyoploca japoniea (Koike, 1954), and for the mealworm Tenebrio inolitor (Fraenkel," 1955). A few years ago the present authors became aware of the fact that the midgut homogenate of the silkworm is able to hydrolyze salicin, but the digestive fluid gave scarcely the same reaction. Recently, this problem was re-investigated to obtain more detailed results. This report is mainly concerned with the occurrence of /3- glucosiclase in the silkworm midgut, its characterization, and partial purification. A comparison of the enzyme activity of the normal larvae was also made with amylase-free mutants, and with jaundice-diseased larvae. MATERIALS AND METHODS Practical methods of obtaining midgut homogenates have been previously re- ported (Tto. Horie and Ishikawa, in press; Ito and Horie, in press). Homogenates made in water were used directly in some experiments, but the acetone powder made with midgut homogenates was used for most enzyme preparations. Midgut homogenates made from middle fifth instar larvae were dehydrated by mixing with 7 volumes of chilled acetone and the precipitates were collected in a Biichner funnel under suction. The precipitates were subsequently re-suspended in chilled acetone, then separated from acetone with funnel as above. The precipitates were washed by running alcohol-ether mixture (1:1) and brought to dryness in a vacuum desicca- tor. The dried, pale-yellow cake was ground in a mortar and the acetone powder thus made was used for enzyme tests. The powder was kept in racuo at 5 C. at least for 8 months without any loss of /}-glucosidase activity. Preparing the enzyme solu- tion, the powder was suspended in water, allowed to stand for two hours at 5 C., 95 96 TOSHIO ITO AND MOTOZO TANAKA and the supernate, obtained after centrifugation at 10,000 X g for 10 minutes, was used for the experiments. Digestive fluid was collected from middle fifth instar larvae by applying a weak electric shock to them. The fluid was either used for enzymic measurements directly after dialysis against water at 5 C. for 48 hours, or after conversion to an acetone powder. Enzyme activity was assayed by measuring the amount of glucose liberated from /?-glucoside in the reaction system. Salicin was used as a substrate in most ex- periments, and cellobiose or phenyl /3-glucoside in some. Unless otherwise indicated, each reaction mixture contained 200 pM citrate buffer (pH 5.4), 48 p.M salicin and 1.0 ml. acetone powder solution (total volume 4.0 ml.) and was incubated at 30 C. for two hours. The reaction was stopped at intervals by adding an aliquot to Ba(OH) 2 and ZnSO 4 , or Na 2 WO 4 . When cellobiose was used as the substrate, the reaction was stopped by Na 2 WO 4 and H 2 SO 4 , and bakers' yeast then applied to the supernate of the reaction mixture in order to remove fermentable sugar. Glucose was determined mainly by the method of Hagedorn and Jensen (1923) and some- times by Somogyi's procedure (1952). Nitrogen was determined by the micro-Kjeldahl method. RESULTS Optimal pH range The supernate obtained from an acetone powder suspension was incubated with various buffers at different pH levels. As shown in Figure 1, almost no alteration of pH optimum was found with different buffers. Optimal pH ranges were 5.0-6.2 for citrate and phosphate, and 5.2-6.4 for acetate. These ranges are more extended than those reported for other insects (Newcomer, 1952, 1954, 1956; Robinson, 1956) and for plants (Veibel, 1950). The enzymic activity was relatively high at a high pH level such as 7.0 or even 8.0, which has not been reported so far for other species of insects. The measurement also showed that /3-glucosidase activity in borate buffer was not reduced to zero at pH 9.4. It has been known that the pH optimum of this enzyme is dependent on the source of the enzyme and to a minor degree on the substrate and the buffer solution (VeibeL 1950). Velocity of hydrolysis The relationship between enzyme concentration and velocity of hydrolysis is shown in Figvire 2, where the enzyme concentration was doubled, respectively, from curve 3 to curve 1 (1:2:4). It is apparent that the rate of glucose libera- tion is proportional to enzyme concentration. Figure 2 also shows that the reaction proceeded at a uniform rate when enzyme concentration was relatively low. In Figure 3 the effect of the concentration of the substrate on the enzyme activity is shown. The curves were plotted according to the procedure of Lineweaver and Burk (1934), i.e., the inverse of the activity against the inverse of salicin concentra- tion. The Km value (the Michaelis constant) is 0.013 M, which is in accord with the value reported for salicin (Veibel and Lilleluncl, 1938). Inhibition by high temperature The effect of high temperature on midgut /?-glucosidase is shown in Table I. The supernate obtained from acetone powder suspension was treated at 40 to 70 C. BETA-GLUCOSIDASE OF SILKWORM MIDGUT 97 0.3 0.2 Q l o ^ < o o 8 FIGURE 1. Relationship between pH and /3-glucosidase activity. Phosphate buffer: . Borate buffer : -X. Citrate buffer : O- Acetate buffer: A 2 3 HOURS 5 24 16 8 Q 4 6 I/S 8 10 xlO FIGURE 2 (left). /3-Glucosidase activity as a function of time for different enzyme concentrations. FIGURE 3 (right). Relationship between /3-glucosidase activity and salicin concentration. Borate buffer (pH 6.0). Total volume, 5.0 ml. Incubation, one hour. Enzyme activity was expressed in terms of glucose liberated per dry matter on the basis of the same weight. 98 TOSHIO ITO AND MOTOZO TANAKA TABLE I Effect of high temperature on f)-glucosidase activity Relative activity (%) 40 C. 50 C. 60 C. 70 C. 5 min. 102.4 100..? 86.4 9.2 If) 100.4 96.1 61.9 4.9 20 94.2 81.6 22.3 4.9 Control 100.0 100.0 100.0 100.0 for 5 to 20 minutes. At 40 C. no effect was observed with a 10-minute exposure and slight inhibition was recognized after exposure for 20 minutes. The treatment at 50 C. for 10 minutes resulted in a slight inhibition and that for 20 minutes in a 20 per cent inhibition. The treatment at 60 C., however, resulted in a markedly increasing loss of the activity according to the prolongation of exposing period up to 80 per cent of inhibition. By applying a high temperature of 70 C., most of the activity was lost within 5 minutes. Inhibition />v lieai'v metals In insects the inhibition of /3-glucosidase by heavy metals has been reported for the ventriculus of the adult cockroach (Newcomer, 1954) . Inhibition of the enzyme solution obtained from the silkworm miclgut with varying concentrations of AgNO 3 or HgCl L , resulted in varying degrees of inhibition, as shown in Table II. Effect of organic acids Inhibition of /8-glucosidase by organic acids has been reported for Pcnicilliuiu. when phenyl /3-glucoside was used as substrate (Murakami, 1950). Malic, fumaric, and citric acids were tested for their inhibitory effects on silkworm midgut fi- glucosidase -at a final concentration of 0.05 M. The results showed that no ap- preciable inhibition was observed, when salicin was used as substrate. Effect of toluene Newcomer (1954) has shown that an activation of /3-glucosidase by toluene does not occur in the cockroach. The effect of toluene on midgut /3-glucosidase in the silkworm was tested and no activation was recognized. Toluene was, there- TABLE II Final concentration (M) 1 X 10~- 2 X 10- 3 1 X 10- 3 2 X 10~ 4 1 X 10~ 4 Inhibition of fi-glucosidase activity by heavy metals Inhibition (%) AgNO.-i 95.3 76.4 57.9 26.4 1.1 0.0 HgCl-. 86.7 52.8 23.5 15.5 0.0 BETA-GLUCOSIDASE OF SILKWORM MIDGUT 99 fore, added to the incubation mixture when a long period of incubation was necessary. Distribution of the actk'itv in the midgut The activity of /8-glucosidase was compared among different parts of the mid- gut, i.e.. anterior, middle, and posterior midguts. The measurement of the activity was carried out with fresh homogenates and the results are shown in Figure 4. It is evident that the majority of the activity is concentrated in the posterior midgut. while a very low activity is found in the anterior and middle midguts. i Z. 1 9 I . i. 2 .0 Z 3 d 0.8 5- 3 2. t \ r\ ^ I U.O > LiJ _ C/} 1- < 3 0.4 o d * 02 2 2 7 n i 3 1 1 ANTERIOR MIDDLE POSTERIOR TOTAL FIGURE 4. Distribution of /3-glucosidase activity in the different parts of the midgut. 1, fourth day; 2, seventh day; 3, eighth day of hfth instar. The change in the activity (hiring larval development The changes in /3-glucosidase activity according to the development were meas- ured with fresh midgut homogenates during fourth and hfth larval instars. The measurements were made with spring silkworms, the rearing temperature ranging approximately from 20 to 25 C.. and with summer silkworms, the rearing tempera- ture ranging approximately from 25 to 30 C. Though the activity expressed by unit glucose freed per nitrogen was higher in spring silkworms than in summer silkworms, the changes in the activity were almost the same in both (Fig. 5). In general, the activity was low during the fourth and early fifth instars. A marked increase in the activity occurred at the middle period of the fifth instar, and was maintained for a few days. Then the activity dropped suddenly and reached the lowest level during cocoon-spinning. Precipitation by ammonium sulfate In a preliminary experiment it was noticed that the majority of the activity was precipitated between 0.3 and 0.5 saturation with ammonium sulfate, when a suspen- 100 i.o - o 0.8 i "- 1 - 1 >o 0.6 * e> O- 4 2 0.2 -A TOSHIO ITO AND MOTOZO TANAKA 0.5^-2 0.4 0.3 0.2 O.I 23450123456789 012340123456789 AGE IN DAYS FIGURE 5. Change in /3-glucosidase activity during larval development. 1, spring rearing ; 2, summer rearing. Fresh homogenates were diluted to ] /l> in spring, and % in summer. A, fourth instar ; B, fourth molting period ; C, fifth instar ; D, cocoon-spinning period ; M, maturity. sion of acetone powder was used. Therefore, an attempt was made to purify /?- glucosidase of the midgut by means of ammonium sulfate precipitation. Subse- quently, the precipitation procedure was repeated several times by increasing the concentration of ammonium sulfate progressively. Table III shows one of the results obtained. Acetone powder made with posterior midguts w r as suspended in water in the cold for four hours ; this suspension was used for the precipitation experiment. The specific activity of this suspension was 1.83 and that obtained with supernate after centrifugation at 12,000 X g for 10 minutes was increased al- most three times, as seen in Table III. About 90 per cent of the original activity was found in the supernate. Until 0.350 saturation, very slight activity was pre- cipitated. Most activity was precipitated between 0.350 and 0.450 saturation and the highest specific activity was obtained between 0.375 and 0.425 saturation. The specific activity was increased to about 4 times that of the supernate, and 10 times that of the original suspension. The application of ammonium sulfate precipitation thus seems to be to some extent useful for the purification of ^-glucosidase. TABLE III Precipitation of 0- glucosidase by ammonium sulfate Saturation of ammonium sulfate Total activity, mg. glucose Specific activity, mg. glucose/mg. N Recoverv. % Suspension Supernate -0.325 360.67 327.88 5.23 1.83 4.37 0.23 100.0 90.91 1.45 0.325-0.350 8.13 0.84 2.25 0.350-0.375 55.73 9.68 15.45 0.375-0.400 92.00 16.61 25.51 0.400-0.425 75.05 14.92 20.81 0.425-0.450 52.50 7.64 14.55 0.450-0.475 12.23 3.71 3.39 0.475-0.500 7.30 3.97 2.02 BETA-GLUCOSIDASE OF SILKWORM MIDGUT 101 Acetone powder used in the present study was recognized to possess amylase and invertase, in addition to /3-glucosidase. An attempt was therefore made to separate /3-glucosidase from amylase or invertase by means of ammonium sulfate precipitation. The result showed that the precipitates at between 0.375 and 0.425 saturation contained all of three activities at almost the same level (/3-glucosidase, 56.5% ; amylase, 41.7% ; invertase, 57.0%). Several methods have been presented for the standardization of /3-glucosidase ( Veibel, 1950) . An enzyme efficiency was obtained with a few fractions precipitated by ammonium sulfate by the use of phenyl /?-glucoside as the substrate (final con- centration, 0.052 If), according to the procedure by Helferich (1933, 1938). A high value of enzyme efficiency, 0.898, was obtained with the precipitate at between 0.375 and 0.425 ammonium sulfate saturation, while 0.170 with the precipitate be- 0.4 O 0.2 I I I 10 FIGURE 6. Distribution of /3-glucosidase activity after paper electrophoresis. was expressed in terms of mg. glucose liberated. The activity tween 0-0.375 saturation, 0.154 with that between 0.425-0.700 saturation, and 0.095 with the original supernate of acetone powder suspension. Furthermore, /3-glucosidase of the silkworm midgut was recognized to hydrolyze cellobiose as the substrate. Purification by paper electrophoresis Robinson (1956) has applied a paper electrophoretic procedure for the separa- tion of /?-glucosidase from /3-glucuronidase in the locust-crop fluid. A similar procedure was also tested with /?-glucosidase from the silkworm midgut. Either an acetone power supernate or the precipitate at 0.425-0.450 saturation of ammonium sulfate was used for the experiment. An enzyme fraction was subjected to elec- trophoresis on filter paper (Toyo No. 51)3 cm. >< 30 cm. in 0.1 AI phosphate buffer at pH 5.8, 150 volts, 5 ma., for 8 hours. Subsequently, the paper was cut into half along the long side of the paper. One of the divided strips was dried and suspended in the staining solution (Amido Black) for proteins, and the other was cut into 102 TOSHIO ITO AND MOTOZO TANAKA one-cm, sections starting from the original spot, each section being chopped into small pieces which were immediately placed into a test tube with 1.0 ml. of phosphate buffer (pH 5.8). The test tube was kept at 5 C. for 5 hours to extract the enzyme, then heated to 37 C. with the addition of an appropriate amount of salicin solution (final concentration, 0.0125 M). After a 16-hour incubation, the amount of sugar liberated was determined. The results of the enzymic test, as well as of the staining test on the precipitate by ammonium sulfate, are shown in Figure 6, where the /3-glucosidase activity is shown in the form of histograms. /3-Glucosidase appeared in the locations corresponding to the staining test on the other strip. Little activity was found at the original point, where a protein band remained. However, when the whole midgut suspension was used, another two protein bands were recognized on the paper, which were considered to have been removed by the procedure of the precipitation with ammonium sulfate. The separation of invertase from /3-gluco- sidase by electrophoresis was unsuccessful. (3-Glucosidase activity in the inidynt oj jaundice-diseased lurrac Two types of polyhedroses are known to occur in the silkworm, one of which is called cytoplasmic polyhedrosis, with the formation of the polyhedral bodies in the midgut cytoplasm. Several days after the infection, midgut tissue becomes white, which is a typical svmptom of this disease. /i-Glucosidase activity was compared between normal and infected larvae. The activity was always lower in diseased larvae reduced to 61 per cent of the normal larvae (incubation period, two hours) and 82 per cent (incubation period, 6 hours ). fl-Glucosidase uetii'itv in the digestive fluid The activity of the digestive fluid per unit nitrogen of early fourth instar larvae was one-third that of the midgut or less, while that of late fourth instar and fifth instar larvae was less than one-tenth that of the midgut. /?-Glucosidase activity was also recognized in the digestive fluid of the amylase-free strain, which is de- ficient in amylase activity in the digestive fluid. The experiment performed at the same time showed that the midgut of the amylase-free strain possessed the same level of activity of /?-glucosidase as the normal strain. DISCUSSION The exact physiological role of /3-glucosidase of the silkworm midgut in diges- tion is at present not well understood. The enzyme activity on the basis of the same unit is, however, higher in the midgut than in the digestive fluid. This seems to suggest that /?-glucosidase in the midgut cells is of rather more importance than that in the digestive fluid. The optimal pH of midgut /^-glucosidase ranges ap- proximately 5.0 to 6.4, while an effort was unsuccessful to determine its range in the digestive fluid. A possible role of /3-glucosidase in the cells of the midgut in digestion is also deduced from the fact that the pH value of the digestive fluid is strongly alkaline, as much as 10.0. The movement of food through the gut is generally fast in the silkworm larva, occurring within a few hours. Thus, even though the degree of participation of this enzyme in digestion as a whole is still BETA-GLUCOSIDASE OF SILKWORM MIDGUT 103 unknown, the possibility remains that the mulberry carbohydrates which have not been completely hydrolyzed in the lumen of the gut might be hydrolyzed after absorption in the midgut tissue. It is interesting from the standpoint of compara- tive physiology that the intercellular enzyme might participate in the digestion. Al- though conclusions drawn from a study of enzyme alone are generally open to question in regard to the physiological role in intact organs, a good correlation was found between pure compounds supporting growth and the presence of digestive enzymes in insects (Day and Waterhouse, 1953). Koike (1954) could not demon- strate cellulase in the digestive tract of the silkworm and Hiratsuka (1917) has shown that cellulose is not utilized by silkworm larvae. This is the same situation as reported for the hepatopancreas of Porcellio (Newcomer, 1956) where an activ- ity of /3-glucosidase was demonstrated without that of cellulase. /?-Glucosidase of the midgut or of the digestive fluid of the silkworm seems to hydrolyze /3-glucosides contained in the mulberry leaves. A few papers have been so far published on glucosidic compounds in the mulberry leaves ; recently Hamamura and Naito (1956) isolated arginine /?-glucoside and the presence of glucosides of the pigment has also been reported (Oshima and Nakabayashi, 1951). There is no doubt that these glucosides and possibly other not yet identified glucosides are utilized by the larvae. The results on the characterization experiments suggest that the /2-glucosidase of the midgut is very much similar to that in plants (Veibel. 1950). The enzyme efficiency of /3-glucosidase of the midgut is rather higher than that obtained with plants (Pigman, 1946). A variation in the digestive enzyme activities of different parts of the midgut, as well as in the ability of the absorption of the nutrients, is well known in insects (Day and Waterhouse, 1953; Waterhouse and Day, 1953). The physiological or diges- tive differentiation in the different portions of the midgut of the silkworm is still not well known in many respects. However, the highest activity of /?-glucosidase was found in the posterior midgut (Fig. 4). Matsumura and Oka (1935) have shown that the activity of amylase or invertase is also the highest in the posterior midgut. The glycogen content is increased most markedly in the posterior midgut after sugar ingestion (Horie and Tanaka, 1957) and the highest phosphorus metab- olism was obtained also in this portion (Ito, Horie and Tanaka, in press). The authors wish to express their thanks to Prof. G. S. Fraenkel of the Uni- versity of Illinois for reading the manuscript. SUMMARY 1. The presence of a /2-glucosidase was demonstrated in the midgut of the silk- worm larva, Bomby.r tnori. 2. The enzyme has a pH optimum of approximately 5.2-6.2 and the Km value was 0.013 with salicin as a substrate. 3. The action of the enzyme was slightly inhibited at a temperature of 40 C., and strongly inhibited at 70 C. An inhibition by silver or mercury salts was also observed, while no inhibition was found by organic acids. No activation by toluene was demonstrated. 4. Most of the activity in the midgut was concentrated in the posterior portion. 104 TOSHIO ITO AND MOTOZO TANAKA 5. The enzyme activity varies according to larval growth, being lower at the beginning of the fifth instar, higher after the middle of the instar, and again lower during cocoon-spinning. 6. The enzyme activity was concentrated 10 times by means of ammonium sulfate precipitation at a saturation of 0.375-0.425. Separation by the paper elec- trophoretic method was successfully applied for this fraction, but it was unsuccessful for separating /?-glucosidase from other enzymes. 7. Virus-infected larvae showed a decrease in enzyme activity, compared with normal larvae. 8. /?-Glucosidase activity in the digestive fluid was much lower than that in the midgut. A mutant, amylase-free strain possessed in the digestive fluid the same level of /?-glucosidase activity as the normal one. LITERATURE CITED DAY, M. F., AND D. F. WATERHOUSE, 1953. The mechanism of digestion. In: Insect Physi- ology, pp. 311-330. John Wiley & Sons, Inc., New York. FRAENKEL, G., 1955. Inhibitory effects of sugars on the growth of the mealworm, Tenebrio molitor L. /. Cell. Comp. Physiol, 45 : 393-408. HAGEDORN, H. C, AND B. N. JENSEN, 1923. Zur Mikrobestimmung des Blutzuckers mittels Ferricyanid. Biochem. Zeitschr., 135 : 46-58. HAMAMURA, Y., AND K. NAITO, 1956. Studies on the micro constituent in mulberry leaves. I. On the isolation of arginine-glucoside from mulberry leaves. /. Agricul. Chem. Soc. Japan, 30: 358-361. (In Japanese with English summary.) HELFERICH, B., 1933. Die Spezifitat des Emulsins. Ergcbn. Ensymforsch., 2 : 74-89. HELFERICH, B., 1938. Emulsin. Ergebn. Ensymforsch., 7 : 83-104. HIRATSUKA, E., 1917. Researches on the nutrition of the silk worm. Bull. Imp. Sericul. Exp. Sta., 2: 353-412. (In Japanese.) HORIE, Y., AND M. TANAKA, 1957. Absorption and utilization of glucose in silkworm larvae, Bombyx nwri. J. Sericul. Sci. Japan, 26: 40-45. (In Japanese with English sum- mary.) ITO, T., Y. HORIE AND M. TANAKA. Phosphorus compounds of the midgut in the silkworm. Proc. 10th Internal. Congr. Entomol. (in press). ITO, T., Y. HORIE AND S. ISHIKAWA. Oxidative enzymes of the midgut of the silkworm Bombyx nwri. J. Insect Physiol. (in press). ITO, T., AND Y. HORIE. Carbohydrate metabolism of the midgut of the silkworm Bombyx mori. Arch. Biochem. Biophys. (in press). KOIKE, H., 1954. Studies on carbohydrases of insects. I. Distribution of carbohydrases in several insects. Zool. Mag., 63: 228-234. (In Japanese with English summary.) LINEWEAVER, H., AND D. BURK, 1934. The determination of enzyme dissociation constants. /. Amer. Chem, Soc., 34: 658-666. LIPKE, H., AND G. FRAENKEL, 1956. Insect nutrition. Ann. Rev. Entomol., 1 : 17-44. MATSUMURA, S., AND T. OKA, 1935. Physiological studies on the carbohydrases of the silk- worm. Bull. Nagano-ken Sericul. Exp. Sta., 31 : 1-32. (In Japanese.) MURAKAMI, H., 1950. The effect of organic acids on /3-glucosidase of Penicillium. Kagakn (Science), 20: 326-327. (In Japanese.) NEWCOMER, W. S., 1952. The occurrence of beta-glucosidase in the digestive juice of Por- cellio and Armadillidium. Anat. Rec., 113: 536. NEWCOMER, W. S., 1954. The occurrence of 0-glucosidase in digestive juice of the cockroach, Periplaneta amcricana L. /. Cell. Comp. Physiol., 43 : 79-86. NEWCOMER, W. S., 1956. Digestive carbohydrates of the wood louse, Porccllio. Physiol. Zool., 29: 157-162. OSHIMA, Y., AND T. NAKABAYASHI, 1951. Studies on tannins and pigments by partition chromatography. I. Analysis of quercetin and its 3-glycosides. /. Agricul. Chem. Soc. Japan, 25: 21-25. (In Japanese with English summary.) BETA-GLUCOSIDASE OF SILKWORM MIDGUT 105 PIGMAN, W. W., 1946. Specificity, classification, and mechanism of action of the glycosidases. Adv. EnsymoL, 4: 41-74. ROBINSON, D., 1956. The fluorimetric determination of -glucosidase : its occurrence in the tissues of animals, including insects. Biochem. /., 63 : 39-44. SOMOGYI, M., 1952. Notes on sugar determination. /. Biol. Chem., 195: 19-23. VEIBEL, S., 1950. /3-Glucosidase. hi: The Enzymes, Vol. I, Part 1, pp. 583-620. Academic Press Inc., New York. VEIBEL, S., AND H. LILLELUND, 1938. t)ber die Standardisierung von /3-glucosidase. Enzymol., 5: 129-136. WATERHOUSE, D. F., AND M. F. DAY, 1953. Function of the gut in absorption, excretion, and intermediary metabolism. In: Insect Physiology, pp. 331-349. John Wiley & Sons, Inc., New York. THE EFFECTS OF THIOUREA AND SOME RELATED COMPOUNDS OX REGENERATION IN PLANARIANS 1 MARIE M. JENKINS 2 Department of Biology, The Catholic University of America, Washington, D. C. During recent years much research has been devoted to the effects of the ad- ministration of various anti-thyroid agents to vertebrate animals. Interest is due to the fact that these agents have been demonstrated to inhibit the activity of the thyroid gland. Only a few studies have been made of the effects of such drugs on invertebrates, and the majority of these deal with the effects of the goitrogens on fertilized eggs and developing embryos. Bevelander (1946), using fertilized sea urchin eggs placed in test solutions of 0.1-1.0% thiourea in sea water, found that at a concentration of 1.0% no cleavage occurred, but cleavage was normal in a similar concentration of urea, indicating the inhibition of cleavage was not due to any osmotic effect. Lower concentrations produced a retardation in over-all growth rate. Rulon (1950), studying the modifications in developmental patterns in the sand dollar by thiourea, reports substantially similar results. The present investigation was undertaken in order to study some comparative effects of varying concentrations of thiourea and related compounds on an inverte- brate beyond the embryonic stage. For this study a species of planarian, a fresh- water flatworm, was chosen. In this animal, when the tail is separated from the body by a dorso-ventral cut posterior to the pharynx, the body will produce a new tail, and the separated tail will regenerate all missing structures, becoming a new and independent organism. A study was made of the rate of growth of a new tail by the body, and of the time required for the appearance and development of the regenerated organs in the newly formed worm. Observations were also made of any modifications in the regenerating structures, due to the action of the goitro- gens, and of pigment loss or lack of development, both in the new tissue and in the old, mature cells. MATERIALS AND METHODS The animals used in this study were specimens of Dugcsia tigrina, collected in a stream near Baltimore, Maryland. Stock animals were fed once a week. Experi- mental animals were taken five days after feeding, and were not fed during the experiment. 1 A contribution from the Department of Biology, The Catholic University of America, Washington, D. C. This paper is based on the author's dissertation submitted in partial ful- fillment of the requirements for the degree of Master of Science. The writer wishes to express her appreciation to Dr. E. G. S. Baker, major professor, now Chairman of the Department of Biology, Drew University, Madison, New Jersey, and to Dr. W. G. Lynn, Professor of Zoology, of The Catholic University of America, for their many helpful suggestions during the course of the investigation. - Present address : Department of Zoology, University of Oklahoma, Norman, Oklahoma. 106 EFFECTS OF GOITROGENS ON PLANARIANS 107 Three chemicals were used in the study : thiourea, phenylthiourea, and thiouracil. Since a comparative study of the effects was to be made, three series of experiments were performed, using concentrations of 0.005%, 0.01%, and 0.02% of each chem- ical. Worms chosen for experimentation were as near the same size as possible, varying from seven to nine millimeters in length. Tails were severed a short dis- tance behind the pharynx, and placed in fingerbowls of the proper solution. These were stacked to prevent evaporation. The bodies were placed similarly in other fingerbowls. The worms were handled with sable-hair brushes, or wide-tipped medicine droppers. Control animals were kept in tap water. In Series I, the experimental animals were placed in 0.005% solutions of the chemicals. No observations were made on the regenerating tails the first day after cutting. Beginning with the second day, the tails were observed every day for ten days, then on the fourteenth, eighteenth, and twenty-fifth days. At the end of seven days, the worms in each chemical were divided into two groups. One group was kept in the chemical until the end of the experiment ; the other group was returned to water to see if any of the effects noted were reversible. For observation, the tails were placed in a drop of the solution on a microscope slide, and observed through the low-power objective of a compound microscope, using a blue filter in a standard lamp. Information was obtained concerning the time in days required for healing to take place, and for eyes, proboscis, and sense lobes to form. Observations were also made concerning the color and appearance of the eyes and of the proboscis, and of such noticeable special effects as might occur. The bodies of the worms were observed every second day for the first week, and every third day thereafter. At the end of seven days the worms in each chemical were divided into two groups. One group was returned to water; the other remained exposed to the goitrogen. The rate of regeneration was observed by measuring the lengths of the worms on successive days. The effect of the chem- icals on the pigmentation was noted. For measuring, a somewhat modified form of the method originated by Wulzen (1927) was employed, and the average length of worms in each solution was com- puted. Graphs of growth rate were made, plotting average lengths, calculated to 0.1 mm., against time in days. In order that a better comparison of growth rates in the different solutions and series might be made, the daily average length in each group was recalculated, using as the original average length on the day of cutting that average exhibited by the water controls, namely, 5.6 units. A second series of experiments, using a concentration of 0.01% of each of the chemicals, was performed. No other change was made in either method or mate- rials. A third series, using a 0.02% concentration was likewise performed, but due to the toxicity of phenylthiourea at this concentration, a comparative study of effects at correspondingly higher concentrations was not attempted. THE EFFECT OF THE GOITROGENS ON SEVERED TAILS Healing. In the normal planarian, when a tail is severed, the cut edge contracts, forming a pronounced, black indentation, semi-circular in shape. Within two to three days, as healing progresses, relaxation occvirs, and the newly forming, unpig- mented flesh is protruded forward in a more or less triangular shape as the worm glides about. In the worms treated with chemicals, the healing process was notice- 108 MARIE M. JENKINS ably slowed. When thiourea was vised, the effect appeared to be in proportion to the concentration used. Worms placed in a 0.005% solution were all healed on the third day, in a 0.01% solution on the fourth day, and in a 0.02% solution on the fifth day. The phenylthiourea was markedly more effective than the thiourea, even in the lower concentrations. It was not until the sixth day that healing occurred in all worms placed in a 0.005% solution, and in a 0.02% solution complete healing did not occur. The effect of the thiouracil solutions on healing, while greater than that of the thiourea, was less than that of the phenylthiourea. Worms placed in a 0.005% and in a 0.01% solution were healed by the third day, but six days were required for complete healing of those placed in the 0.02% solution. Formation of sense lobes. When a head is forming in a regenerating planarian, by the fourth or fifth day the triangular protuberance of unpigmented new flesh has become sufficiently large that the animal, in moving about, exhibits the beginnings of sense lobes by protruding and withdrawing, seemingly at will, a small bit of tissue on either side, just anterior to the healed cut. In this experiment, it was found that the 0.005% solution of each of the three chemicals and the 0.01% con- centration of thiourea and thiouracil were ineffective in retarding this. All the animals in these solutions were able to produce sense lobes by the fifth day. The other concentrations used were more effective in this respect. Sense lobes appeared in all worms in the 0.02% solutions of thiourea and thiouracil on the sixth day, and in the 0.01% concentration of phenylthiourea on the eighth day. It was not until the tenth day, however, that the worms in 0.02% phenylthiourea showed this stage of development. In the worms returned to water from higher concen- trations of the chemicals, the sense lobes appeared within twenty-four hours after return, or by the eighth day. Proboscis development. The first definite sign of a developing proboscis in a severed tail can be seen in a freely moving planarian on the third or fourth day after cutting. A smooth, tan-colored protuberance appears at the point where the two sides of the digestive tract have grown together, and grows caudally until its length is about four times its width. Pigmentation and wrinkling, the latter due to an increase in real but not apparent length, occur on the fifth or sixth day after cutting, in the normal worm. In this experiment both the 0.005% and the 0.01% solution of each of the three chemicals had little effect on the time required for the appearance of the proboscis, or on its subsequent development, but each of the chemicals was effective at a con- centration of 0.02%. At this concentration the organ could be seen in all the animals in thiourea and thiouracil on the fourth day, but it was not until the fifth day that it could be found in all of the worms in phenylthiourea. Further develop- ment of the proboscis was also affected. By the fourteenth day the worms in both thiourea and phenylthiourea exhibited a very immature proboscis, shorter and nar- rower than is normally found on the fourth day. The latter solution was particularly toxic. The animals in thiouracil fared better. In them the proboscis, while less mature in appearance than those in the water controls, was apparently able to func- tion normally. The effect was reversible in the worms returned to water at the end of seven days. In these worms, by the fourteenth day the proboscis was as developed, pigmented, and wrinkled as those of the water controls. Eye formation. Eye formation in the normally regenerating planarian begins EFFECTS OF GOITROGENS ON PLAXARIANS 109 quite early. By the third day definite, tiny, black eyespots can be seen under the low power of the microscope, and by the sixth day the spots have become large and black, curved and smooth in outline on the median side, and concave and slightly granular on the lateral side. In this experiment the effect of the thiourea was quite varied as far as individual worms were concerned, but the concentration did not seem to cause a marked dif- ference. At all three concentrations the developing eyes were somewhat smaller and more granular in appearance than those of the water controls. The black pigment that formed began to disappear irregularly on the sixth day in Series I and II. and on the fifth day in Series III. On the seventh day. before the trans- ference of half the animals to water was made, it could be seen the pigment was disappearing to a greater or lesser extent in the eyes of all the worms at all three concentrations. During the following week a change could be noted daily. All the worms which were kept in the 0.005% solution of thiourea lost all eye-pigment by the eighteenth day. The animals in the 0.02% solution of thiourea lost all eye- pigment by the tenth day of subjection to the chemical, but in each one there per- sisted a distinct, ghost-like outline of the eye shape, very faintly yellowish-pink in color. The 0.01% concentration was variable in its effects. By the twenty-fifth day, in one of the worms there was a nearly normal amount of black pigment, while in the others the pigment was nearly gone, but in no case was it completely absent. In contrast, the worms which were returned to water gained pigment little by little, until by the fourteenth day they closely resembled the water controls. The phenylthiourea, at all concentrations used, inhibited pigment formation completely in the developing eyes, although the eyes themselves could be seen in faint, ghost-like outline, faintly yellowish-pink in color. In Series I, the eyes of the worms which remained in the chemical showed during the second week a faintly brown, smooth outline. By the eighteenth day this was more pronounced, and by the twenty-fifth day reddish-tan granules had begun to appear in the eyes. It is possible that black pigment might have eventually developed, but the regenerat- ing tails, which had been without food over three weeks, had become so small that sustenance was necessary for their continued existence, and the experiment was brought to a finish. In both the two higher concentrations of phenylthiourea, the worms which re- mained in the chemical during the entire experiment showed practically the same effect. After the eye outlines appeared, there was no change until the fourteenth day, when a slightly pinker color began to show. In the worms in Series II, the eyes were full size and very pink in color on the twenty-fifth day, but the worms in Series III had died and disintegrated by the eighteenth day, so that further observa- tion was impossible. In the worms which were returned to water from each of the three concentrations of phenylthiourea, a steady development of pigment followed. The smooth outline became darker and a golden-brown color developed inside. This gradually changed to a reddish-brown, then black. The eye outlines became granular as the darker colors appeared. By the fourteenth day, the eyes of all returned to water appeared like the eyes of the water controls, with the exception that these retained a slightly reddish cast. By the twenty-fifth day these were indistinguishable from the water controls. HO MARIE M. JENKINS Solutions of thiouracil showed much less effect than solutions of either thiourea or phenylthiourea. In all cases the general effect of the chemical was to cause the eyes to become slightly more granular in appearance than is normal, and to become slightly reddish in spots as the pigment partially disappeared. This was more pronounced in the higher concentrations, but in no case did the pigment completely disappear, even after twenty-five days exposure to the chemical. Worms returned to water on the seventh day regained normal eye appearance within three days. Skin pigmentation. During all series careful attention was given to possible effects of the chemicals on skin pigmentation, both in mature cells and in newly forming tissue. No bleaching effect was noticed under the influence of any one of the three chemicals, at any concentration used, up to twenty-five days, when the experiment was terminated. THE EFFECT OF THE GOITROGENS ON GROWTH RATE By a comparison of the average lengths of the worms, as measured on succeeding days, it was found that regenerating planarians in water, at a controlled temperature, exhibit a characteristic growth curve. For the first four days after the tails are severed, rapid growth of the bodies occurs, followed by two days of slower growth. The maximum length is reached on the sixth day. Following this, if food is not given the animal, it must begin to live on its own tissues, and a decrease in length results. After a four- to five-day interval, the graph line begins to level off somewhat. An- other period of rapid decline follows, then another period of levelling-off. The characteristic growth curve of planarians in water is shown in Figure 1, together with a typical response of the animals to the effects of the goitrogens. In this graph, the regenerative growth rate of worms subjected to a 0.02% solution of thiourea, and of those returned to water at the end of seven days, is compared with the curve exhibited by the water controls. It will be noticed the peak of growth occurs on the sixth day for both groups of animals, although the peak attained by the experimentals is lower. The graph line for the planarians returned to water shows the characteristic lessening of retardation of growth. A study of the com- parative effects of thiourea at different concentrations reveals that the 0.005% con- centration is least effective in depressing the growth rate, and recovery from exposure to it follows most rapidly; the 0.01% solution is most effective in depress- ing the growth rate during the first few days of exposure; and the 0.02% concen- tration, while not most effective in depressing the initial growth rate, is much more potent after long exposure. The distinct lessening of retardation of growth in animals returned to water at the end of seven days was quite apparent in all three series with each chemical used. In the majority of cases the lessening of effect was so pronounced that a second growth peak was reached. This was especially noticeable in animals exposed to thiouracil. In Figure 2 the second growth peak is shown to have occurred on the fourteenth day, or seven days after the planarians were returned to water from 0.02% thiouracil. The occurrence of the second growth peaks ranged from the eleventh to the fourteenth day. It was found by a comparison of the effects produced by each of the goitrogens at a concentration of 0.005% that the thiourea affected the rate of growth less at this concentration than did either thiouracil or phenylthiourea, and that the latter EFFECTS OF GOITROGENS ON PLANARIANS 111 was the most effective. This conforms with the findings above of the influence of the chemicals on the regeneration of missing organs in severed tails. A study of the growth rate of planarians in a 0.01% solution of the chemicals showed that, while initial exposure to thiourea at this concentration was not highly effective, continued exposure produced a marked retardation in growth, and a return to water allowed nearly normal growth to be resumed. At this concentra- 7.0 6.5 H 6.0 e> z LJ LL O 5.5 5.0 4.5 68 II 14 17 TIME IN DAYS 20 23 FIGURE 1. Growth rate curve of planarians exposed to 0.02% thiourea (Ta) and of those returned to water at the end of seven days (Ta-W) compared with the characteristic curve of water controls (W). tion both phenylthiourea and thiouracil were found to be quite effective in depressing initial growth, so much so that the peak of growth was not only quite low, but was reached seven to eight days after exposure to the chemical, or one to two days later than the peak observed in the water controls. A comparison of the effects of exposing the experimental animals to a 0.02% 112 MARIE M. JENKINS 68 II 14 17 TIME IN DAYS 20 23 FIGURE 2. Growth rate curve of planarians exposed to 0.02% thiouracil (TL) and of those returned to water at the end of seven days (TL-W) compared with the characteristic curve of water controls (W). concentration of the chemicals revealed that, at this concentration, thiouracil was least effective in retarding growth, while phenylthiourea was most effective. A marked depression, approaching toxicity, followed continued exposure to both thio- urea and phenylthiourea, but a noticeable recovery was made when the animals were returned to water after a week's exposure. This, too, is in accord with the facts noted above. DISCUSSION In the present study, it was found that the normal regenerative powers of the planarians were reduced by the administration of goitrogenic agents in varying concentrations, and that the effect was more pronounced as the concentration was increased. This is in conformity with the findings of Lynn (1948) and Rulon (1950). Lynn, testing two of the thioureas on a toad, Eleutherodactylus ricordii, which possesses no aquatic larval stage, found that a concentration of 0.001% thio- urea was ineffective, a concentration of 0.005% was slightly effective, and that a concentration of 0.05% thiourea caused a definite retardation in development. Rulon reported that continuous exposure of newly fertilized eggs of Dcndrastcr to low concentrations of thiourea resulted in slight inhibition of development, and that with higher concentrations the degree of inhibition increased. EFFECTS OF GOITROGENS ON PLANARIANS 113 In this experiment the depression of growth rate by the goitrogens, noticeable to some extent at all concentrations, was shown not only by the lower peak of growth as exhibited by the graphs, but also by the fact that certain of the concentrations slowed the initial growth sufficiently that the peak was reached after seven to eight days' exposure to the chemical, at a time when the period of rapid decline was apparent in the water controls. A possible explanation of this is that the lowered metabolic rate allowed a longer use of the food present in the animal, before the necessity of subsisting on its own tissues became imperative. The second growth peaks noted in the majority of animals returned to water, which occurred at a time when a levelling-off period was to be found in the water controls, were apparently due to an upsurge of metabolic activity following the release of the animals from the influence of the goitrogens. This effect appears to be similar to that noted in the severed tails, when rapid reconstitution of deficient organs followed the return of the animals to water. None of the chemicals used had any appreciable effect on head formation, the appearance of functioning sense lobes, or the development of the proboscis, when used at a concentration of 0.005%, and only phenylthiourea exhibited a marked modifying action at a concentration of 0.01%. All three chemicals, at a concentration of 0.02%, produced a distinct retardation in all phases of organ development. The results of this study show that not only is the retardation of the metabolic rate of planarians, as evidenced by the rate of regeneration, influenced by the degree of concentration to which the animals are subjected, but that certain goitrogens are more effective than others in this respect. In all phases of the study, phenylthiourea was found to be more potent in repressing the rate of regeneration, and in causing modifications in developing organs, than either thiourea or thiouracil. This, too, is in agreement with the results obtained by Lynn (1948), who found that a 0.005% concentration of phenylthiourea was as effective in retarding embryonic development as was the 0.05% thiourea. Reports of several workers indicate that the development of pigmentation in the animal body is intimately associated with the metabolic process. Lynn (1948), treating leptodactylid embryos with 0.005% phenylthiourea, found that not only was there a definite retardation in development, but that within three days the experi- mentals were noticeably lighter than the controls, and by the sixth day all visible dark pigment, both in the skin and in the retina of the eye, had disappeared. Frieders (1954), studying the effect of the same chemical on fish, found that the animals showed a definite loss of body pigment, and that a gradual but noticeable loss of pigment could be observed in the eyes. At the same time, the growth rate of the experimentals was much slower than that of the controls. While no bleaching effect in regard to skin pigmentation was noted at any time in this experiment, it was found that all three chemicals interfered to some extent with the production of eye-pigment at all concentrations, the effect increasing as the concentration was increased. That the goitrogens inhibited pigment formation, not the development of the eye itself, was shown by the fact that the planarians, particu- larly those in phenylthiourea, developed eye outlines, although pigment did not appear. In this study, as in those cited above, the rate of metabolism of the planarians, as evidenced by the growth rate and by the appearance of new organs appeared to parallel the speed or slowness of pigment formation. It is probable that a funda- 114 MARIE M. JENKINS mental correlation exists between the production of animal pigment and the pro- duction of chemicals which exert a controlling influence on the metabolic rate. The fact that goitrogens affect metabolism and pigment formation similarly in both vertebrates and invertebrates lends support to this view. SUMMARY 1. A study was made of the effects of the three goitrogens, thiourea, phenylthi- ourea, and thiouracil, on Dugesia tigrina, a species of planarian. Observations were made of the effects of the drugs on healing, head formation, proboscis development, eye and skin pigmentation, and regenerative growth rate. 2. Phenylthiourea w r as found to be most effective in preventing healing. Both thiourea and thiouracil retarded the rate of healing. 3. Higher concentrations of all three goitrogens were effective in retarding or suppressing the normal development of sense lobes and proboscis. Phenyl- thiourea was most potent. Lower concentrations were ineffective. The effect was reversible. 4. Phenylthiourea inhibited eye-pigment formation, but not eye formation. The effect was reversible. Thiouracil had little effect on the formation of eye-pigment. The effect of thiourea w j as varied. 5. Bodies with severed tails, placed in water, showed a characteristic growth curve when body length was plotted against time in days. Plotted curves of planar- ians in goitrogens, compared with controls, showed retardation of growth. Notice- able recovery was made upon the return of the experimentals to water. LITERATURE CITED BEVELANDER, G., 1946. Effect of thiourea on the development of the sea-urchin, Arbacia func- tulata. Proc. Soc. Exp. Biol. Med., 61 : 268-70. FRIEDERS, F., 1954. The effects of thyroid-inhibiting drugs on some tropical fish. The Cath- olic University of America Biological Studies, 31 : 1-37. LYNN, W. G., 1948. The effects of thiourea and phenylthiourea upon the development of Eleutherodactylus ricordii. Biol. Bull., 94: 1-15. RULON, O., 1950. The modification of developmental patterns in the sand dollar by thiourea. Physiol Zool, 23 : 248-57. WULZEN,, R., 1927. Nutrition of planarian worms. Science, 65 : 331-32. ALMYRACUMA PROXIMOCULI GEN. ET SP. NOV. (CRUSTACEA, GUMACEA ) FROM BRACKISH WATER OF CAPE COD, MASSACHUSETTS N. S. JONES AND W. D. BURBANCK Marine Biological Station, Port Erin, Isle of Man; and Biology Department, Emory University, Atlanta 22, Georgia An interesting cumacean was collected by W. D. Burbanck in the Pocasset River, Cape Cod, Massachusetts. Specimens were sent to Dr. Thomas E. Bowman at the Smithsonian Institution, who forwarded them to N. S. Jones for identifica- tion. In the following account N. S. Jones is responsible for the description and systematic remarks and W. D. Burbanck for the sections on habitat and general ecology. GENUS ALMYRACUMA GEN. N. Anterolateral angles of the carapace not developed. Second antenna of the male rudimentary, one-jointed, resembling that of the female. Second maxilla with two endites. First maxilliped with four joints, the last very small, and the epipodite w r ith only rudimentary branchiae. Third maxilliped pediform, with an exopodite. Only the first and second peraeopods bear an exopodite in either sex. ALMYRACUMA PROXIMOCULI SP. N. Material examined. Pocasset River, Cape Cod, Massachusetts ; W. D. Bur- banck, collector; 15 March 1958; 11 males. 38 females (12 ovigerous), 8 juveniles. Description. Ovigerous female. Length range from 3.2 to 3.7 mm. Integu- ment thin, finely granulated, with a few scattered hairs. Color yellowish white with dark brown pigment spots specially concentrated on the lower part of the carapace and at the sides of the free thoracic somites. Eyes black. Carapace two-sevenths of total body length, about as high as it is long, and slightly longer than its greatest width posteriorly ; from the side the dorsal outline swells upwards behind the eyelobe and is further elevated at the posterior end ; a dorsal groove is present between the branchial regions ; a prominence is set on each side of the hinder end with a hollow running forwards from below it towards the eyelobe ; the pseudorostrum is short with the lobes divided for about half their length above ; the anterolateral margin is only slightly concave and without any angle. Eyes well developed with corneal lenses, set close together but distinctly separated forming a double eyelobe. Five free thoracic somites clearly visible from above. Brood pouch containing 10-14 ova. Pleon somites smooth, the fifth the longest. Telsonic somite little produced posteriorly. First antenna with the three joints of the peduncle not very different in length, the third joint slightly the shorter ; the flagellum with three joints, the short third 115 116 N. S. JONES AND W. D. BURBANCK FIGURE 1. Paratype ovigerous female from side. FIGURE 2. Paratype adult male from side. FIGURE 3. Female from above. FIGURE 4. Male from above. NEW CUMACEAN FROM CAPE COD 117 joint carrying two aesthetascs ; the accessory flagellum very small, one-jointed. Second antenna rudimentary, one- jointed, bearing two small plumose setae at its end. Mandibles of normal shape, with molar process not styliform. First maxilla with two processes on the palp. Second maxilla normal with tw r o upper lobes. First maxillipeds with a lamellar merocarpus and a small end joint as in Campylaspis ; the merocarpus bears a few flattened bifid spines as well as a number of pointed plumose spines ; only two rudimentary branchial lobes are present on the epipodite. Second maxillipeds six-jointed with the basis curved outwards. Third maxillipeds pediform, bearing an exopodite ; the basis less than half the length of the whole appendage, with its distal end not produced ; the ischium short ; the merus and carpus about equal in length and rather shorter than the propodus ; the dactylus shorter than the propodus, ending in a fairly strong spine. First peraeopods longer and more slender than the third maxillipeds ; the basis stout, a little more than one-third the length of the whole appendage ; the ischium fairly short: the remaining joints successively a little longer; the dactylus ending in a long slender spine. Second peraeopods much shorter than the first pair, with exopodite ; the basis stout, as long as the next four joints together ; the dactylus about twice as long as the propodus. Third to fifth peraeopods without exopodites ; the third and fourth pairs with the basis slender, about as long as the remaining joints together ; the fifth pair with the basis relatively shorter. The uropods as long as the fifth abdominal and telsonic somites together ; the peduncle fairly stout, about the same length as the subequal rami, with three or four setae on the inner edge; the outer ramus two-jointed; the first joint about one-fourth the length of the second ; the second joint with a stout terminal spine and two setae on the outer and one on the inner edge; the inner ramus one-jointed with two strong spines on the inner edge. Adult male. Length 3.8-4.3 mm. Carapace one-fourth of the total length of the body. The pleon relatively longer than in the female and more stoutly built. The dorsal outline of the carapace rather less elevated than in the ovigerous female and the lateral protuberances more prominent. Rather more scattered hairs are present. The appendages are similar to those of the female except as follows: third maxillipeds with all the joints stouter; first peraeopods much more stoutly built, and with the dactylus less than two-thirds the length of the propodus and its ter- minal spine short and stout; the peduncle of the uropods relatively stouter and longer than in the female, about H the length of the rami, with several basal setae and 10-12 stout spines on the inner edge; the outer ramus similar to that of the female but more robust ; the inner ramus broad in the basal half, with 6-8 strong spines on the inner edge and two setae on the outer edge, and with a subterminal plumose spinule. The bases of the appendages bearing exopodites are not specially widened, and it may be noted that the second antennae resemble those of the female, being similarly rudimentary without any trace of a flagellum. Holotype and paratypes. USNM No. 102259-102261. Systematic remarks. A. proximoculi clearly must be placed in the family Nannastacidae for the following reasons : it has no separate telson ; there are three 118 N. S. JONES AND W. D. BURBANCK O-l -J FIGURE 5. Female first antenna. FIGURE 6. Male first antenna. FIGURE 7. Female second antenna. FIGURE 8. Male second antenna. FIGURE 9. Female left mandible. FIGURE 9a. Same, distal and further enlarged. FIGURE 10. Female right mandible. FIGURE 11. Female labium. FIGURE 12. Female first maxilla. FIGURE 12a. FIGURE 13. Female second maxilla. FIGURE 13a. FIGURE 14. Female first maxilliped. FIGURE 14a. FIGURE 15. Female second maxilliped. Same, distal end further enlarged. Same, distal end further enlarged. Same, and joints further enlarged. NEW CUMACEAN FROM CAPE COD 119 pairs of thoracic exopodites in the female ; the male has no pleopods ; the inner ramus of the uropods is one-jointed. It differs from all other described species of Cumacea in the rudimentary state of the male second antenna. There seems to be no doubt that the males are fully adult. They are larger than the females in the collection. They differ from the females in the shape of the carapace and the spinu- lation of the uropods. Ovigerous females were present and some of the males when captured were clasping females. There is some tendency towards reduction of the second antennae in certain species such as those of the genus Lamprops, where these appendages are used to clasp the female, and it is possible that in this species, where the second antennae are rudimentary, the greater development of the third maxillipeds and first peraeopods in the male is an adaptation for this purpose. The male resembles the female and differs from most other members of the Nannastacidae in possessing a similar number of thoracic exopodites. Picrocinna poecilota Hale (1936), placed in the family Nannastacidae (Hale, 1945), has exopodites on the third maxillipeds and the first to third peraeopods in both sexes. It also resembles A. pro.i'imoculi in the absence of an anterolateral angle on the carapace, in the positioning of the eyes, and in the pediform shape of the third maxillipeds. The second antenna of the male has a reduced prehensile flagellum as in Lamprops. It differs considerably in other respects, however, such as the shape of the first antennae, mandibles and uropods. Almyracuma proximocnli shows a combination of characters which excludes it from any previously defined genus of the Nannastacidae. The mouthparts on the whole resemble those of Cuinella, with the exception of the first maxillipeds which are somewhat similar to those of Campylaspis but have only rudimentary branchial lobes. Its affinities are obscure but it is possibly closer to Picrocinna than to any other genus described at present. Habitat. The type locality of the new cumacean is approximately one mile from the mouth of the Pocasset River, Cape Cod, Massachusetts (also known as Bar- low's River) and nearly 100 yards downstream from a dam which separates the brackish part of the river from the last of a series of six confluent ponds (Fig. 26). A constant flow of fresh water from the ponds is appreciably augmented by cold water issuing from numerous springs lying in a semi-circle around the small flat where the cumaceans live. At low tide the flat may be almost out of water (Fig. 27). Water running over parts of it has a pH of 6.0 and readings taken in situ, where cumaceans were living, ran as low as 4.4. The cumaceans live in a substrate of detritus and algae. At low tide the water in which they live has a salinity of less than 1 % . At high tide they are overlain by 3-4 feet of water which has a pH of approximately 8.0 and a salinity of about 30 %c. During the course of the fall, winter, spring and summer of 1957-58 the tem- perature of the substrate ranged from 3-20 C., with water temperatures slightly higher, 3-22 C. Less than 30 yards beyond where the cumaceans were living, Pocasset River froze over during the months January and February of 1958. No cumaceans were found in this area later in the year while they were present in the open areas during the coldest months, with one pair being found clasping in Feb- ruary. Most of the animals apparently breed during the month of March although 120 N. S. JONES AND W. D. BURBANCK 17b. FIGURE 16. FIGURE 17. FIGURE FIGURE 18. FIGURE 19. FIGURE 20. FIGURE 21. Female uropods. Male uropods. FIGURE 17a. Same, spine of peduncle further enlarged. Same, tip of inner ramous further enlarged. Female third maxilliped. FIGURE 22. Male third maxilliped. FIGURE 23. Female first peraeopod. FIGURE 24. Male first peraeopod. FIGURE 25. Female second peraeopod. Female third peraeopod. Female fourth peraeopod. Female fifth peraeopod. NEW CUMACEAN FROM CAPE COD 121 N BUZZARDS BAY FIGURE 26. Tracing of an airplane photograph of the Pocasset River, Cape Cod, Massa- chusetts taken on March 18, 1958 at 10:10 AM Eastern Standard time at an altitude of 2300 feet. The distance from the mouth of the river to the dam is approximately 1.4 miles. The X's in the headwaters indicate the type location of the cumaceans and the O's represent known locations of springs. 122 N. S. JONES AND W. D. BURBANCK a pair was seen in a finger bowl containing algae which had been brought in to the laboratory from the field on August 18, 1958. The composition of the substrate is unusual since it contains not only sand and gravel and plant debris but also a great man}- charcoal fragments and small pieces of iron slag. Supposedly the latter material was residue left from an iron foundry located on this site 73 years ago. Although porous, the substrate underlying the 1-cm. thick algal-detritus layer is quite hard and supports easily the weight of a man standing or walking on it. FIGURE 27. View of the Pocasset River looking upstream in an easterly direction at the type location. The cumaceans were found jn the left foreground in an algal mat beneath shallow water and on the exposed flat above and to the left. These two areas are represented in Figure 26 by the more southern of the two X's. Photograph taken August 4, 1955 at low tide. Although the small cumaceans were noted in collections from Pocasset River made from December 1957 to September 1958, there is no reason to believe that they are not present and active every month in the year. General ecology. Associated with the cumacean and perhaps a source of food for it is the diatom, Mclosira sp., which is "probably the dominant in terms of bulk and general distribution" ('A. J. Bernatowicz, private communication). Also present are the blue-green alga, Anabaena sp., and, on pebbles, Ulothri.r sp. Small numbers of the larger algae, Monostroma sp., Ulra sp., and Enteromorpha sp., are present while Vauchcria sp. lives on the mud among the adjacent Spartina alterni- flora Loisel. NEW CUMACEAX FROM CAPE COD 123 Living with tin- cumacean are two tanaids. The very common one is Lcptochclia dnbia (Krpyer) which has a hreeding cycle similar to that of the cumacean, and less frequent is Lcptochelia rapa.v (Harger). Two gammarids are also present in large numbers during the warmer months and these are Garnmarus tigrinus Sex- ton and Leptocheirus sp. Corophium lacitstrc Vanhoffen is also in association with the cumaceans but it. unlike the gammarids, is quite patchy both as to distribution and numbers. Just under the animal-algal association and sometimes entering it are the isopods, Cyathura sp.. Edotca sp., and Chiridotca almyra Bowman. Of these only CyatJiura sp. was ever found in appreciable numbers ; however, since it is commonly found in densities of 1000-1400 per m.' J , it might be considered to be the dominant form in the upper reaches of Pocasset River where the cumaceans live. Often the am- pharetid worm, Hypaniola gravi Pettibone. was found with the crustaceans as well as the spionid worm, Scolecolepides riridis (Yerrill), which is present in largest numbers during the warmer months. The only vertebrates regularly found with the cumacean were elvers of the American eel, Anguilla rostrata ( Le Sueur). Examination of stomachs of small eels 8-10 cm. in length revealed that they ate cumaceans. Other fish in the same locality which eat small crustaceans and might well feed on cumaceans were the killifish. Fnndnlns hctcroclitns (L.), the four-spined stickleback, Apcltcs qnadraciis (Mitchell), and some small clupeids and other members of the herring family. The black duck and least sandpiper also feed in the area where the cumaceans live. In all months of the year except March the cumaceans are dispersed, with only a few occurring in four-cubic inch cores of the algal-detritus layer. In March, how- ever, as many as 50 were found in a sample of that size. Apparently the large increase is due to aggregation rather than to a sudden seasonal increase in total numbers. In 1955 Bowman described the type habitat for the estuarine isopocl. Chiridotca alinvni. and he also listed the invertebrates living in association with it. The type locality was the Edisto River, S. C, with collections having similar habitats and associations from the Ogeechee River, Ga., and Haverstraw, N. Y. Pocasset River, because it possesses a very similar type of habitat and fauna, may represent a north- ern extension of the same type of tidal-marsh community. It is well known that a number of species of Cumacea, all placed in the Pseudo- cumidae, occur in brackish or almost fresh water in the Caspian and neighboring regions, and species of Cnniclla have been found with other forms in the Black Sea in water of salinity about 18-21 r / tc , but Aliiivracitina pro.vimocnli is the first member of the Xannastacidae to be found in water of such low salinity as exists in its habitat at low tide. A few other species of Cumacea have been found in brackish water on the eastern coast of North America, especially in Chesapeake Bay. in- cluding Mancocitina altcra Zimmer, M. stcllifcra Zimmer. Cyclaspis pustitlata Zim- mer. C . ranatis Caiman, Lcncon aiucricanns Zimmer and Oxyurostylis sinitJii Cai- man (Zimmer, 1941 ), but these are all placed in other families. Acknowledgments. For photographs of the Pocasset River: Airplane view from which tracing was taken. Mr. Carlyle Hayes of the Woods Hole Oceanographic Institution, and the type location, Dr. Charles Ray. Jr.. Dept. of Biology, Emory University. For the identification of Crustacea. Dr. Thomas E. Bowman of the 124 N. S. JONES AND W. D. BURBANCK Smithsonian Institution, U. S. National Museum, Dr. Milton A. Miller, Dept. of Zoology, University of California, Dr. Henry Werntz, The Biological Laboratories, Harvard Universitv. For identification of the pnlvchaete worms. Dr. Marian ., i Pettibone, Dept. of Zoology, University of Xe\v Hampshire. For the identification of algae. Dr. A. J. Bernatowicz, Dept. of Botany, University of Hawaii. LITERATURE CITED FiowMAN, T. E., 1955. The isopod genus Chlridotca Harger, with a description of a new species from brackish water. /. Washington Acad. Sci., 45: 224-229. HALE, H. M., 1936. Cumacea from a South Australian reef. Rcc. S. Australian Mtix., 5: 404-438. HALE, H. M., 1945. Australian Cumacea. No. 9. The family Xannastacidae. Rec. S. Aus- tralian Mus., 8: 145-2 IS. ZIMMER, C., 1941. Cumacea, in H. G. Bronn's Klassen und Ordnungen des Tierreichs, 5 (Aht. 1, Buch 4, Teil 5) : 1-222. PURINES AND PTERIDINES FROM THE REFLECTING PIGMENT OF THE ARTHROPOD RETINA 1 L. H. KLEINHOLZ The Biological Laboratories, Kccd College, Portland, Oreol\'[>Jicnnts. METHODS Eyestalks of Honiants were usually removed before the rest of the animal was turned to other purposes. The eyes of Liiintlus, together with adjacent tissue, were excised from animals immobilized by bleeding. Immediately after removal the eyes were placed in 95 % ethanol for 2 to 4 days for hardening, after which the retinas of Homarus were cut from the stalks while, in Li in n I us, the extraneous tissue was dissected away from the eye. The ethanol was changed frequently until no more color was leached from the retinas. Retinal reflecting pigment in Hoinanis does not undergo photomechanical changes and occurs as a compact layer distal to the fenestrated basement membrane, as well as in substantial deposits proximal to this membrane (Fig. 1). Initially, the reflecting layer was exposed by removing and discarding these proximal de- posits and adjacent tissue; material from the reflecting layer was then scraped free in ethanol and concentrated by centrifugation. After it was found that the chroma- tographic results were qualitatively the same, these deposits of reflecting pigment 1 These studies were made possible by a grant-in-aid from the American Academy of Arts and Sciences, as well as by grants from the National Science Foundation. 125 126 L. H. KLEINHOLZ v%'^w$s3'if$w.' \-*x'^.l*V^W; a, ' - Jt \ *.* pV * . / ^*t" ' -. vf ' '' j '*47r '- 'i - / v..' A Vf'.;^ ' ' ' V s , P'p| ip So - v ' c FIGURE 1. All the photographs are of a longitudinal section through the eye of Homantx and show the proximal portion of the retina. The bottom of each figure is proximal to the body ; the top of the figure is distal from the body. FIGURE 1A. Bright-field illumination ; the proximal pigment and the layer of reflecting pigment above the fenestrated basement membrane surround the rhabdomes. Granules of both proximal pigment and reflecting pigment also occur below the basement membrane, but these are not readily distinguishable from each other. FIGURE IB. Dark-field illumination of the same region seen in Figure 1A. The layer of reflecting pigment distal to the fenestrated basement membrane, and the deposits of this pigment proximal to the basement membrane are now readily evident. Comparison of the distribution patterns of the pigments below the basement membrane in the two prints permits some differentiation between granules of reflecting pigment and of proximal pigment. FIGURE 1C. The rectangular region marked in Figure 1A shown under higher magnification by bright-field illumination. RETINAL PURINES AND PTERIDINES 127 proximal to the basement membrane also were combined with the scrapings from the reflecting pigment layer. In the case of Linntlus, reflecting pigment is located distally in the eye. The intervening retinal melanin was exposed, chipped away with a small scalpel, and discarded. In a few instances most of this retinal melanin was dissolved by im- mersing the eye for an hour in ethylene chlorohydrin ; the treated retinas were then washed in a tew changes of ethanol. Either of these methods of removing the melanin exposed the reflecting pigment which was then scraped free and concen- trated by centrifugation. Masses of white material, similar in appearance to the reflecting pigment, and described by some authors as "rudimentary eyes," are closely associated anatomically witli the lateral and median eyes of Linnilits; these, too, were removed for study. The reflecting pigments and associated tissue were ground and extracted from 1 to 6 hours in a micro-centrifuge tube with 0.1 ml. per retina of one of the following alkaline solutions: \% NaOH ; \% LiOH ; 0.5 N NH 4 OH ; 0.069r Li,CO, ; 0.2 M borate buffer at pH 9.2; or a solution of 50 f /r ethanol containing 2% NH 4 OH. The tubes were centrifuged and samples of the supernatant solution as well as samples of standard purine solutions were applied with a micro-pipette to sheets or strips of Whatman No. 1 filter paper for subsequent chromatography or elec- trophoresis. Either ascending or descending development was used with a wide variety of solvent mixtures, such as are listed by Block, Durrum and Zweig (1955) and by Viscontini, Schmid and Hadorn (1955). The most useful solvent systems were: (1) water-saturated n-butanol: formic acid -- 9:1 ; (2) pyridine: ethyl acetate rwater : 4:3:3; (3) isoamyl alcohol saturated with 5% disodium hydrogen phosphate (with a layer of each in the chromatography chamber) ; (4) water-saturated col- lidine ; (5) 3/c aqueous ammonium chloride; (6) n-butanol: acetic acid : water = 8:2:2, followed by a second development in the same direction with acetone :n- butanol : water == 8:1:1. After development the paper was dried and examined in short-wave ultraviolet light ( Mineralight Model Y-41 lamp, manufactured by Ultraviolet Prod. Inc.) and the spots outlined with pencil. Tentative identifica- tions of the components of the reflecting pigment were made by comparing the distances the component spots migrated with the distances migrated by the spots of reference standards. Spots developed from reflecting pigment were cut out and eluted in 0.1 N NaOH or 0.1 X HC1. The identification was then verified by determining the ultraviolet absorption spectra of these eluates in a Beckman spec- trophotometer and comparing them with spectra of the known standards. In a large number of cases developed chromatograms were also treated according to the method of Vischer and ChargafT (1948) whereby purine spots are made visible as a black mercuric sulfide complex. The latter procedure revealed overlapping or masking of components when the}- occurred, and thus indicated need for develop- ment in different solvent systems. Paper electrophoresis was used primarily in resolving one of the pteridines FIGURE ID. Dark-field illumination of the region shown in Figure 1C. Arrows point to granules of dark proximal pigment intermingled with the reflecting pigment layer. Strands- of reflecting pigment at the bottom of the print aid in recognizing this pigment in Figure 1C. m, fenestrated basement membrane ; />, proximal pigment ; r, reflecting pigment ; s, rhabdome. 128 L. H. KLEINHOLZ which could not be satisfactorily separated from the other components of reflecting pigment by paper chromatography. Samples, about 0.1 ml. in volume, of ethanol- ammonia extract of lobster retina were applied to paper strips which were then developed at 375 volts or 500 volts for 18 to 20 hours in the LKB or the Spinco instrument. The buffer was 0.04 M boric acid and 0.01 M borax at pH 8.6. After the strips were developed and dried, the blue-fluorescent segments, which had migrated toward the cathode, were cut out and eluted in 0.1 N HC1 or in 0.1 N NaOH for subsequent spectrophotometry. Initial studies on solubility of the reflecting pigment of Houiarns were made on histological sections cut at 10 microns from paraffin-embedded retinas. The mounted sections were de-waxed with xylene and re-hydrated before testing with the various solvents. The murexide and enzymatic tests were made on small amounts of reflecting pigment which had been removed as described. The methena- mine-silver reaction of Gomori (1952) was used as a histochemical test for uric acid. RESULTS A. Nature of the reflect! in/ pigment of Houiarns Reflecting pigment was dissolved from sections of lobster retina within 30 minutes after immersion in 1 N solutions of specific acid (hydrochloric, acetic, nitric or sulfuric) or of specific alkali (ammonium hydroxide, sodium hydroxide, 0.1 % aqueous solutions of sodium carbonate or sodium bicarbonate). At 60 C. the reflecting pigment dissolves within an hour in glycerine or ethylene glycol or ethylene glycol monoethyl ether. When, however, these same solvents are used at room temperature, one finds little visible solution in glycerine, partial solution in ethylene glycol monoethyl ether, and complete solution in ethylene glycol. The reflecting pigment is partially dissolved from sections remaining overnight in 95% ethanol but showed no discernible solution in absolute ethanol. These solubilities differ in several important respects from those reported for guanine by Millot (1923). Thus, according to Millot, guanine is insoluble in ammonium hydroxide or acetic acid, whereas the reflecting pigment of Hoinants is soluble in both these solutions. Gwilliam (1950) also reports solubilities of retinal reflecting pigment of the crab, Hemigrapsus oregonensis, that fail to agree with those of guanine. The residue obtained by evaporating to dryness a dilute lithium carbonate extract of Honianis reflecting pigment gives positive murexide but negative or faint, dubiously-positive Weidel reactions. Guanine, uric acid, xanthine and its methyl derivatives give positive murexide reactions (Lison, 1936). Millot (1923) reports that guanine and xanthine, but not uric acid, react positively to the Weidel test ; adenine and hypoxanthine. among the other common purines, are reported to give neither murexide nor Weidel reactions. Comparison of these reported results with the findings for Houiarns casts doubt on the reflecting pigment's being guanine and indicates, instead, that the reflecting pigment of the lobster may be uric acid. A histochemical test depending on an argentaffin reaction between uric acid and methenamine-silver (Gomori, 1952) proved positive for the reflecting pigment of Hojnanis. Argentaffin reactions, particularly in neutral solution, have been criti- cized (Lison, 1936) because positive reactions are also given by calcium carbonate and phosphate, if present. In this study, however, exposure of sections to me- RETINAL PURINES AND PTERIDINES 129 thenamine during incubation is supposed to bring about ready solution of such calcifications. More specific identification of uric acid in the reflecting pigment was made by paper chromatographic resolution of mixtures after incubation with uricase (Nutri- tional Biochemicals Corp.). Preliminary exploration showed that 1 to 5 ^gm. of uric acid in 5 pi. of 0.5^ lithium carbonate solution are detectable when the n- butanol-formic acid solvent system and the Vischer-Chargaff (1948) visualization method are used. Uric acid and 5 pi. of a solution containing the reflecting pigment of one lobster retina in 0.1 ml. showed similar R f indices (0.14 to 0.17) with this same solvent system. The reflecting pigment of 20 lobster eyes, dissolved in 0.5 ml. of dilute lithium carbonate solution, was mixed with 50 mg. of uricase, 0.5 ml. of 0.05 M borate buffer at pH 9.2, and 0.5 ml. of toluene. A 5-ju.l. sample of this mixture was removed for application to paper within 5 minutes (zero time). This mixture was gassed with oxygen and incubated at 38 C. Thereafter, at intervals of 0.5, 1, 2, 4, and 6 hours, 5-pl. aliquots were removed and applied to paper ; a 5-/xgm. sample of uric acid to serve as a reference standard was applied to the same sheet of paper which was then developed in butanol-formic acid solvent. Treatment of the developed chromatogram by the Vischer-Chargaff method revealed the purine as black spots with an R f index of 0.15 for the reference standard and also for those aliquots taken at 0-, 0.5- and 1-hour intervals. The intensity of the spots decreased with time of incubation with uricase. The sample taken after 2 hours of incubation showed an Rf index of 0.14 and was very faint. No spots were present for the 4- hour and 6-hour samples. Because of the specificity of uricase in the oxidation of uric acid, these results may be considered a satisfactory demonstration of the presence of uric acid in the retinal reflecting pigment of Hoinarus. Examination of these chromatograms revealed an additional faint spot distal to each of the corresponding retinal uric acid spots ; this faint spot was not present above the uric acid standard. The possible presence of other purines besides uric acid was in- dicated by this observation. This possibility was explored by first examining chromatograms developed in butanol-formic acid solvent in ultraviolet light, and then using the mercuric nitrate-ammonium sulfide visualization method for purines. When this was done, the results diagrammed in Figure 2 were obtained for the lobster. The diagram shows the presence of three apparent purines. one of which is uric acid, and two fluorescent compounds. For subsequent reference, these spots are labelled, starting from the baseline on the chromatogram, as Fluorescent 1, Absorbent 1 (uric acid). Fluorescent 2, Absorbent 2, and Absorbent 3. B. Further identification of the retinal compounds The two fluorescent compounds of the reflecting pigment were believed to be pteridines which have been reported present in the eyes of vertebrates (Pirie and Simpson. 1946; Kama. 1953) and of crustaceans ( Busnel and Drilhon. 194S). After chromatographic development of retinal pigment samples and aliquots of known purines and xanthopterin as reference standards in a variety of solvent systems, the Rf indices of the components were compared. In this wav, four of the five spots of Figure 2 were identified : Absorbent 1 is uric acid : Fluorescent 2 is 130 L. H. KLEINHOLZ xanthopterin ; Absorbent 2 is xanthine ; and Absorbent 3 is hypoxanthine. The linear sequence of the spots, starting from the baseline on the chromatogram, may vary strikingly with different solvent systems (Fig. 3). Advantage was taken of this property to make the final verification of the above-mentioned identifications. Well-resolved spots, not masked or overlapped by other components, were cut out, eluted in 0.1 N HC1, and the absorption spectrum of the eluate determined. The AB 3 AB 2 FL 2 AB I FL I o I5pl. 1 5jul. FIGURE 2. Tracing of a chromatogram of retinal reflecting pigment of Hotnarus, developed in butanol-formic acid solvent. The resolved spots were first outlined in ultraviolet light and then were treated to make the purine spots visible ; the broken lines indicate boundaries made evident after this latter treatment. The size of the sample applied to the paper is given in microliters. The labels to the left identify and describe the appearance of the spots in ultraviolet light : FL, fluorescent ; AB, absorbent. See text. maxima of the spectra obtained for spots identified as uric acid, xanthine, and hypoxanthine corresponded with those reported by Borough and Seaton (1954). The absorption spectrum of the retinal component identified by R f index as xanthopterin was determined after similar elution from a chromatogram developed in butanol-formic acid solvent. This is compared with the spectrum obtained from eluates of xanthopterin used as a reference standard on a paper chromatogram RETINAL PURINES AND PTERIDINES 131 .50 .40 R F -30 .20 .10 .50 n o o o o .40 .30 .20 N-PROPANOL NH 4 OH U XP XA H .10 N-BUTANOL ACETIC H 2 U XP XA H .80 .60 .40 .20 Q .60 ETHYL ACETATE PYRIDINE H 2 U XP XA H .50 .40 .30 ISOAMYL NA 2 HP0 4 U XP XA H FIGURE 3. Diagrams showing the tentative identification of four of the five components of the reflecting pigment of Homanis arrived at by comparing the Ri indices of the components with those of reference standards. The solvent system is indicated on each diagram. Broken lines represent the boundaries of components not evident on examination in ultraviolet light but which became visible after formation of the mercuric sulfide complex. L, reflecting pig- ment of Homarus; U, uric acid; XP, xanthopterin ; XA, xanthine ; H, hypoxanthine. (Fig. 4). The maxima of the reference xanthopterin at 230 m^, 259 m/x, and 355 m/A agree with the maxima reported for xanthopterin by Elion and Hitchings (1947). The spectrum of xanthopterin from the retina shows similar maxima at 231 m/ji, 261 m/i,, and 355 m/*., although the geometry of the retinal spectrum differs somewhat from that of the reference standard. The basis of this difference is not understood. 132 .4 L. H. KLEINHOLZ - .3 CO z LU o .2 CL o 250 300 WAVE 350 LENGTH 4 Omp FIGURE 4. Absorption spectra of reference xanthopterin (upper curve) eluted from paper chromatogram and of Fluorescent 2 component (lower curve) from reflecting pigment of Homarus. 250 300 350 WAVE LENGTH 400 4 5 Omp FIGURE 5. Absorption spectra of unidentified Fluorescent 1 component of lobster reflecting pigment. Upper curve is the eluate from the paper in 0.1 N NaOH; lower curve is the eluate in 0.1 N HC1. RETINAL PURINES AND PTERIDINES 133 C. The unidentified fluorescent component There remains to be considered the unidentified component of Homarns reflect- ing pigment labelled Fluorescent 1 in Figure 2. The rarity of many pure pteridines limited the number available for use as chromatographic standards ; the Rf indices of the few pteridines used for this purpose failed to give a satisfactory match with the index for Fluorescent 1 in a variety of solvent systems. Attempts to isolate this component in sufficient concentration for subsequent spectrophotometry, as was done with the other retinal components, were generally frustrated by con- tamination due to streaking or tailing of the other constituents. Fluorescent 1 was finally isolated by paper electrophoresis, and was eluted in 0.1 N HC1 or in 0.1 N NaOH, as described in Methods. The absorption spectrum of the eluate in acid showed maxima at 245 m/A and 353 m/j,; in alkali, these maxima were shifted to 255 mp. and 390 m/i (Fig. 5). D. Specific localization within the retina It cannot be stated with complete certainty in which part of the lobster retina the five purines and pteridines are specifically localized. The evidence described above indicates that uric acid is most probably a component of the reflecting layer of retinal pigment, as may also be the two other purines, xanthine and hypoxanthine. The two pteridines may be more widely distributed among the retinal components. Busnel and Drilhon (1948) found several substances, detectable by fluorescence microscopy, in the crustacean retina. These fluorescent materials not only are closely associated with the proximal pigment but also occur in the regions of the reflecting and distal pigments. It is apparent from Figure 1 (C and D) that, although most of the proximal pigment in light-adapted retinas has migrated distal to the reflecting pigment layer, proximal pigment granules still remain intermingled with and below this layer. The preparation of reflecting pigment for chromatography unavoidably in- cluded some of these proximal pigment granules. However, chromatography of preparations of reflecting pigment, previously washed with ethylene chlorohydrin to remove the traces of dark proximal pigment, showed the presence of the two pteridines obtained with untreated reflecting pigment. Thus, while the above ob- servations are presumptive evidence for localization of the pteridines in the reflecting pigment, the possibility of their occurring also in the other retinal pigments cannot be excluded. E. Retinal reflecting pigment in Limuliis Reflecting pigment from the lateral and median eyes of Limulus was obtained as described under Methods. The deposits of white material of so-called rudimen- tary eyes, located in the postero-medial region of each lateral eye, as well as similar material associated with the median eyes, were dissected free. Each of these was dissolved separately in 0.5% NaOH. Samples of the solutions were applied to paper and developed, along with a series of purine reference standards. The solvent systems were butanol-formic acid ; water-saturated collidine ; and butanol-water- morpholine-diethylene glycol. Examination of the chromatogram in ultraviolet light generally revealed a single quenching spot whose R f index was the same as that 134 L. H. KLEINHOLZ of guanine. A faintly bluish-fluorescing spot was also evident in one case but was not observable in any of the other chromatograms. Chromatograms treated by the Vischer-Chargaff method confirm the coincidence of R f indices for the reference guanine and reflecting pigment from lateral, median, and rudimentary eyes. The spots quenching ultraviolet light, obtained with reflecting pigment from a lateral eye, were cut from a chromatogram developed in butanol-formic acid and were eluted overnight in 1% NaOH. The spectrum of this eluate had a maximum at 275 m/x, in agreement with that reported by Hotchkiss (1948) for guanine. I am indebted to Profs. C. M. Williams and J. H. Welsh for helpful suggestions and critical comments on the manuscript. SUMMARY 1 . The chemical nature of the retinal reflecting pigment was studied in Homarus and in Limulus. In crustaceans the reflecting pigment has been thought to be guanine, but the solubility and chemical properties of this pigment from Homarus do not agree with those for guanine. 2. Use of paper chromatographic methods shows the presence of five substances in the reflecting pigment of Homarus, three of which are absorbent or quenching in ultraviolet light and two of which are fluorescent. 3. Histochemical treatment with methenamine-silver and incubation studies with uricase identify one of the three ultraviolet-absorbent compounds as uric acid. Comparisons of R f indices of the other two ultraviolet-absorbent compounds with those of reference purines show them to be xanthine and hypoxanthine. Identifica- tions of all three were verified by determining the ultraviolet absorption spectra of the retinal purines eluted from paper chromatograms. 4. One of the two fluorescent components of Homarus reflecting pigments is xanthopterin, identified both by its R f indices after chromatographic development in a variety of solvent systems, and by its absorption spectrum. The second fluores- cent compound, probably a pteridine, has not been identified, but its absorption spectrum shows maxima at 245 mju, and 353 m/* in 0.1 N HC1 ; in alkali these maxima are shifted to 255 m/t and 390 m/i. 5. Retinal reflecting pigment from Limulus is guanine. LITERATURE CITED BLOCK, R. J., E. L. DURRUM AND G. ZWEIG, 1955. A Manual of Paper Chromatography and Paper Electrophoresis. Academic Press Inc. New York. 484 pp. BROWN, F. A., JR., M. N. HINES AND M. FINGERMAN, 1952. Hormonal regulation of the distal retinal pigment of Palaemonetes. Biol. Bull., 102 : 212-225. BROWN, F. A., JR., H. M. WEBB AND M. I. SANDEEN, 1953. Differential production of two retinal pigment hormones in Palaemonetes by light flashes. /. Cell. Comp. Physiol., 41: 123-144. BUSNEL, R. G., AND A. DRILHON, 1948. Sur les pigments flaviniques et pteriniques des Crustaces. Bull. soc. zool. France. 73 : 142-185. DOROUGH, G. D., AND D. L. SEATON, 1954. A method for the extraction and assay of nucleic acid fragments in tissues. /. Amer. Chem. Soc., 76 : 2873-2877. ELION, G. B., AND G. H. HITCHINGS, 1947. The synthesis of some new pteridines. /. Amer. Chem. Soc., 69 : 2553-2555. GOMORI, G., 1952. Microscopic Histochemistry. Univ. of Chicago Press. Chicago. 273 pp. RETINAL PURINES AND PTERIDINES 135 GWILLIAM, G. F., 1950. On the occurrence and solubility of a reflecting pigment in the eyes of the Brachyura. Anat, Record, 108: 613. HAMA, T., 1953. Substances fluorescentes du type pterinique dans la peau ou les yeux de la grenouille (Rana nigromaculata) et leurs transformations photochimiques. E.vperlentia, 9: 299-300. HOTCHKISS, R. D., 1948. The quantitative separation of purines, pyrimidines, and nucleosides by paper chromatography. /. Biol. Chem., 175: 315-332. KLEINHOLZ, L. H., 1936. Crustacean eye-stalk hormone and retinal pigment migration. Biol. Bull., 70: 159-184. KLEINHOLZ, L. H., 1955. The nature of the reflecting pigment in the arthropod eye. Biol. Bull., 109: 362. LISON, L., 1936. Histochemie Animale. Gauthier-Villars. Paris. 320 pp. MILLOT, J., 1923. Le pigment purique chez les vertebres inferieurs. Bull. Biol. France et Belg., 57 : 261-363. PIRIE, A., AND D. W. SIMPSON, 1946. Preparation of a fluorescent substance from the eye of the dogfish, Squalus acanthias. Biochem. J., 40: 14-20. VISCHER, E., AND E. CHARGAFF, 1948. The separation and quantitative estimation of purines and pyrimidines in minute amounts. /. Biol. Chem., 176: 703-714. VISCONTINI, M., H. SCHMID AND E. HADORN, 1955. Isolierung fluoreszierender Stoffe aus Astacus fluviatilis. Experientia, 11 : 390-392. WELSH, J. H., 1932. The nature and movement of the reflecting pigment in the eyes of crustaceans. /. Exp. Zool, 62: 173-183. WELSH, J. H., 1939. The action of eye-stalk extracts on retinal pigment migration in the cray- fish, Cambarus bartoni. Biol. Bull., 77: 119-125. THE RESPIRATORY ENZYMES OF DIAPAUSING SILKWORM PUPAE: A NEW INTERPRETATION OF CARBON MONOXIDE-INSENSITIVE RESPIRATION 1 CHARLES G. KURLAND - AND HOWARD A. SCHNEIDERMAX DC part in cut of Zoology, Cornel! University, Ithaca, .Yrrc York The respiration of most organisms is inhibited in large measure by carbon monoxide. This indicates that cytochrome oxidase is the main terminal enzyme in electron transfer (Warburg, 1949; Keilin and Slater, 1953). But as is well known to students of insect physiology, the respiration of many diapausing insects is remarkably insensitive to cyanide, carbon monoxide, and other inhibitors of cytochrome oxidase. The significance of this insensitivity was recently discussed by Harvey and Williams (1958a, 1958b) as a result of studies on the heart of diapausing pupae of the Cecropia and Polyphemus silkworms. Quite independently we have carried out a detailed study of another aspect of this phenomenon (Kurland, 1957). Our attention has centered, not on a single organ such as the heart, but on the respiration of the whole insect. These two investigations prove complemen- tary in the analysis of the problem as a whole. Carbon monoxide-insensitive respiration in insects was first detected by Bodine and Boell (1934a, 1934b) who reported that the oxygen consumption of diapausing eggs of the grasshopper Mclanoplus was not inhibited by carbon monoxide. Later, Allen (1940) showed that the cytochrome c oxidase activity of these diapausing eggs was high, despite the insensitivity of their respiration to both carbon monoxide and cyanide. He concluded (p. 162) that the "rates of oxygen consumption of pre- diapause, diapause, and very early post-diapause eggs are independent of the relative amounts of cytochrome oxidase." An important clue to the reconciliation of the CO-insensitivity of diapausing Melanoplus eggs with the simultaneous presence of cytochrome oxidase was provided by Bodine and Boell (1936, 1938). They dis- covered that 2,4-dinitrophenol (DNP) increased the respiration of diapausing eggs and that this increased respiration was inhibited by carbon monoxide and cyanide. Unfortunately, the significance of this observation could not be fully comprehended because the mechanism of DNP action was not explained until a decade later (Loomis and Lipmann, 1948). As the result of an intensive investigation of the CO-insensitivity of pupal res- piration in the giant silkworm Hyalophora cccropia, Schneiderman and Williams (1952, 1954a, 1954b) concluded that the cytochrome c oxidase system was not functioning in most tissues of the diapausing pupa, although it functioned at all other 1 This study was aided by Grant H-1887 from the National Heart Institute, U. S. Public Health Service. Several of the experiments were drawn from a thesis submitted in partial fulfillment of the requirements for the degree of Bachelor of Arts with Honors in Zoology from Cornell University. 2 Present address : Biological Laboratories, Harvard University, Cambridge 38, Massa- chusetts. 136 CARBON MONOXIDE AND RESPIRATION 137 stages in the life history. Their arguments have recently been summarized (Lees, 1956; Schneiderman. 1957). They suggested that pupal respiration was mediated by an autoxidizable flavoprotein or a heme-containing enzyme insensitive to carbon monoxide. This explanation was supported by the observations of Shappirio and Williams (1953), Shappirio (1954), and Pappenheimer and Williams (1953, 1954) who reported the existence of a new autoxidizable cytochrome component (e or b 5 ) in Cecropia pupae. Also, Chefurka and Williams (1952) reported an in- creased amount of flavoprotein in pupal tissues. However, there was no evidence to indicate that the new cytochrome or the flavoprotein functioned as a terminal oxidase in the pupal respiratory chain. The experiments reported here continue these earlier studies and were prompted, in part, by recent advances in our understanding of electron transfer in the cytochrome system (cj. review by Chance and Williams, 1956). We examined the effects on respiration of two inhibitors of cytochrome oxidase, carbon monoxide and sodium azide, and also of antimycin A, a potent inhibitor of the DPNH-cyto- chrome c reductase system. In addition we studied the effects of 2,4-dinitrophenol which dissociates phosphorylation from oxidation. It was hoped that a study of the action of these rather specific inhibitors on pupae in various metabolic states might permit a decisive definition of the terminal oxidase of diapausing pupae. This ob- jective has been achieved. The results of the present study, coupled with the re- cent findings of Harvey and Williams (1958b), have enabled us to identify this oxidase as cytochrome oxidase and thus contradict earlier conclusions. The ex- periments also reveal some new biochemical peculiarities of the diapause condition. MATERIALS AND METHODS 1. Experimental animals Diapausing pupae of Hyalophora (Platysaniia) cecropia (4to6gm.), Callosamia promethea (li/^ to 2y 2 gm.), Samia cynthia (iy 2 to 3VL> gm.) and Antheraea (Telca) polyphcinus (4 to 6 gm.) were used as experimental animals. In our experience diapausing pupae of these four species of closely related saturniid moths behave in virtually identical fashion in respiration experiments and hence we have used them interchangeably. The animals were reared under field conditions or collected in nature and were stored at 25 C. for a minimum of four months before use in experiments. One group of Cynthia and Promethea pupae was maintained at 5 C. for several months and then returned to 25 C., whereupon their brains were removed. This brain removal put the pupae in a state of permanent diapause (Williams. 1946) and, after three months at 25 C.. these animals behaved in experi- ments like normal unchilled diapausing pupae. Only pupae displaying a relatively constant respiratory rate over a period of at least six hours were used in experi- ments. Also, since it was shown by Schneiderman and Williams (1954a) that cellular respiration of the pupal abdominal muscles is mediated by cytochrome oxi- dase, pupae showing excessive muscular activity were excluded. 2. Measurement of respiration The present investigation is based on more than 2000 respiratory measurements performed on about 500 pupae. Rates of oxygen consumption were determined 138 CHARLES G. KURLAND AND HOWARD A. SCHNEIDERMAN manometrically according to techniques described previously (Schneiderman and Williams, 1953a). Measurements were carried out in 50-cc. vessels equipped with venting plugs and adapters for use with standard Warburg manometers. 3. Gas mixtures In some experiments, pupae were exposed to various gas mixtures while enclosed in the Warburg vessels. Commercial gases were purified and gas mixtures pre- pared and analyzed by methods described previously (Scheiderman and Williams, 1954a). All of the experiments were performed at atmospheric pressure. The vessels were periodically re-flushed during the course of the experiments, a maneuver which prevented any significant reduction of oxygen tension within the vessels. Appropriate control vessels were run in all experiments to take into account the manometric effect of reactions between carbon monoxide and the alkali. 4. Reagents Sodium azide and 2,4-dinitrophenol were reagent grade. Crystalline antimycin A, obtained from the Wisconsin Alumni Research Foundation, was dissolved in aqueous ethanol. The final dilutions of antimycin A injected into the pupae were uniformly in 1% ethanol solutions. Previous to injection, the pupae were anesthetized with carbon dioxide. In our experience the respiration of diapausing pupae is not significantly affected by thirty minutes of carbon dioxide anesthesia. Approximately 0.1 cc. of solution was in- jected via a 26-gauge needle into each pupa. The final concentrations within the animal were calculated on the basis of a pupal water content of 70 per cent. Res- piration was measured for a minimum of three hours after injection. 5. Interpretation of inhibitor experiments The act of piercing merely the skin of a diapausing pupa with a fine hypodermic needle causes a prompt stimulation of respiration for several hours. This is followed by a subsequent slow rise in respiration injury respiration (Schneiderman and Williams, 1953a, 1953b). Hence in interpreting inhibitor experiments, it is neces- sary to separate the effects of injury from those of the chemical injected (Scheider- man and Williams, 1954a). This can best be accomplished by comparing experi- mental pupae with control pupae injected with a corresponding volume of the solvent used, e.g., 1 % ethyl alcohol, distilled water, etc. Furthermore, it is simplest to make comparisons soon after injection, before injury respiration increases to high levels and possibly before the injected chemical is detoxified or otherwise metabolized. In most of the inhibitor experiments to be reported, the pupae had a very low basal metabolic rate and simple injection commonly doubled their oxygen consumption. 6. Injury Pupae were anesthetized with carbon dioxide. Injuries were made either by removing a rectangle of pupal cuticle and underlying hypodermis from the face or by excising the pupal legs. The wounds were then covered with plastic windows sealed in place with paraffin. A few crystals of streptomycin sulfate and phenyl- CARBON MONOXIDE AND RESPIRATION 139 thiourea (a 1:1 mixture) were placed in the wounds to prevent infection and to prevent darkening of the blood by tyrosinase (Williams, 1952; Schneiderman and Williams, 1953a). EXPERIMENTAL RESULTS 1. Diapause respiration A. The development of CO -insensitive respiration after pupation The effects of carbon monoxide on the respiration of newly pupated Cecropia silkworms were observed at intervals over a ten-day period. The pupae were ex- posed first to a nitrogen-oxygen mixture and then to a carbon monoxide-oxygen mixture. The results, as well as details of the procedure, are recorded in Table I. As the pupae aged they exhibited a gradual decrease in their respiratory rate which TABLE I The development of CO-insensitive respiration in four newly molted Cecropia pupae* Respiration in Age after pupation nitrogen mixture % insensitive (hrs.) (mm. 3 /gm./hr.) respiration 5 34 59 29 26 49 197 7 92 6 37 51 30 28 69 198 7 86 19 26 47 43 25 74 211 7 80 19 26 57 43 23 55 211 10 80 * Pupae were exposed for three hours to an atmosphere of 6 per cent oxygen and 94 per cent nitrogen, and then for three hours to an atmosphere of 6 per cent oxygen and 94 per cent carbon monoxide. To calculate per cent insensitive respiration, oxygen consumption in the carbon monoxide mixture was compared to oxygen consumption in the nitrogen mixture. was accompanied by a marked decrease in the fraction of respiration sensitive to carbon monoxide. Thus while immediately after pupation half their respiration was inhibited by carbon monoxide, 200 hours later less than 20 per cent was CO-sensitive. B. The CO-insensitiz'ity of respiration of diapausing pupae Figure 1 records the per cent of CO-insensitive respiration for a large number of pupae with different basal rates of oxygen consumption. The data show that as oxygen consumption increases, respiration becomes increasingly sensitive to carbon monoxide. However, it is of special interest that even when carbon monoxide in- hibited the respiration of diapausing pupae it rarely inhibited more than 20 per cent of their total respiration, and for pupae whose basal respiration was between 15 and 20 mm. 3 /gm. live wt./hr., the respiration appeared to be unaffected by car- bon monoxide. It is also noteworthy that carbon monoxide appeared to stimulate 140 CHARLES G. KURLAND AND HOWARD A. SCHNEIDERMAN the respiration or at least the gas uptake of pupae whose basal oxygen consumption was less than 15 mm. 3 /gm. live wt./hr. We have duplicated these results in numer- ous experiments with Cynthia, Polyphemus and Promethea pupae. In all cases the apparent stimulation was greatest for pupae with low basal respiratory rates and possible explanations for this phenomenon will be offered in the Discussion. But soo r z jl 400 cr Q. Itl E 300 20O o o too 120 r < 100 O O 00 80 60 S 40 13 in o o c? 20 CO '/. CO-INSENSITIVE RESPIRATION C < 10 20 30 40 2 CONSUMPTION (MM 3 /GM. LIVE WT./HR.) z O 0. in TO ( LL> > 60 UJ 100 Q UJ t < _l o 50 a. o 5X10 *M. ONP IXIO~*M. DNP 10 20 30 40 50 BASAL 2 CONSUMPTION (MM / GM. LIVE WT./HR.) A 10 20 30 40 BASAL 2 CONSUMPTION (MM 3 /GM. LIVE WT./HR.) B FIGURE 5. The stimulation of O2 consumption by DNP as a function of basal respiratory rate. (A) The percentage stimulation of O 2 consumption of Cynthia pupae after injection of DNP to internal concentrations of 5 X 10~* M and 10~* M is plotted as a function of basal respiration. (B) The total DNP-stimulated respiration of the pupae in (A) is plotted as a function of the basal respiration. (b) the development of an "energy debt" metabolism (analogous to an "oxygen debt repayment" (Kurland et al., 1958)) as the result of prolonged uncoupling of phosphorylation by DNP. Comparable results were obtained with diapausing pupae of Cecropia, Promethea and Polyphemus. The time course of the respiratory changes recorded in Figure 4 is also typical of pupae receiving 5 X 10~ 4 M DNP but the pattern differed somewhat in pupae that received lower concentrations. Because the initial stimulation of respiration was less, the fall in respiration recorded in Figure 4 was commonly absent. The 144 CHARLES G. KURLAND AND HOWARD A. SCHNEIDERMAN 5 X 10~ 4 M concentration is apparently close to the lethal level and occasional in- dividuals died about a week after receiving that amount. Further analysis of the effects of DNP disclosed that pupae with high initial basal respirations were proportionately less stimulated by DNP than were pupae with low basal metabolic rates. Figure 5 A shows that a Cynthia pupa with a basal metabolic rate of 5 mm. 3 /gm. live wt./hr. experienced a 16-fold stimulation of respiration after injection of DNP whereas a similar pupa with a basal respiration of 30 mm. 3 /gm. live wt./hr. experienced only a 6-fold stimulation of respiration. Thus, there is a steep decline in the per cent of DNP-stimulated respiration as basal respiration increases. Figure 5B further reveals that DNP-stimulated respira- tion approaches a limit as the basal respiration approaches 25 mm. 3 /gm. live wt./hr. The significance of this limit will be considered in the Discussion. B. The effect of carbon monoxide, azide and antimycin A on DNP-stimulated respiration Diapausing Cynthia pupae were injected with DNP and then exposed to carbon monoxide. The results, summarized in Figure 6, reveal that about half the DNP- stimulated respiration was inhibited by carbon monoxide. Further analysis of the data from this experiment revealed that CO-sensitivity increased slightly as the rate of oxygen consumption increased. Thus pupae with a DNP-stimulated res- piration of 90 mm. s /gm. live wt./hr. had only 45 per cent of their respiration in- hibited by carbon monoxide, whereas pupae with a DNP-stimulated respiration of 125 mm. 3 /gm. live wt./hr. had nearly 70 per cent of their respiration inhibited by carbon monoxide. The effect of azide on DNP-stimulated respiration of diapausing Cynthia pupae is recorded in Figure 7. There was no significant initial inhibition of the respiration when sodium azide alone was injected (see Section ID), but some of the DNP- stimulated respiration was inhibited by this reagent. Indeed, as Figure 7B shows, more than three-fourths of the DNP-stimulated respiration was inhibited by 5 X 10~ 4 Jl/ sodium azide. However, in group B only half of the pupae receiving injections of DNP and none of the pupae receiving sodium azide survived for more than a week, indicating these high concentrations of antimetabolites were ultimately toxic. Comparable results were obtained with Cecropia pupae. Experiments appraising the antimycin A-sensitivity of DNP-stimulated respira- tion were conducted on a series of 15 Cynthia pupae which received 10~ 4 M DNP and 10~ 6 M antimycin A. About 30 per cent of the DNP-stimulated respiration was inhibited by this concentration of inhibitor. Thus, the respiration of DNP- stimulated pupae is no more sensitive to antimycin A than the respiration of normal pupae. 3. Injury-stimulated respiration A. The C0-sensitivit\ of injury-stimulated respiration As mentioned previously, integumentary injuries to pupae dramatically ac- celerate respiration for one to three weeks ( Schneiderman and Williams, 1953a, 1953b). Moreover, this accelerated respiration is proportional to the extent of injury and seems to be caused in part by diffusible substances released at the site CARBON MONOXIDE AND RESPIRATION 145 of injury (Jankowitz, 1955; Schneiderman, 1957). Although the respiration in- duced by a small incision into Cecropia pupae was not inhibited by carbon monoxide, repair of extensive wounds was prevented by this gas (Schneiderman and Williams, 1953b, 1954b), suggesting that the cytochrome oxidase system was functioning in injured pupae. To investigate this possibility the following experiments were carried out. Four Cynthia pupae were given a large injury by removing their pupal legs; two pupae AIR UJ jj* eoo H O) < CD 400 - Z o 0. 200 -^ in a o CSJ o AIR CO 5X10 4 M. DNP XlCf 4 M. DNP FIGURE 6. The CO-sensitivity of DNP-stimulated respiration. Two groups of five dia- pausing Cynthia pupae were injected with DNP to internal concentrations of 5 X 10~* M and 10" 4 M. All the pupae were exposed to 5% O 2 and 95% N 2 and then to 5% O 2 and 95% CO (CO/Os^ 19:1). The average respiration over a one-hour period is recorded. were immediately placed in 7 per cent oxygen and nitrogen, and the other two were placed in a corresponding atmosphere of oxygen and carbon monoxide. The pupae were maintained in their respective gas mixtures for one week, and the gas mixtures were renewed thrice daily. As Figure 8 shows, injured pupae in the nitrogen mixture developed a charac- teristic injury respiration; on the other hand, those in carbon monoxide mixtures did not. Indeed, five days after injury both of the pupae maintained in carbon monoxide had died. Thus carbon monoxide apparently caused death by preventing 146 CHARLES G. KURLAND AND HOWARD A. SCHNEIDERMAN the development of injury respiration. Similar results were obtained with Prome- thea pupae. These results are in general agreement with those of Schneiderman and Williams (1954b), who reported that the repair of injury was CO-sensitive. How- ever, their experiments failed to detect the CO-sensitivity of the respiration as- sociated with repair of injury, presumably because they employed only small injuries. Such CO-sensitivity was demonstrated by Harvey and Shappirio (Harvey, 1956) who pointed out that after very large injuries respiration becomes sensitive to car- bon monoxide. This is confirmed in the following experiment summarized in UJ 1200 - 1100 - < 1000- < m 900 - 800 - ^ 700 - g 600 - 2 500 - O j= 400 - Q. 300 - (/> 20 - 5XIO 4 M.DNP o CM O 100 - IO~*M.DNP IO~ 4 M.DNP AZIDE 2.5 XfO AZIDE B FIGURE 7. The azide-sensitivity of DNP-stimulated respiration. (A) Four diapausing Cynthia pupae were injected with 10~ 4 M DNP, four with 2.5 M sodium azide, and four with both reagents. The average Oa consumption over a three-hour period is recorded. (B) Five pupae were injected with 5 X It)" 4 M DNP, five with 5 X It)" 4 M sodium azide and five with both reagents. The average O 2 consumption over a three-hour period is recorded. The average initial oxygen consumption of the pupae in (A) and (B) was 16.8 mm. 8 /gm. live wt./hr. Figure 9. Four brainless Cynthia pupae were injured by removing the pupal legs and after three days, when they had developed a large injury respiration, the CO- sensitivity of their respiration was determined. About two-thirds of the injury respiration was inhibited by carbon monoxide. Similar results were obtained with Cecropia pupae. It can also be seen in Figure 9 (as well as in Figure 6) that the oxygen uptake of pupae respiring at a rapid rate was limited by the low oxygen tension. This contrasts with the respiratory behavior of pupae with low metabolic rates, where 5 per cent oxygen and 95 per cent nitrogen commonly stimulated oxygen consumption (see Section 1C). CARBON MONOXIDE AND RESPIRATION 147 400 -i UJ cc _1 (f) CD 300 200 CO < 100 CO z. o o cvi O I 2 DAYS AFTER INJURY FIGURE 8 (left). The effect of injury and simultaneous exposure to CO on respiration. Two injured Cynthia pupae were maintained continuously in 7% O plus Ns and two were maintained in 7% O plus CO (CO/O = 13:1). The average respiration of each pair over a 3-hour period is recorded as a function of time. The day of injury is denoted as day "0". FIGURE 9 (right). The CO-sensitivity of injury respiration. The average respiration over a 4-hour period of four brainless Cynthia pupae 3 days after injury in air, in 5% Oa and 95% N, and in 5% O 2 and 957* CO (CO/O 2 ratio = 19: 1). 400 r < E 2 300 h u. o tO < 200 Q. o o 100 5XIO 5 M.NoN 3 .. IX IO 4 M,NoN 3 sxio M.NON, < tr 600 CO < m 400 - 2 200 Z) co O o 01 2 3 4 DAYS AFTER INJURY FIGURE 10 (left). The effect of azide injection on the O= consumption of four groups of five diapausing Cynthia pupae over a five-day period. The day of injection is denoted as day "0". FIGURE 11 (right). The effect of DNP on injury respiration. The average O 2 consump- tion over a 3-hour period of two injured diapausing Cynthia pupae prior to and after the in- jection of water, and of two injured pupae prior to and after the injection of 5 X IO"" 4 M DNP. 148 CHARLES G. KURLAND AND HOWARD A. SCHNEIDERMAN B. Injury-stimulated respiration in newly pupated Cecropia Four Cecropia silkworms were injured within one day after pupation by re- moving a rectangular window of pupal cuticle from their faces. No significant stimulation of respiration was observed. Since injury respiration is characteristic of diapausing pupae, and since the respiration of newly pupated Cecropia is much greater than the respiration of pupae firmly in diapause, this result suggests that the production of injury respiration is intimately associated with the extremely low respiration of the diapausing insect (see Discussion). C. The aside-sensitivity of injury-stimulated respiration The effect of azide on injury respiration was examined by injecting a series of diapausing Cynthia pupae with sodium azide at several concentrations. In this experiment the injection itself served as the injury. The average daily respiration of each group of pupae over a five-day period is plotted in Figure 10. All pupae treated with 5 X 10~ 4 M sodium azide died within 10 days after injec- TABLE 1 1 Effect of simultaneous injury and injection of DNP on the respiration of six diapausing Cynthia pupae Basal respiration, mm.Vgrn./hr. Treatment Max. resp. as % basal rate Day of max. 25.0 Injury + H 2 O 258 2 12.0 Injury + H 2 O 521 2 31.0 Injury + H 2 O 234 3 26.5 Injury + DNP 449 6 40.5 Injurv + DNP 496 6 30.5 Injiirv + DNP Died tion, indicating either (a) that this concentration had a simple toxic effect, or (b) that the development of injury-stimulated respiration was inhibited by azide and this caused death, as was the case when injured pupae were exposed continuously to mixtures of carbon monoxide and oxygen (see Section 3 A above). In lower concentrations of azide, the inhibition of injury respiration was proportional to concentration. D. The effects of DNP on injury-stimulated respiration The pupal legs were removed from a group of four Cynthia pupae. Three days after wounding, when injury respiration had reached its maximum, two of the pupae received injections of DNP to an internal concentration of 5 X 10~ 4 M, and the remaining two received injections of water. The data summarized in Figure 11 show that 5 X 10~ 4 M DNP caused a significant acceleration of maximum injury respiration ; however, the increase was proportionately much less than that en- countered in DNP-treated diapausing pupae. Comparable results were obtained with Cecropia and Promethea pupae. In another experiment, six diapausing pupae were injured by removing their CARBON MONOXIDE AND RESPIRATION 149 pupal legs ; half these pupae immediately received water injections, while the remainder received injections of DNP to an internal concentration of 5 X 10" 4 M. The respiration of these pupae is summarized in Table II. The maximum respira- tion of injured pupae treated with DNP was reached six days after the injury, while those receiving injections of water displayed maximum respiration two or three days after injury. Thus DNP delayed the development of injury respiration. DISCUSSION 1. A new explanation for the insensitivity of pupal respiration to carbon monoxide Studies noted in the Introduction have shown that the onset of pupal diapause in giant silkworms is accompanied by a precipitous fall in the rate of oxygen con- sumption, and that the low respiration of the diapausing pupa is virtually uninhibited by carbon monoxide and cyanide. As judged by its insensitivity to these inhibitors, nearly all of the respiration of the diapausing pupa appeared to proceed via path- ways independent of cytochrome oxidase. Hence it was suggested that the respira- tion of the diapausing pupa was mediated by a terminal oxidase other than cyto- chrome oxidase, possibly a flavoprotein or an autoxidizable cytochrome of the b type (Schneiderman and Williams. 1954a. 1954b). This suggestion was taken up by various investigators (Cotty, 1956; Ito, 1955). The present experiments provide an alternative explanation for the CO-insensitivity of pupal respiration ; namely, that it is due to a great excess of cytochrome oxidase relative to trace amounts of cytochrome c in most of the tissues of the diapausing pupa. This limitation of cytochrome c leads to an unsaturation of cytochrome oxidase, and this in turn leads to the insensitivity of pupal respiration to carbon monoxide and azide. Under this view the principal factor underlying the lozv respiration of the diapausing pupa is the limitation of cytochrome c in most of the pupal tissues, while the prin- cipal factor underlying the CO- and aside-inscnsitivity of pupal respiration is the excess of cytochrome c o.ridase in most of the pupal tissues. Thus, quantitative changes in the relative amounts of respiratory enzymes after pupation are respon- sible for both the low over-all respiration of diapause and for CO-insensitivity. In other words, the basic differences between the respiratory enzyme systems of diapausing and non-diapausing insects are quantitative, but they lead to qualitative differences in the response of the insect to certain inhibitors. Contrary to earlier opinions, cytochrome oxidase appears to be the principal terminal oxidase during diapause as well as during all the other stages of the life history. 2. Preliminary theoretical considerations It can be shown that an excess of cytochrome oxidase may lead to a virtual CO-insensitivity of respiration that is actually mediated by cytochrome oxidase, and in the final section of this discussion a brief theoretical analysis of this asser- tion is presented. The argument offered is that when cytochrome oxidase is in great excess and thus not saturated, a large fraction of the cytochrome oxidase may be inhibited by carbon monoxide without affecting the rate of electron transfer from cytochrome c. Stated in another way the greater the "saturation" of cyto- chrome oxidase by cytochrome r. the greater the CO-sensitivity of respiration; the less the "saturation" of cytochrome oxidase by cytochrome c, the less the CO- 150 CHARLES G. KURLAND AND HOWARD A. SCHNEIDERMAN sensitivity. This conclusion seems intuitively acceptable and is proven in Section 9 (below). Recognizing this relation between CO-sensitivity and saturation of cytochrome oxidase it is not difficult to interpret the several experimental results. 3. Carbon monoxide experiments Evidence presented previously has shown that the specific target of carbon monoxide in the insect at all stages is reduced cytochrome c oxidase (Schneiderman and Williams, 1954a, 1954b). A principal factor determining the impact of carbon monoxide on cytochrome oxidase is the CO/CX ratio: the higher this ratio, the greater the proportion of reduced cytochrome oxidase molecules inhibited. The CO/O 2 ratios employed in the present experiments were usually 16:1 or 19:1 and ambient oxygen tensions were maintained at 6 or 5 per cent. Direct analysis of the composition of the tracheal gas of normal diapausing pupae kept at these oxygen tensions by a precise microgasometric method (Levy and Schneiderman, 1957, 1958) revealed that the actual oxygen tension within the tracheal system, and hence within the insects' tissues, was about 1 per cent lower than ambient, that is, about 5 or 4 per cent. Therefore in the present experiments the actual CO/O, ratios within the pupal tissues approached 24:1. Since it has been shown that a CO/Oo ratio of 16:1 causes a 50 per cent light-reversible inhibition of the cyto- chrome oxidase activity of homogenates of the thoracic muscles of Cecropia moths (Pappenheimer and Schneiderman, unpublished), we may conclude that the CO/0,, ratios used in the present experiments were capable of inhibiting no less than 50 and probably as much as 75 per cent of the reduced cytochrome oxidase activity of homogenates of the insect's tissues. However, as we have already noted in the previous section, the inhibition in a homogenate where cytochrome oxidase is satu- rated by added cytochrome c may be quite different from the inhibition observed in the intact insect where the cytochrome oxidase may not be saturated by cyto- chrome c. Let us now consider what our several experiments tell us about the saturation of cytochrome oxidase in the diapausing pupa. Perhaps the most crucial result is recorded in Figure 2. As the figure shows, when the oxygen tension is reduced to 2 per cent in a mixture of oxygen and nitrogen, the oxygen consumption of the pupa remains about the same as in air, but the CO-sensitivity of the respiration is enhanced. The simplest interpretation of this result is that cytochrome oxidase is present in excess over some rate-limiting link in the respiratory chain, and only at low oxygen tensions does the cytochrome oxidase-oxygen reaction become the limiting step in the respiratory chain, subject, as a consequence, to inhibition by carbon monoxide. The reasons for stimulatory effects of carbon monoxide on pupae with low metabolic rates (cf. Fig. 1) are not yet clear. Similar stimulatory effects of carbon monoxide have been reported by Bodine and Boell (1934a) for Melanoplus, by Klein and Runnstrom (1940) for unfertilized eggs of the sea urchin, and by others (cf. review by Needham, 1942, p. 496). Possibly it does not represent stimulation of respiration but is simply gas uptake due to an actual oxidation of CO by the tissues to CO,, (cf. review of Lilienthal, 1950). Perhaps it is some- thing different altogether, such as an uncoupling action (Thimann et al., 1954). For our present purposes suffice it to say that the phenomenon, although not yet ex- plained, does not affect our interpretation of the basic action of carbon monoxide CARBON MONOXIDE AND RESPIRATION 151 cytochrome oxidase and the argument that cytochrome oxidase is only partially saturated in pupal tissues. Further evidence supporting this argument derives from studies with DNP and azide which are considered in Sections 4 and 5 below. Significant data revealing the degree of saturation of cytochrome oxidase in the diapausing pupa are also to be found in the observation that CO-sensitivity of pupal respiration increases with increasing basal respiration and is instantly enhanced by DNP, and in the fact that the increased respiration that follows injury or the initia- tion of adult development is inhibited by carbon monoxide. Moreover, we have found that the increased respiration that follows a prolonged period of anoxia is also sensitive to carbon monoxide. These results, which are summarized in Table III, TABLE III Summary oj tine effects of metabolic inhibitors on the respiration of diapausing pupae in various physiological states and on developing adults Effect ^ylnhibitor Physio- >. logical condition \ CO CO 'O2=about 20:1 DNP Azide Antimycin A Diapause respiration Stimulation at low basal rates Slight or no inhibition at modest basal rates. Inhibition increases as basal respiration in- creases Up to 50% inhibition at low oxygen tensions 5 X 10- M stimulates respiration an average of 12-fold and as much as 16-fold Stimulation less at high basal rates No immediate effect at concentrations up to 5 X 10-4 M 30% inhibition at 10-6 M DNP-stimulatert respiration An average of 50% in- hibition 30 to 70% inhibition depending on concen- tration of azide 30% inhibition at 10~ 6 \I Injury-stimulated respiration No or slight inhibition after small injury; up to 60 per cent inhibition after large injury Exposure immediately after injury prevents de- velopment of injury- stimulated respiration Stimulation by DNP in- versely proportional to size of injury-stimulated respiration. After large injuries, about 2-fold stimulations by DNP. Injection of DNP im- mediately after injury delays development of injury respiration Inhibition propor- tional to concentra- tion of azide Developing adult More than 50% inhibi- tion (Schneiderman and Williams. 1954a) 5 X 10-< M stimulates respiration about 2-fold lead to the conclusion that the fraction of respiration sensitive to carbon monoxide is a function of the rate of oxygen consumption of the silkworm at all stages. This implies that virtually any process which increases the rate of pupal respiration in- creases the saturation of cytochrome oxidase and that, in the pupa, cytochrome oxidase is in great excess and hence very unsaturated. Recognizing the importance of low over-all respiratory rate as a factor in CO-insensitivity, it is worthwhile considering certain diapausing insects whose res- piration is not resistant to carbon monoxide or cyanide. Two species whose respira- tion continues to be inhibited by carbon monoxide or cyanide during diapause are prepupae of the larch sawfly. Pristophora (McDonald and Brown, 1952), and larvae 152 CHARLES G. KURLAND AND HOWARD A. SCHNEIDERMAN of the horse hot fly, Gastrophilus (Levenbook, 1951). It is of considerable signifi- cance that the respiration of these insects at 25 C. is many times greater than the respiration of diapausing silkworm pupae. Thus the respiratory rate of Pristophora is about 165 mm. 3 /gm. live wt./hr., while that of Gastrophilus is more than 100 mm. 3 /gm. live wt./hr. 3 This compares with a respiratory rate for diapausing silkworm pupae of 8 to 20 mm. 3 /gm. live wt./hr. Furthermore, in diapausing silkworm pupae only the skeletal muscles, of which there are few, have a saturated cytochrome c oxidase, and these account for only a small fraction of the insect's total respiration. In diapausing species with high respiratory rates like Pristophora and Gastrophilus it appears likely that ( 1 ) they have more muscular tissue and this accounts for a larger fraction of their total respiration than do the muscles of diapausing pupae, and (2) some of their non-muscular tissues may have a saturated cytochrome oxidase. These factors could easily account for their sensitivity to carbon monoxide. 4. The significance of DNP-stimulated respiration The experiments with DNP demonstrate that in diapausing pupae cytochrome oxidase is not fully saturated. As is well known, DNP increases the turnover of the respiratory carriers, presumably because it is able to uncouple phosphorylation from electron transfer, and so increases the demand for oxygen (Chance and Wil- liams, 1956). The data in Section 2 reveal a striking 12- to 16-fold acceleration of pupal oxygen consumption by 5 X 10~ 4 M DNP. This may be one of the largest DNP stimulations ever recorded. It contrasts with the finding of Bodine and Boell (1938) that the respiration of diapausing Melanoplus eggs was accelerated a maximum of only 3.5 times by 3 X 10~ 5 M DNP, while further increase in concen- tration produced a submaximal response. De Meio and Barren (1934) and Maroney et al. (1957) have reported DNP stimulations in various invertebrate tissues of only about two-fold. Aside from the magnitude of DNP-stimulated respiration (which by itself suggests unsaturation of cytochrome oxidase), the CO- sensitivity of DNP-stimulated respiration is of special interest. It indicates that DNP accelerates the turnover of several carriers of the respiratory chain but has a lesser effect on the turnover of cytochrome oxidase. This conclusion arises from the fact that CO-sensitivity is a function of the saturation of cytochrome oxidase. The CO-sensitivity of DNP-stimulated respiration tells us that DNP increases the saturation of cytochrome oxidase. Thence it follows that DNP not only accelerates over-all respiratory rate but alters the quantitative relationship between cytochrome oxidase and the intermediate carriers in the respiratory chain. One of these accelerated carriers is almost certainly cytochrome c, which may be the most important rate-limiting carrier in the respiratory chain, a point we shall consider further in Section 6 (below). Dinitrophenol appears to increase in some way the effective turnover of this enzyme and increases thereby the saturation of cytochrome oxidase. It is significant that both the absolute magnitude and the CO-sensitivity of DNP-stimulated respiration were lower for pupae with low basal respiration. Thus in the present experiments, although pupae with low basal metabolic rates were proportionately more stimulated by DNP than pupae with high basal metabolic rates, the latter developed a greater over-all respiration under 3 This last value was calculated from values obtained at 37 C. by assuming a Q of about 2.5. CARBON MONOXIDE AND RESPIRATION 153 the influence of DNP. The data also reveal that CO-sensitivity reached a maximum of about 70 per cent when DNP-stimulated respiration reached its maximum. We interpret these findings to mean that pupae with low basal respiration have less cytochrome c available to be turned over and, as a result, these pupae are not capable, even under the influence of high concentrations of DNP, of completely saturating their cytochrome c oxidase and thereby achieving maximum CO-sensitivity. These DNP studies provide support for the argument that the low over-all respiration of diapause is due to a low concentration of some respiratory component, probably cytochrome c, whereas the CO-insensitivity is the result of the relatively high con- centration of cytochrome c oxidase. In our experience the respiration of developing adults is accelerated by DNP to a much lesser extent than that of diapa vising pupae, usually about two-fold. This fact suggests that in the developing adult, as contrasted with the diapausing pupa, cytochrome oxidase is virtually saturated. Also, although development may be delayed, developing adults survive concentrations of DNP which are toxic to diapausing pupae, possibly because their higher metabolic rate enables them to metabolize the DNP (cf. Cross et al. 1949). 5. The significance of aside-insensitive respiration In these insects it seems safe to identify cytochrome oxidase as the main target of azide CHorecker and Stannard, 1948; Stannard and Horecker, 1948). The experiments summarized in Section ID disclosed that azide had no immediate effect on diapause respiration at concentrations as high as 5 X 10~ 4 M. This re- sult supports the conclusion drawn above, that cytochrome oxidase does not limit pupal respiration. On the other hand, the sensitivity to azide of DNP-stimulated respiration was quite striking. This is consistent with the argument that, under the influence of DNP, cytochrome oxidase becomes more saturated. 6. The limiting link in the pupal respiratory chain The present experiments provide only one clue to the identity of the limiting link in the pupal respiratory chain. This is the fact that antimycin A a potent inhibitor at the concentrations we employed of the DPNH-cytochrome c reductase system had only a minor effect on normal pupal respiration and DNP-stimulated respiration. This inhibitor is said to have as its specific target the Slater factor which mediates the transfer of electrons from flavoprotein to cytochrome c (Potter and Reif, 1952; Reif and Potter, 1953; Chance and Williams, 1956). The insensitivity of pupal respiration to this reagent suggests that the limiting link in the pupal respiratory chain lies between the Slater factor and cytochrome oxidase, e.g., cytochrome c. Recent studies of Shappirio and Williams (1957a, 1957b) indicate that the limiting link is very likely cytochrome c, for with very sensitive spectroscopic techniques they were unable to detect this enzyme in most pupal tissues although cytochrome oxidase was easily demonstrated. They also showed that in homogenates of pupal tissues, cytochrome c is a rate-limiting link in the oxidation of DPNH. Hence it seems safe to identify limiting concentrations of cytochrome c as a principal cause of the unsaturation of cytochrome oxidase in pupal tissues. 154 CHARLES G. KURLAND AND HOWARD A. SCHNEIDERMAN 7. Injury-stimulated respiration The increased sensitivity to carbon monoxide and azide shown by pupae sup- porting an injury respiration (Sections 3 A and 3C) indicate an increased satura- tion of cytochrome oxidase after injury. The observation (Section 3D) that 5 X 10"* M DNP failed to accelerate injury respiration to the same degree as diapause respiration supports the conclusion that cytochrome oxidase is virtually saturated when injury respiration is at its maximum. What brings about this in- creased saturation of cytochrome oxidase is not known with certainty but the pres- ent experiments suggest that it is caused by a gradual synthesis of cytochrome r which is provoked by injury. Recall that injured pupae treated with DNP were delayed in developing maximum injury respiration when compared with injured pupae receiving water injections. This suggests that integumentary injury in- itiates some process which requires a supply of phosphate bond energy which was uncoupled by DNP. The gradual development of maximum injury respiration over a three-day period suggests further that this energy-demanding process in- volves, in part, the synthesis of one or more of the respiratory chain components and does not simply reflect increased turnover of pre-existing enzymes. We in- terpret the increased CO-sensitivity of injury-stimulated respiration to indicate that more cytochrome c is being synthesized than cytochrome oxidase. That augmented protein synthesis does in fact follow injury has been demonstrated by Telfer and Williams (1955), who showed that the incorporation of OMabelled glycine into the pupal proteins was stimulated by injury to about the same extent as respiration. It is not without interest that the synthesis of these respiratory components ap- pears to be obligatory. Indeed, the data in Section 3A suggest that when synthesis is prevented by prolonged exposure to carbon monoxide, the pupae fail to develop an injury respiration and die. This obligatory synthesis of new respiratory com- ponents may be imposed upon diapausing pupae because their capacity for wound repair is restricted by their low metabolic rate. Apparently this repair process is able to compete with the "maintenance" processes of the diapausing pupa, thereby causing death when total energy production is reduced by carbon monoxide. In this connection, it is noteworthy that newly molted pupae, whose respiratory rate is considerably larger than that of pupae firmly in diapause, fail to show an injury respiration. This reflects their capacity to underwrite the energy requirements of injury without augmenting the respiratory chain. This capacity is also present in developing Cecropia adults and we have also shown it in all stages of non-diapausing species such as the bee-moth Galleria mellonella. 8. Conclusions The several lines of evidence considered in the preceding sections persuade us that earlier conceptions of the respiratory enzyme system of diapausing silkworms need re-evaluation. The basic differences between the respiratory enzyme chains of the diapausing pupa and the non-diapausing stages appear to be quantitative dif- ferences and not qualitative differences as was suggested earlier (Williams, 1951 ; Schneiderman and Williams, 1954a, 1954b). The CO-insensitivity of pupal respira- tion does not stem from the activity of a CO-insensitive terminal oxidase, but re- sults from a great excess of cytochrome oxidase relative to other components of the CARBON MONOXIDE AND RESPIRATION 155 respiratory chain. None of our findings supports the renewed suggestions of Wojtczak (1955) and Ito (1955) that tyrosinase functions as a terminal oxidase in insects. Indeed, in view of the failure of potent inhibitors of tyrosinase like phenylthiourea to inhibit respiration (Schneiderman and Williams, 1954a) and the light-reversibility of the carbon monoxide inhibition of silkworm growth (Schneider- man and Williams, 1954b) and respiration (Pappenheimer and Schneiderman, un- published) this is not likely. The present data, coupled with the recent spectroscopic findings of Shappirio and Williams (1957a, 1957b) and with the studies of Harvey and Williams (1958a, 1958b) on the pupal heart, indicate that cytochrome oxidase is the terminal oxidase during pupal diapause and cytochrome c is the limiting com- ponent in the pupal respiratory chain. In this perspective, the increased respiration following integumentary injury and initiation of adult development reflects an increase in cytochrome c content which occurs at a faster rate than any increase in cytochrome oxidase. Possibly the in- crease in cytochrome c reflects its adaptive synthesis in response to changes in the energy requirements of the tissues. These changes were induced on the one hand by injury and on the other by the prothoracic gland hormone which initiated adult development. Such an adaptive synthesis of cytochrome c has been suggested in the case of regenerating rat liver by Drabkin (1955). However, while the data sup- port the view that cytochrome c is the limiting link in the pupal respiratory chain, they do not rule out the possibility that other factors, such as phosphate acceptors, may exert short-term effects on pupal respiration. In conclusion, it is worth recalling that many animals other than diapausing pupae of the silkworm have a low respiration that is insensitive to carbon monoxide. Moreover, in many of these, such as diapausing eggs of grasshoppers and silkworms and unfertilized eggs of sea urchins, cytochrome oxidase is clearly present. The usual explanation for CO-insensitivity has been that respiration proceeded along tracks alternative to the cytochrome oxidase system (cf. Needham, 1942, p. 567). It is noteworthy, however, that in interpreting some of the very first experiments which showed this CO-insensitivity, Runnstrom (1930) suggested that cytochrome oxidase was not saturated with its substrate and this was the reason for CO- insensitivity in the sea urchin egg. In retrospect, it seems likely that this idea was sound and that the CO-insensitivity of the respiration of many systems is probably the result of an excess of cytochrome oxidase relative to some other component of the respiratory chain. 9. Final theoretical considerations of carbon monoxide-insensitive respiration The basic premise underlying the arguments offered in the earlier sections of this discussion is that an excess of cytochrome oxidase can lead to a virtual carbon monoxide-insensitivity of a cytochrome oxidase-mediated respiratory chain. This is shown as follow r s. It is well known that carbon monoxide combines only with the reduced form of cytochrome oxidase (also called a 3 ) : (1) CO + a 3 ++ ^ C0-a,++ ; K ( C(>a: < ++) (C0)(a 3 ++ y Equation (1) is the simple chemical equilibrium with a characteristic equilibrium constant that describes the interaction of reduced cytochrome oxidase (a s ++ ) with 156 CHARLES G. KURLAND AND HOWARD A. SCHNEIDERMAN carbon monoxide. This equation tells us that at a given concentration of carbon monoxide the amount of CO-a 3 ++ complex formed is determined solely by the steady-state concentration of reduced cytochrome oxidase. Now the steady-state concentration of reduced (and oxidized) cytochrome oxidase is determined by the rate of electron transfer to cytochrome oxidase, and this, of course, is measured by the rate of oxygen consumption. /^\ 4__i_ /-\ +++ I /"k \) Q-3 I ^2 ~~ #3 ~T~ ^J% Equation (2) describes this steady-state between reduced and oxidized cytochrome oxidase. It is important to note that equation (2) does not describe a simple chemical equilibrium but a steady-state where the "apparent equilibrium constant" depends on the rate of electron transfer through the respiratory chain. Thus, if the rate of electron transfer to a s +++ from the previous component in the chain is very slow, most of the cytochrome oxidase will be in the oxidized state and the ratio of a 3 ++ to a 3 +++ will be small. Since the rate of electron transfer is measured by the rate of oxygen consumption, the "apparent equilibrium constant" for equation (2) will vary with the rate of oxygen consumption. This fact, incidentally, rules out the use of the usual Warburg formulation to describe quantitatively the effects of carbon monoxide on respiration, namely N CO o~ ^"oT where "N" is the fraction of respiration not inhibited by carbon monoxide (War- burg, 1927). For, this formulation assumes that all the oxidase is in the reduced state, and hence that "the observed respiration is proportional to the amount of enzyme not combined with carbon monoxide'' (Warburg, 1949. p. 78). Indeed, Warburg points out that in view of this assumption it is remarkable that there are cells for which his equation applies (p. 79). When carbon monoxide is used as an inhibitor of cytochrome oxidase. the de- gree of inhibition of respiration depends upon the new steady-state reached by the system, in which both oxygen and carbon monoxide compete for reduced cytochrome oxidase. In this steady-state, some of the cytochrome oxidase is in the oxidized state, some is reduced and complexed with carbon monoxide, and the remainder is reduced and transferring electrons to molecular oxygen, i.e., playing a role in res- piration. The effect of carbon monoxide on respiration depends on the degree to which carbon monoxide decreases the concentration of reduced cytochrome oxidase that is transferring electrons to molecular oxygen. Since a s ++ must satisfy the equili- brium conditions of equation (1) and the steady-state conditions of equation (2), it becomes apparent that the amount of a 3 +++ plays a major role in determining how much a 3 ++ remains to function in respiration. We thus see that the effect of carbon monoxide on respiration depends on the fraction of the total cytochrome oxidase in the reduced state. In other words, the effect of carbon monoxide on respiration depends upon the ratio of the actual rate of uninhibited respiration (as measured by the concentration of reduced cytochrome oxidase) to the maximum potential rate of respiration when virtually all the oxidase is kept in the reduced state (as measured by the total concentration of cytochrome oxidase). This ratio, ++ _U /r ..+++* a,++ + a-. CARBON MONOXIDE AND RESPIRATION 157 the fraction of the total oxidase in the reduced state, is what we ordinarily refer to as the "saturation" of cytochrome oxidase. When the saturation of cytochrome oxidase is high, the carbon monoxide sensitivity is high, and when the saturation is extremely low, the effect of carbon monoxide on respiration is insignificant. This can easily be seen when we consider two extreme cases, bearing in mind equations (l)and(2). Let us examine a system in which the initial steady-state concentrations of aS + and a, +++ are about equal (i.e., a high saturation). In such a system, with a 20:1 CO/O 2 ratio an appreciable amount of CO- 3 ++ can form. When the new steady- state is established in the presence of carbon monoxide, the ratio of the concentra- tions of a ?i ++ to o, +++ is the same as before. However, the absolute concentration of both these components has been reduced considerably since a large part of the cytochrome oxidase is complexed with the carbon monoxide. As far as respiration is concerned, the significant reduction in a, ++ leads to a significant inhibition of respiration by carbon monoxide. By contrast, consider a system in which the initial steady-state concentration of a n +++ is much greater than the concentration of a, 4+ . The presence of a CO/O., ratio of 20: 1 will lead to the formation of only a small concentration of CO-a 3 ++ because of the low concentration of a., ++ . Indeed, when the difference between the concentra- tions of rt 3 +++ and <7., ++ is very great (i.e., a very low saturation), the total pool of cytochrome oxidase will not be significantly affected by carbon monoxide. As a result, the steady-state concentration of a.^ + will not be significantly diminished by the presence of carbon monoxide. Thus the CO-sensitivity of such a system is small. From the above analysis we learn that an excess of cytochrome oxidase relative to other components of the respiratory chain will lead to CO-insensitivity of respira- tion. The same conclusion was reached independently by Harvey and Williams (1958b) using a different system and method of analysis. One further theoretical consideration is crucial to the explanation offered above for CO-insensitivity. If the inhibition of cytochrome oxidase by carbon monoxide is a function of the total cytochrome oxidase present, then it must be possible for the transfer of electrons from the carrier part of the respiratory chain to proceed independently of a sterically specific arrangement of the chain components. In their review. Chance and Williams (1956) have discussed this possibility. They concluded that it was highly improbable that the chain components were fixed in position, and they presented two alternatives. Either the chain components were free to act by random collisions according to a modified law of mass action ; or, they were fixed in such a manner that the prosthetic groups were free to rotate on an axis and be brought into apposition with adjacent chain components. In either case, electron transfer could proceed across chain components that were not im- mediately adjacent to one another. Therefore, it seems possible for the carriers of the respiratory chain of the dia pausing pupa to transfer electrons to a "pool" of cytochrome c oxidase. This pool of cytochrome o.ridase can manage all of the oxidations, even in the presence of inhibitors, as long as there is sufficient uninhibited enzyme present to meet the needs of electron transfer. In short, it appears possible for an excess of cytochrome oxidase in tissues to account for the CO- and azide- insensitivity of respiration and of various physiological functions such as heart-beat. 158 CHARLES G. KURLAND AND HOWARD A. SCHNEIDERMAN The arguments presented in the previous sections persuade us that this is the situa- tion in most of the tissues of diapausing silkworm pupae. The experiments reported in Section 1A were performed in collaboration with Dr. Roger D. Smith. We gratefully acknowledge the helpful criticisms of Dr. David P. Hackett, Dr. Conrad S. Yocum, Dr. Carroll M. Williams, and Dr. Howard M. Lenhoff. SUMMARY 1. To characterize the respiratory enzyme chain that functions during diapause, the respiration of diapausing pupae of the Cecropia, Cynthia, Promethea and Poly- phemus silkworms was measured in the presence of specific mixtures of oxygen, nitrogen and carbon monoxide, after injection of various metabolic inhibitors and after injury. 2. Pupal respiration is at best only slightly inhibited by carbon monoxide and is often stimulated. Whatever CO-sensitivity there is occurs only in pupae with high basal metabolic rates. Moreover, when respiration is accelerated by injecting dinitrophenol (DNP), or by injury, this evokes an enhanced sensitivity to carbon monoxide. Indeed, it appears that the fraction of respiration sensitive to carbon monoxide is a function of the rate of oxygen consumption of the silkworm at all stages. 3. Reducing external oxygen tension to 2% fails to inhibit oxygen consumption, but increases markedly the CO-sensitivity of pupal respiration. Thus low oxygen tensions seem to unmask CO-sensitivity. 4. Pupal respiration is insensitive to azide concentrations as high as 5 X 10~ 4 M. However, the azide-sensitivity, like the CO-sensitivity, increases markedly when pupal respiration is stimulated by DNP or injury. 5. Antimycin A at a concentration of 10~ 6 M inhibits less than one-third of normal pupal respiration or DNP-stimulated respiration. Compared to other or- ganisms diapausing pupae are resistant to this inhibitor of the cytochrome c re- ductase system. 6. Dinitrophenol at a concentration of 5 X 10~ 4 M stimulates pupal respiration an average of 12-fold and as much as 16-fold. These are among the largest DNP- stimulations ever recorded. Although pupae with high basal metabolic rates are less stimulated proportionately by DNP than are pupae with low basal metabolic rates, they develop a greater over-all respiration under the influence of DNP. 7. Dinitrophenol-stimulated respiration is inhibited by carbon monoxide. The higher the DNP-stimulated respiration, the greater the inhibition by carbon mon- oxide. From this and other evidence it appears very likely that DNP accelerates the turnover of one or several components of the respiratory chain while having a lesser effect on cytochrome oxidase. 8. Dinitrophenol delays the appearance of injury-stimulated respiration, sug- gesting that the development of this increased respiration requires phosphate bond energy. Furthermore, exposure to carbon monoxide causes the death of injured pupae indicating that injury respiration is obligatory and involves the synthesis of new respiratory components. 9. Newly molted pupae not yet firmly in diapause do not respond to wounding with an injury respiration and their respiration is sensitive to carbon monoxide. These findings are correlated with their high respiratory rate. CARBON MONOXIDE AND RESPIRATION 159 10. The modes of action of the several inhibitors within diapausing, injured, and developing insects are considered in detail and a new explanation is proposed to account for the CO-, azide-, and cyanide-insensitivity of pupal respiration. 11. It is concluded that the insensitivity of diapausing pupae to inhibitors of cytochrome oxidase results from an excess of this enzyme over its functional require- ments in the pupal respiratory chain. This concept is examined in detail and found to be theoretically sound. Evidence is presented that the limiting link in the res- piratory chain is cytochrome c. Thus, contrary to earlier conceptions, it appears that cytochrome oxidase is the principal terminal oxidase during diapause as well as during all the other stages of the life history, and that the CO-insensitivity of pupal respiration stems from a great excess of cytochrome oxidase relative to cytochrome c. 12. The increased CO- and azide-sensitivity of pupal respiration after injection of DNP or injury results from an increase in the saturation of cytochrome oxidase provoked on the one hand by an increase in the turnover rate of cytochrome c, and on the other by the synthesis of cytochrome c. 13. It is suggested that the CO-insensitivity of the respiration of other organisms may be the result of an excess of cytochrome oxidase relative to some other com- ponents of the respiratory chain. LITERATURE CITED ALLEN, T., 1940. Enzymes in ontogenesis (Orthoptera). XI. 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Heavy Metal Prosthetic Groups and Enzyme Action. Oxford at the Clarendon Press. WILLIAMS, C. M., 1946. Physiology of insect diapause : the role of the brain in the production and termination of pupal dormancy in the giant silkworm Platysamia cecropia. Biol. Bull, 90 : 234-243. WILLIAMS, C. M., 1951. Biochemical mechanisms in insect growth and metamorphosis. Fed. Proc., 10 : 546-552. WILLIAMS, C. M., 1952. Physiology of insect diapause. IV. The brain and prothoracic glands as an endocrine system in the Cecropia silkworm. Biol. Bull., 103 : 120-138. WOJTCZAK, L., 1955. Terminal oxidases of insects. Proc. 3 me Congres International de Biochimie. Bruxelles. Abstract 12-31. PERIODICITY OF MITOSIS AND CELL DIVISION IN THE EUGLENINEAE 1 GORDON F. LEEDALE Department of Botany, The Durham Colleges in the University of Durham, England In the course of an investigation into the division cytology of flagellates of the class Euglenineae, it became necessary to determine the time and rate of mitosis for each of the forty species under examination. The present paper deals with the periodicity of mitosis revealed in the twenty species studied in detail for this feature, and relationship of the periodicity to the day-night cycle. An account of the structure and division of the cell and nucleus will be published separately (see Leedale, 1958a, 1958b). MATERIAL AND METHODS 1. Species studied - The three main sources of material have been my own wild collections, the Cam- bridge Culture Collection of Algae and Protozoa, and the Sammlung von Algenkul- turen, Gottingen. All species have been isolated by Professor E. G. Pringsheim or myself, with the exception of Trachelomonas grandis which was isolated by Singh (Singh, 1956) and sent to me by Professor H. C. Bold. The names of species are corrected according to Pringsheim (1956) for the genus Euglena, and to Huber-Pestalozzi (1955) for the remaining genera. Color- less species are indicated by an asterisk. * Astasia klebsii Lemmermann Colacium mucronatum Bourrelly Cryptoglena pigra Ehrenberg * Distignia proteus Ehrenberg em. Pringsheim Euglena acus Ehrenberg Euglena deses Ehrenberg Euglena gracilis Klebs (strain "T," green form) * Euglena gracilis Klebs (strain "T," colorless form) Euglena gracilis Klebs (strain "Z," green) Euglena spirogyra Ehrenberg Euglena viridis Ehrenberg Eutreptia pertyi Pringsheim Eutreptia viridis Perty 1 From a study carried out in the Botany Departments of Queen Mary College, London, and The Durham Colleges; some of the results were included in a thesis approved for the degree of Doctor of Philosophy in the University of London. My thanks are due to Dr. M. B. E. Godward of Queen Mary College and Professor E. G. Pringsheim of the University of Gottingen for their help and advice. 2 I would like to thank Professor E. G. Pringsheim, Mr. E. A. George of Cambridge and Professor H. C. Bold for supplying me with material. 162 MITOTIC RHYTHMS IN THE EUGLENINEAE 163 * Hyalophacus ocellatus Pringsheim Lepocinclis ovum var. buctschlii (Conrad) Huber-Pestalozzi Lepocinclis steinii Lemmermann em. Conrad * Menoidium cultellus Pringsheim * Peranema trichophorum (Ehrenberg) Stein Phacus pusillus Lemmermann Phacus pyrum (Ehrenberg) Stein Trachclomonas India Stein em. Deflandre Trachelomonas grandis Singh 2. Cultivation Cells were isolated from wild collections by the micropipette method (Prings- heim, 1946a). All species except Peranema trichophorum were grown in soil-water tubes (biphasic culture, Pringsheim, 1946a, 1946b) with a wheat grain, starch or ammonium magnesium phosphate beneath the soil. Eutreptia spp. were grown in tubes with 50% sea-water. Peranema trichophorum was grown in soil extract containing 0.5% milk. In addition to the biphasic cultures, green and colorless forms of Euglena gracilis (strain "T") were cultivated in 0.2% Difco beef extract, or "SATBY" (0.1% sodium acetate, 0.2% Difco tryptone, 0.1% Difco beef extract, 0.2% Difco yeast extract, in distilled water). Cultures were hung in a north-facing window or in temperature-controlled cab- inets with either incandescent or fluorescent lighting on a time-switch. The cul- tures were grown at a standard temperature of 20 C. GENERAL FEATURES OF THE CULTURES A biphasic culture of any species of the Euglenineae has a typical growth pattern. Sub-culturing to a new tube with a heavy inoculum is followed by a lag-period of two to three days during which time there are few or no divisions. This is followed by a period of multiplication which is eventually slowed and halted by overcrowding of the medium. There is an upper limit of number of cells per ml. of medium (the "culture saturation point") at which cell multiplication falls to a low rate. This effect is not caused by exhaustion of the medium ; if the cells of a "saturated" culture are centrifuged off and the medium re-inoculated, the culture builds up as quickly as before, and this can be repeated several times. According to the size of the inoculum, the division rate, and the "culture satura- tion point" of the species concerned, the increase in cell numbers may continue for one to twelve months. It is the mitotic rhythms occurring during this period of multiplication which are the subject of this paper. MITOTIC PERIODICITY IN GREEN SPECIES Fixations made at two-hourly intervals for several (not successive) 24-hour periods showed that all green species of the Euglenineae had mitosis confined to the dark period when growing in biphasic culture under natural light conditions. The restriction of nuclear division to the dark period was examined in detail in 164 GORDON F. LEEDALE 4O- o "Seo O I/) 13 O -^40 CJ -4 A B o cr> 6 (/>' -4 -2 8p.m. Time FIGURE 1. fifteen green species. Fixations were made at half-hourly intervals from the onset of darkness on one, two or three consecutive nights, the material for any one series being taken from the same culture tube. Five hundred cells were counted in each of two preparations from each fixation and the number of cells in mitosis and cell division noted. The results of these counts were similar for all species and are recorded graphically for six representative species in Figures 1 and 2. Mitosis began one to two hours after the onset of darkness. In Euglena spirogyra (Fig. 1, A), Euglena viridis (Fig. 1, B) and Eutreptia pertyi, mitosis began at the same time on each of three successive nights. The mitotic maximum MITOTIC RHYTHMS IN THE EUGLENINEAE 165 Time FIGURES 1 and 2. The number of cells per thousand in mitosis at half-hourly intervals during one, two or three consecutive nights, plotted as mitosis percentage against time. All results are for biphasic cultures growing in the natural day-night cycle, the dark period be- ginning at 8 PM. FIGURE 1. Green species : A, Euglena spirogyra; B, Euglena viridis. FIG- URE 2. Green species : A, Phacus pusillus; B, Phacus pyrum; C, Trachelomonas bulla; D, Trachelomonas grandis. occurred from 2% to 4^ hours after the onset of darkness in all species. The maxima for Euglena viridis (Fig. 1, B), Eutreptia pertyi and Phacus pusillus (Fig. 2, A) occurred at the same time on three successive nights; those for Euglena spirogyra (Fig. 1, A) covered a two-hour period within three nights. The span 166 GORDON F. LEEDALE of the nightly period during which mitosis occurred ranged from three to six hours in the different species. The mean maximum percentage of cells undergoing mitosis each night is recorded in Table I. Recording the number of cells at each stage of mitosis in each fixation produced a more detailed picture of the periodicity. The results for Euglena spirogyra for one dark period (Fig. 3) illustrate the complete restriction of nuclear and cell division to within a five-hour period, beginning approximately two hours after the onset of darkness. Successive maxima of the mitotic stages occur, a wave of pro- phases being followed by waves of metaphases, anaphases, telophases and cell cleavage. This pattern was repeated in other cultures of the same species and by other species, the relative size and span of the maxima varying according to the duration of the stages of mitosis in the different species. TABLE I The mean maximum percentage of cells undergoing mitosis each night in green species of the Euglenineae in biphasic culture at 20 C. Species Mean maximum % Colacium mucronatum 2.6 Cryptoglena pigra 1.8 Euglena acus 1.9 Euglena deses 2.2 Euglena gracilis "T" 4.2 Euglena gracilis "Z" 5.7 Buglena spirogyra 3.4 Euglena viridis 4.9 Sutreptia pertyi 3.5 Sutreptia viridis 2.3 Itepocinclis ovum var. buetschlii 6.8 Itepocinclis steinii 1.3 PJiacus pusillus 3.4 Rhacus pyrum 3.1 T*rachelomonas butla 1.8 Trachelomonas grandis 2.9 Further series of fixations over a period of one year showed that no matter at what time of the clock the natural dark period began, mitosis began one to two hours later, the percentage of cells dividing each night being of the same order for any one species (at 20 C.). There was no variation in the mitotic rate in relation to day-length. Examination of the same culture over a period of several months showed the multiplication period to be discontinuous. Weeks with divisions occurring every night were interspersed with occasional days when no divisions occurred. The introduction of an artificial dark period during the natural light period affected mitotic periodicity in all the green species. If the artificial dark period was begun three hours or less before the natural one, mitosis occurred, but in a lower percentage of cells than usual. When the artificial dark period was introduced six hours or more before the natural one was due to begin, divisions rarely occurred. The shortest day-length after which mitosis would occur was approximately twelve hours. No mitosis or cell division could be induced in any green species in biphasic MITOTIC RHYTHMS IN THE EUGLENINEAE 167 culture in any conditions or intensity of artificial lighting, either direct or diffused, incandescent or fluorescent. Attempts to reverse the mitotic periodicity in a tem- perature-controlled cabinet with lighting on a time-switch were unsuccessful, the cells becoming quiescent with no divisions occurring. Similarly, no mitosis occurred in either continuous light or continuous darkness. Returned to natural light con- ditions after such treatment, the cells recovered their full division rate within a day if the treatment had been short, but less quickly if the treatment was prolonged. C o (fl o O 6- 4- 2- 10pm 12 M. 2a.m. Ti me FIGURE 3. Mitosis in Euglena spirogyra. The number of cells per thousand in prophase (P), metaphase (M), anaphase (A), telophase (T) and cell cleavage (Cl) at half -hourly intervals during one night, plotted as percentage of each mitotic stage against time. The results are for a biphasic culture growing in the natural day-night cycle, the dark period be- ginning at 8 PM. 168 GORDON F. LEEDALE 80- 6am 12N. 6pm 12M. 6am darkness. Time FIGURE 4. Once mitosis had begun, it proceeded to conclusion even if the dividing cell was then subjected to light. However, if light was introduced less than an hour after the onset of darkness, no mitosis occurred. If a dark period of more than one hour followed a full-length day and artificial light was then introduced, some cells underwent a complete mitotic division, though on first examination no cells could be found in mitosis, not even in prophase. Euglena gracilis was the only species in which the time and rate of mitosis in biphasic culture could be compared with those in a rich liquid medium. The MITOTIC RHYTHMS IN THE EUGLENINEAE 16') (J 30- 20- 60f 40- C O ^20 o -C ^20- 10- A B C iA i^. 43 2 41 -6 -4 -2 2 41 6a.m. 12N. 6p.m. 12M. 6am. darkness T me FIGURES 4 and 5. The number of cells per thousand in mitosis at half-hourly intervals during one, two or three (not consecutive) 24-hour periods, plotted as mitosis percentage against time. All results are for biphasic or milk cultures growing in the natural day-night cycle. FIGURE 4. Colorless species : A, Astasia klebsii ; B, Distigma protcus. FIGURE 5. Colorless species : A, Hyalophacus ocellatns; B, Menoidium cultellus ; C, Pcranema tricho- phorum. mitotic rhythm shown by the green form of strain "T" in biphasic culture was absent in 0.2% beef extract or "SATBY" medium. During the period of rapid multiplica- tion prior to crowding of the culture, a fixation at any time of day or night showed from 5-6% (beef extract) or 8-10% ("SATBY") of the cells undergoing mitosis 170 GORDON F. LEEDALE (at 20 C.)- At 30 C. the mitotic rate of Euglena gracilis "T" in "SATBY" was 25-30%. In biphasic culture, maximum division rates were obtained at 20 C. ; raising or lowering the temperature by five degrees resulted in a fall in division rate. MITOTIC PERIODICITY IN COLORLESS SPECIES Fixations made at half -hourly intervals over 24-hour periods showed that a constant rate of mitosis was not maintained in any colorless species of the Eugleni- neae in biphasic culture, bursts of mitotic activity alternating with periods when mitosis was almost completely absent. The results for 24-hour series of half-hourly fixations are recorded for the five species studied in Figures 4 and 5. In addition to these series where a division maximum occurred at some time during the 24-hour period, numerous series con- tained no divisions or a few divisions scattered throughout the period. Many single fixations at different times of day or night contained cells in mitosis. TABLE 1 1 The maximum percentage of cells recorded in mitosis at any one time in colorless species of the Englenineae in biphasic or milk culture at 20 C. Species Maximum % Astasia klebsii 8.0 Distigma proteus 3.9 Hyalophacus ocellatus 1.9 Menoidium cultellus 4.7 Peranema trichophorum (in milk) 2.1 Mitotic maxima occurred at any time of the clock. In none of the five species did the periods of major mitotic activity bear any relationship to the alternating light and darkness of the natural day-night cycle. The recorded maxima for Astasia klebsii (Fig. 4, A) occurred at 10 AM, 4:30 PM and 10 PM ; those for Hyalophacus ocellatus (Fig. 5. A) at 9 AM, 3 PM and 9:30 PM. The time-spans of the major periods of mitotic activity ranged from 3y 2 to 8^2 hours. The highest percentage of cells obtained dividing at any one time is recorded for each species in Table II. The percentages of cells dividing at different times on different dates were of the same order for some species (Fig. 5, A and B) but not for Astasia klebsii (Fig. 4, A). The irregularly spaced bursts of major mitotic activity in the colorless species continued in alternating artificial light and darkness, in continuous light, and in continuous darkness. The colorless form of Euglena gracilis "T" growing in 0.2% beef extract or 'SATBY" medium behaved as did the green form in these media, exhibiting no periodicity of mitosis, regular or irregular. A continuous division rate of 6-7% was maintained in "SATBY" at 20 C., the rate increasing to 30-35% at 30 C. DISCUSSION Mitotic rhythms have been recorded for higher plants by Lewis (1901), Kellicott (1904), Karsten (1915), Laughlin (1919), Stalfelt (1919), Friesner (1920), Tischler (1921), Abele (1925), Brown (1951) and Jensen and Kavaljian (1958). MITOTIC RHYTHMS IN THE EUGLENINEAE 171 The rhythm, in most cases thought to be endogenous, has been related to the onset of germination, the balance between cell elongation and division, or light periodicity. Lewis (1901) and Karsten (1915) found that the times of the maxima altered when light conditions were changed, but Friesner (1920) found the maxima were inde- pendent of light changes. Stalfelt (1919) and Brown (1951) state that the mitotic rhythm of higher plants is exogenously imposed by the day-night cycle, disappearing when the plants are grown in continuous darkness. No evidence of mitotic rhythms in higher plants was found by Winter (1929) or Gray and Scholes (1951). Mitotic rhythms in animals have been recorded by Ortiz-Picon (1933), Carleton, (1934), Cooper and Schiff (1938), Cooper and Franklin (1940), Blumenfeld (1942, 1943), Bullough (1948) and Milletti (1950). The rhythm has been related to the activity cycle, a higher division rate occurring when the animal is at rest (Cooper and Schiff, 1938; Bullough, 1948; Milletti, 1950). Kalmus (1935) has recorded an exogenous rhythm of cell division for Paramecium. Twenty-four-hour rhythms of mitosis have been recorded for a number of algae. Division occurring exclusively at night has been recorded for species of the genera Cladophora and Stigeoclonium (Braun, 1851), Spirogyra (Braun, 1851; Famintzin, 1867; Sachs, 1874; Strasburger, 1880). Zygnema (Kurssanow, 1912), and Vauch- cria, Hydrodictyonand Ulothri.v (Sachs, 1874), whilst Karsten (1918) found three maxima in each 24-hour period for species of Closterium, Cosmarium and Mesotacn- hmi. Wildeman (1891) found no mitotic rhythm in Spirogyra. The present author has found mitosis almost entirely confined to the dark period in species of Hydrodictyon, Ulothri.v, Mougcotia, Spirogwa, Zygnema, Closteriwn, Cosmarium and Staurastrum in biphasic culture. The rhythm was exogenous in these species, and mitosis could be produced at any time of the clock by adjusting the time of the dark period in a culture cabinet. Some species of Spirogyra and Zygnema under- went mitosis in continuous light. A nocturnal periodicity of mitosis in euglenoid species has been mentioned by Dangeard (1902) for species of Euglena, Phacus and Trachelomonas, Baker (1926) for Euglena gracilis in a split pea infusion, Ratcliffe (1927) for Euglena spirogvra in modified Doflein's medium, S. R. Hall (1931) for the parasitic Euglena leucops Hall when in its host, a species of Stenostomum, Gojdics (1934) for Euglena deses in 0.1^ beef extract, Johnson (1934) for Colacium vesiculosum Ehrbg. and Chu (1946) for Euglena spp. in biphasic culture. Only sparse growth was possible in several of the media recommended by these authors. Lackey (1929) has made the only record of a division maximum at night in a colorless species (Entosi- phon sulcatum (Duj.) Stein, grown in a cracked wheat medium) and suggests it might be explained on phylogenetic grounds. This is to be doubted since the nocturnal rhythm of the green species is not endogenous and no such rhythm is present in the five colorless species investigated in the present study. Lackey re- cords some divisions during the day and it is probable that his division maxima occurred during the dark period by chance, without being related to it. The mitotic maxima recorded for higher plants and animals are increases over a continuous low division rate. As would be expected, in organisms composed of many cells arranged in tissues, some of which are specifically concerned with cell division, mitosis occurs throughout the 24-hour period in the division sites. Diurnal rhythms, whether in areas directly affected by light or not (root-tip meristems in 172 GORDON F. LEEDALE plants, bone marrow in animals), can be related to metabolic rhythms, maximum mitosis occurring during the period of minimum activity. In unicellular organisms the division of labor between cell growth and mitosis is often in time rather than in space. A cell tends to divide during a period of minimum activity of that particular cell. Thus green unicells and filamentous green algae often have a rhythm of mitosis which is closely related to the rhythm of photosyn- thetic activity in the day-night cycle. Such a relationship is exhibited by the Euglenineae. Green species, when living autotrophically, divide only in the dark, and an almost full period of natural day- light is necessary before mitosis will occur in the ensuing dark period. A threshold period of darkness is required for the induction of mitosis, but once induction has occurred, the mitotic process will begin and proceed to completion, even though the cell be subjected to light before its nucleus has begun the anterior migration which is the first sign of approaching mitosis. This induction precedes prophase by a period of up to one hour, since the threshold period for induction is approximately one hour after the onset of darkness, whilst the first prophases in all species appear one to two hours after darkness. The final inductions would then be occurring approximately three hours later, since the last prophases appear four to five hours after darkness (Fig. 3). It has been shown in Euglena gracilis that the nocturnal periodicity of mitosis is removed by the stimulus of a rich food supply, the heterotrophic ("chemotrophic") mode of nutrition of this species in beef extract or "SATBY" being unrelated to the day-night cycle. Mitosis in colorless species of the Euglenineae in biphasic culture shows an irregular periodicity which is not related to the natural day-night cycle. The heterotrophic mode of nutrition of the colorless species is also independent of light. The factor deciding which cells in a culture divide during any one period of mitosis is probably cell age (reflecting cell size and cell maturity) in both green and colorless species. If 5% of the cells of a biphasic culture of a green species divide each night in turn, the span of a generation will be 20 days. In Euglena gracilis in "SATBY" at 30 C., with a division rate of 25-35%, the generation span cannot be more than 8-12 hours. The nocturnal rhythm of mitosis is presumably present in green species in the wild when the supply of nutrients is low. An influx of rich organic nutrients will remove the periodicity and, when combined with optimum temperature and pH, may result in the sudden euglenoid "blooms" which often occur in bog-pools, farm- yards, ponds and lakes. SUMMARY 1. The periodicity of mitosis and cell division has been investigated in 15 green and 5 colorless species of the Euglenineae. 2. Green species in biphasic culture under natural light conditions have mitosis confined to the dark period. Mitosis begins one to two hours after the onset of darkness, each species having a predictable percentage of cells dividing each night. There is a threshold period at the beginning of the dark period after which mitosis cannot be inhibited by light. The mitotic rhythm is exogenous, being removed by growth in artificial light or darkness (resulting in no mitosis), or in a rich organic medium (resulting in continuous mitosis at a constant rate). MITOTIC RHYTHMS IN THE EUGLENINEAE 173 3. Colorless species in biphasic culture under any light conditions have an ir- regular mitotic periodicity, bursts of mitosis occurring at any time of the clock and alternating with periods in which mitosis is almost absent. 4. 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SCHEER Department of Biology, University of Oregon, Eugene, Oregon, and Laboratoire Arago, Banyuls-sur-Mcr Recent reviews of the metabolic events in the intermoult cycle of decapod crusta- ceans, and of the hormonal control of these events, have emphasized the fragmentary nature of our present knowledge (Knowles and Carlisle, 1956; Scheer, 1957). Particular interest centers around the metabolism of carbohydrate, which is known from the work of Renaud (1949) to undergo considerable changes in the course of the intermoult cycle. The present report is based on a study of a laboratory population of approximately 100 specimens of the crab Carcinides ( = Carcinus] maenas, in which the content of total carbohydrate, total soluble polysaccharide, blood carbohydrate, blood lipochromes, and total non-protein nitrogen was deter- mined on the individuals in samples drawn at intervals from the population. MATERIALS AND METHODS The animals were taken from a lagoon north of Banyuls-sur-Mer and brought into the laboratory on October 11 : they were maintained throughout the experiment in large aquaria in running sea water, and fed regularly on mussels. Examination of the animals showed them all to be in the hard-shelled condition (stages Co through D., of Drach, 1939), but closer determination of intermoult cycle stage was not made until the animals were killed for analysis. Most of the animals were males, and only males were used for the studies reported, to avoid complications arising out of sexual differences. On October 14, November 13, and December 1, samples of 20 to 30 crabs, selected at random, were drawn from the group, and the eyestalks were removed from every second animal. Mortality was very low. It is probable that a few animals moulted during the period of the experiment, but cannibalism prevented any certain determination of this. Eight to ten days after the sampling, the animals were extracted for analysis. The stage in the intermoult cycle was carefully determined, using the criteria of Drach (1939). For this study, an exact determination of the division between the end of the intermoult period (C 4 ) and the beginning of the premoult period (D : ) was essential. Accordingly, microscopic examination of the external branchial epipodite of the first maxilliped was made to determine the presence of newly formed setae beneath the old integument of this appendage. The presence of even the most rudimentary new setae was taken as an index of the beginning of stage D a . These rudimentary setae can be detected only by careful microscopic examination under good illumination by transmitted light. 175 176 BRADLEY T. SCHEER A blood sample was taken by bleeding from a cut walking leg. The animal was then quickly cut up into 50-75 ml. of 5% trichloracetic acid; Renaud (1949) had already shown that the elaborate precautions to prevent glycolysis which are neces- sary in mammals are not as important in crabs. The mixture of acid and tissue was transferred to an electric blender (Cadillac Atomixer), and blended at 8000 rpm. for 3 minutes. The mixture was rapidly filtered with suction, and the residue returned to the blender with a second portion of acid for a second extraction. The blender and residue were washed with a third portion of acid. The combined filtrates were then diluted in a volumetric flask, usually to 250 ml., and stored in the refrigerator until analyzed, always within a few days. Blood carbohydrate was determined on some samples by the anthrone method of Roe (1955). One ml. of blood was collected by dripping from a cut walking leg, into a calibrated tube. One ml. of 5% trichloracetic acid was added with mix- ing, and the mixture was centrifuged. One ml. of the supernatant was then trans- ferred to a second tube for colorimetric determination. Blood lipochromes were determined on other samples. To 1 ml. of blood. 5 ml. of acetone were added. The acetone solution was then extracted with 2 ml. of petroleum ether, the ether layer was washed with water, dried with solid KOH, and diluted to 5 ml. with petroleum ether. The concentration of lipochromes was then read in the spectro- photometer at 450 m/x against a petroleum ether blank. The measurements are given as optical densities, since the exact nature of the lipochromes involved is not known. Total carbohydrate was determined by the anthrone method (Roe, 1955). One hundred microliters (/xl) of the extract were transferred to a tube with a micro- pipette, and diluted to one ml. for colorimetric determination. Polysaccharide was determined by the same method. To 1 ml. of extract, 5 ml. of 95% ethyl alcohol were added, and the mixture allowed to stand overnight in the refrigerator. The tubes were centrifuged, the precipitate carefully drained, and suspended in 10 ml. of distilled water. A one-mi, sample of this suspension was used for colorimetric analysis. The anthrone method has the advantage for this study that it determines a variety of carbohydrates, and relatively few other naturally occurring compounds. All results are expressed in terms of glucose equivalents. Non-protein nitrogen (NPN) was determined on 10-ml. samples of the extract, using the micro-Kjeldahl digestion method of Hiller et al. (1948) and distilling the digested mixture into 0.1 N HC1 in an all-glass still. Ammonia nitrogen (NH 3 N) was separated by distilling the undigested extract in the same still. The final de- termination of ammonia in both cases was colorimetric, using the Nessler reagent. RESULTS In the first sample, examined 11 to 19 days after collection, 10 out of 16 animals (63%} were in the C 4 stage (late intermoult) of the intermoult cycle; the remainder were in the D t stage (early premoult). In the second sample, examined 43 to 45 iays after collection, the proportion of C 4 animals was 52% (12 C 4 , 8 D x , 3 D 2 ). In the third sample, the proportion was 32% (7 C 4 , 15 D x ). These values suggest that the population from which the samples were drawn was undergoing a steady progression towards the moult. The x 2 test shows that the proportion of C 4 animals in the third sample is significantly less than in the first, at the 5% level of probability. HORMONAL CONTROL OF METABOLISM 177 In the first sample, only one of the 10 C 4 animals had blood clearly pigmented with lipochromes, while 5 of the 16 Dj animals had blood so pigmented; no quanti- tative determinations were made in this series. In the second sample, 5 of the 12 C 4 animals and 6 of the 1 1 D 1 animals had lipochromes clearly evident in the blood ; quantitative measurements were made on these 1 1 animals, and are presented in Table I. For the third sample, quantitative measurements were made on all the animals, and are presented in Table I. From the results on the third sample, in which traces of lipochrome are found in nearly all specimens, it appears that the level for qualitative detection of lipochromes lies at about 0.05 on the density scale used to express concentrations. On this basis, we would conclude that only one of the 7 C 4 animals of the third sample had substantial amounts of lipochrome in the blood, while 7 of the 15 Dj animals had such amounts. If we apply the x" test to TABLE I Lipochromes in the blood of Carcinides maenas. Optical density at 450 m/j. of a petroleum ether extract, volume 5 nil., from 1 ml. of blood. The values for sample 2 (see text) represent only animals in which blood lipochromes were qualitatively evident C4 Di .Stage condition Normal Eyestalkless Normal Eyestalkless Animal Density Animal Density Animal Density Animal Density Sample 2 22 0.072 24 0.064 31 0.150 35 0.070 36 0.065 20 0.050 34 0.077 23* 0.157 33 0.112 40 0.088 42* 0.076 Sample 3 45 0.063 44 0.016 47 0.000 48 0.000 40 0.010 46 0.020 55 0.035 50 0.055 51 0.015 54 0.010 57 0.030 52 0.020 53 0.012 50 0.026 56 0.050 61 0.105 58 0.090 63 0.015 60 0.010 65 0.066 62 0.210 * Stage D 2 . these values, we find that the frequency of occurrence of easily observable amounts of lipochrome in the blood is not significantly different from 1 in 10 animals for the C 4 stage in samples 1 and 3, but is significantly different, at the 10% level of probability or better, for all the other groups. The 1:10 ratio observed in C,, sample 1, is also significantly different from the 1:2 ratio observed in D lt sample o. The mobilization of lipochromes from the digestive gland to the integumentary tissues is an important part of the preparation for the moult, and all of the D, animals in this study showed deposits of pigment in the region of the membranous layer of the integument; indeed, this characteristic appears to be a fairly reliable means of detecting the beginning of the premoult period. The appearance of lipo- chrome in substantial amounts in the blood may therefore be taken as an indication of the beginning of preparations for moulting. It is clear from the results presented that this mobilization begins before the first morphological signs of premoult (initia- 178 BRADLEY T. SCHEER tion of new setae) appear. Moreover, we may conclude that the C 4 animals in the second sample were further advanced towards the premoult stage than were those in the first or third samples. There is no conclusive evidence that eyestalk removal has any effect on the mobilization of lipochromes. The results of the carbohydrate determinations are presented in Table II. We may first note the rather striking difference in carbohydrate content of normal animals in stage Q between sample 2 and the other two samples. The mean values TABLE II Total carbohydrate and polysaccharide content (mg. glucose equivalent per gm. body weight] of three samples from a population of Carcinides maenas, and the effect of eyestalk removal C 4 D, Stage Normal Eyestalkless Normal Eyestalkless condition No. Carb. Poly- No. Carb. Poly- No. Carb. Poly- No. Carb. Poly- sac. sac. sac. sac. Sample 1 7 2.14 1.31 6 5.64 4.04 11 3.63 3.00 13 3.34 2.06 11-19 days 8 0.59 0.24 9 9.50 7.48 19 2.93 1.85 18 6.48 5.42 12 2.38 1.70 10 8.59 7.67 20 6.10 5.24 21 6.19 5.05 15 2.18 1.12 14 13.5 11.0 16 3.03 1.96 17 4.52 4.01 Sample 2 22 8.88 8.48 24 9.45 9.20 31 4.83 4.83 35 15.7 13.9 43-45 days 25 12.7 8.57 27 9.53 9.53 32 16.4 16.4 41 5.45 5.25 26 1.13 1.13 29 7.40 7.32 34 8.84 8.52 43 14.4 12.8 28 15.7 14.8 30 12.8 12.3 40 14.4 13.7 23* 15.0 15.0 36 12.2 11.6 33 16.5 15.8 38* 14.8 14.2 42* 23.6 10.9 37 3.18 3.18 39 19.3 16.8 Sample 3 45 1.36 1.36 44 8.13 8.13 47 1.45 1.45 48 1.72 1.41 67-70 days 49 2.73 2.21 46 9.40 7.06 55 9.41 8.48 50 3.52 2.98 51 4.10 4.10 54 6.11 5.03 57 16.0 15.2 52 4.90 4.90 S3 2.28 1.88 59 12.8 11.1 56 12.7 11.0 61 24.4 21.6 58 11.4 11.4 63 15.7 11.9 60 2.49 1.95 65 10.7 9.53 62 10.6 8.33 64 18.9 16.3 * Stage D 2 . for samples 1 and 3 are 2.06 and 2.62 mg. per gm. for total carbohydrate, while the corresponding mean for sample 2 is 10.12 mg. per gm. The difference between the means for sample 1 and 2 is significant at the 5% level on the basis of the t test. This difference in means arises from the fact that all but one of the values from sample 2 are greater than 8 mg. per gm., while none of the values from samples 1 and 3 is as great as 5 mg. per gm. Moreover, the single low value in sample 2 was obtained from one of the animals (no. 26) which had no obvious lipochrome in the blood. If our earlier conclusion, that a substantial fraction of the animals in stage C 4 of the second sample were well on their way toward stage D lf is correct, then we can further conclude that one characteristic of this transition is a marked increase in HORMONAL CONTROL OF METABOLISM the carbohydrate content of the body. This conclusion is confirmed by the values, for normal animals in stage D a , which are nearly all well above those for the C 4 animals of groups 1 and 3. The difference between mean values for sample 3 for C 4 and D 1 is significant at the 1% level on the basis of the t test. Renaud (1949) had already observed a similar change in Cancer pagunts with a mean glycogen content of 2.09 mg. per gm. for animals in C 4 . rising to 4.43 mg. per gm. by the end of Dj. We may therefore conclude that the increase in carbohydrate content which is characteristic of the transition from intermoult to premoult may occur during the latter part of stage C 4 , before any morphological evidence of the transition is apparent. TABLE III Non-protein nitrogen (NPN) and ammonia nitrogen (NH 3 N) in normal and eyestalkless Carcinides maenas (nig. per gm. body weight) C4 Di Stage condition Normal Eyestalkless Normal Eyestalkless No. NPN NH 3 N No. NPN NH 3 N No. NPN NH 3 N No. NPN NH 3 N Sample 1 8 2.40 0.25 6 2.45 0.10 11 2.44 0.13 10 2.56 0.10 12 2.89 0.17 9 2.52 0.08 19 2.84 0.05 13 3.07 0.12 15 2.88 0.20 14 3.58 0.08 20 3.39 0.06 18 3.08 0.05 16 2.58 0.07 17 2.65 0.08 21 3.82 0.04 Sample 2 22 2.90 0.16 24 2.68 0.24 31 2.91 0.19 35 2.96 0.30 25 3.21 0.20 27 3.96 0.24 32 3.97 0.28 41 3.17 0.29 26 2.03 0.13 29 3.22 0.23 34 3.13 0.24 43 3.30 0.25 28 4.74 0.23 30 3.51 0.26 40 3.48 0.28 23* 3.17 0.32 36 2.82 0.25 33 4.07 0.27 42* 3.69 0.42 37 2.72 0.19 38* 3.03 0.26 39 2.64 0.27 * Stage D... The second item to be noted from Table II is the fact that the carbohydrate content of the eyestalkless animals in C 4 is throughout at levels characteristic of D, animals. Indeed, there was no eyestalkless C 4 animal with a carbohydrate content as low as 3 mg. per gm., and in all but two, the value was higher than 5 mg. per gm. The differences in means for normal and eyestalkless animals in C 4 were significant at the 5% level for both samples 1 and 3, on the basis of the t test. We may there- fore conclude that the operation of eyestalk removal causes an increase in carbohy- drate content from the low values characteristic of C 4 animals to the higher values characteristic of the next stage in the cycle, D 1 . The same operation is clearly without effect upon animals already in stage D t if for some reason these animals have low carbohydrate content, since there are several eyestalkless D l animals with rela- tively low carbohydrate values, and the distribution of values in normal and eye- stalkless specimens in this stage is substantially the same. We may further infer from our results, though conclusive evidence is lacking, that some endocrine factor is secreted in the eyestalk during stage C 4 , and that secretion of this factor stops towards the end of that stage. One effect of this factor would be the maintenance 180 BRADLEY T. SCHEER of carbohydrate content at relatively low levels. Since Renaud (1949) has shown a steady increase in glycogen content beginning in stage C,, we may suppose that the secretion of the factor concerned decreases gradually rather than suddenly. In general, the polysaccharide values follow the carbohydrate values rather closely, and 80% or more of the carbohydrate is precipitated by alcohol. However, in the C 4 animals of the first sample, the polysaccharide averages only 62 % of the total carbohydrate ; the eyestalkless individuals, and indeed all of the other groups, had a higher ratio. Blood carbohydrate was measured for the animals of sample 1 only. The results are presented in Table III. Since it appears that the carbohy- drate content of the blood does not reflect changes in the total carbohydrate of the body, and is not influenced by any of the other factors considered here, we utilized the blood samples from the second and third group for lipochrome studies. The observation of Needham (1955) that increased nitrogen excretion follows eyestalk amputation, led us to examine the nitrogen content of some of the extracts. The results are presented in Table IV. There appears to be no systematic variation in either NPN or NH 3 N, except that both sets of values, and especially the NH.,N values, are generally lower in the animals of sample 1 than in those of sample 2. No obvious explanation for this difference appears. In both samples, the extracts were prepared 7 to 14 days after eyestalk removal, by which time Needham (1955) found that nitrogen excretion had returned to normal levels. We conclude that no long- lasting modification in nitrogen metabolism evident from NPN or NH 3 N content of the animals is related to the variables considered here. DISCUSSION Perhaps the most important finding of this study is that metabolic changes (mobilization of lipochromes, increased carbohydrate content) preparatory to the moult precede in time the morphological changes (formation of new setae). This may not be surprising, but it has not been emphasized before. We cannot on the basis of the evidence available conclude that the metabolic changes are causally related to the subsequent structural changes, but this is a reasonable inference. However, the two metabolic changes observed do not seem to be directly related one to the other. There is in general no complete correlation between increased blood lipo- chrome and increased carbohydrate. Moreover, the increase in carbohydrate which follows eyestalk removal is not in general associated with increased blood lipochrome. The increase in carbohydrate content as the animal approaches a moult was al- ready known from the study of Renaud (1949) on Cancer pagurus. Moreover, Schwabe et al. (1952) had observed a marked increase in total glycogen, represented by deposition in the digestive gland and epidermis, in the transition to the premoult stage in spiny lobsters ; their data also suggest that eyestalk removal in stage C increases the total glycogen of the body, while the same operation in stage D re- sults in no change. However, they did not determine this quantity directly, and neither of their methods, for determination of glycogen or for determining intermoult cycle stage, was entirely satisfactory. The demonstration of an increased carbohy- drate content following eyestalk removal therefore comes as a definite addition to the long list of metabolic and other changes which are consequences of this operation (Knowles and Carlisle, 1956; Scheer, 1957). The absence of any changes in blood carbohydrate was something of a surprise. HORMONAL CONTROL OF METABOLISM 181 Renaud (1949) found a steady increase in the reducing power of the blood from C 3 through D 1 in Cancer payurus, but this increase was not evident when the blood was treated with cadmium sulfate and sodium hydroxide, a procedure supposed to eliminate non-glucose reducing substances. Recent studies in my laboratory by McWhinnie (unpublished) on the blood of Hciniyrupsus mains have shown that the blood carbohydrate, like the total carbohydrate in acid extracts of the body, in- cludes several components, of which glucose is a relatively minor one. Using the highly specific hexokinase glucose-6-phosphate dehydrogenase method, she found glucose concentrations averaging below 2 mg. per 100 ml., with a maximum about 2.5 mg ( /c in stage Q, and a slight decrease in stage C 3 , but no change as a result of eyestalk extirpation. We had earlier found a decrease in the reducing substances of spiny lobster blood (Schee^and Scheer, 1951) following eyestalk removal, but others (Abramowitz ct al., 1944; Kleinholz and Little, 1949) found no such change in crabs. It is clear that the problem of blood sugar regulation in crustaceans re- quires further careful study with particular attention to specificity of methods. The question now arises, what is the source of the increased carbohydrate in late intermoult, and what alterations in metabolism are responsible for the increase. Related to this is the question of the endocrine factors which we presume to be re- sponsible for the increase. The evidence on which we can base hypotheses re- mains fragmentary. Injection of eyestalk extracts increases blood reducing sub- stances, and specifically the fermentable reducing substances (Abramowitz et al., 1944; Kleinholz and Little, 1949; Scheer and Scheer, 1951). It would be unwise at present to equate fermentable reducing substances with glucose, and we do not know the metabolic relations among the various carbohydrates found in the blood, nor indeed the identity of these substances. Scheer and Scheer (1951) showed that injected glucose was removed from the blood more rapidly in eyestalkless than in normal spiny lobsters, and that most of the carbon of this glucose could be recovered in the water- and alcohol-soluble fraction of tissue extracts. From the work of Hu (1958) we know that this fraction may contain, besides glucose, several oligosac- charides of the maltose series. But the relation of these substances to synthesis of polysaccharides or other aspects of carbohydrate metabolism remains obscure. Present evidence, from plants and animals alike, indicates that glycoside linkages in general are formed by adding one monosaccharide unit at a time to existing nuclei by the agency of nucleotide coenzymes. Hu ( 1958) has shown that nucleo- tides are present in crabs, and that carbon from administered glucose appears in these compounds. Whatever the intermediate steps, the increased carbohydrate content of late inter- moult crabs may be derived ultimately either from protein or carbohydrate or both. The evidence that, in fed crabs, there is no change in the non-protein nitrogen, suggests that there is no fundamental alteration in the intensity of protein metabo- lism. On the other hand, the evidence of Neiland and Scheer ( 1953) that, in fasting crabs, protein is used in preference to carbohydrate, and in eyestalkless crabs, the amount of protein used is greater, together with the evidence of Needham (1955) that under conditions (trauma) in which protein breakdown is increased eyestalk removal leads to a further increase, suggest that, in eyestalkless animals there may be an increased conversion of protein to carbohydrate. The fact that, in such animals, the utilization of glucose is also increased (Scheer and Scheer, 1951) 182 BRADLEY T. SCHEER would lead one to place the site of the presumed endocrine effect in the process of glycogenesis, rather than in that of gluconeogenesis. We may, therefore, postulate that, during the C 4 stage, there is a gradual transition from carbohydrate oxidation to polysaccharide synthesis as a major pathway of carbohydrate metabolism. This offers a possible explanation for the difference in glucose oxidation observed by Hu (1958) and Scheer and Scheer (1951) using similar procedures with different animals. The crabs used by Hu may have been in the early part of stage C 4 or even in C 3 , when carbohydrate oxidation is dominant ; the spiny lobsters used by Scheer and Scheer (1951) may have entered into the phase in which carbohydrate synthesis predominates. Neither author determined the intermoult stage with great accuracy. Or, if we accept the view of Carlisle (Knowles and Carlisle, 1956) that the intermoult cycle is qualitatively different in animals with prolonged inter- moults (diecdysis, as in crabs in winter) from the intermoult in animals which moult regularly throughout the year (anecdysis, as in Hawaiian spiny lobsters), we might suppose that carbohydrate oxidation is primarily characteristic of diecdysis and that in anecdysis polysaccharide synthesis predominates. These suggestions can be tested by careful comparison of the fate of carbon from administered labeled glucose in the various stages of moult cycles of both types. The question of hormonal control is likewise a difficult one. Carlisle (Knowles and Carlisle, 1956) has summarized evidence suggesting that two separate hormones are concerned in the control of the intermoult period. Carlisle attributes diecdysis to the action of the well-known moult-inhibiting hormone, and cites his own ob- servations and some of ours (Scheer and Scheer, 1954) to the effect that this hor- mone is not active in certain crustaceans, including British populations of Carcinides maenas in the summer. We have no way of knowing whether the animals studied in the present investigation undergo a cycle of the type characterized by anecdysis, or one characterized by diecdysis. It would appear that, to make much further progress with these problems, it will be essential to work with at least partly purified hormone preparations, and to have full information about the type of cycle and stage of the animals in the cycle. We earlier postulated ('Scheer and Scheer, 1951) an eyestalk factor which restrains carbohydrate utilization for polysaccharide, and specifically chitin, synthesis. This factor would be the same as the "diabetogenic" factor of Abramowitz ct al. (1944), and we suggested that it might also be the moult-inhibiting factor. The results reported here do not give us any reason to alter this hypothesis, nor do they substantially strengthen it. However, at present it seems best to consider that the effects of eyestalk removal noted by Neiland and Scheer (1953) on protein catabolism, and the related effect noted by Needham (1955), might result from the action of this same factor. For conclusive evidence concerning this hypothesis, it is essential to have a hormone preparation of reason- able purity which can be tested for its specific metabolic effects. This work was done during tenure of a John Simon Guggenheim Memorial Fellowship. The author wishes to thank the Fellowship Board for the grant which made this work possible, and especially to express his appreciation of the generous provision of materials and facilities for this work at Laboratoire Arago, and of the many kindnesses and the helpful assistance of the director. Prof. G. Petit, and his staff. HORMONAL CONTROL OF METABOLISM 183 SUMMARY 1. A laboratory population of Carcinides maenas was sampled three times over a period of 70 days, and the blood carbohydrate, blood lipochromes, total body carbohydrate, total body polysaccharide, non-protein nitrogen, and ammonia nitrogen were determined ; the effect of eyestalk removal on these quantities was also examined. 2. During the course of the observations, there was a progression within the population from late intermoult (C 4 ) to early premoult (D) stages. 3. The change from intermoult to premoult was signalized by the appearance of relatively large amounts of lipochromes in the blood and integument, and by an in- crease in total body carbohydrate content. These biochemical changes preceded any morphological signs of preparation for moult. 4. Eyestalk extirpation caused an increase in the body carbohydrate, but did not alter the blood lipochromes. The increase in carbohydrate was observed only in those animals which had not undergone the change spontaneously. 5. The other quantities measured showed no variation attributable either to the stage in the intermoult cycle or to eyestalk removal. 6. The results are discussed with relation to the possible mechanisms of the effects observed, and the hormonal factors concerned. LITERATURE CITED ABRAMOWITZ, A. A., F. L. HISAW AND D. N. PAPAXDREA, 1944. The occurrence of a diabeto- genic factor in the eyestalks of crustaceans. Biol. Bull., 86: 1-5. DRACH, P., 1939. Mue et cycle d'intermue chez les crustaces decapodes. Ann. hist. Occanogr. Paris, 19: 103-391. HILLER, A., J. PLAZIN AND D. D. VAN SLYKE, 1948. A study of conditions for Kjeldahl de- termination of nitrogen in proteins. J. Biol. Chcm., 176: 1401-1420. Hu, A. S. L., 1958. Arch. Biochem. Biophys. (in press). KLEINHOLZ, L. H., AND B. C. LITTLE, 1949. Studies in the regulation of blood sugar concen- trations in crustaceans I. Normal values and hyperglycemia in Libinia emarginata. Biol. Bull, 96: 218-227. KNOWLES, F. G. W., AND D. B. CARLISLE, 1956. Endocrine control in the Crustacea. Biol. Rev., 31 : 396-473. NEEDHAM, A. E., 1955. Nitrogen-excretion in Carcinides maenas (Pennant) during the early stages of regeneration. /. Embryol. Exp. Morphol., 3 : 189-212. NEILAND, K. A., AND B. T. SCHEER, 1953. The influence of fasting and sinus gland removal on body composition of Hcmigrapsus midus. Part V of the hormonal regulation of metabolism in crustaceans. Physiol. Comp. Occol., 4 : 321-326. RENAUD, L., 1949. Le cycle des reserves organiques chez les crustaces decapodes. Ann. Inst. Oceanogr. Paris, 24: 259-357. ROE, J. H., 1955. The determination of sugar in blood and spinal fluid with anthrone reagent. /. Biol. Chem,, 212 : 335-343. SCHEER, B. T., 1957. Recent Advances in Invertebrate Physiology, pp. 213-227. Univ. of Oregon, Eugene. SCHEER, B. T., AND M. A. R. SCHEER, 1951. Blood sugar in spiny lobsters. Part I of the hormonal regulation of metabolism in crustaceans. Physiol. Comp. Oecol, 2 : 198-209. SCHEER, B. T., AND M. A. R. SCHEER, 1954. The hormonal control of metabolism in crus- taceans VII. Moulting and colour change in the prawn Lcandcr serratus. Pubbl. Stas. Zool. Napoli, 25 : 397-418. .SCHWABE, C. W., B. T. SCHEER AND M. A. R. SCHEER, 1952. The molt cycle in Pamtlirus japonicits. Part II of the hormonal regulation of metabolism in crustaceans. Physiol. Comp. Oecol, 2 : 310-320. THE LIFE-CYCLE OF THE DIGENETIC TREMATODE, PROCTOECES MACULATUS (LOOSS. 1901) ODHNER, 1911 fSYN. P. SUBTENUIS (LINTON, 1907) HANSON, 1950], AND DESCRIPTION OF CERCARIA ADRANOCERCA N. SP. HORACE W. STUNKARD 1 AND JOSEPH R. UZMANX U. S. Fish and ITildlifc Service The genus Proctocccs was erected by Odhner (1911) to contain Distomum maculatum Looss, 1901, from Labrus memla and Crenilabrus spp. at Triest. Odhner had found the parasite in Blennius ocellaris at Naples. One adult specimen from Chrysophrys bifasciata and two immature specimens from lulis lunaris taken in the Red-Sea, were described as a new species, Proctoeces erythraeus. Dawes (1946) listed P. erythraeus as a synonym of P. maculatus (Looss), but the species was recognized by Manter (1947) on the basis of six specimens he had collected from Calamus calamus and Calamus bajonado at the biological laboratory of the Carnegie Institution at Dry Tortugas, Florida. Several additional species have been de- scribed. Fujita (1925) reported a metacercaria from the Japanese oyster, Ostrea gigas, as a new species, Proctoeces ostreae. The paper was translated by R. Ph. Dollfus who noted (p. 57), "II est a souhaiter que des recherches chez les poissons mangers de Lamellibranches, sur les cotes de la prefecture d' Hiroshima, permettent de decouvrir des exemplaires completement adultes de Proctoeces ostreae Fuj., chez lesquels 1'extension des vitellogenes et les dimensions des oeufs puissent etre observees avec precision ; il sera alors possible de savoir definitivement si P. ostreae Fuj. doit ou non tomber en synonymic avec P. maculatns (Looss)." Yamaguti (1934) described P. maculatns from Spams arics, Spams macrocephalus, Pagroso- mus auratus, and Epinephelus akaara in Japan. Several specimens from Pagroso- mus auratus, which differed from P. maculatus in larger size, larger eggs, and trilobed ovary, he described as a new species, Proctoeces major. Yamaguti (1938) reported P. maculatus from Semicossyphus reticiilatus and described a larva from the liver of the pelecypod mollusk, Brachidontes senhausi, as an unidentified member of the genus Proctoeces. Manter (1940) described Proctoeces magnorus from a single specimen found in the intestine of Caulolatilus anomalus, taken at Cerros Island, Mexico. Hanson (1950) identified two specimens collected from Calamus sp. at Bermuda by the late F. D. Barker as Distomum subtenue Linton, 1907, a species described originally from Calamus calamus in the same area. Comparison of these specimens with those from Tortugas identified by Manter as P. erythraeus estab- lished their identity, and P. erythraeus was suppressed as a synonym of Proctoeces subtenue (Linton, 1907). Hanson corrected the statement of Manter (1947), noting that it is the vitellaria, not the uterus, which never extends into the post- testicular region. Yamaguti (1953) predicated that Xenopera Nicoll, 1915 is a 1 Mailing address : American Museum of Natural History, Central Park West at 79th Street, New York 24, N. Y. 184 LIFE-CYCLE OF PROCTOECES 185 synonym of Proctoeces, and Xcnopcra insolitus from S pants australis was listed as Proctoeces insolitus (Nicoll, 1915). Winter (1954) described Proctoeces mac- rovitellus from the intestine of Cyinatogaster aggregatits, taken off the coast of southern California. It is notable that the final hosts of these trematodes are porgies and labroid fishes of temperate and warm seas ; hard-mouthed, bottom forms that feed on mollusks. Uzmann (1953) described Cercaria miljordensis, a microcercous trematode larva from Mytilus edulis in both intertidal and subtidal areas of Long Island Sound and along the cost of Connecticut. About seven per cent of the mussels were infected in the years 1951 and 1952. Although the infection was heavy in the area around Milford, Connecticut, Uzmann noted that the parasite had not been reported from higher latitudes despite intensive study of M. edulis over a period of many years. The sporocysts develop in the venous sinuses of the mussel, beginning in the late fall and continuing during the winter, with the release of the cercariae in greatest numbers in the late winter and spring. The infection largely destroys the gonad of the host and development of the sporocysts precludes normal gametogenesis. The intensity of the infection seriously impairs the vitality of the mollusk and may be lethal under temporary or sustained periods of ecological conditions unfavorable to the host. Uzmann described the behavior of the cercariae and reported un- encysted progenetic larvae referable to the genus Proctoeces in mussels harboring C. miljordensis infections. He stated (p. 449), "Morphological comparison of the two forms is favorable, and if the apparent relationship truly exists, an abbreviated life-cycle may be possible since the larval Proctoeces contain many eggs with well developed, motile miracidia. Experimental studies are projected and it is hoped that decisive information can be presented at a later date." Shortly thereafter, Uzmann was transferred to the Seattle, Washington Laboratory of the U. S. Fish and Wildlife Service. Further significant information was provided by the work of Hopkins (1954) who described infection of the hooked mussel, Brachidontcs recurvus (syn. Mytilus recuri'its} taken in Barataria Bay, Louisiana by Cercaria brachidontis n. sp., a species so similar morphologically to C. miljordensis that their relationship was im- mediately apparent. Cercaria brachidontis develops in orange-pigmented sporocysts which completely destroy the gonad of the mussel. Immature cercariae have small, knob-like tails, similar to those of C. miljordensis, but they are not present on fully developed larvae. Hopkins referred the species to the family Fellodistomatidae but without generic designation. After the text of this paper was written, the account by Freeman and Llewellyn (1958) appeared, announcing the discovery of the adult stage of a digenetic trema- tode in the renal organs of the lamellibranch mollusk, Scrobicularia plana taken from the mud-flats of the Thames estuary, at Chalkwell in Essex and Whitstable in Kent. The worms were identified as Proctoeces subtenuis (Linton, 1907) Hanson, 1950, a species which was known previously only as a parasite of the hind-gut of marine fishes belonging to the families Labridae and Sparidae, which occur chiefly in tropical and subtropical seas. The asexual generations were not discovered and since the adult stages had not been recorded from fishes of the English coast, the authors concluded that in British waters the life cycle had been abbreviated and restricted to invertebrate hosts. Possible methods were considered by which the parasite had been introduced. They reported (p. 446) that, "The eggs are enclosed 186 HORACE W. STUNKARD AND JOSEPH R. UZMANN w -J O. LIFE-CYCLE OF PROCTOECES 187 in a thin, light-brown capsule." This statement appears confusing, since the "cap- sule" is obviously the egg-shell and an egg comprises the shell and its contents, ovum, embryo, or miracidium. Although many eggs contained active miracidia, they varied much in size (from 0.026-0.073 by 0.015-0.030 mm.). The use of his- tochemical techniques disclosed the presence in the vitellaria of dihydroxy-phenols and protein, which on oxidation combine to form the quinones of the egg-shell, but the corresponding phenol-oxidase was not demonstrated. Deficiencies in the egg- making apparatus may account for the small and abnormal eggs. Freeman and Llewellyn gave a detailed account of the morphology of the parasite and noted the extent of individual variation. They stated (p. 447), "Several hundred specimens were examined, and it is apparent that many of the characters thought to indicate specific differences probably represent intraspecific variations of the kind emphasized by Stunkard (1957)." As a result of their investigation, P. erythraeiis Odhner, 1911 and P. inagnonts Manter, 1940 were suppressed as synonyms of P. subtcnuis. Furthermore, they stated (p. 455) that, "The differences between P. insolitus (Nicoll, 1915) and P. subteniiis, and between P. maculatiis (Looss, 1901) and P. subtcnuis, require reexamination." The findings of Freeman and Llewellyn amply confirm the postulate of Uzmann (1953) and constitute an important contribution to knowledge of the biology of the digenetic trematodes. The studies begun by Uzmann at Milford were continued at Woods Hole, Massachusetts by the appointment of Stunkard to investigate the parasites of clams and their predators in New England. Infections by C. milfordcnsis were found in M. cdnlis taken in the Woods Hole area, although the incidence of infection was low, about 0.5 per cent. However, the findings of developmental stages, from cercariae to adults, confirmed the prescience of Uzmann that C. inilfordcnsis is the larval stage of a species of Proctoeces. DESCRIPTIONS Adults. (Figs. 3. 4) The general morphology of the worms is portrayed in the figures. The cuticula is unarmed ; the suckers large and powerful. The digestive tract shows no unusual features. The excretory vesicle bifurcates at the level of the posterior testis ; both the stem and crura are lined with a simple epithelium which is flattened when the wall is distended. The flame-cell pattern of the adult worm was not studied. The genital pore is lateral, situated usually between the acetabulum and the pharynx. The testes are diagonally tandem, either adjacent or somewhat separated. Sperm ducts arise at the anterior ends, pass forward and join to form a common duct just before entering the cirrus sac. In the posterior end of the cirrus sac it forms a coiled seminal vesicle, filled with spermatozoa, and then opens into a straight, thick- walled muscular canal. This structure is lined with high, secretory cells, whose distal PLATE I Drawings of P. macitlatits from M. edulls; made from fixed and stained specimens and at the same magnification. FIGURE 1. Juvenile specimen; length 1.20 mm. FIGURE 2. Specimen just reaching sexual maturity, 6 eggs in uterus ; length 2.00 mm. FIGURE 3. Gravid specimen, eggs small and mostly misshapen ; length 2.65 mm. FIGURE 4. Gravid specimen, eggs normal with developing miracidia, length 2.62 mm. 188 HORACE W. STUNKARD AND JOSEPH R. UZMANN PLATE II LIFE-CYCLE OF PROCTOECES 189 ends are filled with chromatic granules, and terminates in the cirrus which protrudes into a long, hermaphroditic atrial duct. The area between the wall of the cirrus sac and the thick-walled canal is filled with secretory cells, whose ducts pierce the thick wall and discharge into the narrow lumen. The ovary is pretesticular, in the anterior part of the posterior one-half of the body. The oviduct arises at its posterior face and turns ventrad and mediad where it expands into a fertilization space, from which Laurer's canal emerges and continues dorsad and anteriad to open at the surface above the ovary. The oviduct then enters Mehlis' gland where it receives the common vitelline duct and expands into the ootype, where the egg is formed. The uterus passes posteriad to the end of the body where coiled loops on either side are followed by a median trunk which passes forward below the cirrus sac to open into the ventral side of the hermaphroditic duct, six to ten microns before the genital pore. In many of the specimens the eggs are malformed, of varying sizes, often about one-third as large as in more normal individuals. Average measurements in millimeters of ten gravid, mounted specimens ; limits in parentheses: length, 2.74 (2.4-3.2); width, 0.81 (0.6-0.92); acetabulum, 0.38 X 0.43 (0.35-0.46) ; oral sucker, 0.24 )< 0.30 (0.21-0.32) ; pharynx, 0.18 (0.16- 0.20) ; ovary, 0.19 (0.16-0.22) ; anterior testis, 0.18 (0.15-0.20) ; 'posterior testis, 0.19 (0.16-0.23) ; eggs, 0.055 X 0.026 (see text). Juveniles. (Figs. 1,2) Figure 2 shows a specimen just reaching maturity, which has 6 eggs in the uterus. It is somewhat flattened as a result of pressure during fixation. Measure- ments in millimeters are: length, 2.00; width, 0.80; acetabulum, 0.34x0.37; oral sucker, 0.18x0.22; pharynx, 0.125x0.150; ovary, 0.15x0.14; anterior testis, 0.17 X 0.15; posterior testis, same size. Figure 1 shows a smaller and less mature specimen, also flattened during fixa- tion. The acetabulum is almost exactly in the middle of the body ; the post-acetabular region increases relatively in size with the development of the reproductive organs. Measurements are: length, 1.2; width, 0.56; acetabulum, 0.21 X 0.275; oral sucker, 0.128 X 0.15 ; pharynx, 0.125 X 0.125 ; ovary, 0.057 ; anterior testis, 0.079 < 0.072 ; posterior testis, 0.092 X 0.079. Sporocysts and Cercariae. (Figs. 5, 6, 7, 8) Descriptions of the sporocysts and cercariae were given by Uzmann (1953). His observations have been confirmed and additional data are presented. There are at least three generations in the mollusk. Figure 5 shows a sporocyst with two PLATE II FIGURE 5. P. inacit/atits, mother sporocyst with daughter sporocysts containing germinal cells of the next generation ; length 0.34 mm. FIGURE 6. P. macidatns, daughter sporocyst with developing cercariae; length 0.54 mm. FIGURE 7. P. macitlatus, large daughter sporocyst with developing cercariae, Cercaria milfordcnsis, length 1.18 mm. FIGURE 8. P. maculatus, cercaria, from stained and mounted specimen, excretory system added from sketches of living worms ; length 0.26 mm. FIGURE 9. Cercaria adranoccrca n. sp., daughter sporocyst from G. gemma; length 0.48 mm. This drawing is at the same magnification as Fig. 7. FIGURE 10. Cercaria adranocerca n. sp., stained and mounted specimen, excretory system .added from sketches of living worms ; length 0.21 mm. 190 HORACE W. STUNKARD AND JOSEPH R. UZMANN daughter sporocysts and in each of them there are heavily staining germinal cells of the next generation. The cercariae are subcylindrical and taper toward anterior and posterior ends. In addition to the three pairs of cephalic gland ducts reported by Uzmann, two additional pairs are sometimes visible ; the cell bodies are lateral, in the preacetabular area, but so far it has been impossible to demonstrate them with certainty, either by the use of vital dyes or in permanent preparations. The flame- cell pattern has been worked out and is shown in Figure 8. On either side a duct, which contains patches of cilia, emerges from the excretory vesicle near its anterior end ; it passes forward, loops backward and here receives the two collecting ducts. The anterior one of these ducts receives the fluid from four flame-cells and capillaries located in the anterior quadrant of the body; the posterior one from flame-cells and capillaries in the posterior quadrant. The flame-cell formula is 2\(2 + 2) + (2 + 2)]. Cercaria adranocerca n. sp. (Figs. 9, 10) In addition to the specimens from M. edulis, described above, dissection of some three hundred Gemma gemma from the region of Boothbay Harbor, Maine, in Au- gust and September 1957, disclosed two infections by sporocysts and microcercous cercariae, similar to those from M. edulis and from Brachidontcs rccuruus. The sporocysts were relatively few, oval to sausage-shaped ; the largest one measured 0.42 mm. long and 0.11 mm. wide after fixation while smaller ones, no larger than a cercaria, contained a few germ-balls. The end that bears the birth-pore may be extended as a long, tapering protrusion. The cercariae were studied alive, unstained and after staining lightly with Nile blue sulphate and with neutral red ; also after fixation as stained and cleared per- manent mounts. Frequently, one adhered to the slide by the posterior end and extended the body in all directions. The body is subcylindrical, typically more rounded anteriorly than posteriorly. When extended, contraction of the circular muscles may produce an annulate appearance. The body wall is relatively strong for so small a larva. The cuticula bears rows of closely set, flattened spines. Ducts of cephalic glands were sometimes visible in the region of the oral sucker, but their number and the location of the cell bodies were not determined. Alive, cercariae measured from 0.16 to 0.33 mm. in length and 0.044 to 0.09 mm. in width. The tail is terminal, spherical, and measures 0.01 mm. in diameter; it is easily detached. The oral opening is subterminal, the sucker is 0.032 to 0.043 mm. in diameter. When the anterior end of the body is extended, the prepharynx is about one-half the length of the pharynx which measures 0.014 to 0.018 mm. in diameter. The ceca are relatively long, extending about midway between the acetabulum and the posterior end of the body. The acetabulum is situated in the posterior portion of the anterior half of the body and protrudes, although it is not stalked. It measures 0.025 to 0.036 mm. in diameter: the ratio in size between the oral and ventral suckers is about 4:3; although the size increased greatly over the figures given above when the cercaria was subjected to extreme pressure for the study of the excretory system. The region between the excretory vesicle and the acetabulum contains cells which stain deeply ; they are the rudiments of the reproductive organs. The excretory pore is terminal, at the base of the tail ; the vesicle is oval and may extend forward more than half the distance to the acetabulum. On either side, from the anterior end of the vesicle, a collecting duct passes forward to the level of the LIFE-CYCLE OF PROCTOECES 191 bifurcation of the digestive tract where it turns backward ; the recurrent portion contains tufts of cilia and near the level of the acetabulum it receives anterior and posterior collecting tubules. The arrangement is portrayed in the figure and the flame-cell formula is 2 [(2 + 2) + (2 + 2)]. In flame-cell formula and vestigial tail, this species is similar to Cercaria mil- ford ensis and Cercaria brachidontis. In form of the excretory vesicle and presence of cuticular spines it more closely resembles C. brachidontis and although neither can be included in the genus Proctoeces, it is probable that they are larvae of some member of the family Fellodistomidae. The species, described as ne\v in this paper, is designated Cercaria adranocerca (adrano, inactive, feeble). Type and paratype specimens are deposited in the U. S. Nat. Museum. Helminth. Collection, No. 56236. DISCUSSION The discover}'- of unencysted metacercariae and of developmental stages from cercariae to gravid adults demonstrates that C. miljordens'is is the larval stage of a species of Proctoeces. However, the progenetic worms are often not entirely normal. In some of the specimens (Fig. 3). the eggs are misshapen and of varying sizes, often not more than one-third as large as in other individuals. A similar situation was reported by Freeman and Llewellyn (1958). It appears that the female organs, especially the vitellaria, may be deficient or that the ova are not fertilized, and such abnormal eggs do not contain miracidial larvae. For this reason, the extent and development of the vitelline follicles may not provide sound data for specific criteria. Identification of these specimens presents disturbing problems. The descrip- tion of P. maculatus by Looss ( 1901 ) is based on the largest of his specimens and is illustrated by a good figure. The characterization of P. crythraeus was very inadequate ; Odhner gave no figure or measurements and the species was distin- guished from P. maculatus because in the single mature specimen the acetabulum was one-third smaller, the eggs were smaller, and the vitellaria did not extend as far posteriad, a condition which might be expected in a specimen just reaching maturity. For this reason, Dawes (1946) suppressed P. er\thraeus as a synonym of P. maculatHs. The six specimens taken by him from Calamus spp. at Tortugas agreed with Odhner 's account and Manter (1947) recognized P. erythraeus as a valid species, but there was no figure and as yet there is no complete description of P. crythraeus. In the (1947) paper. Manter stated that his (1940) listing of Proctoeces and Tergestia in the family Monorchidae was an error, since the family name, Fellodistomatidae, was accidentally omitted. In a report on parasites of Bermuda fishes, Linton (1907) published the descrip- tion of a new species, Distomum subtenue, from Calamus calamus. Smaller, im- mature specimens were found in other hosts, two in Iridio bivittatus, and one each in Harpe rufa and Lachnolaimus ma.rimus. Although two small specimens are reported on p. 106 from H '. rufa, the table on p. 87 shows that only one trematode was found in this host. In the "Food notes" on the fishes, which accompanied his account of their parasites, Linton stated that C. calamus feeds on mussels and crabs ; the others on mollusks, crabs, sea urchins and annelids. All are bottom feeders and Breder (1929), in describing these fishes, stated that the mouths of porgies (C. calamus is the saucer-eye porgy) are (p. 180), "armed with strong jaw teeth,"' 192 HORACE W. STUNKARD AND JOSEPH R. UZMANN and that the memhers of the Labridae are (p. 202), "usually provided with strong canine teeth. . . . These fishes are provided with powerful pharyngeal teeth with which they crush mollusks." Comparison of Linton's description and figure of Distomum subtcnue with the two specimens from Calamus sp. taken at Bermuda by Barker and the six specimens taken from Calamus spp. at Tortugas by Manter, led Hanson to the conviction that all were conspecific and accordingly she (1950) announced the specific identity of Dist. subtcnue Linton, 1907 and P. erythracus Odhner, 1911. The species was designated Proctocccs subtcnuc (Linton, 1907). Again, there was only a scanty description and no figure. Manter (1954) identified five specimens from Latridop- sis ciliaris, taken near Wellington, New Zealand, as Proctocccs subtenuc (Linton, 1907) Hanson, 1950. and listed the soecies from the Red Sea, Bermuda, Tortugas, and New Zealand. If this determination is correct, the parasite is widely distributed and infects different kinds of fish. The latter point is probably not significant since the worms are progenetic and voting mature specimens could be taken from the digestive tract of any fish which had recently ingested an infected host-mussel. Dollfus fin Fujita. 1925) was undoubtedly correct in the prediction that mollusk- eating fishes would be found to harbor the adult stage of Proctoeces ostreac, the unencysted metacercaria discovered by Fujita. Since members of the genus Proctoeces develop and may actually mature in bivalve mollusks, it seems certain that fishes may acquire the infection by eating these mollusks, although another method is of course not precluded. Linton's (1907) description of P. subtcnuis is accompanied by a figure and al- though done over fifty years ago it was, until the paper by Freeman and Llewellyn (1958), the most complete account of the species available. The length and width of the specimens and the sizes of the oral and acetabular suckers as given by Linton are actually greater than the corresponding measurements given by Looss (1901) for P. maculatus. Although P. sitbtemds may be specifically distinct, there is at present no adequate basis for distinguishing between it and P. maculatus. The progenetic specimens described in the present paper are almost certainly identical with those described by Linton, and until they can be distinguished from P. maculatus, should be assigned to that species. Proctoeces is clearly a member of the family Fellodistomidae, the name of which was confirmed in a letter by the late Charles W. Stiles and published in Stunkard and Nigrelli (1930). Cable (1953) recognized four subfamilies: Fellodistominae Nicoll, 1909; Gymnophallinae Odhner, 1905; Haplocladinae Odhner, 1911; and Tandanicolinae Johnston, 1927. Dollfus (1947), however, had maintained that Monascus Looss. 1907 has priority over Haplocladus Odhner, 1911 and that the correct name of the subfamily is Monascinae. Finally Freeman and Llewellyn (1958) pointed out that the excretory vesicle in members of the genus Proctocccs, which has an epithelial lining, controverts the thesis of La Rue (1957) that in the Anepitheliocystidia, in which the family Fellodistomidae is included, the definitive bladder is not epithelial. ABSTRACT-SUMMARY Sexually mature worms from Mytilus cditlis, taken in Connecticut and Massa- chusetts, are identified as Proctoeces maculatus (Looss, 1901). The specimens are often sterile, which reflects the abnormal conditions of development in the molluscan LIFE-CYCLE OF PROCTOECES 193 host. Similar worms were reported by Freeman and Llewellyn (1958) from Scrobicularia plana taken in the Thames estuary, England, and identified as Proc- toeccs subtenuis (Linton, 1907), but we regard P. subtenuis as identical with P. inaculatits. Evidence is presented to show that Cercaria milfordcnsis Uzmann, 1953 is the larval stage of P. maculatns. The taxonomy of the species is discussed. Cercaria adranoccrca n. sp. is described from Gemma gemma taken at Boothbay Harbor, Maine. It is not congeneric with P. maculatns, but is referred tentatively to the family Fellodistomidae. LITERATURE CITED BREDER, C. M., JR., 1929. Field Book of Marine Fishes of the Atlantic Coast from Labrador to Texas. G. P. Putnam's Sons, New York and London. CABLE, R. M., 1953. The life-cycle of Parvatrema borinquehae gen. et sp. nov. ( Trematoda : Digenea) and the systematic position of the subfamily Gymnophallinae. /. Parasitol., 39: 408-421. DAWES, B., 1946. The Trematoda. Cambridge Univ. Press. DOLLFUS, R. P., 1947. Sur Monascns filiformis (Rudolphi, 1819) A. Looss, 1907, trematode de 1'intestin de Ccpola ntbcsccns (L.) en Mediterranee. Ann. Parasitol.. 22: 319-323. FREEMAN, R. F. H., AND J. LLEWELLYN, 1958. An adult digenetic trematode from an inverte- brate host: Proctocccs subtenuis (Linton) from the lamellibranch Scrobicularia plana (da Costa). /. Marine Biol. Assoc., 37: 435-457. FUJITA, T., 1925. Etudes sur les parasites de 1'huitre comestible du Japon Ostrca gigas Thun- berg. Ann. Parasitol., 3: 37-59. Translation by R. Ph. Dollfus. HANSON, MARY L., 1950. Some digenetic trematodes of marine fishes of Bermuda. Proc. Helm. Soc. ll'ashington, 17: 74-89. HOPKINS, S. H., 1954. Cercaria brachidontis n. sp. from the hooked mussel in Louisiana. /. Parasitol., 40: 29-31. LA RUE, G. R., 1957. The classification of digenetic Trematoda: a review and new system. Exp. Parasitol., 6: 306-349. LINTON, E., 1907. Notes on parasites of Bermuda fishes. Proc. U. S. Nat. Museum, 33 : 85-126. Looss, A., 1901. Ueber einige Distomen der Labriden des Triester Hafens. Zentrbl. Bakt., 29: 402-404. MANTER, H. W., 1940. Digenetic trematodes of fishes from the Galapagos Islands and the neighboring Pacific. Rep. Coll. A. Hancock Pacific E.rped., 1932-1938, 2(16) : 531-547. MANTER, H. W., 1947. The digenetic trematodes of marine fishes of Tortugas, Florida. Amcr. Midi. Naturalist, 38: 257-416. MANTER, H. W., 1954. Some digenetic trematodes from fishes of New Zealand. Trans. Rov* Soc. N. Z.. 82: 475-568. NICOLL, W., 1915. The trematode parasites of North Queensland. Parasitol., 8: 22-41. ODHNER, T., 1911. Zum naturlichen System der digenen Trematoden. III. Steringophoridae. Zoo/. Ans., 38: 97-117. STUNKARD, H. W., 1957. Intraspecific variation in parasitic flatworms. S\stem. Zool., 6 : 7-18. STUNKARD, H. W., AND R. F. NIGRELLI, 1930. On Distomum vibe.v Linton, with special refer- ence to its systematic position. Biol. Bull., 58 : 336-343. UZMANN, J. R., 1953. Cercaria milfordcnsis nov. sp., a microcercous trematode larva from a marine bivalve, M\tilus cdulis L., with special reference to its effect on the host. J. Parasitol., 39: 445-451. WINTER, H. A., 1954. Proctocccs inacroi'itcllus n. sp. de un pez embiotocido del Oceano Pacifico del Norte (Tremat, Fellodistom.) . Ciencia, Mex., 14: 140-142. YAMAGUTI, S., 1934. Studies on the helminth fauna of Japan. Part 2. Trematodes of fishes. I. Jap. J. Zool., 5: 249-521. YAMAGUTI, S., 1938. Studies on the helminth fauna of Japan. Part 21. Trematodes of fishes, IV. Kyoto, Japan : pp. 1-139. YAMAGUTI, S., 1953. Systema Helminthum. Part I. Digenetic trematodes of fishes. Tokyo, Japan : pp. 1-405. Vol. 116, No. 2 April, 1959 THE BIOLOGICAL BULLETIN PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY TWO NEW GENERA OF DINOFLAGELLATES FROM CALIFORNIA 1 ENRIQUE BALECH - Scripps Institution of Oceanography. University of California, La Jolla, California The coastal waters in the San Diego region support fairly abundant popula- tions of phytoplankton. Armored dinoflagellates of this region were studied extensively by Kofoid and his associates (1907-1933), but there are still numerous undescribed or little known representatives especially among the smaller species. In the present paper, two new genera and species are described. These were originally isolated by Dr. Beatrice M. Sweeney in 1956-57 from coastal water at La Jolla, Calif., and have since been maintained as laboratory cultures. Acknowledgment is here made to Prof. Francis T. Haxo and Prof. Martin \V. Johnson for their interest and for providing research facilities. The author is indebted to Dr. Beatrice M. Sweeney whose cultures made this study possible, to Mrs. Anne Dodson for valued technical aid and to Dr. K. A. Clendenning for assistance in the preparation of the manuscript. METHODS The dinoflagellates were first examined alive. Fixed material was then studied under an oil immersion objective and by phase contrast. To derive the general plate formulae of the thecae, an individual cell was isolated under a cover-glass. A drop of concentrated sodium hypochlorite solution was then passed slowly under the cover-glass to destroy the protoplasm and to remove the cement which unites the plates. This process was assisted by applying very gentle pressure to the cover-glass, but great caution was necessary because of the fragility of the speci- mens. With Scrippsiella, it proved helpful to store droplets of the cultures in a wet chamber for a few hours. Under these conditions many of the cells shed their thecae, to which the hypochlorite treatment was then applied. After testing other methods, the following technique was adopted for the examination of Fragilidiiim. Actively swimming individuals were killed by transferring them into 5% formaldehyde with a micropipette. Individual specimens were next isolated, and by applying gentle pressure to the cover-slip, the protoplasm was 1 Contribution from the Scripps Institution of Oceanography, New Series. 2 Permanent address : Casilla de Correo 64, Necochea, Argentina. This work was conducted during the tenure of a John Simon Guggenheim Fellowship, 1957-58, at the Scripps Institute of Oceanography, University of California, La Jolla. 195 Copyright 1959, by the Marine Biological Laboratory 196 ENRIQUE BALECH Scrippsiella sweencyi n. gen., n. sp. a FIGURE 1. Scrippsiella sivecneyi. a) A typical individual, ventral view, b) Ventral view of the epitheca. c) Dorsal view of the hypotheca. d) Apical view of the epitheca. e) Antapical view of the hypotheca. f) Sulcal region (S.a. : Anterior sulcal. S.i. : left sulcal. S.d. : right sulcal. S.p. : posterior sulcal). All figures about X 1500. TWO NEW DINOFLAGELLATES 197 forced out of the theca through the cingular region. The hypochlorite treatment was then applied to the empty Fragilidium theca, especially in studies of the sulcus and cingulum. Diagnosis. Small-sized, conical epitheca, rounded hypotheca, without horns. Cingulum wide, cavazone, descendent, with displacement equal to two-thirds of its width, without lists. The cingulum has six plates, five equal, preceded at the left by a transitional one. Sulcus deep, of medium width, slightly curved to the right. a FIGURE 2. Scrippsiella siveeneyi, atypical plate patterns, a and b) Two epithecae, apical view, c) Antapical view of a hypotheca. All figures about X 1500. The sulcus has four plates, with the posterior plate largest. The pattern of the major body plates is the same as that of an Orthoperidinium with three inter- calaries. Cell length, 2432.5 ^; transdiameter, 19-24 /t, chromoplasts numerous, elliptical, generally brown-yellow. La Jolla, California. Description. This organism resembles Peridinium trochoideum in its general shape and size, and to some degree in its plate formula: 4', 3a, 7", 6c, 5" ', 2" ", and 4s. Its epitheca is high and conical, most individuals deviating from a 198 ENRIQUE BALECH rectilinear outline by a concavity near the apex, as shown in Figure la. The hypotheca is almost hemispherical, and slightly shorter in length than the epitheca. In the region of the girdle, there is a slight dorsiventral compression. In apical view, the cells normally appear almost circular. The sulcus indents slightly into the epitheca, is very deep, and of medium width. It does not reach the antapex when in true frontal view. The plate pattern of the major body plates is the same as that of an Ortho- peridinium with three intercalaries. In the epitheca, the first apical plate (!') is very narrow, with an asymmetrical rhombic shape and upwardly curved base. Attached to its anterior end, there is an extremely narrow ventral apical plate. The apex of the theca is horizontal, and is closed by a circular plate (apical pore platelet) which indents the pentagonally shaped third apical plate (3'). Plates 2' and 4' are comparatively large, and generally 2' is a little wider than 4'. There are three dorsal intercalaries. Plate 2a is usually pentagonal but is sometimes hexagonal. In the hypotheca, there are five post-cingulars and two antapicals. Plates 1" and 5" ' are wide, and 3" ' is very asymmetrical ; its border with 2" ' is very long in comparison with its border with 1" ". The two antapicals have a very restricted connection with the end of the sulcus. The cingulum has five plates of similar size, plus a transitional plate at the left end which is somewhat different in shape and also a little higher than the other cingular plates. The sulcus of dinoflagellates is not easily examined, and has been neglected by most protistologists for that reason. The sulcus of Scrippsiella sweeneyi is exceptionally difficult to analyze, being about as difficult to study as that of Hetcrocapsa triquetra. The anterior sulcal plate (S.a.) is narrow and a little curved. It borders 7". Posterior to this plate are two smaller plates (S.i. and S.d.). The shorter and broader of these two is the left plate (S.i.), which extends very slightly beyond the distal end of the girdle. The right border of this plate (S.i.) is thickened and ref ringent ; it is provided with poroids and at the extreme anterior end there are two closely spaced pores. The right sulcal plate (S.d.) narrows toward the posterior. The posterior plate (S.p.) is the largest, forming the greatest part of the sulcus. Its right anterior border is strongly oblique to the axis of the plate and articulates with S.i. The posterior right border of S.p. is thickened. The nucleus is round and located at the girdle level. Its diameter is about one-third of the total cell length. The chromatin strands are less evident than in most dinoflagellates. The chromoplasts are elliptical and numerous, sometimes yellow-green but normally brown-yellow. Food is apparently stored as small granules and also around the chromoplasts in bodies that resemble pyrenoids. There is no pusule nor stigma. The first external evidence of cell division is the formation of two discrete longitudinal flagella with separate points of attachment. During division, the cell escapes from the theca but retains a tough cellular membrane. The two daughter cells remain attached to each other in an oblique plane. The posterior cell is usually the smallest. Locomotion is normally rapid, with a strongly rotatory motion. There is usually TWO NEW DINOFLAGELLATES 199 one complete rotation of the organism during an advancement of one or two cell lengths. Sometimes, when 5. sweeneyi cells reach the border of a drop, they suddenly cast off their flagella. Generally they lose the transverse ribbon-like flagellum first, which continues to beat in the detached state for a few seconds and then vacuolizes. The longitudinal flagellum is about three times as long as the cell ; it does not beat or vacuolize after detachment. Occurrence. This organism was originally isolated on March 15, 1956, from water collected off the S.I.O. pier at La Jolla, California, and has since been observed frequently in locally collected water samples. It seems to be a year-round inhabitant of the San Diego region, thriving especially in the summer months. This species has also been observed in plankton net samples, its relative scarcity in these being caused by its small size and poor retention on plankton silk. Variations. The cingular and sulcal formula has been constant in laboratory and field specimens : 6C and 4S. The cells varied in size, and in the laboratory clonal culture used in the description, the cells also varied in shape and in plate pattern. Deformed or aberrant forms of 5". szveeneyi were numerous in old laboratory cultures, but these were not observed in plankton samples. The plate formulae of the thecae from plankton samples were established in only a few cases, so we do not yet know how much the plate formula of this organism varies in nature. On the whole, the plate pattern has shown an amazing range of variation. The normal plate formula is as stated above : 4', 3a, 7", 6c, 5" ', 2" ", and 4s. It is generally assumed that the hypotheca is more conservative than the epitheca, and this is true of the present organism. Deviations from the normal epithecal plate configuration were observed in about 10 per cent of the specimens examined. The range of variation encountered in the atypical specimens of 5\ szveeneyi was rather exceptional for dinoflagellates, although similar variations occur in Pyro- phacus Jwrologiciiiu. Plate formulae in these atypical specimens were: (1) 4', 2a, 6"; (2) 4', 3a. 5"; (3) 4', 3a, 4"; A single specimen with 3', 3a and 5" showed an exceptional overgrowth of 1", which reached the apical pore thus transforming 2' into la. No alteration of hypothecal formula has been noticed in actively growing cultures. In old cultures, I have observed hypothecal formulae of : 5" '. 2" ' and one intercalary ; 4" ', 2" " ; and 3" ', 2" " and one intercalary. However, the plate variation of the hypotheca has not exceeded two per cent in all examined specimens. Discussion. The general characteristics of this organism place it in the Peridiniaccae. If it were classified solely on the basis of its major body plates, it would be included in the genus Peridinium. The cingular formula and sulcus plates are characteristically different, however, from those of Peridinium. The cingular and sulcal plates are conservative and important structural features con- nected with the most dynamic parts of the cell. Undoubtedly this organism belongs to a new genus. The species is also new. The only other known species which bear general resemblances to the present organism are Peridinium subsalsum Ost., and especially P. trochoideum (Stein) Lemm. Laboratory cultures of these species \vere provided by Dr. Sweeney for comparative studies. Their assignment 200 ENRIQUE BALECH to the genus Peridinium was clearly correct, but they bore only superficial re- semblances to Scrippsiella sweeneyi. The genus is named after the institution at which it was discovered, and the species is dedicated to Dr. Beatrice M. Sweeney who made the original isolation and whose cultures made this study possible. Fragilidium heterolobum n. gen. n. sp. G FIGURE 3. Fragilidium heterolobum. a) A typical individual, ventral view, b) Apical view of the epitheca. c) Sulcal plates, d) Antapical view of the hypotheca. All figures about X 1000. Diagnosis. Medium-sized, roughly roundish pentagonal in ventral view. Epitheca dome-shaped ; hypotheca asymmetrically bipedal, the left lobe being the largest. Cingulum deeply impressed, subcentral, descending, displaced distally about one girdle width, without lists. The cingulum has eleven sub-equal rec- tangular plates plus a transitional plate at the right end. Sulcus narrow, only slightly excavated, with six plates. Theca easily exuviated. Cell length 53-56 ^, TWO NEW DINOFLAGELLATES 201 transdiameter 48-54 ^. Chromoplasts numerous, elliptical, brown. Genus char- acterized by the high number of precingular, postcingular and cingular plates. La Jolla, California. Description Plate pattern. The epithecal formula is 4', 9" and a "pore platelet." The plate 1' is in general large and it has the most irregular form. It has connections with seven plates: 1", 2", the pore platelet, 4', 8" and 9"; its border for 2" is the smallest. Plate 1' is asymmetrically located, most of it being on the right side of the epitheca; its width decreases gradually to the left. The other three apicals are more regular. Apical 2' has six edges (for 1', 3', the pore platelet, 2", 3", 4")- The plate 3' touches 2', 4', 4", 5", 6" and the pore platelet. The apical 4' touches 3', 1', 6", 7", 8" and the pore platelet. The so-called pore platelet is relatively large, oval sigmoid, and placed obliquely, to the median plane, i.e., the plane which passes through the sulcus, the joint of 1" and 9", and the apex. This plate is variable but generally it has a convex left side subdivided into two edges for 2' and 3', a concave side which touches 1', a major pole for 4', and a minor one for the suture between 1' and 2'. The most characteristic feature of this plate is a long and narrow reinforcement at the middle of the plate, sigmoid, with a dorsal hook to the right ; it is variable sometimes double. Along it there are sometimes a few very small pores. The most characteristic precingulars are 1" and 9". The first, trapezoidal in shape, is the smallest. The precingular 9", pentagonal, has two edges at the left : the superior one for 1' and the posterior, reinforced, forms a part of the right border of the sulcus. The precingulars 3", 5", and 7" are more or less quadrangular. The hypothecal formula is 7" ', 2" " and Ip. The narrowest postcingular plates are V" and 7"'. The latter is the smaller and is somewhat displaced posteriorly. The antapicals are very asymmetrical, the left one being much longer. The suture between 1" ' and 2" ' is irregular. Antapical 2"" contacts the sulcus more than 1" ". which just barely touches it. The intercalary (p) is a large irregular plate, bordered by the antapicals, 3" ', 4" ' and 5" '. All plates of the epi- and hypotheca are smooth. Some spots of different optical densities could be seen in a few specimens, especially in plate 1", with oil immersion and phase contrast. There are sometimes pores located on the cingular border of this plate. The cingulum is formed by eleven subequal retangular plates, plus another different plate at its right end. This C 12 is curved, irregular, extending somewhat into the sulcus, with a narrow left-posterior or sulcal end. For that reason this plate could be named "transitional." The cingular plates lack sculptures. The sulcus is narrow, has six plates, and is only slightly excavated. The anterior sulcal plate has a very characteristic "boomerang" shape, with a posterior concavity and a longer and narrower right arm. In its sinus there are two very tiny platelets; the right one is the smaller. Behind the anterior plate and in contact with 1" ' there is a long plate, with a little sinus at the middle of its right border, where C 12 ends. In connection with the latter, there is another small plate. Finally, there is a posterior sulcal plate. Protoplasm. The protoplasm is surrounded by a strong membrane and contains more than one hundred elongate-elliptical chromatophores which are dark 202 ENRIQUE BALECH yellowish-brown. Food is stored as numerous granules of variable shape, which are generally small and located most abundantly in the peripheral layer. The nucleus is large and compact, and is surrounded by a strong membrane. It is elongated in the equatorial plane, is somewhat curved, and has very dense thin threads of granular chromatin more or less perpendicular to the major axis of the nucleus. At the concavity, I sometimes observed large masses that were not distinctly granular. The longitudinal flagellum extends beyond the antapex about two and a half cell lengths ; it has a fast vibratory movement of short amplitude. The transverse flagellum, very slightly flattened, is long and completely encircles the girdle. The organism swims with a predominantly rotating motion. Dimensions (in fixed and slightly distorted cells). Length 53-56 //,; trans- diameter 48-54 ju,. In an individual with a length of 55.5 /A, the epitheca was 27.5 ju, and the hypotheca was 24 p in length. Variations. I have observed some variation in form (cell length more or less short in comparison with the transdiameter) and also in the plate pattern. Sometimes 4" appears divided into two plates; thus the postcingular series some- times has eight instead of seven plates. Occasionally there are eight instead of nine precingulars, and in one individual, a very narrow 1' was observed fused with the pore platelet. The normal formula is: 4', 9". a pore platelet, 12c, 7'", 2"", Ip and 6s. Discussion The only difficulty encountered in the tabulation of this organism was the rapidity with which it exuviated its plates. Most of the individuals were found in a quiet state, short ellipsoidal in form, and without theca. The actively swimming cells were of course difficult to measure and draw. Any attempt to stop them for a moment led to cell deformation and ecdysis. This is accomplished in a very peculiar way : all the plates separate from each other, but in general, they remain surrounding the cell at a short distance, forming a regular assemblage. The plates are very delicate. The plate pattern of this species is fundamentally different from all of those previously known (Balech, 1956; Biecheler, 1952; Dangeard, 1927; Graham, 1942; Kofoid, 1907-33; Lindemann, 1928; Schiller, 1933, 1937). The differences are in both the epi- and hypotheca. Since very little is known regarding the cingulum and sulcus of most dinoflagellates, we cannot discuss the differences concerning these regions. Nevertheless, it should be pointed out that the structure of the sulcus of this species is different from that of all sulci already studied, and no other genus is known with such a high number of girdle plates. Other genera without epithecal intercalaries and with four apical plates are Diplopsalis, Dolichodiniwn, Goniodinium, Glenodiniwn, Cladopyxis, and Ceratiuin. The two latter are very different in form, bearing strong horns or arms, and with many differences in plate pattern. Diplopsalis as defined by Lindemann (1928) is actually an assemblage of several genera. But even on these terms, Diplopsalis never has more than seven precingulars, five postcingulars and it lacks the posterior intercalary. Glenodiniitm, as defined by Schiller (1933-37), is another very heterogeneous assemblage with a very large variation of plate patterns. None of its TWO NEW DINOFLAGELLATES 203 species has so many pre- and postcingular plates, and they also lack posterior intercalaries. Dolichodinium seems to have only six girdle plates and has six precingulars and six postcingulars instead of nine and seven. Goniodimmn is perhaps the genus most closely related to Fragilidium, but it has only six precingu- lars and six postcingulars ; instead of one posterior intercalary it has three inter- calaries in the hypotheca. Until the discovery of Fragilidium, Goniodinium was the genus with the highest known number (nine) of cingular plates (this number, however, was not stated with certainty). The high number of precingular, postcingular and cingular plates is sufficient to characterize this new genus. The only other genera with seven postcingulars are Glenodiniopsis and Heterodinimn. Pyrophacus is the only genus with a higher number of these plates. Fragilidium heterolobum was isolated from plankton at La Jolla (San Diego. California) on March 20. 1957. SUMMARY 1. Two new genera and species of dinoflagellates are described. Both were originally isolated from plankton samples collected at La Jolla (San Diego, California). 2. Scrippsiclla sweeneyi is a small species with the general tabulation of an Orthoperidinium, but it differs in having six cingular plates. The structure of the sulcus is also different. A great deal of variation in plate pattern was ex- hibited by this organism. 3. Fragilidinm hetcrolobuin is a medium-sized species having a tabulation that is quite different from all previously described dinoflagellates. It has a very high number of cingular plates (twelve). The generic name refers to the characteristic frequency and suddenness with which it sheds its plates. LITERATURE CITED BALECH, E., 1956. Etude des Dinoflagelles du sable de Roscoff. Revue Algologiquc Nile., Serie2(l-2) : 29-52. BIECHELER, B., 1952. Recherches sur les Peridinicns. Supp. an Bull. Biologiquc dc France et dc Belgiqnc. 36 : 1-149. DANGEARD, P., 1927. Phytoplankton de la croisiere du Sylvana. Ann. Inst. Occanog., 4 (8) : 285-407. GRAHAM, H. W., 1942. Studies in the Morphology, Taxonomy and Ecology of the Peridiniales. Scientific results of Cruise VII of the Carnegie during 1928-29. Carnegie Inst. of Washington Publication 542. KOFOID, C. A., 1907. New species of dinoflasellates. Bull. Mus. Comp. Zool. Harvard, 50 : 163-207. KOFOID, C. A., 1911. Dinoflagellata of the San Diego region. V. On Spiraulax, a new genus of the Peridineae. Univ. Calif. Pub. Zool, 8 : 295-300. KOFOID, C. A., AXD A. M. ADAMSON, 1933. The Dinoflagellata: the family Heterodiniidae of the Peridinioidiae. Mem. 3fns. Comp. Zool. Harvard, 54 (1), 136 pp. KOFOID, C. A., AXD J. R. MICHEXER, 1911. New genera and species of Dinoflagellates. Bull. Mus. Comp. Zool. Harvard, 54: 265-302. LINDEMANN, E., 1928. Pcridiuialcs. In: Engler and Prantl, Die Pflanzenfamilien. 2nd Ed., Vol. 2, 104 pp. SHILLER, J., 1933, 1937. Dinoflagcllatae (Peridineae). In: L. Rabenhorst's Kryptogamenflora. Vol. 10, Leipzig. PHOTORECEPTION IN THE OPOSSUM SHRIMP, MYSIS RELICTA LOVEN x ALFRED M. BEETON 2 Department of Zoology, University of Michigan, Ann Arbor, Michigan Little information is available on the physiology of photoreception in the Mysidacea other than a few studies of phototaxis. Practically nothing is known of their spectral sensitivity, dark-adaptation, or lower limits of vision. The two observations that have been made on the response of the opossum shrimp, Mysis relic ta Loven, have shown that they swim downward when subjected to light (Dakin and Latarche, 1913) and are especially sensitive to a combination of high temperature and bright light (Larkin, 1948). The present study of the spectral sensitivity, dark-adaptation, and phototaxis of M. relicta was undertaken to add to our information on the physiology of photoreception. MATERIALS AND METHODS Mysis relicta is ideal for a study of this nature, since its large size (average length, 15.0 mm.) facilitates observation, and a laboratory population can be easily maintained. The animals used in this study were collected in Lakes Huron and Michigan. All experiments were conducted in a cold-room under controlled light con- ditions and at a constant temperature of 10 C. The dark-adaptation and special sensitivity studies were carried out in an all-glass aquarium 12.0 by 8.0 by 3.0 inches, filled to 1 inch from the top. A 24-inch glass tube, with a 1-inch diameter, was used in the experiments on phototaxis. Under conditions of continuous darkness or light, the mysids normally rested on the bottom of the tank, periodically making short excursions off the bottom. If the experimental light was turned on when the mysids were swimming upwards, they hesitated momentarily, turned, and swam rapidly to the bottom of the aquar- ium. This momentary hesitation was found to be a reliable indicator for the photic response. The difficulty of observation in a dark room was met by using an infra-red viewer and infra-red light source or a large Fresnel lens to focus low-intensity red light (approximately 0.001 foot-candle) on the observer's eye. The red light was produced by a neon glow lamp and a number 2404 Corning glass filter, transmitting wave-lengths of 620 m//, and greater. It was permissible to use this light for viewing, since preliminary studies had established that M. relicta was almost completely insensitive to the red region of the visible spectrum in the 620- 1 Based on a thesis submitted in partial fulfillment of the requirements for the Ph.D. degree, University of Michigan, 1958. 2 Present address of the author is : U. S. Department of Interior, Fish and Wildlife Service, Ann Arbor, Michigan. 204 PHOTORECEPTION IN MYSIS RELICTA 205 450 400 450 500 550 600 650 Wave- length (mu) 700 750 FIGURE 1. Spectral distribution of a tungsten filament lamp (color temperature 2300 K) and the spectral transmission of monochromatic filter combinations. to 700-nijU. wave-band. Mysid behavior was the same when viewed by either of the above methods. A General Electric 7C7 tungsten filament lamp suspended 3 inches above the water surface provided the light source for the spectral sensitivity studies. This lamp has an average color temperature of approximately 2300 K and its lumen output averages 45. Although the exact distribution curve of spectral energy was not available for the lamp, a reasonably accurate curve was constructed by extrapola- tion from data supplied by the Xela Park Laboratory of General Electric (Fig. 1). 400 450 500 550 600 Wave-length ( 650 700 750 FIGURE 2. Radiant energy output from a tungsten filament lamp (color temperature 2300 K) through various monochromatic filter combinations. 206 ALFRED M. BEETON Nine monochromatic-filter combinations were made with Corning glass color filters and interference filters; Figure 1 gives the spectral transmission of these filter combinations. The total energy output of a given filter combination was calculated by multiplying the energy available (in 10-m/x-wide wave-bands) from the lamp by the percentage transmission of the filter combination (Fig. 2). The percentage transmission of the various filter combinations was obtained either from data supplied by Corning (1948) or with a Beckman DU Spectrophotometer. The range of intensities for a particular wave-band was obtained by the use of evaporated-metal, neutral-density filters, having optical densities of 0.6, 0.9, and 1.0. The reaction times of the mysids to the various intensities of radiant energy were measured by a stop watch. Sufficient time was allowed between successive tests to keep the mysids completely dark-adapted. In the dark-adaptation experiments, the mysids were first subjected for 3 minutes to the intense light of a 1000- watt photoflood lamp. At the end of the 3-minute period all lights were turned off except the viewing light. Then the mysids were subjected to flashes of light, approximately 0.1 second in duration, of a given intensity. These flashes were spaced at 1- to 3-minute intervals. The experimental light was a 6-watt, 9-foot-candle, 115-volt. tungsten-filament lamp suspended 3 inches above the water surface. The intensity of the light was altered by interposing various numbers of evaporated-metal, neutral-density filters, each having an optical density of 1.0. The time in the dark, prior to first stimula- tion, was measured by stop watch. In the experiment on the phototactic response of M. relic ta, six individuals were placed in a 24-inch glass tube, with a 1-inch diameter, lying horizontally to eliminate any gravitational effects. The experimental light, a 7C7 lamp, was suspended 1 foot above the midpoint of the tube. After the mysids had been subjected for measured intervals to total darkness or light, one-half of the tube was shaded and the number of mysids in the unshaded half of the tube was recorded at 30-second intervals for a 5-minute period. First the right and then the left half of the tube was shaded to detect any bias in the distribution of the mysids. Control runs, with neither half being shaded, were made at frequent intervals. SPECTRAL SENSITIVITY Earlier studies of spectral sensitivity It has been well established that the first step in the response to light in any animal is a photochemical reaction. Hecht's (1919, 1920, 1921) work on the clam, Mya arenaria Verrill, contributed much toward establishing the photo- chemical nature of photoreception. He demonstrated that the fundamental concept of photochemistry, the Bunsen-Roscoe reciprocity law which holds that the photo- chemical effect is equal to the product of time and intensity, could be applied to the data of his studies. Before a photochemical reaction can occur, light must be absorbed. The absorption spectra of the visual pigments will therefore determine the effects on the photoreceptor and in turn the response of the organism. The behavioral response of an organism to different regions of the radiant-energy spectrum has PHOTORECEPTION IN MYSIS RELICTA 207 given rise to the concept of "action spectra." For example, if a visual pigment has its absorption peak at a wave-length of 500 111/1, the animal possessing this visual pigment would be most sensitive to this wave-length. This concept has led to a number of action-spectrum studies on invertebrates. Mast (1917) re- TABLE I Reaction time (seconds) of Mysis relicta to various wave-lengths of light at several intensities ( Unless noted otherwise the neutral-density filters transmitted 10 per cent of the light) Wave-length of peak transmission (HIM) Number of neutral-density filters Energy output of 7C7 lamp through filters (microwatts/10 m/i/ lumens) Average reaction times for 10 trials (seconds) Standard deviation 395 2.20 0.77 0.18 395 *1 0.26 0.88 0.12 395 2 0.02 1.16 0.39 430 11.19 0.72 0.06 430 **1 2.80 0.84 0.06 430 *1 1.34 0.86 0.31 430 ***2 0.34 0.92 0.16 430 **** 3 0.03 1.15 0.39 485 7.63 0.87 0.14 485 1 0.76 1.04 0.12 485 2 0.08 1.18 0.10 488 13.63 0.86 0.19 488 2 0.14 1.13 0.20 496 14.93 0.77 0.24 496 1 1.49 0.82 0.11 496 2 0.15 0.98 0.14 515 32.98 0.64 0.13 515 1 3.30 0.71 0.20 515 2 0.33 0.83 0.21 515 3 0.03 1.01 0.19 515 4 0.003 1.42 0.36 540 29.85 0.84 0.15 540 1 2.99 0.92 0.06 540 2 0.30 0.95 0.22 540 3 0.03 1.33 0.25 610 108.94 1.10 0.17 610 1 10.89 1.10 0.09 610 2 1.09 1.33 0.36 640 256.11 1.13 0.31 640 1 25.61 1.22 0.43 640 2 2.56 1.32 0.45 640 3 0.26 (No reaction) * Neutral-density filter transmitting 12 per cent of the light. ** Neutral-density filter transmitting 25 per cent of the light. *** Filters 1 and 2 combined. * Neutral-density filter transmitting 10 per cent of the light combined with filters 1 and 2. ported peak sensitivity to light at the following wave-lengths : 483 in/A in Euglena, Arenicola, Trachelonwnas, and Lmnbricus ; 524 m/x in Pandorina and Eudorina; 503 nijit in Chlamydonionas, and blowfly larvae. Hecht (1921) established the basis for future studies through his experiments on Mya arcnaria. This clam 208 ALFRED M. BEETON had a maximum sensitivity at 490 m^u. Most insects apparently have two peaks of maximum sensitivity, 365 m/*, and 492 niju, (Weiss, 1943). Results obtained through the methods of these investigators are similar to those secured by in vitro studies of the spectral absorption of squid rhodopsin (Bliss, 1948). An electrical method has been employed to determine the spectral sensitivity of the eyes of Limulus (Graham and Hartline, 1935), a grasshopper, Melanoplus (Jahn, 1946), and the diving-beetle, Dytiscus (Jahn and Wulff, 1948). These studies demonstrated, as did the behavior method of Weiss, that the arthropod eye has a peak sensitivity in the green region of the spectrum. 100 10 en x_ OJ c I o> Eo. o.oi - 0.001 0.6 0.8 1.0 1.2 1.4 Reaction time (seconds) FIGURE 3. Reaction rate of Mysis rclicta to certain wave-lengths (m/ct) of light at various intensities. (Each point represents the average reaction time for 10 trials.) The only source of information on the sensitivity of mysids to various regions of the spectrum is Hess' (1910) study of the phototactic response of a marine mysid to certain regions of the visible spectrum. Sixty-four mysids were placed in the dark and then subjected to a continuous spectrum of light. The mysids swam toward the light and aggregated in certain regions of the spectrum : 40 in the yellow-green, 19 in the blue and violet, and 5 in the red. When the position of the spectrum was altered the mysids followed the yellow-green band. PHOTORECEPTION IN MYSIS RELICTA 209 Spectral sensitivity in M. relicta The dark-adapted mysids reacted to the different regions of the spectrum with the typical photic response but the time of reaction varied with wave-length. The mysids reacted most quickly at wave-lengths in the vicinity of 515 m/A and 395 m^t. (Table I). The reaction times at wave-lengths of 610 m^ and 640 m/A were much slower (Fig. 3) despite the fact that considerably more energy was available in the red region of the spectrum than in the blue region (Fig. 2). If the mysid eye were equally sensitive to all regions of the spectrum, the faster reaction time 0.7 0.8 en T3 0.9 O> CD E c o o . Biol. Med., 7: 332-335. NOWIXSKI, W. W., 1948. Influence of anti-organ sera upon metabolic processes: Reticulo endothelial-immune-serum (REIS) and the oxygen uptake of rat spleen. Texas Rep. Biol. Mcd., 6 : 493. NOWINSKI, W. W., 1949. Influence of anti-organ sera upon metabolic processes : Influence of chick anti-brain serum upon the oxygen consumption of chick brain homogenates. Texas Rep. Biol. Mcd.. 7 : 230-236. PERLMAN, P., 1954. Study on the effect of antisera on unfertilized sea urchin eggs. Exp. Cell Res., 6: 485-490. PERLMAN, P., 1957. Analysis of the surface structure of the sea urchin egg by means of anti- bodies. I. Comparative study of the effects of various antisera. Exp. Cell Res., 13: 365-390. PERLMAN, P., AND H. PERLMAN, 1957. Analysis of the surface structures of the >ca urchin egg by means of antibodies. II. The J- and A-antigens. Exp. Cell Res., 13: 454-474. SEVAG, M. C, AND R. E. MILLER, 1948. Studies on the effect of immune reactions on the metabolism of bacteria. I. Methods and results with Ebcrtliella tvpliosa. J. Bact., 55 : 381-392. TYLER, A., 1949. Properties of fertilizin and related substances of eggs and sperm of marine animals. Amcr. Nat.. 83 : 195-219. TYLER, A., 1956. Physico-chemical properties of the fertilizins of the sea urchin Arbacia pitnctiilata and the sand dollar, Ecliinarachnius panna. Exp. Cell Res., 10 : 377-386. TYLER, A., AND J. W. BROOKBANK, 1956a. Antisera that block cell division in developing eggs of sea-urchins. Proc. Nat. Acad. Sci., 42: 304-308. TYLER, A., AND J. W. BROOKBANK, 1956b. Inhibition of division and development of sea-urchin eggs by antisera against fertilizin. Proc. Nat. Acad. Sci., 42: 308-313. TYLER, A., AND N. H. HOROWITZ, 1937. Glycyl-glycine as a sea water buffer. Science, 86 : 85-86. WARBURG, O., 1908. Beobachtungen iiber die Oxydationprozesse im Seeigelei. Hoppe-Seyler's Zeitschr. f. pliysiol. Chcni., 57: 1-16. A BICOLORED GYNANDROMORPH OF THE LOBSTER, HOMARUS AMERICANUS FENNER A. CHACE, JR., AND GEORGE M. MOORE Division of Marine Invertebrates, U. S. National Museum, Smithsonian Institution, Washington 25, D. C., and Department of Zoology, University of Neiv Hampshire, Durham, Neiv Hampshire Lobsters with sharply defined, bilateral color differentiation have been described by several authors. Herrick (1896) mentioned the following variations of this pattern in both the American and the European lobster : half normal color and half light sky blue ; half normal and half pale red ; half greenish black and half light orange ; half blue and half white ; and half light yellow and half bright red. Schaanning (1929) gave a color figure of a European lobster that was light red and dark blue. Templeman (1948) added records of two more bicolored American lobsters, one normal and red, the other whitish red and purplish blue. Such color variants have occasionally been referred to as gynandromorphs or hermaphrodites, but there is no evidence that any of the previously recorded bicolored specimens were also bisexual. Only two cases of possibly complete hermaphroditism have been recorded here- tofore for Homarus. Nicholls (1730) described and figured a specimen of the European lobster, H. gaminarus, received from Newgate- Market, London, that displayed all of the external and internal female characters on the right side and all of the male structures on the left. Halkett (1919) collected a specimen of H. americanus at Bay View, Pictou County, Nova Scotia, November 1917 "which was absolutely male on the left side and absolutely female on the right side" ; this specimen was sent to Queen's University, Kingston, Ontario, but apparently no complete description of it has been published. Gordon (1957) described a speci- men of H. gammarus from off Seahouses, Northumberland, that had all of the characters of a perfect gynandromorph female on the right side, male on the left except that there was no male opening on the left fifth pereiopod but, instead, an imperforate indication of an opening on the coxa of the left third pereiopod ; this specimen was not dissected, but Dr. Gordon suggests that "it probably has a normal ovary on the right side and part ovary, part testis on the left side or a testis with ova in the anterior position." Herrmann (1890) discovered that eggs are occasionally developed during spermatogenesis in the lobster testis but he gave no indication that this was associated with any unusual external charac- teristics. Finally, Ridewood (1909) recorded an ovigerous specimen of H. gam- marus, presumably from off the Orkney Islands, that had genital openings on the third right pereiopod and on the fourth and fifth left pereiopods, but dissection disclosed only a normal paired ovary with apparently three oviducts, two of them on the left side leading to the abnormally placed openings. The specimen described below (U. S. Nat. Mus. Cat. No. 102241) seems to be the first lobster to be recorded in which a color anomaly was associated with 226 LOBSTER GYNANDROMORPH 227 gynanclromorphism. The specimen was alive when presented to the Fish and Wildlife Service at Woods Hole, Massachusetts, during the summer of 1954 by a dealer operating between Boston and Cape Cod. Its place of origin is unfor- tunately unknown ; it probably came from Massachusetts Bay but it could have been shipped from New Hampshire, Maine, or even Canada. It died while being photographed by John P. W T ise, who offered it to the junior author for description. After remaining in a freezer for about six months, it was transferred to formalin for dissection ; the dissected portion is stored in ethyl alcohol, and the carapace, abdominal tergites, tail fan, and chelipeds have been dried. The specimen was about 10 inches (25 cm.) long from the tip of the rostrum to the end of the telson. The carapace measures 86 mm. from the eye socket to the hind margin. To the left of the midline of the animal, as well as on most of the gastric and hepatic regions on the right side, the ground color was orange, mottled and spotted with dark, greenish brown. The right side, posterior to the mesogastric and hepatic regions, was similarly spotted and mottled but in shades of blue over a lighter blue ground color, reminiscent of the colors of willow-pattern china. The color pattern is indicated in Figure 1. The spotting and mottling pattern seems to be continuous across the midline ; only the color is different. The color transparency made from the living animal suggests that blue pigment was largely lacking on the left side, and red, yellow, and possibly black pigments were missing on the right side, although there is a greenish cast to the dark mottling on the left and a pinkish tinge in some of the light blue areas on the right. The left, crusher cheliped was colored like the left side of the body for the most part, but the color photograph suggests that there were blue patches at the outer, distal end of the merus, on the dorsal surface of the carpus, on the thick portion of the hand, near the base of the fixed finger, and on the base of the dactyl. The spines on this cheliped were bright red. The right, cutting cheliped was blue, with a tinge of brown near the base of the fixed finger, and the spines were almost pure white. The other pereiopods on the left side were orange with greenish brown shadings similar to those on the adjacent portion of the carapace. Those on the right side were very light, pinkish blue with darker blue shadings. The left uropods were orange with greenish brown margins, and those on the right, pale, pinkish blue with dark blue marginal bands. The fringe of setae on the left uropods and on the telson to the left of the midline were reddish orange, those on the right side, yellowish orange. As in the three previously described gynandromorph lobsters, the specimen dis- played female characters on the right side and male structures on the left. The body was skewed to the right anteriorly and to the left posteriorly as shown in Figure 1. This was almost certainly a result of the differential growth of male and female lobsters. Templeman (1944) stated: "At all commercial sizes the relative length of the carapace is less in the female than in the male. It remains constant when the lobsters are small, but shows a definite and progressive increase for those localities for which large lobsters were available for measurement. . . . The ratio of the greatest width of the carapace to total length is in the smaller relatively immature lobsters approximately the same for both males and females, while in the larger animals it increases with size and more so in the male than in the female." The skewing of the axis and the more swollen left (male) side 228 FENNER A. CHACE, JR., AND GEORGE M. MOORE FIGURE 1. Dorsal surface of body of gynandromorph of Homarus aincricantis shuuing asjTnmetry and color pattern. Drawn from color transparency of living animal. FIGURE 2. Ventral surface showing opening of oviduct on coxa of third right pereiopod and of vas deferens on fifth left pereiopod, asymmetrical thelycum, and characteristic female and male first pleopods. FIGURE 3. Abdominal somites viewed from the right (female) side. The left (male) pleura are shown as if viewed from the inside. LOBSTER GYNANDROMORPH 229 of the carapace are therefore readily understandable. The reverse skewing of the abdomen probably also resulted from this differential growth, accentuated by the proportionately larger abdominal pleura in the female, as shown in Figure 3. The greater apparent length of the left (male) pleura of the fourth, fifth, and sixth somites in this figure is misleading and is caused by the fact that the left (male) pleura curve downward nearly vertically, whereas those on the right (female) side extend obliquely outward, as shown in Figure 1. The distortion is less striking than in Gordon's larger (11-inch) specimen of H. gaiiiinarns and in Nicholls' specimen of the same species, the size of which was not given. This might be expected from Templeman's (1944) findings that the differential growth of males and females becomes progressively more marked with age. The most noticeable disparity in our specimen is found on the ventral surface. As shown in Figure 2. there is a female opening on the coxa of the right third pereiopod and a male opening on the left fifth pereiopod, which is represented only by the coxa and basis. The thelycum is distinctly asymmetrical, the right (female) part being broad and nearly bare and the left (male) part narrower and provided with long hairs. Neither element of this structure corresponds exactly with its form in normal males and females, but the similarity to the con- ditions in the appropriate sexes is more than superficial. Even the median plate extending forward from the last thoracic sternite is modified as would be expected : the right portion is longer and acute, like half of a typical female plate, and the left portion is shorter and rounded as in the male. The first right pleopod is typically female, flexible and long-haired, while the left one is a rigid male intromittent organ. There is a well-developed appendix masculina on the endopod of the left second pleopod, but none on the right member of this pair. The second, third, fourth, and fifth pleopods on the left (male) side are 37.5, 35.3, 33.5, and 29.0 mm. long, respectively, from the basal articulation to the end of the endopod. Those on the right (female) side have corresponding lengths of 37.2, 38.5, 38.0, and 33.3 mm. These figures agree remarkably well with the proportionate lengths of these appendages in normal males and females, as determined by Templeman (1944) : "In the male the swimmerets (including protopodite and endopodite) on the second abdominal segment are the longest and in the female those on the third and fourth are the longest and approximately equal. The second swimmerets are not greatly different in length in males and females. . . ." The sternal spines of the gynandromorph are about 2.5 mm. long on the second, third, and fourth abdominal somites and about 2.0 mm. long on the fifth somite. Data given by Templeman (1944) for New Brunswick speci- mens indicate that the average length of the spine on the second somite in specimens of comparable size is about 3.75 mm. in males and 0.5 mm. in females, and the spine on the fifth somite is about 2.5 mm. long in males and 0.5 mm. in females. The spines in the gynandromorph therefore seem to be intermediate in size, per- haps more nearly approaching the male than the female condition. Careful removal of the carapace and abdominal tergites of the specimen disclosed a well-developed ovary filled with maturing eggs on the right side and a normal testis on the left, as shown in Figure 4. An oviduct led from beneath the ovary to the opening on the right third pereiopod and a typical vas deferens connected the testis with the left fifth pereiopod. A few of the eggs in the ovary appeared slightly discolored. Herrick (1911) stated that the presence of orange flecks 230 FENNER A. CHACE, JR., AND GEORGE M. MOORE in the ovary, representing degenerating eggs that were not shed, is conclusive evidence that a lobster has already spawned at least once, but the spots in our specimen were not sufficiently distinct to permit an unequivocal determination that spawning had occurred. As can be seen in Figure 4, a lobe of the ovary, probably representing the connection between the two halves of the organ in a normal female, was found just anterior to the heart. At this point the testis was inter- rupted and the two parts of it were continuous with the intermediate portion of the ovary. It appeared that the two portions of the testis were differentiated parts of a single organ. There is no doubt that the testis was functional, for sections showed active spermatogenesis. Normal, fully formed spermatozoa were extracted from the vas deferens. It is, of course, impossible to determine whether this specimen could have func- tioned reproductively as either or both a male and a female lobster. Viable sperma- tozoa and eggs were probably produced, but the unpaired intromittent organ and the deformed thelycum might have prevented successful copulation with normal males and females. The specimen must have been nearly or quite mature. As testis hepatopancreas FIGURE 4. Dissection of lobster gynandromorph after removal of heart and stomach showing well-developed ovary on right side and testis and vas deferens on left. mentioned above, we were unable to determine whether or not it had spawned. If it was caught off the Massachusetts coast, it was probably six or seven years old according to Herrick (1911). Herrick (1896) also maintained that "very few lobsters under 9 inches in length have external eggs, while only few have attained the length of lO 1 /^ inches without having them." Templeman and Tibbo (1945) concluded from the examination of New Brunswick specimens that the length of males at sexual maturity is at least 5 cm. less than that of females. One can hardly assume, however, that the size of a gynandromorph is directly com- parable with that of a normal individual of either sex. Female lobsters probably grow more slowly than males (Herrick, 1911), and one might therefore expect the present specimen to be smaller than a normal male and larger than a normal female of the same age, but the growth rates of abnormally bisexual crustaceans may be complicated by hormonal or other factors. We wish to thank John P. Wise for calling our attention to this unusual speci- men. We also wish to acknowledge the assistance of members of the staff of the LOBSTER GYNANDROMORPH 231 Division of Marine Invertebrates, U. S. National Museum, during the preparation of the paper. Special thanks are due Charles E. Cutress of that staff for the histological preparation and study of the reproductive organs of the specimen. LITERATURE CITED GORDON, I., 1957. A pseudo-hermaphrodite specimen of the lobster, Homants ganimarus (Linnaeus). Ann. Mag. Nat. Hist., (12) 10: 524-528. HALKETT, A., 1919. An hermaphrodite lobster. Canad. Field-Nat., 33 : 40. HERRICK, F. H., 1896. The American lobster : A study of its habits and environment. Bull. U. S. Fish Comm., 15 : 1-252. HERRICK, F. H., 1911. Natural history of the American lobster. Bull. Bur. Fish., 29: 149-408. HERRMANN, G., 1890. Notes sur la structure et le developpement des spermatozo'ides chez les decapodes. Bull. sci. Fr. Belg., 22: 1-59. NICHOLLS, F., 1730. An account of the hermaphrodite lobster presented to the Royal Society on Thursday, May the 7th, by Mr. Fisher of Newgate-Market, examined and dissected, pursuant to an order of the Society. Phil. Trans. Roy. Soc. London, 36: 290-294. RIDEWOOD, W. G., 1909. A case of abnormal oviducts in Honiarus I'ttlgaris. Ann. Mag. Nat. Hist., (8) 3 (13) : 1-7. SCHAANNING, H. T. L., 1929. En eiendommelig varietet av hummer (Homants vulgaris). Staranger Mus. Aarsh.. 1925-28, pt. 5 : 1-3. TEMPLEMAN, W., 1944. Sexual dimorphism in the lobster (Homarus americanus). J. Fish. Res. Bd. Canada, 6: 228-232. TEMPLEMAN, W., 1948. Abnormalities in lobsters. Bull. Newfoundland Govt. Lab., No. 18: 3-8. TEMPLEMAN, W., AND S. N. TIBBO, 1945. Lobster investigations in Newfoundland, 1938-1941. Dept. Nat. Res. Newfoundland Res. Bull., No. 16: 1-98. RE-EXAMINATION OF AN INHIBITOR OF REGENERATION IN TUBULARIA CHANDLER FULTON The Rockefeller Institute, Nnt' }'ork, New York, and the Marine Biological Laboratory, Woods Hole, Massachusetts In the marine hydroid Tnlndaria the presence of hydranth structures has been thought to prevent the development of new hydranths in nearby stem tissue. Two preparations have been made from adult hydranths which inhibited the regeneration of new hydranths on isolated stem segments. One of these (inhibitor ^vatcr > Rose and Rose, 1941) was made by agitating adult hydranths in aerated sea water for from 12 to 24 hours, while the other (hydranth extract, Tardent, 1955) was found in the supernatants of homogenates of adult hydranths. These inhibitors of re- generation were specific to hydranth tissue in that they were not obtained when stems were treated in the same manner. They have been compared (Tweedell, 1958) and found to differ in a number of properties. The regeneration-inhibiting substances in inhibitor water have been considered by a number of authors to represent the substances normally responsible for physiological dominance in Tnlndaria, and inhibitor water has been employed by Steinberg (1954) in an experiment to indicate the mechanism of physiological dominance. In the present investigation it was found that active inhibitor water could not be prepared in the absence of bacterial growth, and as a consequence a re-examina- tion of this inhibitor was undertaken. MATERIALS AND METHODS Freshly-collected Tubular ia crocca, provided by the Supply Department of the Marine Biological Laboratory, was used in all experiments. Sea water was filtered through paper shortly before use. In preparing inhibitor water, an attempt was made to use methods comparable to those used by previous workers (cf. Tweedell, 1958). Populations of adult hydranths with 5 mm. of stem attached were isolated and washed thoroughly, and then aerated in sea water for 24 hours at 17-22 C. After aeration, the hydranths and debris were removed by filtration and the preparation was tested for its effect on regeneration. The bacterial population was estimated subjectively in early experiments by turbidity and microscopical examination, and in later experiments was determined using a Petroff-Hauser bacteria-counting slide. The bacterial population of filtered sea water was found to be approximately 10 r> per ml., which is too low for accurate estimation with a counting slide. It was assumed that no bacterial proliferation had occurred during the preparation of any given solution if the bacterial population did not exceed this order of magnitude. It should be cautioned that if mature male hydranths are used to prepare inhibitor water, turbidity may in part result from the release of large numbers of sperm into the water. 232 TUBULARIA REGENERATION INHIBITOR 233 When it was desired to remove bacteria, the preparations were filtered through an HA millipore filter (Millipore Filter Corp., Watertown, Mass.) or centrifuged for 5 minutes at 30,000 g. Bacterial growth was prevented by the addition of antibiotics. Penicillin and streptomycin were used at 100-125 ju,g./ml. ; sulfadiazine was used at about 0.001 per cent (or saturation in sea water). At these con- centrations, and in the cases of penicillin and streptomycin even at four-fold higher concentrations, the antibiotics did not have any significant effect on the rate or course of regeneration. The solutions to be described were tested immediately after preparation for their effect on the regeneration of freshly-cut, 7-mm. stem segments. Virtually all of the stems in control groups regenerated, although as is usual with Tubularia there was a considerable variation in the rate, even within a single group. A preparation was considered to have inhibited regeneration if, during the time required for the complete regeneration of the controls (emergence), all or a significant fraction of the experimental group either disintegrated or healed but did not begin re- generation. Stems in inhibitor water which regenerated were usually but not always retarded. RESULTS Populations of 1.5-2 hydranths per ml. aerated in sea water regularly pro- duced an inhibitor water which completely prevented regeneration. Occasional batches of inhibitor water prepared at these or lower hydranth densities were in- active, while populations of more than two hydranths per ml. usually gave a preparation which caused disintegration of the stem tissue. However, considerable variability was found in the activity of preparations made at the same hydranth densities and under the same conditions (temperature, time, etc.), suggesting that some factor other than those controlled was involved. A number of observations suggested that the activity of inhibitor water was due to bacterial growth. Preparations became quite turbid during the course of aeration, and the condition of the hydranths deteriorated rapidly. The solution developed a putrid odor. Hydranths killed by exposure to 30 C. for 15 minutes rapidly disintegrated, but nevertheless produced active inhibitor water. When active preparations were examined microscopically, a large, heterogeneous popula- tion of bacteria was found. Removal of these bacteria often resulted in a reduction but never in an elimination of the inhibitory activity of a preparation. An estimate of the amount of bacterial growth which occurs in inhibitor water preparations was obtained by preparing a series of 5 inhibitor waters at a density of 1.5 hydranths per ml. and making counts with a bacteria counting slide at the beginning and end of aeration. In this series, the bacterial density increased during aeration from about 3 X 10 5 bacteria per ml. to about 10 8 bacteria per ml. Bacteria were removed by centrifugation and each preparation tested for its effect on the regeneration of 10 stems. The results are given in Table I. The increase in bacterial number represents a minimum of 9 generations of bacterial growth. It should be noted, however, that counts made at the beginning of aeration do not include bacteria which are present in the hydranths and are released into the water during aeration as the hydranths disintegrate. In order to determine whether or not hydranths could produce active inhibitor water in the absence of bacterial growth, hydranths were agitated in sea water 234 CHANDLER FULTON containing antibiotics at concentrations sufficient to maintain bacteriostasis. The results of three of the experiments with penicillin and streptomycin are given in Table II. In the first two experiments shown (A and B), the amount of bacterial growth was estimated subjectively. No detectable bacterial growth occurred in any of the preparations in experiment A, and in spite of the fact that the hydranth density was more than twice that necessary to produce complete inhibition in the absence of antibiotics, the stems in both experimental groups all regenerated at the same rate as the control stems. In experiment B, the hydranth density was over four times that necessary to produce complete inhibition without antibiotics. The preparation agitated at this hydranth density with antibiotics (B4) produced some delay in the rate of regeneration. This delay may have been due to the cytolysis of some of the hydranth tissue releasing the same sub- stances present in hydranth extracts (see discussion). These experiments indicate that in the presence of penicillin and streptomycin in concentrations which suppress bacterial growth, inhibitor water cannot be col- lected. However, it may be argued (Tweedell, 1958) that the antibiotics used either prevent the hydranths from producing an inhibitor of regeneration or destroy this inhibitor as it is produced. Three observations appear to exclude these TABLE I The number of bacteria present in five similar preparations of inhibitor water and the effect of these preparations on regeneration. Observations were made at intervals for 4.3 days Preparation Control 1 2 3 4 5 Bacteria/ml. X 10" 0.0 2.3 3.3 3.7 5.5 7.4 Effect of preparation on stems 10/10 emerged within 2.4 days 10/10 did not begin regeneration 10/10 did not begin regeneration 10/10 disintegrated within 3.6 days 10/10 disintegrated within 1.4 days 10/10 disintegrated within 1.0 day alternatives. ( 1 ) That penicillin and streptomycin do not destroy the inhibitors was demonstrated by experiments in which bacteria were removed by centrifugation and these antibiotics added to inhibitor water after preparation. In one such experiment, there was no measurable reduction in the activity of the preparation when antibiotics were added (Table II, C3) ; in another (not listed) there was a slight reduction but not an elimination of the inhibition produced by the prepara- tion (similar to that observed in other preparations when they were sterile filtered). (2) Particularly important are three experiments with penicillin and streptomycin and one with sulfadiazine in which bacterial growth occurred in the preparations even though antibiotic was added at the beginning of aeration. Presumably bacteria resistant to the antibiotics used developed in these preparations. In these cases, the preparations inhibited regeneration in proportion to the amount of bacterial growth which occurred in them (e.g., Table II, C5, 6), showing that, in spite of the antibiotics, if bacterial growth occurred, an active regeneration inhibitor was produced. (3) Three antibiotics penicillin, streptomycin and sulfadiazine dif- fering greatly in chemical structure and presumed mode of action, were used alone or in pairs to maintain bacteriostasis. Regardless of which antibiotic was used, if bacterial growth was prevented the preparation failed to inhibit regeneration. TUBULARIA REGENERATION INHIBITOR 235 To see if hydranths were a necessary component of the system, experiments were done in which bacterial growth was allowed to occur in sea water in the absence of hydranths. Dilute proteose-peptone solutions in sea water, aerated for 24 hours, and then sterilized by millipore filtration followed by the addition of antibiotic, were potent inhibitors of regeneration, while control solutions in which TABLE 1 1 Selected experiments which illustrate the activity of inhibitor water prepared with penicillin and streptomycin. Bacterial density was either estimated (number represented by pluses in the table) or counted directly using a bacteria counting slide (represented by number per ml.). Abbreviations: pen., penicillin; strep., streptomycin Experiment Components added to sea water Bacteria per ml. Stems regenerated vs. total Mean time of emergence in days Al Pen. and strep. 10/10 2.5 2 4 hydranths/ml. + pen. and strep. 10/10 2.6 3 4 hydranths/ml. + pen. and strep. 10/10 2.5 Bl None 10/10 2.3 2 8 hydranths/ml. + + + 0/10 3 Pen. and strep. 10/10 2.4 4 8 hydranths/ml. + pen. and strep. 10/10 3.1 Cl None ra. 10 5 10/10 2.4 2 2 hydranths/ml. 5 X 10 s 0/10 3 2 hvdranths/ml., pen. and strep, added after aeration* 0/10 4 Pen. and strep. ; 10* 10/10 2.3 5 2 hvdranths/ml. + pen. 2 X 10' 5/10 4.8 6 2 hydranths/ml. + strep. 1 X 10 8 2/10 2.3 7 0.1% proteose peptone + pen. and strep. ca. 10 r ' 10/10 3.1 8 0.1% proteose peptone, pen. and strep, added after aeration 3 X 10 8 0/10 * Penicillin and streptomycin were added to a portion of solution C2. bacterial growth was prevented by the addition of antibiotic at the beginning of aeration, at most, slightly retarded regeneration (e.g.. Table II, C7, 8; compare Cl, 2). To make certain that the inhibition produced as a result of bacterial growth was not dependent on the presence of specific bacteria, preparations were made 236 CHANDLER FULTON using Escherichia coli. Cultures were grown in a minimal medium (Davis and Mingioli, 1950) from a small inoculum to 10 9 cells per ml. The bacteria were removed by centrifugation, and the used medium diluted 1 : 5 in sea water containing antibiotic. Such a preparation completely inhibited regeneration, while control stems placed in a 1:5 dilution of sterile minimal medium with antibiotic regenerated normally. From these data it is clear that the activity of inhibitor water can be explained on the basis of the bacterial growth which occurs in the medium, and that no other inhibitors can be collected when bacteriostasis is maintained with antibiotics. TABLE III Summary of all experiments which indicate that inhibitor water is a by-product of bacterial growth. Refer to the text for explanations of each experiment. Abbreviations: pen., penicillin; strep., streptomycin; sulfa., sulfadiazine Components added to sea water before aeration Number of experiments Bacterial growth Inhibition of regeneration None 17 Hydranths 17 + + Hydranths* 6 + + Hydranths** 2 + + Heat-killed hydranths 4 + + Pen., strep., or sulfa. 14 Hydranths + pen., strep., or both pen. and strep. 7 3 + + Hydranths + sulfa. 2 1 + + Stem lengths 2 Proteose peptone, pen. and strep. 3 Proteose peptone** 4 + + * Preparation sterile filtered after aeration. * Preparation centrifuged after aeration, penicillin and streptomycin added to the super- natant. As an argument for the specific role of hydranth structures in producing in- hibitor water it has been noted that a population of stems, aerated in sea water, does not produce an inhibitor (Tweedell, 1958). After cutting, the ends of a stem rapidly heal and secrete a thin layer of perisarc, so that very soon a cut stem is entirely covered with chitin. Since no tissue is exposed, a preparation of stems could not be expected to be a good medium for bacterial growth, and this might be TUBULARIA REGENERATION INHIBITOR 237 the reason why no inhibitor was produced. Experiments were done in which populations of clean stems were cut, washed, and aerated in sea water. Such preparations did not support the growth of significant numbers of bacteria, and, when tested on stems, permitted regeneration at the same rate as the controls. A summary of the experiments which have been described, together with the number of cases of each type, is presented in Table III. Cases in which verv slight bacterial growth occurred in the preparations or in which the preparations only produced a slight delay in regeneration (such as case B4, Table II) are recorded as negative ( ) in the table ; only definite cases of bacterial growth or regeneration inhibition are recorded as positive ( + ). As the table indicates, the inhibition of regeneration was always correlated with the growth of bacteria. It is pertinent to mention certain experiments done with the regeneration in- hibitor found in Tnbularia hydranth extracts. Such extracts were prepared by homogenizing a population of adult hydranths and collecting the supernatant, as described by Tardent (1955) and Tweedell (1958). It was found that the in- hibition of regeneration produced by such extracts was not a result of bacterial growth, in that when penicillin and streptomycin were added to the extracts to maintain bacteriostasis the activity of the extracts was not affected in terms of the proportion of stems inhibited by a given dilution of extract. It was found. however, that in contrast to the original report of Tardent (1955), the inhibition produced by Tnbularia tissue extracts was not specific to hydranth tissue. The supernatant of homogenates from equivalent quantities of stem tissue also sup- pressed the regeneration of stems. Tardent (personal communication) has ob- tained the same result recently with Tnbularia larynx. Preliminary comparisons on a wet weight basis indicate that hydranth tissue is about twice as active a source of inhibitor as stem tissue. The lack of specificity of this inhibitor makes it impossible, however, in the absence of further data, to adequately evaluate the normal physiological role of the substances involved. DISCUSSION The results of the experiments with inhibitor water may be summarized as follows. (1) Hydranths agitated in sea water produce bacterial growth and inhibitors of regeneration. (2) If bacterial growth is suppressed with antibiotics, re- generation inhibitors cannot be collected. (3) If antibotics are added at the begin- ning of aeration but bacterial growth is not prevented, inhibitors can be collected. (4) Bacterial growth in the absence of hydranths produces regeneration inhibitors. These results, together with the appropriate controls, demonstrate that inhibitor water as prepared in these experiments is a by-product of bacterial growth for which the hydranths serve as inoculum and nutrient source. The results, however, should not be taken to indicate that hydranths cannot produce any inhibitors of regeneration, but rather that inhibitor water prepared as described by previous workers contained no inhibitors which could not be accounted for as the products of bacterial rather than hydranth metabolism. If hydranths are agitated with antibiotics at densities several-fold higher than those used to prepare inhibitor water (rf. Tweedell, 1958), occasionally such preparations (e.g.. Table II, B4) retard regeneration even though bacteriostasis has been maintained with antibiotics. It is interesting to note that in such cases 238 CHANDLER FULTON bulbous outgrowths appear at one or both ends of many of the stems. These out- growths are similar to those found in stems placed in Tubularia tissue extracts (Tweedell, 1958; author's unpublished observations), suggesting that the cytolysis of some of the hydranth tissue has released the substances found in hydranth extract into the water. Since this manuscript was originally submitted for publication, a paper by Tweedell (1958) has appeared in which the results described in the present paper are discussed. The results of this work were presented incompletely by Tweedell ; the results as presented here answer the objections raised in his discussion. In particular, the possibility that the antibiotics used had significant effects other than that of maintaining bacteriostasis has been excluded by the results described above. Tweedell notes that although bacteria were removed from some of his prepara- tions by sterile filtration the preparations still inhibited regeneration. It is clear from the present work that it is not the bacteria themselves, but rather the metab- olites they release into the medium, which are primarily responsible for the activity of inhibitor water. Removal of the bacteria from inhibitor water or proteose-peptone solutions after aeration by filtration or centrifugation, or the addition of penicillin and streptomycin to such preparations, in some cases reduced the inhibitory activity of the preparation but in no case eliminated it. SUMMARY 1. Rose and Rose (1941) found that adult Tubularia hydranths agitated in sea water produced a solution, inhibitor water, which prevented regeneration. They and subsequent workers have ascribed to this inhibitor a role in normal physiological dominance. In the present investigation it has been found that considerable bacterial growth occurs in the solution during the preparation of inhibitor water by the usual methods, and that when antibiotics have been added to maintain bacteriostasis no inhibitor can be collected. Experiments have excluded the possibilities that the antibiotics used are preventing the production of the inhibitor or destroying it as it is produced. It has been shown that metabolites produced by bacterial growth in the absence of hydranths inhibit regeneration. 2. These data lead to the conclusion that inhibitor water represents the by- products of bacterial growth for which the hydranths serve as source of inoculum and as nutritive medium. LITERATURE CITED DAVIS, B. D., AXD E. S. MIXGIOLI, 1950. Mutants of Eschcrichia coli requiring methionine or vitamin B12. /. Bact., 60: 17-28. ROSE, S. M., AND F. C. ROSE, 1941. The role of a cut surface in Tubularia regeneration. Physiol. Zool., 14 : 323-343. STEINBERG, M., 1954. Studies on the mechanism of physiological dominance in Tubularia. J. Exp. Zool., 127 : 1-26. TARDENT, P., 1955. Zum Nachweis eines regenerationshemmenden Stoffes im Hydranth von Tubularia. Rev. Suisse Zool., 62: 289-294. TWEEDELL, K. S., 1958. Inhibitors of regeneration in Titbitlaria. Biol. Bull., 114: 255-269. STUDIES ON THE STRUCTURE AND PHYSIOLOGY OF THE FLIGHT MUSCLES OF BIRDS. 4. OBSERVATIONS ON THE FIBER ARCHITECTURE OF THE PECTORALIS MAJOR MUSCLE OF THE PIGEON J. C. GEORGE AND R. M. NAIK Laboratories of Comparative Anatomy and Animal Physiology, Department of Zoology, M. S. University of Baroda, Baroda, India Denny-Brown (1929), studying the red and white muscles of vertebrates, made some observations on the "light" and "dark" muscle fibers in the breast muscle of the pigeon. The later works on these two types of fibers have been reviewed by George and Naik (1957). More recently, George and Naik (1958a, 1958b) have shown that the red narrow fibers are rich in fat and mitochondria in sharp contrast to the white, broad, glycogen-loaded fibers, which contain only a negligible amount of fat and mitochondria. George and Scaria (1958a) histochemically demonstrated higher lipase activity in the red narrow fibers. The Krebs' cycle enzymes, too, seem to be localized in the narrow fibers (George and Scaria, 1958b). These findings have stimulated considerable interest and called for a basic under- standing of the nature and disposition of the fiber components of this muscle as a whole. The present study, therefore, is an attempt to provide a comprehensive picture of the pattern of fiber distribution and the nature of the metabolite load in the different regions of the muscle. MATERIALS AND METHODS In order to obtain uniformly well developed pectoralis major muscle, only fully grown wild pigeons, either shot or trapped from a single locality, were used through- out for the present study. Mapping the distribution of the two types of fibers in the muscle Due to the bipectinate arrangement of the fasciculi, it was found convenient to divide the muscle into twelve regions, each one extending to 10 mm. in length along a hypothetical line, drawn midway between the origin of the muscle fasciculi and the centrally placed tendon (as shown in Fig. 1 ) . From each of these regions at the level of the aforesaid line, fresh frozen transverse sections were cut on a freezing microtome. Subsequently the sections were treated in the following manner. Transferring a fresh frozen section into distilled water or even saline or isotonic sucrose solution resulted in uneven curling up of the section. Again, the size of the muscle piece handled being large, some difficulties which were encountered in the beginning in obtaining a good entire section, were completely avoided by transferring the section directly into chilled 50% glycerol and mounting it on a microslide in the glycerol solution. In the preparations thus made the 239 240 J. C. GEORGE AND R. M. NAIK arrangement of the fibers in the section, however large, was faithfully maintained with no distortions taking place. The glycerol-impregnated sections were thus found to he ideal to manipulate. Moreover, the sections left in glycerol solution and maintained at C. can remain for more than a week without any perceptible defect and thus could be utilized for future observations. The desired region of the mounted section was projected on the screen of a microphotographic camera at a magnification of 47 X and the photographic print- ing paper exposed directly to the image. "Normal" bromide papers were found FIGURE 1. Dorsal view of the pectoral is major muscle of the pigeon showing the hypo- thetical lines 0-120 along which the distribution of broad fibers is recorded in Figure 2. The squares A and B indicate the regions of the muscle used for studying the variation in metabolite load and the structure at different depths of the muscle. suitable. Using the sliding vernier on the stage of the microscope, continuous photographic records of the distribution of the broad fibers were made (Fig. 5). From such records by the method of random sampling, the mean value of the number of broad fibers per square mm. was determined for every mm. depth of the muscle. A survey of all the twelve regions was thus completed and a graph plotted illustrating the continuous distribution of broad fibers per square mm. at the distance of every 5 mm. along the line 0-120 (Fig. 1). The lines demarcating the areas containing 30-50. 50-70, 70-90, 90-100, 100-120 and 120-140 and 120150 broad fibers per square mm. were, drawn. The entire procedure was ARCHITECTURE OF PIGEON PECTORALIS 241 _ 10 D - R 20 30 40 D - F 50 60 O DISTANCE ALONG HYPOTHETICAL LINE (mm) 110 120 D.F. D.F. FIGURE 2. Cross-sectional view of the pcctnralis major along the line 0-120 drawn in Figure 1. The figures in the chart show the number of broad fibers per square mm. D.F., dorsal face of the muscle ; V.F., ventral face of the muscle. NUMBER OF B.F. PER mnV 200 400 600 800 NUMBER OF N.F. PER mm* FIGURE 3. Relation between the number of broad fibers and the number of narrow fibers per square mm. of transverse section of the muscle. 242 J. C. GEORGE AND R. M. NAIK repeated on the f^ectoralis of three pigeons. The results obtained are summarized in a graphical representation as shown in Figure 2. Since the individual varia- tions in the pectoralis of different pigeons are considerable, the lines demarcating different areas in the figure are not claimed to be absolute, but they do show the % AGE OF FAT 12 10 % AGE OF GLYCOGEN 3 - 6 3.2 RATIO OF 3 - AREA 12 OCCUPIED BY B.F. a9 TO THAT 0.6 OF N.F./rrm? 0.3 2 A 6 8 DEPTH OF MUSCLE (mm) 10 FIGURE 4. Variation in the percentage of glycogen and fat, in relation to the ratio of the area occupied by the broad fibers to that of the narrow fibers per square mm., at different depths of the muscle. The regions of the muscle marked A and B in Figure 1 were used. generalized pattern of the distribution of the broad fibers in the pectoralis major muscle of the pigeon. For counting the broad as well as the narrow fibers in one and the same region, the same procedure was adopted, except that the image of the section projected on the screen was magnified to about a hundred times, and the sections from the different typical regions of the muscle were used. FIGURE 5. Negative prints of the transverse section taken from the region A (Fig. 1) showing the continuous distribution of broad fibers (darker in color) at different depths of the muscle. The numbers 1-10 on microphotographs indicate the depth in mm. from the ventral to the dorsal face of the muscle. 243 244 J. C. GEORGE AND R. M. NAIK Estimation of fat and glycoycn at different depths of the muscle For the sake of convenience, the region of the muscle (marked A in Fig. 1) on the posteriormost end of the keel was used throughout. In this region the thickness of the muscle is only about 10 mm. and the variation in the distribution of the broad fibers at the different depths of the muscle is gradual. From this region A, a piece about 10 cubic mm. in size was cut out for the estimation of glycogen and a somewhat bigger piece for the estimation of fat. From a region B lateral to A, another piece was cut out and transferred to the freezing chamber of the refrigerator and used later on for studying the distribution of the broad fibers in this region by the method already described. The muscle piece cut out from region A was mounted on the stage of a freezing microtome so as to obtain horizontal sections. It was frozen hard, the outermost epimysium was peeled off with a pointed forceps or sliced off by a superficial stroke of the microtome knife, and 1-mm. thick slices of the muscle were serially cut. Since all these horizontally cut sections were of uniform and known thick- ness, each could be said to represent the nature of the muscle tissue at a known depth. The thickness of the sections was not actually measured since the micro- tome used was a brand new "Sartorius" model and all the possible precautions, such as avoiding the fluctuations in the temperature, were taken so as to obtain sections of uniform and accurate thickness. Each frozen section was immediately transferred to a weighing bottle and dehydrated. The sections to be used for the estimation of glycogen were dehydrated in a vacuum-desiccator at one atmosphere pressure and maintained at C., whereas for fat extraction, sections were de- hydrated in an air-oven at 80 C., and finally in vacuum. The dehydrated sections were weighed and their glycogen content was estimated according to the method of Kemp ct al. (1954). For the quantity of the muscle used for estimation (about 20-30 mg. per dry weight) it was found necessary to dilute the glycogen extract in the deproteinizing solution to 10 ml. The color developed was measured on the Beckman spectrophotometer (DU model) at 520 ^. For the estimation of fat the dehydrated material was ground and, after weighing, transferred to a fat-extraction thimble. The fat was extracted in the Soxhlet ap- paratus with 1:1 ethanol-ether mixture (George and Jyoti, 1955). About 70-100 mg. of dehydrated muscle were used for each estimation. The estimation of glycogen in the two types of fibers Small pieces from the breast muscle of a decapitated pigeon were cut out and dropped in previously chilled 80% methanol and left undisturbed at - - 10 C. for 24 hours. The fibers from the muscle thus preserved were teased out in methanol under a binocular dissection microscope with watch-maker's forceps. The two types of fibers were isolated and transferred to two separate containers containing methanol and fitted with air-tight glass lids and stored in the refrigerator. Suffi- cient numbers of fibers which would yield about 2-5 mg. in dry weight were iso- lated and collected for each estimation. These fibers were then removed from the methanol solution, dehydrated in vacuum and weighed on a microbalance. Glycogen was estimated, as already mentioned, by the micromethod of Kemp et al, (1954). ARCHITECTURE OF PIGEON PECTORALIS 245 RESULTS Figure 5 presents a typical picture of the distribution of broad fibers in the muscle. In each fasciculus the broad fibers are mainly concentrated towards the periphery. This pattern is maintained throughout the muscle. In regions of the muscle where there are larger numbers of broad fibers or lesser numbers of narrow fibers, the fasciculi have a smaller cross-sectional area with broad fibers closely packed along their borders without any intervening narrow fibers. The number of broad fibers per square mm. in the different regions of the muscle is shown in Figure 2. The relation of the number of broad fibers to that of the narrow ones per square mm. is shown in Figure 3. From both these, the number of broad fibers, as well as the number of narrow fibers per square mm., in any region of the muscle could be approximately determined. The variation in the metabolite load and the number of broad fibers per square mm. at different depths of the muscle are indicated in Table I, while in Figure 3 the same data are utilized to show the relation between the structure of the muscle TABLK I The number of broad fibers per square //. and the percentage of fat and g/ycogen at different depths of the breast muscle of the pigeon. (The portion of the muscle marked A in Fig. 1 was used. The figures indicate the average values of six sets of readings) Depth of the muscle in rnin. (starting from the ventral face) Number of broad fibers per square mm. S.D. Percentage per dry weight of the muscle S.D. Glycogen Fat 0-2 90 14 3.655 0.275 10.289 1.942 2-4 63 8 .U75 0.054 12.095 1.056 4-6 48 3 3.102 0.127 14.632 1.752 6-8 51 4 3.409 0.184 13.250 0.571 8-10 72 9 3.588 0.236 11.743 0.572 and the metabolite load. The number of narrow fibers for the corresponding number of broad fibers was calculated by using the formula of the regression line in Figure 3 and the ratio of the area occupied by the broad fibers to that of the narrow fibers in square mm. was determined by using the mean value of the diameter of these fibers. The diameter of the broad fibers is 69.00 14.00 /j. ( 1000) and that of the narrow fibers is 30.11 6.56 p (2000). The figures given in parentheses indicate the number of fibers measured from the fresh frozen sections taken from the various regions of the muscle. The values of the glycogen content of the broad and narrow fibers, calculated on the dry weight of the muscle preserved in methanol, are, respectively, 10.240 0.093// and 2.464 0.311% (each value is the mean of three readings). Methanol removes much of the fat (mainly from the narrow fibers) and some of the amino acids. DISCUSSION It has been known that in many active muscles, the muscle fibers towards the periphery become larger in diameter and lighter in color, compared to those in 246 J. C. GEORGE AND R. M. NAIK the interior. In such muscles even in the individual fasciculus, the light fibers are situated towards the periphery. In the pigeon breast muscle, the white broad fibers and the red narrow fibers show a somewhat similar distribution pattern but these fibers differ from the light and dark fibers of the other muscles in that they are sharply differentiated into two distinct types without any intermediate forms. The broad fibers are glycogen-loaded and poor in fat inclusions and mitochondria, whereas the narrow fibers are fat-loaded and have a high mitochondria! content and are poor in glycogen (George and Naik, 1958a, 1958b). In a single muscle uneven distribution of metabolites has been long since realized. To reduce such localized variation to the minimum, customarily a large piece of muscle is utilized for the estimation of metabolites. Present work shows that in a muscle like the pectoraHs major of pigeon having heterogeneous cellular elements, variation in metabolites in the different regions of the same muscle and even in a single fasciculus is quite large. Needless to say, what applies to glycogen and fat might equally apply to other chemical constituents in which the two types of fibers differ. A general belief that the muscle fibers towards the periphery of the muscle are more active than those in the interior and, due to higher activity, increase in diameter, does not seem to hold good, at least in the case of the pcctoralis of pigeon. Undoubtedly, the red fibers of pigeon breast muscle, due to their re- markably well developed enzyme systems, play a major role in effecting the sus- tained contractions of the muscle. In white fibers, on the other hand, the oxidative processes are not developed or developed only to a negligible extent, in that the dehydrogenase activity in these fibers, as shown by histochemical method, is neg- ligible or nil (George and Scaria, 1958b). All the same, the white filters are not inactive elements of the pigeon breast muscle. In the normal animal they show no signs of atrophy. A glycerinated white fiber of pigeon breast muscle contracts in the same manner as a glycerinated red fiber of the same muscle on the addition of ATP. The study on the reactions of these two types of fibers to experimentally induced disuse atrophy has yielded significant results. When the movement of the humerus is restricted for three months by a plaster cast, the white fibers in the deeper layer of the muscle show acute sign of atrophy whereas the red fibers appear practically unaffected (George and Naik, unpublished data). These find- ings suggest the possibility of some differences in the mechanical properties of the two types of the fibers and in that case some physical factors may underlie the distribution pattern of the two types of fibers in the muscle. Denny-Brown (1954), has shown that a single nerve in the breast muscle of pigeon can innervate both, the red as well as the white fibers. Since the activity of these muscle fibers must be conditioned by the fundamentally different chemical system in them, it is difficult to believe that the amount and the mode of activity performed by these two types of fibers are the same. In what exact manner the white fibers contribute to the activity of the muscle is far from clear and as a prelude to such an understanding, an extensive study of these fibers is essential. For such a study Figure 2 can be a useful guide. Moreover, the method used in the present work to study the variation in the metabolite load in relation to the variation in the fiber make-up of the muscle, can be used for studying the distribu- tion of various constituents such as enzymes, amino acids and minerals in the muscle. ARCHITECTURE OF PIGEON PECTORALIS 247 \Ye are grateful to the members of the staff and the technicians of the Depart- ments of Chemistry and Statistics, Faculty of Science, Baroda, for their unfailing assistance in completing this work. One of us (R. M. N.) is indebted to the Ministry of Education, Government of India, for the award of a Senior Research Scholarship. SUMMARY 1. The relative distribution pattern of the red and white muscle fibers in the breast muscle of the pigeon is studied. 2. There exists a direct relation between the distribution of metabolites and that of the two types of fibers in the different regions of the muscle. 3. Quantitative estimation of glycogen in the two types of filters confirms the higher concentration of glycogen in the white fibers. LITERATURE CITED DEX \v-BRO\vx, D., 1929. The histological features of striped muscle in relation to its func- tional activity. Proc. Roy. Soc. London. Ser. B, 104: 371-411. DEXXV-BRUWX, D., 1954. As cited by Adams, R. D., ct al. in Diseases of Muscle. Paul B. Hoeber, Inc., New York ; pp. 38 and 40. GEORGE, J. C., AXD D. JVOTI, 1955. The lipid content and its reduction in the muscle and hver of birds and bat during long and sustained activity. /. Aniin. Morph. Phvsiol., 2: 38-45. GEORGE, J. C., AXD R. M. NAIK, 1957. Studies on the structure and physiology of the flight muscles of birds. 1. The variations in the structure of the pectoral is major muscle of a few representative types and their significance in the respective modes of flight. /. Anim. Morph. Pliysiol., 4: 23-32. GEORGE, J. C., AXD R. M. NAIK, 1958a. The relative distribution and the chemical nature of the fuel store of the two types of fibres in the pectoralis major muscle of the pigeon. Nature, 181 : 709-710. GEORGE, J. C., AXD R. M. NAIK, 1958b. Relative distribution of the mitochondria in the two types of fibres in the pectoralis major muscle of the pigeon. Nature. 181 : 782-783. GEORGE, J. C., AXD K. S. SCARIA, 1958a. Histochemical demonstration of lipase activity in the pectoralis major muscle of the pigeon. Nature, 181 : 783. GEORGE, J. C., AXD K. S. SCARIA. 1958b. A histochemical study of the dehydrogenase activity in the pectoralis major muscle of the pigeon and certain other vertebrate skeletal muscles. Quart. J. Micro. Sei. Cm press). KEMP, A., J. M. ADRIEXXE AXD KITS VAX HEIJXIGEX, 1954. A colorimetric method for the determination of glycogen in tissues. Biochcin. J.. 56: 646. THE EFFECT OF OSMOTIC STRESS ON THE IONIC EXCHANGE OF A SHORE CRAB WARREN J. GROSS Division of Life Sciences, University of California, Riverside, California The decapod Crustacea have received considerable attention with regard to their ability to regulate the inorganic ions of their blood (Krogh, 1939; Robertson. 1949, 1953, 1957; Prosser ct al, 1950). Prosser ct al. (1955) studied responses of the shore crab Pachygrapsus crassipes to different concentrations of sea water. The chief concern of their study was to determine the changes in the ionic con- centrations of blood and urine which were effected by altering the concentration of the external medium from normal. Determinations on the total losses and gains of the respective ions between animal and medium were not made nor were the effects of desiccation on ion concentrations in urine or blood determined. This in- formation would be of special interest in the case of a semi-terrestrial crab such as Pachygrapsus. Gross (1958) demonstrated that when Pachygrapsus crassipes was placed under osmotic stress, the principal exchanges of potassium were between the medium and a source of potassium other than the blood, not mainly between blood and ex- ternal medium. Also, evidence was produced that an extra-vascular pool partici- pates in sodium exchanges between crab and medium. This paper will produce further evidence that extra-vascular salt pools in Pachygrapsus contribute to ionic exchanges with the medium, special attention being paid to calcium and magnesium. The effects of desiccation on the ionic concentration of urine and blood in Pachy- grapsus will be revealed and data confirming the findings of Prosser ct al. (1955) will be produced. MATERIAL AND METHODS The subject of this investigation, Pachygrapsus crassipes Randall, was collected at Laguna and Dana Point, California. All specimens were between molts, and were mature, none being smaller than 20 gm. Urine was sampled by inserting a micropipette into the excretory pores. Blood was obtained by puncturing the cuticle of the leg joints with a micropipette. Sodium and potassium were measured by means of a Beckman flame photometer. Urine and blood were measured and diluted appropriately before being used directly in the flame photometer (Gross, 1958). Samples as small as 0.05 ml. thus could be analyzed to an accuracy of 2R 7 fi} 80 25 Medium change (mEq./l.) I- Blood change (mEq./l.) S(S 4i ?0 1 00 70 24 Medium change (mEq./l.) Pn Blood change (mEq./l.)** 03 2 77 7 t n 78 66 78 i^a Medium change (mEq./l.) ~\l\rr Blood change (mEq./l.)** n QC XO 71 fi-i -10 1 () Mg Medium change (mEq./l.) * Change in medium for all ions is corrected to a volume equal to the weight of the crab. ** Blood change for calcium and magnesium equals the difference between mean of normal crabs and the observed blood concentration after treatment for each crab. Medium change is the observed concentration change in the medium after treatment for each crab. Analyses of blood potassium and sodium were made before and after desiccation on individual crabs. RESULTS Table I presents the urine and blood concentrations of sodium, potassium, calcium, and magnesium after the following treatments: a) immersion in normal sea water; b) immersion in 50% sea water; c) immersion in 150% sea water and d) desiccation for a water loss of about 7% body weight. Comparing the blood values after immersion in 100% sea water with those of Prosser et al. (1955),. sodium and calcium appear in agreement. However, the potassium (7.43 mEq./l.) and magnesium (20.0 mEq./l.) values are considerably less than those reported by the above workers (12.1 mEq./l. and 58.4 mEq./l., respectively). On the other hand Schlatter (1941) reported blood ion concentrations for this same species which agree closely with the values of the present investigation. It should be emphasized that the indicated stress media (Table I) represent only the initial sea water concentrations, and that these necessarily were altered by exchanges of salts with the animal. However, an accurate knowledge of the sustained osmotic gradient and the final blood concentrations is of little meaning in this investigation, since as described above, the animals were able to raise them- IONIC EXCHANGES IN A CRAB 251 selves out of the water. The main objectives of this study are to demonstrate : 1) the degree to which a blood ion change is reflected in the external medium and 2) the role of the antennary glands in controlling the ion content of the animal. It also should be pointed out that in this crab alterations in the blood concentration in aqueous media are effected by salt exchanges, not water (Gross, 1957). Data in Table I, however, do reveal something of the ability of Pachygrapsus to regulate ions in the different sea water concentrations. Thus blood sodium is held above the sodium concentration of the dilute medium and normal sea water, but below the concentration of the hypertonic medium. Blood potassium is held above the concentration of the dilute medium, but below the concentration of normal sea water or the concentrated medium. Gross (1958) reported that when Pachv- grapsus was immersed in a small volume of 50% sea water, the blood potassium remained less concentrated than the medium potassium. However, these animals were immersed for longer periods than those reported in the present studies (Table I) during which time the animal lost more potassium and the medium gained potassium. Table I also show r s that the blood calcium remains more concentrated than the medium calcium for all treatments. Blood magnesium, on the other hand, is less concentrated than the medium magnesium for all aqueous conditions. All four ions increase under conditions of desiccation. The ratios, urine concentration/blood concentration (U/B ratio), for each respective ion suggest the role of the antennary glands in the ion regulatory mech- anism. Values in Table T are means of U/B ratios observed in individual speci- mens, not ratios of means. Thus all the mean U/B ratios for sodium are less than one, indicating that the antennary glands do not regulate sodium under this set of conditions. That is, sodium is not eliminated effectively \vhen the gradient between blood and medium favors a gain ; nor is it conserved effectively when the gradient favors a loss to the medium (mean U/B ratio in 50% sea water == 0.96). With respect to potassium the mean U/B ratio is less than one when the crab is immersed in 100% or 150% sea water. Thus the antennary gland does not regulate potassium for this set of conditions. In 50% sea water the mean U/B ratio is 1.45 which means, if anything, potassium is being wasted when it is needed. However, for conditions of desiccation the mean U/B ratio is 1 .34 which is signifi- cantly greater than one. P < 0.01. If then there were sufficient production of urine under conditions of desiccation, the antennary glands would tend to keep the blood concentration of potassium at a normal level. ^'ith respect to calcium the mean U/B ratios for crabs immersed in 50% sea water or subjected to desiccation are not significantly different from unity. Thus the antennary glands are ineffective as regulators of calcium for these two con- ditions. On the other hand, after immersion in 150% sea water the mean U/B ratio is 1.32 which is significantly different from one, P < 0.01. In normal sea water the U/B ratio is 1.17, again being significantly greater than one, P < 0.01. Thus, the antennary glands might have a small role in regulating calcium, but in no sense as large a role as they have for magnesium. Data in Table I demonstrate that the mean U/B ratios for magnesium under all conditions studied are much greater than unity. Even after immersion in 50% sea water, the mean ratio is 5.62. However, it should be pointed out that even in this diluted sea water the gradient betw r een blood and external medium favors the 252 WARREN J. GROSS uptake of magnesium. Also, it will he noted that the mean ratio under conditions of desiccation is 23.6 which suggests that the urine concentration depends on the blood concentration, not entirely on the rate of influx from the external medium. The data presented in Table I concerning the treatments in aqueous media are qualitatively in general agreement with the findings of Prosser ct a I. (1955), particularly with regard to the role of antennary glands in the regulation of magnesium. Quantitatively the data presented in Table I differ somewhat from those reported by Prosser ct al. ( 1955). However, precise comparison should not be attempted because of differences in experimental procedure. For example, crabs of the present investigation were immersed directly in small volumes of stress media for a maximum of about 24 hours. The data presented by the above workers were obtained on animals gradually acclimated to osmotic stresses for a period of at least 5 days in relatively large volumes of media. On the other hand there are certain differences which warrant attention. Normal blood potassium and magnesium differences already have been mentioned above. It will be observed that blood calcium after immersion of the animal in 50% sea water ( 34.S mEq./l.) is higher than it is for animals from normal sea water (29.6 mEq. /I.). These means are significantly different ; P =0.01. Prosser ct ul. (1955) showed decreases in blood calcium in 50% sea water which, of course, would be expected. It was thought that perhaps the increased blood calcium resulting from immersion in dilute sea water was an effect of the small volume of medium. Therefore, blood calcium of crabs immersed in large volumes (about 700 ml.) of 50% sea water for 24 hours was determined. The mean blood calcium of 24 crabs thus treated was 30.9 mEq./l., S.D. = 9.0. This is not significantly different from the mean (34.8) obtained by the other treatment; nor is it signfi- cantly different from the average blood calcium of normal crabs. These workers also called attention to the inverse relationship between the urine sodium con- centration and the blood sodium concentration. That is, the urine sodium of animals immersed in concentrated sea water was less concentrated than that of animals immersed in normal sea water, which in turn was less concentrated than that of animals immersed in dilute sea water. The means for urine sodium after treatment in the three aqueous media (Table I) cannot be shown to be significantly different, but the U/B ratios do suggest the same phenomenon. That is, the ratios decrease as the animal is placed in increasing concentrations of sea water. These ratios are all significantly different from each other; P < 0.01. The U/B ratio for the desiccated crabs is not significantly different from the U/B ratio in crabs exposed to concentrated media, but is significantly different from the ratios ob- tained for crabs given the other treatments; P < 0.01. Data in Table II demonstrate the ionic changes that occur in the medium when a given change in the blood is effected. The measurement of calcium exchanges with stress media was complicated by the fact that this ion is lost in significant amounts when the animal is immersed in normal sea water. Such was not the case for the other ions. It became necessary, therefore, to apply a correction to the calcium exchanges, based on an average loss to normal sea water by 30 crabs. This amounted to 0.5 mEq./l. per gram of crab for a 24-hour period in 50 ml. of medium. It was thus necessary to assume that this normal loss is constant in all concentrations of sea water, an assumption which subjects the values for calcium change in the medium to considerable error. IONIC EXCHANGES .IN A CRAB 253 The values for sodium and potassium have been reported previously (Gross, 1958) and represent means of the ratios, blood change (mEq./l.) /medium change (mEq./l.), in individual crabs where the blood change is the difference between the concentration before treatment and the concentration after treatment. For cal- cium and magnesium the values in Table II also represent means of the ratios, blood change (mEq./l.) /medium change (mEq./l.), in individual crabs, but since only one sample of blood could be extracted from single specimens for calcium and magnesium determinations, the blood change (mEq./l.) in the ratio for calcium and magnesium equals the difference between the observed blood concentration after treatment and the average blood concentration for crabs from normal sea water. With respect to sodium, the mean ratios are greater than 2.5 in both 50% and 150% sea water. The response to hypertonic stress and hypotonic stress seems to be symmetrical. \Yith respect to potassium the ratio is unity or less ; while it is 0.56 for crabs immersed in 50 r /c sea water, it is 1.00 for crabs immersed in 150% sea water. However when ion exchanges were measured in crabs transferred from 50% to 150% sea water or vice versa, a symmetrical response for potassium TABLE 1 1 1 Ion in c reuse in blood caused by desiccation Mean change in concentra- No. crabs tion (% original) per 1% body weight loss S.D. by evaporation \a 84 + 2.20 0.71 K 50 + 8.68 11.75 Ca 34 +5.47 4.23 Mg 35 +3.87 9.42 exchanges is observed, the mean ratio, change in blood (mEq./l.) /change in medium (mEq./l.), being about unity in both extreme stresses (Gross, 1958). The mean ratio for calcium and magnesium is less than one for all treatments. Attention should be called to the large variance for the calcium ratio, following immersion in 50% sea water. It also should be mentioned that the ratio, mean + 5.2 blood change (mEq./l.) /mean medium change (mEq./l.), is - -, ^ -- 2.87, the signs of the numerator and denominator being opposite to expectation. Not only does the average value for the blood calcium increase after treatment in dilute sea water, but the medium apparently loses rather than gains calcium. The difference between the mean of the ratios (0.93) and the ratio of the means (2.87) can be explained on the basis of the large variance. Table III reveals ionic changes that occur in the blood when Pachygrapsus is desiccated for a loss of about 7% body weight. The sodium and potassium values, again, have been reported previously (Gross, 1958) and represent averages of changes in individual crabs, where the blood concentration change was determined by before- and after-treatment readings on the same individual. The values for calcium and magnesium are means of blood concentration changes for individual 254 WARREN J. GROSS crabs, but since only after-treatment blood samples were taken, the blood change for these two ions is represented by the difference between the observed concentra- tion in an animal following desiccation and the mean blood concentration of the respective ions in crabs from normal sea water. In Table III it can be seen that the average change for sodium is less than the values for the other ions. While the potassium and calcium changes are significantly greater than the sodium change, P C 0.001, the mean magnesium change cannot be considered significantly different from the sodium change. It will be explained below that blood ions which increase more in concentration than blood sodium probably shift from a salt pool (perhaps the intra-cellular space) into the blood when the animal is desiccated. DISCUSSION The ratios, blood change (mEq./l.) /medium change (mEq./L), presented in Table II suggest that the principal exchanges of potassium, calcium, and mag- nesium between animal and medium are not ultimately between blood and external medium. A ratio of unity means that the concentration change in an external medium which is equal in volume to the animal is identical to the concentration change in the blood. Of course, much of the animal's volume is isolated from the osmotic and ionic processes which occur in the blood. Thus for a ratio of unity, the actual loss or gain of ions with the medium would be greater than the loss or gain of ions in the blood. Therefore a source other than the blood must be con- tributing to these exchanges. These ratios also can be expressed as "apparent volume of distribution," using the equation F - M/P X 100 (Gross, 1958) where: V "apparent volume of distribution" in % body weight; weight of medium M = P = weight of animal change in blood ion concentration (mEq./l.) change in medium ion concentration (mEq./l.)' Thus, the "apparent volume of distribution" for sodium is 38.5% body weight and for potassium, calcium and magnesium more than 100% body weight, which only can be interpreted as an aggregation of these three ions in some sort of pool where they are much more concentrated than they are in the blood. This also means that the extra-vascular pools ultimately contribute more to potassium, calcium and magnesium exchanges with the medium than does the original blood supply (more than twice as much). At least, in the case of potassium, the pool probably lies mainly in the intra-cellular space, because it is well known that intra-cellular potassium concentrations are high. In the crab Carcinus the relative muscle con- centrations of sodium, potassium, calcium and magnesium are 50, 120, 11 and 32 (mEq./kg. water), respectively (Shaw, 1955). If this were representative of intra-cellular concentrations, it would seem unlikely that the intra-cellular space harbors the pool for magnesium and calcium. Although the nature of the pools is unknown, it becomes apparent that a change of a blood ion concentration can occur without a loss or gain in the medium. Or exchanges between animal and medium can occur without being reflected in the blood. The probable exception to IONIC EXCHANGES IN A CRAB 255 this is sodium. The "apparent volume of distrihution" for sodium was calculated to be 38.5% body weight for the moderate stresses of 50% and 150% sea water. Webb (1940) estimates the blood volume of the crab Carcinus as 36% body weight. Thus the calculated volume, 38.5% body weight, which seems close to a reasonable value for blood space, means that the major sodium exchanges are between the blood and external medium. Though there is evidence that a sodium pool contributes to such exchanges when the animal is exposed to extreme osmotic stress, its role is relatively small percentage-wise, compared with the other ions (Gross, 1958). On the other hand sodium contributes about half the ions of the blood ; thus the small percentage effect of a sodium pool would nevertheless affect significantly the total osmotic pressure of the blood. Burger (1957) immersed lobsters in media of abnormally high magnesium con- centrations and noted that neither the blood nor the urine magnesium elevated. On this evidence he concluded that the animal was impermeable to magnesium. However, he did not consider the possibility that the magnesium could enter the animal and be fixed outside of the vascular system, a phenomenon which obviously occurs in Pachygrapsus. The variance for the mean of the calcium ratios, blood change/medium change, when the stress was 50% sea water is high. Nevertheless this ratio for calcium (0.93) is significantly less than the mean ratio for sodium (2.56), F < .025. It should be emphasized that the mean blood calcium after immersion in 50% sea water was more concentrated than that for crabs from normal sea water. Also, the corrected average change for calcium in the medium indicated a loss rather than the expected gain. Now, it was revealed above that crabs in normal sea water tend to lose calcium, and the average loss in normal sea water was applied as a correction to the medium measurements, assuming that a loss of calcium (probably by way of the gut) would be the same in a stress as in a normal medium, but if there were a curtailment of normal calcium output in dilute sea w^ater, then the correction would be too large and falsely could make the sign of the change in the medium negative. It should be mentioned that the observed changes in the medium without correction were all positive. If the sign of the corrected medium change is in error, then the increase in the blood calcium concentration after im- mersion in 50% sea water could be caused only by contributions from a calcium reservoir. Data in Table III demonstrate that for a given weight loss by evaporation the average increase in the blood sodium concentration is less percentage-wise than the increase for the other ions. It was concluded by Gross (1958) that such a differ- ence in increase between sodium and potassium under conditions of desiccation could not be explained on the basis of sodium exclusion from the blood. Rather, it was concluded that it represented a shift of potassium ions from extra-vascular spaces into the blood space. Data for calcium presented in Table III suggest that the same phenomenon happens in the case of this ion ; values for magnesium are questionable. No adaptive significance can be assigned to such a phenomenon ; rather it is interpreted as a physiological failure which imposes a limitation on the terrestrial habits of this crab. The U/B ratios presented in Table I suggest the role of the antennary gland as an, ion regulator. It has been established previously (Prosser ct ai, 1955) 256 WARREN J. GROSS that this organ is ineffective as an osmotic regulator. Thus, it seems probable that a principal function of the antennary gland is the regulation of magnesium. That is, the U/B ratio with respect to magnesium is much greater than unity. Yet the effectiveness of the antennary glands as magnesium regulators for each ex- perimental condition cannot be known for certain until the volume of urine pro- duction is known for each osmotic situation. Thus, even though the urine mag- nesium is high when the animal is desiccated, it is possible that little or no urine is produced when the animal is removed from an aqueous medium. Nevertheless, the antennary glands may effectively remove magnesium ions from the blood, thus tending to keep the blood levels normal, even though no ions are ejected from the animal. These studies were aided by a contract between the Office of Naval Research, Department of the Navy and the University of California, NR 104-309. I wish to thank Mr. David Allison for his able technical assistance. Also I wish to express my gratitude to all those students who assisted in collecting the experimental animals ; to Professor Theodore Holmes Bullock for reading the manuscript ; to Professor Timothy Prout for his advice concerning the statistical handling of the data and to Dr. Frank Bingham for suggesting the method for the calcium and magnesium determinations. SUMMARY 1. The effects of osmotic stress on the ion concentration in the blood of the crab, Pachygrapsus crassipes, were investigated. Stresses imposed were 50% sea water, 150% sea water and desiccation to a water loss of about 7% body weight. 2. The observed ratios, blood change (mEq./l.) /medium change (mEq./l.), for sodium, potassium, calcium and magnesium after the crab was transferred from normal sea water to 50% or 150% sea water yielded values for "apparent volume of distribution." The average value for sodium was 38.5% body weight, but for the other three ions w r as at least 100% body weight. 3. The large values for "apparent volume of distribution" in the cases of potas- sium, calcium and magnesium indicate that these ions are contained in extra- vascular pools in greater concentrations than they are in the blood and that these pools participate in ion exchanges between animal and medium. Thus, a con- centration change can occur in the blood without being reflected in the medium or vice versa. 4. Calcium is lost to the medium by PacJiygrapsits when it is immersed in normal sea water. Blood calcium increases when a crab is transferred from normal sea water to dilute sea water. 5. When Pachygrapsus is desiccated, the blood concentrations of potassium, calcium and magnesium average greater increases than does the sodium concentra- tion. This suggests that potassium, calcium and possibly magnesium shift from an extra- vascular pool into the blood space. The phenomenon is interpreted as a physiological failure and a factor which may limit the terrestrial life of this species. 6. The ratio, urine concentration (mEq./l.) /blood concentration (mEq./L), for the respective ions suggests the role of the antennary glands as ion regulators IONIC EXCHANGES IN A CRAB 257 tinder the various stress conditions. Thus the antennary glands were found to he relatively ineffective as regulators of sodium, potassium and calcium for all conditions studied. The U/B ratio for magnesium averaged 5.62 when the crab was immersed in 50% sea water; 13.6 for normal sea water; 15.4 for 150% sea water and 23.6 when the crab was desiccated. These high ratios suggest that a principal role of the antennary glands is magnesium regulation. 7 '. The volumes of urine production which have not been measured must be known before the effectiveness of the antennary glands as magnesium regulators can be determined. LITERATURE CITED BURGER, I. YY., 1957. The general form of excretion in the lobster Homanis. Biol. Bull., 113: 207-223. GROSS, W. J., 1957. An analysis of response to osmotic stress in selected decapod Crustacea. Biol. Bull., 112: 43-62. GROSS, W. J., 1958. Potassium and sodium regulation in an intertidal crab. Biol. Bull.. 114: 334-347. KNIGHT, A. G., 1951. Estimation of calcium in water. Chemistry and Industry, 1951, 1141. KROGH, A., 1939. Osmotic Regulation in Aquatic Animals. Cambridge at the University Press. PROSSER, C. L., D. W. BISHOP, F. A. BROWN, JR., T. L. JAHN AND V. WULFF, 1950. Com- parative Animal Physiology. W. B. Saunders Co., Philadelphia. PROSSER, C. L., J. W. GREEN AND T. S. CHOW, 1955. Ionic and osmotic concentrations in blood and urine of Pachygrapsus crassipes acclimated to different salinities. Biol. Bull.. 109: 99-107. ROBERTSON, J. D., 1949. Ionic regulation in some marine invertebrates. /. .r/>. Biol., 26: 182-200. ROBERTSON, J. D., 1953. Further studies on the ionic regulation in marine invertebrates. /. Ex p. Biol., 30: 277-296. ROBERTSON, J. D., 1957. Osmotic and ionic regulation in aquatic invertebrates. Recent Ad- vances in Invertebrate Physiology. University of Oregon Publications, pp. 229-246. SCHLATTER, M. J., 1941. Analyses of the blood serum of Cambants clarkii, Pachygrapsus crassipes and Paiiulinis interruptus. J . Cell. Coinf*. Physiol., 17 : 259-261. SCHWARZENBACH, G., W. BlEDERMANN AND F. BANGERTER, 1946. KompleXOllC VI. NeUC einfache Titriermethoden zur Bestimmung der Wasserharte. Hch'. Chini. Acta. 29: 811-818. SHAW, J., 1955. Ionic regulation in the muscle fibres of Carciuus niacnas. II. The effect of reduced blood concentration. /. Ex p. Biol.. 32 : 664-680. WEBB, D. A., 1940. Ionic regulation in Carcimts inaenas. Proc. Roy. Soc. London, Scries B. 129: 107-136. HISTOLOGY AND METABOLISM OF FROZEN INTERTIDAL ANIMALS x JOHN KANWISHER Woods Hole Occanografihic Institution, Woods Hole, Massachusetts Many invertebrate animals are normally exposed to environmental temperatures far below the freezing point of their body fluids. Although supercooling may sometimes be a factor in survival (Salt, 1950; Ditman et al., 1942 ; Scholander et al., 1953), freezing occurs in nature among insects (Asahina ct al., 1954; Scholander et al., 1953), shore animals (Kanwisher, 1955), and other groups (Luyet and Gehenio, 1940.) On the shore during winter, for example, freezing and thawing occurs twice a day when the animals are exposed to the cold air by the tide. Intertidal animals in the Arctic may be frozen for as long as 6 months (Kanwisher, 1955). The survival of these animals depends on their being able to have most of their body water turned to ice. It is remarkable that no injury is produced in a living system when more than half of its bulk is changed to a crystalline solid. I am reporting here some investigations on the histology and metabolism of these intertidal animals. HISTOLOGY In the freeze-drying histological technique, tissue is cooled very quickly with liquid nitrogen. Freezing occurs so fast that ice crystals do not have time to grow very large and cellular organization is very little disturbed. The water is removed by vacuum while the sample is kept cold. The resulting dehydrated tissue matrix is imbedded, sectioned, and stained in a conventional manner. I have used the method here to capture the situation in tissue from shore animals frozen to relatively mild natural temperatures. Comparison with material from unfrozen animals has shown the distortions caused by the freezing. Animals were collected from the shore at Woods Hole in January and moved to a --10 cold room without thawing. Sections of tissue about 1 mm. thick were cut with a cold knife, held with cold tweezers, and plunged into a vial of isopentane suspended in a container of liquid nitrogen. The isopentane allows a faster heat transfer because it does not boil and form an insulating gas layer. The hard frozen samples were quickly transferred to the already cold dehydrating chamber and vacuum applied for 24 hours at about -45. The dehydrated tissue was then imbedded in de-gassed paraffin already in the chamber with the vacuum still applied. Photomicrographs of 10-micron sections are shown in Figure 1. The unfrozen controls were tissue taken from identical animals that had thawed at room tem- perature for an hour. 1 Contribution Number 1013 from the Woods Hole Oceanographic Institution. This study was aided by a contract between the Office of Naval Research and the Arctic Institute of North America. 258 FROZEN INTERTIDAL ANIMALS 259 B D FIGURE 1. Photomicrographs of unfrozen and frozen tissue. Figure 1, A is the unfrozen foot of the shore snail Littorina littorea. The purpose of the randomly arranged muscle fihers is related to the snail's type of locomotion. In the frozen tissue in Figure 1, B. the ice forms in large pockets with a resulting shrinkage and distortion of the cells. The extreme distortions indicated in the initial results were surprising enough 260 JOHN KANWISHER to warrant the following procedure. The frozen muscle slice was cut in two pieces. One was used as the frozen specimen. The other was warmed for less than a minute on the palm of my hand and then hard frozen in the liquid nitrogen. When sectioned it appeared nearly the same as tissue from an unfrozen animal. Figures 1, A and 1, B are actually sections from this run. A transverse section of the unfrozen adductor muscle in the oyster, Crassostrea virgimcus, is shown in Figure 1, C. The parallel muscle fibers are viewed end-on. In its frozen counterpart in Figure 1. D, the fibers are clumped into groups to make room for the intervening ice. The prominent elements that resulted are all about the same size. There may be membranes not visible in the unfrozen muscle to account for this regularity. The same regular clumping was seen in the ad- ductor muscle of two mussels, Modiolus modiolus and Mytilus cdulis. Figure 1, E is of the eggs in the unfrozen ovary of the blue mussel Mytilus ednlis. When frozen as in Figure 1, F, the detail is much less distinct but the eggs clearly have shrunken during the formation of the large amounts of inter- cellular ice. Comparable distortions were seen in other tissues from these and other species. METABOLISM OF FROZEN ANIMALS Scholander ct al. (1953) measured respiration at freezing temperatures by following the decrease in oxygen concentration in a closed volume containing the animal. The same method has been used here. Manometric and volumetric techniques can not be used because of the volume change when water turns to ice. The snails to be used were frozen in 20-nil. syringes in a cold bath. Only those in which the snail froze while fully extended from its shell were used. A short section of tubing on the tip of the syringe extended above the surface of the liquid and was closed with a pinch clamp. A sample of gas could be withdrawn without removing the syringe from the bath. The plunger was free to move up and replace the volume lost in sampling. Allowance was made for the decreased volume in calculating the rate of oxygen removal. Duplicate oxygen analyses good to 0.02 per cent were made with the half-cc. analyzer of Scholander (1947). Serial samples were plotted against time and the slope was used in computing the oxygen consumption. The concentration was never allowed to go below 18 per cent in any run. Respiration was assumed to be independent of tension over this small range. After the snails were placed in the cold bath, at least 6 hours were allowed for phase equilibration between ice and water in the tissues. Previous experience (Kamvisher, 1955) had shown that there was no appreciable increase in ice after this length of time. The syringe was then flushed with cold outside air. A series of oxygen determinations showed that such air did not vary appreciably from 20.94 per cent so this was considered the starting concentration. At intervals ranging from 2 to 120 hours samples were withdrawn with a mercury gas sampler. Volumetric respirometers (Scholander ct a!., 1952) were used above 0. One ml. of sea water was included in the vial with the animals. At such values w r ere in good agreement with those made by gas analysis which is specific for oxygen. The often used and rarely proven hypothesis is thus confirmed that the volume decrease is due to oxygen being consumed. The respiration temperature data from 10 to +30 C. are plotted in Figure 2. Between and +20, oxygen consumption shows the usual logarithmic increase FROZEN INTERTIDAL ANIMALS 261 \vith a Q 10 of 2 to 3. Above this respiration decreases, probably due to thermal injury. Below the metabolic activity drops sharply with an apparent Q 10 of about 50. At -10, respiration was so low it took 6 days for the snails to consume a measureable amount of oxygen. Even in this length of time the concentration change was smaller than desired for accurate determination. This may account for the greater spread of values at this low temperature. At -15 the empty syringes gave blank values of one-third the oxygen decrease in those containing snails. This may be due to oxidation of grease used on the syringe plunger. It 100 - I o 10 o 8 o o 9 8 1 8 8 o 8 o 8 o o o o RESPIRATION vs. TEMPERATURE LITTORINA LITTOREA FIGURE 2 TEMP. C. -10 -5 5 10 15 20 FIGURE 2. Variation of oxygen uptake with temperature. 25 30 did not seem that this technique could be trusted on the slower rates to be expected at still lower temperatures. SALINITY EFFECT ON RESPIRATION Scholander et al. (1953) have given several reasons why the respiratory gas exchange of a frozen animal drops so much more rapidly with temperature than it does above when no ice is present. The ice may act as a diffusion barrier to the gases. The increased viscosity of the body fluids may slow the reaction rates. Finally the increased salinity may directly inhibit the animal's metabolism. No way could be devised to test the first two hypotheses. The respiratory response to increased salinity above can be determined independently of any ice effects. 262 JOHN KANWISHER Higher than normal salinities were made by freezing sea water and using the brine. Dilution with fresh water gave lower than normal salinities. Freshly collected snails were placed in jars containing the different salinities for a minimum of 6 hours before being used. At very high and low values the snails withdrew into their shells. Experience had shown that the operculum blocks respiratory gas exchange so these could not be used. For the respiration measurements single snails were placed in 20-ml. syringes filled with the desired salinity. The syringes were kept in a constant temperature bath except when sampling. One-mi, samples were removed at convenient intervals and analyzed gasometrically for oxygen by the method of Scholander et al. (1955). 200r 100 50 20 10 o o. o CM O / O % SAL \ o \ o \ RESPIRATION vs. SALINITY LITTORINA \ LITTOREA \ \o \ \ FIGURE 3 INITY 2.5 5.0 7.5 FIGURE 3. Response of oxygen uptake to different salinities. Since this is a physical extraction of the gases it could be relied on in spite of the water sometimes becoming cloudy with waste products. As in the low temperature gas analysis method several serial readings were used to indicate the rate of oxygen removal by the snails. Low oxygen tensions were avoided by working in the range of 2.5 to 6 mm. 3 of oxygen per ml. The curves showed that respiration was independent of tension over this range. The variations of oxygen consumption with changes in the external salinity are shown in Figure 3. High salinity depresses the respiration of Littorina littorca. This is a reversible effect since the rate increases again when the snail is returned to normal salinity. When the snails withdrew into their shells at higher salinities than shown, no oxygen consumption could be detected. They are apparently able to subsist for long periods anaerobically. FROZEN INTERTIDAL ANIMALS 263 Since freezing occurs throughout the animal, the remaining body fluids in all parts of the animal are concentrated. If any effect of external salinity above is to be related to freezing, it must be shown that the animal is not osmotically regulating. Increased salinity could conceivably be effective in only altering the absorption of oxygen at the surface. Tissues of snails from water of different salinities were analyzed for chlorides. By carefully cracking the shell, the animal could be re- moved whole. Excess water was mopped off and the weight quickly taken. The water was removed by drying for several hours in a 100 oven. The dry weight then gave the total water by difference. The dry tissue was then digested and titrated for the amount of total chlorides present. This amount was considered dispersed in all the animal water in order to calculate the concentration in the live animal. From the resulting normalities at different external salinities it was clear that internal chloride concentration was proportional to that outside the animal. Any result of externally varied salinity can reasonably be viewed as arising from a corresponding change throughout the animal. DISCUSSION Chambers and Hale (1932) observed plant and animal cells freezing under the microscope. They found that ice formation inside the cellular membrane always resulted in the death of the cell. The detailed studies of Asahina and his colleagues (1954) have described the freezing process in the blood and isolated organs of insect pre-pupae and in the intact insects themselves. They also found that intra-cellular freezing is lethal to the cell or tissue. Such results are responsible for the general belief that all freezing in animals and plants takes place outside the cells. The impressive liquid air freezing experiments of nematodes by Luyet and Gehenio (1940) have almost certainly been an instance of ice within cells. Lack of injury here has been attributed to the very rapid rate of freezing. This vitrifica- tion does not allow time enough for ice crystals to grow to a size where they can damage protoplasmic structures. It forms the basis of the histology used here. This phenomenon probably has little to do with the normal ecology of these animals in nature. Shore animals that are exposed to freezing are in shells. This impedes heat transfer and gives ice crystals time to grow. One can readily see them in an opened animal. The tissues of such an animal are similar in texture and appearance to a frozen piece of meat. It is not surprising when one considers that water makes up three-fourths of the bulk of the animal and four-fifths or more of it may be ice. The photomicrographs presented here show the large amount of distortion necessary at the cellular scale to make room for this ice. Yet this can change back to a more or less normal appearing tissue in 30 seconds as shown in Figures 1, A and B. Siminovitch and Briggs (1949) have related frost hardiness in plants to the ability of water to migrate rapidly in and out of the cells. Unfortunately no equivalent test could be devised to apply this to shore animals. Littorina littorea was found to remain out of its shell and behave normally in salinities of 2 to 7 per cent. It sharply decreased its metabolism in response to a salinity increase. In Figure 3. the data indicate that doubling the salinity above the optimum decreases the oxygen demand to about a third. As the salinity of 264 JOHN KANWISHER the body fluids is increased by the freezing out of water, oxygen uptake must drop in the same fashion. From the freezing curves in a previous paper (Kan- wisher, 1955) 70 to 80 per cent of the water in this species is frozen at 10. This would result in a Q 10 from the salinity of about 10. Above the Q 10 due to the usual temperature effect on reaction rates is between 2 and 3. Combining these one would expect a Q lf , in the range of 20 to 30 below 0. The actually observed one is closer to 50. The effect of ice as a gaseous diffusion barrier and that from the loss of water itself may account for the difference. It is felt that the present data do not warrant a more vigorous interpretation. Similar work with intertidal algae (Kan wisher, 1957) has shown that the drying effect of freezing was chiefly responsible for a similar large decrease in respiration. A three-times increase in salinity had little effect on the oxygen uptake of these plants. Freezing in shore animals to the extent shown here is a normal occurrence twice daily in the winter with no obvious injury to the animal. This freezing hardiness is probably connected with the ability to stand the internal distortions and high salinities that result. The greatly lowered metabolism may be of adaptive significance in severe locations where shore animals are frozen into the ice for months at a time. As such it could represent a considerable saving in food reserves. SUMMARY 1. Histology of frozen shore animals has shown large pockets of intercellular ice with consequent shrinkage and distortion of the surrounding cells. 2. The Q 10 drops precipitously in the region of ice formation and may be as high as 50. 3. High tissue salinity without freezing decreases oxygen uptake. Thus the salinity increase that results from freezing is responsible for a large part of the high Qio- LITERATURE CITED ASAHINA, E., K. AOKI AND J. SniNOZAKi, 1954. The freezing process of frost-hardy cater- pillars. Bui. Entomological Research, 45: 329-339. CHAMBERS, R., AND H. P. HALE, 1932. The formation of ice in protoplasm. Proc. Roy. Soc. London, Ser. B, 110: 336-352. DITMAN, L. P., G. B. VOGT AND D. R. SMITH, 1942. The relation of unfreezable water to cold hardiness in insects. /. Economic Entomology, 35: 265-272. KANWISHER, J. W., 1955. Freezing in intertidal animals. Biol. Bull., 109: 56-63. KANWISHER, J. W., 1957. Freezing and drying in intertidal algae. Biol. Bull.. 113: 275-285. LUYET, B. J., AND P. M. GEHENIO, 1940. Life and Death at Low Temperatures. Biodynamica, Normandy, Missouri. SALT, R. W., 1950. Time as a factor in the freezing of undercooled insects. Canadian J . Res., 28: sect. D: 285-291. SCHOLANDER, P. F., 1947. Analyzer for accurate estimation of respiratory gases in one-half cubic centimeter samples. /. Biol. Chcin.. 167 : 235-250. SCHOLANDER, P. F., C. LLOYD CLAFF, J. R. ANDREWS AND D. F. WALLACH, 1952. Micro- volumetric respirometry. /. Gen. Physio!., 35 : 375395. SCHOLANDER, P. F., W. FLAGG, R. J. HOCK AND L. IRVING, 1953. Studies on the physiology of frozen plants and animals in the Arctic. /. Cell. Coinp. Physio!., 42: supplement 1, 1-56. SCHOLANDER, P. F., L. VAN DAM, C. L. CLAFF AND J. W. KANWISHER, 1955. Micro-gasometric determination of dissolved oxygen and nitrogen. Biol. Bull., 109 : 328-334. SIMINOVITCH, D., AND D. R. BRiGGS, 1949. The chemistry of the living bark of the black locust tree in relation to frost-hardiness. Arch. Biochcm., 23 : 8-17. CHROMATOGRAPHIC ANALYSES OF AMINO ACIDS IN THE DEVELOPING SLIME MOLD, DICTYOSTELIUM DISCOIDEUM RAPER l JEROME O. KRIVANEK AND ROBIN C. KRIVANEK Department of Zoology, Neivcomb College of Tulane University, New Orleans 18, Louisiana The slime mold, Dictyostelium discoideum Raper, is a relatively simple biological system in which to study the processes of differentiation and morphogenesis. From a seemingly homogeneous mass of cells (the aggregation mass), there are eventually formed in the mature sorocarp two basic cell types the stalk cell and the spore cell. The developmental cycle of D. discoideum has been described in detail by Bonner (1944) and Raper (1935, 1940) and will not be repeated here. In the recent literature, studies have been reported that suggest correlations between nitrogen metabolism and the processes of differentiation and morphogenesis in this slime mold. Gregg, Hackney and Krivanek (1954) detected the evolution of ammonia and described changes in several nitrogenous fractions during the life cycle of this organism. In this same study, they suggested that the cellulose of the mature sorocarp was synthesized at the expense of a protein precursor and pointed out that the major nitrogen changes took place while the spore and stalk cells were being formed, i.e., during the culmination process. In addition, Krivanek and Krivanek (1958), using the histochemical technique devised by Francis (1953), demonstrated the occurrence of amine oxidase activity in various regions of the slime mold undergoing differentiative changes. The simultaneous occurrence of changes in nitrogen metabolism and of differentiative and morphogenetic phe- nomena prompted the present study. MATERIALS AND METHODS The method as outlined by Block, Durrum and Zweig (1955) was used for ascending paper chromatographic determinations of amino acids in the slime mold. Chromatograms, using hydrolyzed and unhydrolyzed tissues, were made of four representative stages of development migrating pseudoplasmodium, pre-culmina- tion, culmination, and mature sorocarp. In the case of hydrolyzed tissue, in- dividuals in the desired stage of development were isolated and homogenized in 6 N HC1, hydrolyzed for 18 hours, and evaporated over a boiling water bath. The residue was placed in a soda lime desiccator for 48 hours and then taken up in 2 cc. of warm glass-distilled water and filtered. After evaporating the water filtrate, the residue therefrom was taken up in 1 cc. of iso-propanol, the vehicle used in the application of the spot. In the case of the unhydrolyzed tissue, 1 This research was supported in part by Research Grant E 1453 from the National Insti- tute of Allergy and Infectious Diseases, National Institutes of Health, U. S. Public Health Service. 265 266 JEROME O. KRIVANEK AND ROBIN C. KRIVANEK homogenates were made with water and evaporated. The residue was taken up in 1 cc. of iso-propanol and applied to the paper. The microhomogenizer described by Gregg, Hackney and Krivanek (1954) was used for the preparation of the tissue homogenates. All homogenization took place at room temperature (22 C.). Depending upon the stage of development to be analyzed, the homogenization procedure lasted from thirty minutes to an hour. All evaporation took place over a boiling water bath with the evaporation lasting ALL STAGES (HYDROLYZED) o PHENOL FIGURE 1. Diagram of the results of two-dimensional chromatography on hydrolyzed tissue of D. discoideum. Spots are identified as the leucines (1), phenylalanine (2), methionine (3), proline (4), tyrosine (5), alanine (6), threonine (7), histidine (8), glycine (9), glutamic acid (10), serine (11), asparagine (12), unknown (13), cystine (14), and aspartic acid (15). no more than five minutes in any case. Rupture of virtually all cells was insured by means of periodic microscopic examination of the homogenate. For both types of analyses, i.e., hydrolyzed and unhydrolyzed, two-dimensional chromatograms were made on Whatman No. 1 filter paper. For the first dimension, n-butanol, acetic acid and water (250, 60, 250 v/v/v) were used as the solvent mixture. For the second dimension, an 80% solution of phenol in water was used as solvent. Development of the spots was accomplished by means of spraying the chromatograms with a solution of 0.3% ninhydrin in 95% ethanol. After AMINO ACIDS IN DICTYOSTELIUM 267 spraying, the chromatograms were allowed to dry in complete darkness for 18 hours. No less than 6 and no more than 10 runs were made for each analysis. In the majority of cases, consistent spot patterns were achieved and only 6 runs were made. However, in those few cases where slight inconsistencies in the patterns were evident, additional runs were made to achieve reproducibility. Identification of the spots was achieved in two ways. Firstly, Rf values were calculated and compared with the R f values of known amino acids. Secondly, one- dimensional as well as two-dimensional "control" runs were made using solutions of known amino acids, both singly and grouped, and the loci of spots were com- pared between the control and experimental series. RESULTS Hydrolyzcd tissue. Results of the chromatographic studies of amino acids in hydrolyzed tissues of D. discoidcinn are shown in Figure 1. With the exception MIGRATING PSEUOOPL ASMODIUM (UNHYDROLYZED) FIGURE 2. Diagram of the results of two-dimensional chromatography on unhydrolyzed tissue of D. discoidcwn in the migrating pseudoplasmodium stage. Identified spots are the leucines (1), methionine (2), tyrosine (4), alanine (5), threonine (6), glycine (9), serine (10), glutamic acid (11), aspartic acid (13), and cystine (15). Spots 7, 14, 16, and 17 are unknowns. of one spot (no. 13), all spots were identified. The identified spots included the leucines (1), phenylalanine (2), methionine (3), proline (4), tyrosine (5), alanine (6), threonine (7), histidine (8), glycine (9), glutamic acid (10), serine (11), asparagine (12), cystine (14), and aspartic acid (15). The same spot pattern persisted throughout the four analyzed stages of develop- ment. Although no quantitative determinations of the amino acids were made, comparisons of the relative spot intensities afforded some degree of quantification. Glutamic acid presented the most intense color in each stage. Also quite intense, but not to the degree of glutamic acid, were the spots of the leucines, methionine, alanine, threonine, serine, and asparagine. Medium light spots resulted from 268 JEROME O. KRIVANEK AND ROBIN C. KRIVANEK O PRE-CULMINATION STAGE (UNHYDROLYZEO I O PHENOL FIGURE 3. Diagram of the results of two-dimensional chromatography on unhydrolyzed tissue of D. discoideum in the pre-culmination stage. Spots as in Figure 2, plus spot 12, an unknown. phenylalanine, tyrosine, glycine, and histidine. The faintest spots were those of proline, cystine and aspartic acid. In addition to these well-formed spots, a very faint, vaguely-defined spot was occasionally found in the approximate locus of cysteine. Because of its vagueness O CULMINATION STAGE (UNHYDROLYZED) PHENOL FIGURE 4. Diagram of the results of two-dimensional chromatography on unhydrolyzed tissue of D. discoidcum in the culmination stage. Spots as in Figure 2, plus spots 3 and 12, unknowns. AMINO ACIDS IN DICTYOSTELIUM 269 and the failure of our controls to show a clear cysteine spot, we cannot state positively either the presence or absence of cysteine. Unhydrolyzed tissue. Results of the chromatographic studies of amino acids in unhydrolyzed tissue of D. discoidewn are shown in Figures 2, 3, 4 and 5. Whereas a consistent spot pattern occurred throughout the developmental cycle in the case of hydrolyzed tissue, considerable variability in the spot patterns oc- curred between the several stages in the case of unhydrolyzed tissue. A total of 17 spots appeared in all or nearly all of the stages of development. However, only ten were identified. They were the spots of the leucines (1), methionine (2), tyrosine (4), alanine (5), threonine (6), glycine (9), serine (10), glutamic acid (11), aspartic acid (13), and cystine (15). The remaining seven spots 3, 7, 8, 12, 14. 16, and 17- were not identified. Presumably these ninhydrin-positive o MATURE SOROCARP (UNHYDROLYZED) 03 o ar FIGURE 5. Diagram of the results of two-dimensional chromatography on unhydrolyzed tissue of D. discoidewn in the mature sorocarp stage. Spots as in Figure 2, plus spots 3, 8, and 12, unknowns. spots were simple peptides. It is possible that these spots were the products of partial hydrolysis by enzymes derived from the cells. However, in view of the rapidity with which the tissues were prepared, this would seem unlikely. Those spots which were evident in all stages of development were 1, 2, 5, 6, 7, 9, 10, 11, 13. 14, 15, and 16. Although spot 12, an unknown, did not appear in the migrating pseudoplasmodium. it did appear in the succeeding three stages of development. Spot 3, also unknown, appeared only in the culmination and mature sorocarp stages, while spot 8, a third unknown, appeared only in the mature sorocarp stage. Spot 4, identified as tyrosine, was present in all stages except the mature sorocarp, and spot 17 appeared erratically being present in all but the pre-culmination stage. As in the case of hydrolyzed tissue, cysteine could not be definitely ascertained as either being present or absent. 270 JEROME O. KRIVANEK AND ROBIN C. KRIVANEK DISCUSSION In their quantitative studies of the nitrogen metabolism in the slime mold, D. discoideum, Gregg, Hackney and Krivanek (1954) demonstrated a decrease in the total nitrogen/dry weight during the transition from the migrating pseudoplas- modiuni to the mature sorocarp. They attributed this decrease to a decrease in the total extractable protein nitrogen and total unextractable nitrogen components of the slime mold. In addition, they found that ammonia was being given off by the slime mold during its life cycle. In a subsequent study, Gregg and Bronsweig (1956) found a steady increase in the total amount of reducing sub- stances (presumably carbohydrates) as the life cycle progressed. On the basis of these data, it was suggested that the protein fraction of the slime mold served as a precursor for the carbohydrate of the mature sorocarp. However, no indica- tion was made of the possible pathway (s) involved in this conversion. The present study may be suggestive in this respect. Glutamic acid invariably presented the most intense spot of any of the deter- mined amino acids. This was evident in both hydrolyzed and unhydrolyzed tissues. The deamination of glutamic acid to a-ketoglutaric acid with the cor- responding release of ammonia is known. Because of the reversibility of this reaction, it is considered to be one of the prime mechanisms responsible for the interconversion of ammonia and a-amino group nitrogen. The reaction is catalyzed by glutamic acid dehydrogenase, requiring either DPN or TPN as a coenzyme (Meister, 1957). The importance of this reaction, as it relates to D. discoideum, lies in the fact that not only has glutamic acid been detected to a high degree in the slime mold, but, also, the liberation of ammonia during the life cycle suggests such a deamination reaction. Further, Krivanek and Krivanek (1958) demon- strated non-specific dehydrogenase activity in the pre-stalk area of the pre-culmina-- tion and culmination stages stages in which the future sorophore sheath (con- sisting primarily of polysaccharides) is being secreted by the stalk cells as they move apically to become eventually enclosed within the sorophore sheath. This non-specific dehydrogenase activity could logically be attributed to glutamic acid dehydrogenase. By virtue of the relationship between glutamic acid, a-ketoglutarate, areas of dehydrogenase activity, and sites of carbohydrate secretion, there thus can be postulated this link between carbohydrate metabolism and protein metabolism in the slime mold. The glutamic acid-ketoglutarate relationship, if actually operative in the slime mold, need not be the only link between carbohydrate metabolism and protein metabolism. Aspartic acid, also demonstrated in hydrolyzed and unhydrolyzed tissues of the slime mold, can be deaminated to fumarate, another intermediate in the citric acid cycle (Meister, 1957), thus creating a second possible link between the two types of metabolism. Further, there is the possibility that alanine can undergo deamination forming the Krebs cycle intermediate pyruvate as has been sug- gested by Meister (1957), and serine, as well as cysteine, can undergo the same process yielding ammonia and pyruvate. The suggested relationships already discussed do not preclude the possibility of other mechanisms relating carbohydrate metabolism to protein metabolism, such as clecarboxylation and transamination. There is as yet, however, no evidence to indicate the presence of these mechanisms in the slime mold. AMINO ACIDS IN DICTYOSTELIUM 271 Several of the amino acids of the hydrolyzed tissues appear as well in un- hydrolyzed tissue. Consequently, it is not possible to determine whether these amino acids occur as free amino acids only, or also as bound amino acids. How- ever, four amino acids appear only in the hydrolyzed tissue (phenylalanine, proline, histidine, and asparagine). They are considered therefore to exist only in the bound form. The significance of these amino acids with respect to the differentia- tive process in Dictyosteliuni is at present not apparent. Studies have recently been initiated to test the validity of the above postulates. These correlative studies will embrace the use of the analogs of the amino acids shown to be present in D. discoideum. SUMMARY 1. The amino acids in hydrolyzed and unhydrolyzed tissue of the slime mold, Dictyosteliuni discoideum Raper, have been determined by means of two-dimen- sional ascending paper chromatography. Analyses were made on four stages of development migrating pseudoplasmodium, pre-culmination, culmination, and mature sorocarp. 2. Unhydrolyzed tissue contained the leucines, methionine, tyrosine, alanine, threonine, glycine, serine, glutamic acid, aspartic acid, cystine, and seven unidentified spots, presumably simple peptides. Not all these spots were present in all tested stages. 3. Hydrolyzed tissue contained in addition to the amino acids identified above, phenylalanine, proline, histidine, asparagine, and one unknown spot. All tested stages were identical. 4. The postulate is presented that glutamic acid (and possibly also to a lesser degree aspartic acid, alanine, serine, and cysteine) through deamination may enter the Krebs cycle and form a link between protein and carbohydrate metabolism, the change in balance between protein and carbohydrate being one of the most prominent features of differentiation in this organism. LITERATURE CITED BLOCK, R. J., E. L. DURRUM AND G. ZWEIG, 1955. A Manual of Paper Chromatography and Paper Electrophoresis. Academic Press, Inc., New York. BONNEK, J. T., 1944. A descriptive study of the slime mold, Dictyostelium discoideum. Anier. J. Bot., 31 : 175-182. FRANCIS, C. M., 1953. Histochemical demonstration of amine oxidase in liver. Nature, 171 : 701-702. GREGG, J. H., A. L. HACKNEY AND J. O. KRIVANEK, 1954. Nitrogen metabolism of the slime mold Distvostcliiim discoideum during growth and morphogenesis. Biol. Bull., 107 : 226-235. GREGG, J. H., AND R. D. BRONSWEIG, 1956. Biochemical events accompanying stalk formation in the slime mold, Distyostelium discoideum. J. Cell. Comp. Physiol., 48: 293-300. KRIVANEK, J. O., AND R. C. KRIVANEK, 1958. The histochemical localization of certain bio- chemical intermediates and enzymes in the developing slime mold, Dictyostelium discoideum Raper. /. Exp. Zool, 37: 89-116. MEISTER, A., 1957. Biochemistry of the Amino Acids. Academic Press, Inc., New York. RAPER, K. B., 1935. Dictyostelium discoideum, a new species of slime mold from decaying forest leaves. /. Agric. Res., 50: 135-147. RAPER, K. B., 1940. Pseudoplasmodium formation and organization in Dictyostelium discoideum. J. Elisha Mitchell Sci. Soc., 56 : 241-282. SOME ASPECTS OF OSMOREGULATION IN TWO SPECIES OF SPHAEROMID ISOPOD CRUSTACEA J. A. RIEGEL 1 Department of Zoology, University of California, Davis, California The internal effects of osmoregulation were studied in two euryhaline species of isopod crustaceans, Gnorhnophaeroma orcgonensis (Dana) and Sphaeroma pcntodon Richardson. Although a large literature exists on the subject of osmoreg- ulation in Crustacea, only a little of it concerns isopods. Therefore, the present study was undertaken to gain more information in this relatively unexplored area. Bogucki (1932) studied the ionic composition of the body fluid in Mesidotea cntomon, which according to Ekman (1953) is an inhabitant of the Baltic and Arctic Seas and several fresh-water lakes in the land area of the northwest Pacific, Siberia, and northern Europe. Bogucki found the body fluid concentration to be hypertonic to the medium in lower salinities, becoming isotonic as the medium approached sea water. Lockwood and Croghan (1957), studying the brackish- and fresh-water races of the same species, found the osmotic behavior to be similar in both races, except that the brackish-water animals could not survive in fresh water. They concluded that the fresh-water race has developed a more effective osmoreg- ulatory mechanism that enables it to maintain the high haemolymph concentrations of the brackish-water race in fresh water. Bateman (1933) found that Ligia oceanica maintained its body fluid hypertonic to a medium of about 80 per cent sea water, but swelled and died in 50 per cent sea water. However, Parry (1953), working with Ligia exotica, found that in well-aerated sea water, specimens of the species could survive 17 to 30 days in salinities ranging from 50 to 125 per cent sea water. In very concentrated media (A = 3.46 C.), the body fluid was maintained hypotonic to the medium. Menzies (1954), in addition to splitting Gnorimosphacroma orcgonensis into two subspecies, lute a and oregonensis, per- formed preliminary experiments to test the ability of the two subspecies to survive in various salinities. Specimens of G. o. oregonensis taken from 25 per cent sea water and placed in tap water were all dead after one day. Specimens of G. o. lutea taken from 1.6 per cent sea water and placed in tap water died slowly over a period of three days. Menzies concluded that G. o. oregonensis is probably re- stricted to sea water, but he was puzzled as to why G. o. lutea could survive in sea water, but not in tap water (salts equivalent to 0.3% sea water), which was not very much less saline than the normal habitat water (1.6% sea water). He postulated that G. o. lutea required a slight concentration of salts, greater than the tap water used, or that there were toxins present in that medium. 1 Present address : Department of Zoology, State College of Washington, Pullman, Washington. 272 OSMOREGULATION IN SPHAEROMID ISOPODS 273 MATERIALS AND METHODS Experimental animals Gnorimosphaeroma oregonensis is widely distributed over the west coast of North America from Alaska to central California (Menzies, 1954). It also occurs in Hawaii (M. A. Miller, unpublished report). It may be collected intertidally in bays, in estuarine conditions and occasionally populations of the species are found in fresh-water creeks and ponds. Because of its ability to inhabit such a wide eco- logical range, it was considered to be a suitable experimental type for the further elucidation of osmoregulatory adaptations which enable a marine animal to live in brackish and fresh water. The following designations will be used for the three habitat groups of Gnorimosphaeroma oregonensis. Animals taken from fresh water will be called G. oregonensis (FW) ; those taken from estuarine populations will be called G. oregonensis (EF = estuarine form), and those animals taken from intertidal bay populations will be designated G. oregonensis (BF = bay form). G. oregonensis (FW and EF) equal the subspecies G. o. lute a of Menzies. G. oregonensis (BF) equals the subspecies G. o. oregonensis of Menzies. Sphaeroma pentodon is known only from San Francisco Bay (Richardson, 1905), Tomales Bay (new locality), and Bolinas Lagoon (new locality), California. It lives intertidally in burrows, which it constructs in mud, wooden logs and pilings, and sandstone. The salinity of the habitats from which it was collected ranged from about 11 per cent sea water to about 96 per cent sea water. 6\ pentodon was included in the study because it is related to Gnorimosphaeroma oregonensis, and its range overlaps that of the latter species in parts of its distribution. Methods Four principal types of studies were made : ( 1 ) Changes in the total osmotic pressure of the body fluid after three, 24, and 48 hours exposure to the experimental salinities were made in order to determine the relative degree and pattern of osmoregulation exhibited by the animals. (2) The animals were weighed before and after exposure to the experimental salinities for 24 hours in order to detect possible changes in weight indicating water gain or loss. (3) Survival tests were run to determine the length of time the experimental animals could live in the experimental salinities. (4) Field checks were made by measuring changes in the body fluid of all but Gnorimosphaeroma oregonensis (FW) during a portion of a tidal cycle. The laboratory experiments were conducted at 16 C., a temperature to which all forms were accustomed. The animals were placed in 60 per cent sea water for 24 hours prior to the start of the experiments. The 60 per cent sea water permitted a common starting salinity for all experimental series, facilitating comparisons. E.vperimen tal salinities The experimental salinities used in this study were 125, 100, 75, 50, and 25 per cent sea water, and fresh water (salts equivalent to 0.25% sea water by chloride determination). The 100 per cent sea water (salinity = 34.44%o) was collected off 274 J. A. RIEGEL the Marin County coast away from the influence of fresh-water streams. The 25, 50, and 75 per cent sea water solutions were made by diluting normal sea water with distilled \vater. The 125 per cent sea water solution was prepared by boiling normal sea water, taking care not to precipitate salts. The pH was checked before and after boiling to ascertain that any loss of carbon dioxide was regained by exposure to air. The fresh water was soft creek water collected at Pilarcitos Creek, San Mateo County, California. Salinity determinations Salinity determinations on sea water concentrations greater than 25 per cent sea water were made by a short method described by Welsh and Smith (1953). The salinity of sea water diluted to less than 25 per cent normal sea water and fresh water was determined by the standard silver nitrate titration method using the Knudsen Tables (1901). Melting point determinations A method devised by Gross (1954) was used for determining the melting point of body fluids. From repeated runs on standard samples, it was found that the concentration of the body fluids could be obtained within an error of about two per cent sea water (0.04 C). Body fluid samples (ca. 1-2 mm 3 .) were collected into prepared melting point capillaries (ca. 1 mm. ID X 3 cm. length) which were previously marked with a coded series of dots corresponding to the experimental salinities to which the animals had been exposed. Collection of the body fluid was facilitated by the use of a hand control. After collection, both ends of the capillary were sealed with petroleum jelly and the sample quick-frozen on dry ice. Survival tests The ability of the experimental animals to survive for extended periods of time in the experimental salinities was tested as follows : Seventy animals of each experi- mental group were placed, ten each, in six jars containing the experimental salini- ties, and one jar containing filtered habitat water. The jars were checked daily for 21 days, and the number of survivors recorded. Field tests Changes in the body fluid concentration of Gnorinwsphaeroma oregonensis (EF), G. oregonensis (BF), and Sphacronm pcntodon during a 7 1 /o-hour period from low to high tide in the field were measured as follows : In the case of G. oregonensis (EF), which remains immersed in water during low tide, five body fluid samples and one sea water sample were taken at 114 -hour intervals. In the case of G. oregonensis (BF) and 5". pcntodon, which remain out of the water during low tide, five body fluid samples and five samples of water around the pleopods were collected. The body fluid and pleopod water samples were frozen on dry ice and returned to Davis for determination. OSMOREGULATION IN SPHAEROMID ISOPODS 275 RESULTS The term "gradient" will be used in the following pages to indicate the differ- ence in concentration (expressed in percentage sea water) between the body fluid and the medium. Melting point determination of body fluid concentrations The results of melting point determination of body fluid concentrations are shown in Figure 1. In general, changes in the body fluid concentrations seemed Exposure Time (hours) Exposure Time (hours) co O c CD C O O O rruo Exposure Time (hours) Exposure Time (hours) FIGURE 1. Body fluid concentration changes with time in the experimental salinities. The dotted line represents the body fluid concentration changes of animals kept in habitat salinities (controls) indicated. to be rapid the major changes occurred within the first three hours of exposure to the experimental salinities. After 48 hours' exposure, the fresh-water and estuarine forms of Gnorimosphae- roma oregoncnsis maintained their body fluids hypertonic to the medium in 50 per 276 J. A. RIEGEL cent sea water and less, and hypotonic in 75 per cent sea water and above. How- ever, in 75 per cent sea water after 24 hours' exposure, the body fluid concentration values of G. oregonensis (FW) were quite variable, ranging between hypotonicity and hypertonicity. Possibly that salinity is close to the medium concentration where the "switch" from hyper- to hypo-osmotic regulation occurs. G. oregonensis (BF) maintained its body fluid hypotonic to the medium in 75 per cent sea water and above, and hypertonic to 50 and 25 per cent sea water. Apparently, there was no maintenance of the body fluid concentration in fresh water. In that medium, the body fluid concentration steadily dropped, and after 48 hours, all of the animals were dead. Comparing the above results with those of Menzies (1954) above it can be seen that in both studies, Gnorimosphaeroma oregonensis (BF) (= G. o. oregonensis of Menzies) could not survive in fresh water. However, in Men- zies' study, G. oregonensis (EF) (= G. o. lutca of Menzies) were not surviving after three days in tap water, while in the present study, that form lived for several days in fresh water. It is possible that the tap water used by Menzies (unchlorinated well water) contained some unknown toxic substance or had an imbalance of ions. Its ion analysis is as follows : HCOs, 0.241%c ; SC>4, 0.037%c ; Cl, 0.029% ; Ca, 0.01 \% \ Mg, 0.020^-,; and Na. 0.078% . After 48 hours, Sphaeroma pentodon maintained its body fluid hypotonic to the medium in 100 and 125 per cent sea water and hypertonic in the lower salinities. It is interesting to note that 6". pentodon and Gnorimosphaeroma oregonensis (BF) have extremely wide viability limits in terms of the concentration and dilution of their body fluids surviving within a concentration range (of their body fluids) of over 70 per cent sea water ! Weight changes in the experimental media No weight changes were detected in any of the experimental animal groups, except Gnorinwspliacronia oregonensis (BF) in fresh water. In that salinity, the majority of the animals were very close to death at the end of the 24-hour period, and the weight changes were considered to be subnormal. Those animals that were still active at the end of the 24 hours did not lose weight. It was possible to weigh the animals within an average error of one per cent of their body weight. Survival tests The survival experiment was terminated after 21 days. At termination, the estuarine and fresh-water forms of Gnorhnosphaeronia oregonensis were surviving in all salinities. G. oregonensis (BF) was surviving in all salinities except fresh water, where the LD ; - )0 value (average survival time) was less than two days. Sphaeroma pentodon was surviving in all experimental salinities, except fresh water, where the LD 50 value was 1 1 clays. No unusual mortality was noted among the controls. Field tests The results of the field test of body fluid concentration changes during a tidal cycle showed that no significant changes in concentration of the body fluid or water OSMOREGULATION IN SPHAEROMID ISOPODS 277 surrounding the pleopods were detected in Gnorimosphaeroma oregonensis (BF) or Sphaeroma pentodon. In G. orcgonensis (EF), however, changes were rather characteristic. Starting at low tide, when the animals were exposed to fresh water, the body fluid concentration was 50 per cent sea water. This concentration did not change until over five hours later, when the salinity of the habitat had reached 42 per cent sea water, at which time the body fluid concentration was 58 per cent sea water. Then, by the time of the extreme high tide, 1^4 hours later, the body fluid concentration had changed again to 70 per cent sea water, while the medium concentration had changed to 65 per cent sea water. DISCUSSION Comparative osmoregulatory abilities Figure 2 shows the 48-hour body fluid concentrations of the experimental animals in the experimental media. It was assumed that all major changes in body fluid concentration had occurred by 48 hours. In hypotonic media, Sphaeroma pentodon appears to be a strong regulator, at least in 50 and 75 per cent sea water. There is no apparent reason for the animals to maintain such high body fluid con- centration in those salinities when they can live, at least for several days, in fresh water and 25 per cent sea water with (presumably) much lower body fluid con- centrations. Gnorimosphaeroma oregonensis (BF) has only limited regulation in all media and appears to be the greatest conformer of the group, maintaining a relatively small gradient between its body fluid and the medium in all salinities. G. oregonensis (EF) and G. orcgonensis (FW) are the most able regulators in terms of the ability to maintain their body fluid concentrations relatively constant in hypotonic media. The body fluid concentration differences between the two forms seen in fresh water, 25 per cent sea water, and 50 per cent sea water, are statistically significant (t 6.15, 3.87, and 12.3, respectively, with 11, 10, and 9 degrees of freedom). The ability of G. orcgonensis (FW) to maintain its body fluid more concentrated in the hypotonic media perhaps represents the major osmoregulatory difference between the two forms. The estuarine form is inter- mediate between the bay and fresh-water forms in osmoregulatory ability. Comparing the osmoregulatory abilities of the isopods in this study with those of other crustaceans, a similarity can be seen to species inhabiting similar salinity ranges. From the results of Lockwood and Croghan (1957), it appears that Mesidotca entomon is similar in its osmotic regulation to Gnorimosphaeroma oregonensis. The former species consists of two "races" which have adapted to brackish- and fresh-water. As in G. oregonensis (FW), the fresh-water M. entotnon is able to live in salinities up to normal sea water. However, unlike G. oregonensis (EF), the brackish-water "race" of M. cntomon cannot live in fresh water. The brackish-water M. cntomon is thus closer to G. oregonensis (BF) and Sphaeroma pentodon in its osmoregulatory abilities. However, M. entomon does not show the high degree of hypo-osmotic regulation seen in the isopods in the present study. Beadle and Cragg (1940) reported a difference in the ability to retain chloride between the brackish- and fresh-water forms of the amphipod, Gammarus duebeni, when placed in distilled water. The fresh-water form retained sufficient chloride to survive for several days in distilled water, whereas the brackish- J. A. RIEGEL GNORIMOSPHAEROMA OREGONENSIS (EF) I I GNORIMOSPHAEROMA OREGONENSIS (FW) 2-- ---2 SPHAEROMA PENTODON 3--- 3 GNORIMOSPHAEROMA OREGONENSIS (BF) 4 4 50 75 Medium Concentration (%SW) 125 FIGURE 2. Relation of the body fluid concentration to the medium concentration of animals exposed for 48 hours to the experimental salinities. water form lost chloride and died rapidly in that medium. It appears that the osmoregulatory abilities of the isopods in this study are intermediate between those of one group of crustaceans which can hyper-regulate in dilute sea water, but become isosmotic, or nearly so, in salinities approaching normal sea water {e.g., Carcinus maenasj Schlieper, 1929; Hemigrapsus oregonensis and H. nudus, Jones, 1941) and a second group of crustaceans, which hyper-regulate in dilute sea water and hypo- regulate in salinities approaching normal sea water (e.g., Heloecius cordiformis, Edmonds, 1935 ; Uca crenulata and Pachygrapsus crassipes, Jones, 1941 ; Palae- monetes varians, Panikkar, 1941; and Palaemon serratus, Parry, 1954). All but the last two members of the latter group are primarily semi-terrestrial, which has led Prosser ct al. (1950a, 1955) to suggest that hypo-osmotic regulation may be associated with the semi-terrestrial habit. The isopods in this study are able to survive for extended periods out of water, but they cannot be classified as semi- terrestrial. OSMOREGULATION IN SPHAEROMID ISOPODS 279 The mechanism of osmoregulation Although there is little direct evidence elucidating the actual mechanisms of osmotic regulation of the body fluid of the experimental animals, it is possible to make certain hypotheses concerning that phenomenon based on data obtained in the present study and in studies (unpublished) which were made prior to the present study. a. Evidence for water movement There were no detectable weight changes in the experiments conducted at 16 C., which indicates that there was no net gain or loss of water from the experimental animals' bodies. It is probable that the maintenance of a zero net water flux (that is, no imbalance of the water gain/loss ratio) is dependent upon the ability of the animal to maintain its metabolic rate at a normal level. Duplicate experiments done at 5 C. (see Riegel, 1958) resulted in weight gains by the experimental animals in the dilute salinities and weight losses in the more con- centrated salinities. These results may be interpreted as being due to an inter- ference by the low temperature with the normal metabolism of the animals. b. Evidence for salt movement Since there were no weight changes in the experiments conducted at 16 C., it must be assumed that body fluid concentration changes were due to salt move- ment. In dilute media (fresh water to 50 per cent sea water), the salt concentra- tion of the body fluid was actively maintained against a gradient. In more con- centrated media (75 to 125 per cent sea water), salts were prevented from entering the body (or were eliminated as fast as they came in), since after the initial concentration of the body fluids (generally by 24 hours) the body fluid was maintained hypotonic to the medium. This mechanism could possibly involve, at least in part, an arrest of the mechanism for active salt absorption. Except for Gnorimosphaeroma orcgonensis (FW) there was a rapid loss of salts (within three hours) in the more dilute salinities. Whether this loss was due to an active elimination of salts by the animal, thus reducing the concentration gradient between their body fluids and the medium, or a passive loss from the body is not known. There is some evidence suggesting an active elimination of salts in the more dilute salinities, shown especially by G. orcgonensis (EF) and Sphaeroma pentodon after three hours' exposure to fresh water. In those two forms, the body fluid concentrations dropped more rapidly at 16 C. than at 5 C. (see Riegel, 1958). Whatever the mechanism for the maintenance of the body fluid concentrations in lower salinities, low temperatures interfere with the metabolism of the animals, causing variations in osmoregulation not seen at the higher temperature. In all cases, except Gnorimosphaeroma oregonensis (BF) in fresh water, the animals were able to maintain their body fluid concentrations within viable limits after 48 hours' exposure at 16 C. But at 5 C., G. oregonensis (BF) was dead after 24 hours' exposure to fresh water and 48 hours' exposure to 25 per cent sea water, and Sphaeroma pentodon died after 48 hours' exposure to fresh water. Further, the body fluid concentration of G. oregonensis (FW) and G. oregonensis (EF) dropped 280 J. A. RIEGEL to subnormal values in fresh water at the lower temperature, but remained within normal limits at the higher temperature. Wikgren (1953) studied the effect of low temperature on various poikilotherm- ons animals (a crayfish, a lamprey, and a bony fish) and concluded that low temperatures have their chief effect in interfering with the ion-absorbing mechanism of the animals. In the lamprey, urine production was decreased by low tem- perature, which may have resulted in a weight gain, although Wikgren did not indicate that such was the case. David (1925) performed experiments on the living kidney of the frog, which indicated that that organ's urine diluting and con- centrating activity was not affected by temperature. However, Wikgren (1953) recalculated David's data and stated that the diluting capacity of the frog's kidney was reduced by low temperature. Thus, evidence may be inferred from the review by Wikgren (1953) that low temperature adversely affects the ability of cold- blooded animals (at least, cold-blooded vertebrates) to rid the body of water. The changes in body fluid concentration seen in the present study at 16 C. were undoubtedly due to salt movement. Since there were demonstrated water losses and gains at 5 C., the question arose as to whether the body fluid concentra- tion changes which occurred at that low temperature were due entirely to water movement or were partly due to salt movement. Because the usual procedures for determining body fluid volume were hardly applicable to animals of such small size as used in this study, that component was estimated in the following manner. Ten animals of each experimental group were weighed, and all the body fluid removed from their bodies that could be collected into capillaries of 1-mm. bore. The animals were then re-weighed. Average collectable body fluid weights as a percentage of total body weight were 9.5, 9.7, 11.1, and 6. 8, respectively, for Gnorimosphaeroma oregonensis (FW), G. oregonensis (EF), G. oregonensis (BF), and Sphaeroma pentodon. These values established the minimum possible weight of the body fluid. Ten animals of each experimental group were weighed and dried to constant weight in a calcium chloride desiccator. The average values for total body water as a percentage of the total body weight were 56.5, 55.6, 56.4, and 53.8, respectively, for G. oregonensis (FW), G. oregonensis (EF), G. oregonensis (BF), and 5". pentodon. These values established the maximum possible weight of the body fluid as a percentage of the total body weight. Table I compares the calculated and actual dilution and concentration of the body fluids in fresh water and 125 per cent sea water [using a 40-milligram speci- men of Gnorimosphaeroma oregonensis (EF) as an example] based on estimates of the body fluid weight ranging from ten to 50 per cent of the total body weight. A sample calculation follows : Referring to Table I, it can be seen that a 40-milligram animal, with a body fluid concentration of 50 per cent sea water (column 5), when placed in fresh water would gain 11.3 per cent of its body weight (column 3) after 24 hours. If the weight gain is due entirely to water entry into the body, the incoming water would dilute the body fluids by a factor, X, given by the relation : wt (= original body fluid weight) . . a ., . CA X = - If the body fluid comprises 50 per wt 2 4 (= 24-hour body fluid weight) cent of the total body weight (column 1), its dilution by the gain of 4.5 milligrams OSMOREGULATION IN SPHAEROMID ISOPODS 281 v^ j. ( of water (column 4) would result in a body fluid concentration of X-SQ 20 \ ^T-I 50 I, or 40.8 per cent sea water (column 6) . When a 40-milligram animal whose initial body fluid concentration is 50 per cent sea water is placed in 125 per cent sea water, if the body fluid comprises 50 per cent of the total weight, the body fluid would be concentrated by the factor X I - _ 1. Thus the body fluid will be concentrated to 57.8 per cent sea water \ 1 / . 5 / (column 10). TABLE I Comparison of actual and calculated concentration and dilution of the body fluids* (BF) at 5 C. based on several estimates of the body fluid weight (BF Wt.) as a percentage of total body weight (B W) and assuming the concentration and dilution to be due entirely to water movement 1 2 3 4 5 6 7 8 9 10 11 Est. BF Wt. (% BW) Est. BF Wt. (mg.) % BW gain FW BF Wt. after 24 hrs. FW Start. BF cone. (% SW) Calc. BF cone. FW Actual BF cone. 24 hrs. FW % BW loss 125% SW BF Wt. 24 hrs. 125% SW Calc. BF cone. 24 hrs. in 125% Actual BF cone. 24 hrs. in 125% SW SW 50 20 11.3 24.5 50 40.8 42 6.7 17.3 57.8 112 40 16 11.3 20.5 50 39.0 42 6.7 13.3 60.2 112 30 12 11.3 16.5 50 36.4 42 6.7 9.3 64.5 112 20 8 11.3 12.5 50 32.0 42 6.7 5.3 75.4 112 18 7.2 11.3 11.7 50 30.8 42 6.7 4.5 80.0 112 16 6.4 11.3 10.9 50 29.4 42 6.7 3.7 86.5 112 14 5.6 11.3 10.1 50 27.7 42 6.7 2.9 96.6 112 13 5.2 11.3 9.7 50 26.8 42 6.7 2.5 104.0 112 12 4.8 11.3 9.3 50 25.8 42 6.7 2.1 114.3 112 11 4.4 11.3 8.9 50 24.7 42 6.7 1.7 129.4 112 10 4.0 11.3 8.5 50 23.5 42 6.7 1.3 153.8 112 * A 40-mg. specimen of Gnorimosphaeroma oregonensis (EF) was used as an example. From Table I it can be seen that the calculated body fluid concentrations and dilutions in 125 per cent sea water and fresh water, based on estimates of the body fluid weight percentage, do not completely match the actual results. If the estimated body fluid weight of 50 per cent total weight is correct, the calculated dilution in fresh water is close to the actual value. However, the calculated con- centration in 125 per cent sea water is much lower than the actual value. If the estimated body fluid weight of 12 per cent total body weight is correct, the calcu- lated body fluid concentration in 125 per cent sea water is close to the actual value, but the calculated body fluid concentration in fresh water is much lower than the actual value. Therefore, it is likely that the actual body fluid weight lies somewhere between 10 and 50 per cent of the total body weight. If a reasonable estimate of 20 to 30 per cent is close to the actual value for the body fluid component of the total body weight, it appears that the actual body fluid concentrations in fresh water and 125 per cent sea water at 5 C. are not due entirely to water movement. That is. it is probable that there is a retention or reabsorption of salts in fresh water and an absorption of salts in 125 per cent sea water. J. A. RIEGEL These results are in general agreement with those of Hukuda (1932) who compared the theoretical and actual change in weight with the observed change in osmotic pressure of the blood in P or tunas puber when that marine animal was. immersed in % normal sea water. Gross (1957) found in Emerita analoga that a weight change of less than two per cent of the body weight resulted in a body fluid concentration change equivalent to 25 per cent sea water. Based on the assumption that osmotically active water comprised 40 per cent of the body weight,, he calculated that the weight change, if due entirely to water movement, would have changed the body fluid concentration by less than six per cent. The estimate of 20 to 30 per cent as the haemolymph component of the body weight in Gnorimosphaeroma oregonensis (EF) only partially agrees with similar estimates of that value in other crustaceans. A body fluid value of 50 per cent of body weight was assumed by Lockwood and Croghan (1957) for Mesidotea cntomon. Similarly, a body fluid of % body weight was assumed for Palaemonetcs antennarius by Parry (1957). Gross (1957) made actual calculations of the "solute space" in Pachygrapsus crassipcs and Emerita analoga which were, re- spectively, 56 and 40 per cent of body weight. However, solute space would be expected to be greater than the body fluid volume and less than the total body water. Approximate measurements of blood volume of various crustaceans have been made using sodium thiocyanate. Nagel (1934) found a blood volume of 37 per cent of body weight in Carcinus maenas. Krogh (1939) measured a blood volume of 33 per cent of body weight in Eriocheir sinensis. Prosser and Weinstein (1950) measured the body fluid volume of the crayfish. Orconectes virilis, obtaining values of 25.6 per cent and 25.1 per cent, respectively, with sodium ferrocyanide and a dye, T-1824. The isopods in the present study seemed to have large amount of exoskeleton relative to soft tissue. This fact was further borne out by the relatively low total water values, and in the writer's opinion, supports the estimate of 20 to 30 per cent of total body weight as the body fluid component. To summarize, it is probable that the osmoregulatory abilities of the experimental animals include a mechanism for active salt uptake and retention. In the experi- ments conducted at 16 C., the body fluid concentrations and dilutions were not accompanied by detectable weight losses or gains, sviggesting that the concentration and dilution are due to salt movement. Since concentrations and dilutions of the body fluids could not be explained purely on the basis of water movement (weight losses or gains), in experiments conducted at 5 C., there is evidence that con- centration changes, especially in the higher salinities (75 to 125 per cent sea water) were also due to salt movement at the low temperature. There is some evidence that the experimental animals actively maintain the normal water content of the body fluid. Though body fluid concentrations were well-marked at 16 C., no weight changes were detected. Rather than propose that no water enters or leaves the bodies of the experimental animals upon exposure to the experimental salinities, it might be more reasonable to assume that the normal body water component is actively maintained by pumping water out as fast as it comes in in hypotonic media and by active water uptake and/or salt elimination in hypertonic media. The fact that weight changes were well-marked in experiments conducted at 5 C. and non-existent in experiments conducted at 16 C. indicates that the mechanism for active maintenance of the water balance of the body is depressed or inactivated by- low temperature. OSMOREGULATION IN SPHAEROMID ISOPODS 283 The writer wishes to express his gratitude to Professor Milton A. Miller of the University of California, Davis, for his guidance during the writer's period of graduate study. Appreciation is expressed to Dr. Ralph I. Smith, of the Uni- versity of California, Berkeley, for suggestions and helpful criticism during the balance of the research embodied in this paper. Sincere thanks go to Dr. A. H. Smith, of the University of California, Davis, for technical aid and advice and critical review of the manuscript, and to the Committee on Research of the Uni- versity of California for a Graduate Student Research Grant (DG-6) which made a greater part of this work possible. Finally, a special note of thanks to Professor C. Ladd Prosser, of the University of Illinois, who contributed much to the form of the paper presented here by his generous comments and criticism. SUMMARY 1. Osmoregulatory requirements were analyzed and compared in Menzies' two subspecies of Gnoriinosphacroma orcgoncnsis (G. o. orcgoncnsis and G. o. lutca) and Sphaeroma pcntodon Richardson. 2. The mechanism of osmoregulation was studied by measuring changes in the total osmotic concentration of the body fluid after three to 48 hours' exposure to various experimental salinities ranging from fresh water to 125 per cent sea water. Changes were also measured in the field during a partial tidal cycle. The principal findings and conclusions are as follows : a.) The body fluids of the experimental animals became either diluted or con- centrated in the experimental salinities. Generally, in more dilute media (50% sea water or less), the body fluids were maintained hypertonic to the medium, while in more concentrated media (75 to 125% sea water), they were usually maintained hypotonic to the medium. b.) The lack of weight changes in experimental salinities in experiments conducted at 16 C. indicates that dilution and concentration of the body fluid at normal temperatures are caused primarily by salt movement. c.) Pronounced weight changes that occurred in experiments conducted at 5 C. suggest that the normal water component of the body fluid is actively main- tained and that low temperature interferes with this active maintenance, which normally permits excess water to leave the body in diluted media and to enter in more concentrated salinities. However, the fact that the degree of concentra- tion and dilution of the body fluids at the low temperature could not be explained solely on the basis of water movement suggests concurrent salt gains or losses. LITERATURE CITED BATEMAN, J. B., 1933. Osmotic and ionic regulation in the shore crab, Carcinus maenas, with notes on the blood concentration of Gammanis locusta and Ligia oceanica. J. E.rp. Biol, 10 : 355-372. BEADLE, L. C., AND J. B. CRAGG, 1940. Osmotic regulation in fresh-water animals. Nature, 146 : 588. BOGUCKI, M., 1932. Recherches sur la regulation osmotique chez 1'isopod marin, Mesidotea entomon (L.). Arch. Int. Physiol, 35: 197-213. DAVID, E., 1925. Ueber die Harnbildung in der Froschniere. VI Mitteilung. Ueber den Ein- fluss der Temperature auf die Funktion der iiberlebenden Froschniere. Pfliig. Arch. gcs. Physiol., 208: 146-176. 284 j. A. RIEGEL EDMONDS, E., 1935. The relations between the internal fluid of marine invertebrates and the water of the environment, with special reference to Australian Crustacea. Proc. Linn. Soc. N. S. W., 60: 233-247. EKMAN, S., 1953. Zoogeography of the Sea. Sidgwick and Jackson, London. 417 pp. GROSS, W. J., 1954. Osmotic responses in the sipunculid, Dendrostomum zostericolum. J. Exp. Biol, 31 : 402-423. GROSS, W. J., 1957. An analysis of response to osmotic stress in selected decapod Crustacea. Biol. Bull, 112: 43-62. HUKUDA, K., 1932. Change of weight of marine animals in diluted media. /. Exp. Biol., 9 : 61-68. JONES, L. L., 1941. Osmotic regulation in several crabs of the Pacific Coast of North America. /. Cell. Comp. Physiol., 18: 79-91. KNUDSEN, S., 1901. Hydrographical Tables. Copenhagen: G. E. C. Gad; 63 pp. LOCKWOOD, A. P. M., AND P. C. CROGHAN, 1957. The chloride regulation of the brackish and freshwater races of Mesidotea cntomon (L.). /. Exp. Biol., 34: 253-258. MENZIES, R. J., 1954. A review of the systematics and ecology. of the genus "Exosphaeroma,"' with the description of a new genus, a new species, and a new subspecies (Crustacea; Isopoda, Sphaeromidae). Amcr. Mus. Nov., 1683: 1-24. NAGEL, H., 1934. Die Aufgaben der Exkretionsorgane und der Kiemen bei der Osmoregulation von Carcinus maenas. Zcitschr. f. vcrgl. Physiol., 21 : 468491. PANIKKAR, N. K., 1941. Osmoregulation in some palaemonid prawns. J. Mar. Biol. Assoc., 25 : 317-359. PARRY, G., 1953. Osmotic and ionic regulation in the isopod crustacean Ligea occanica. J. Exp. Biol., 30 : 567-574. PARRY, G., 1954. Ionic regulation in the palaemonid prawn Palaemon ( Leandcr) scrratus. J. Exp. Biol., 31 : 601-613. PARRY, G., 1957. Osmoregulation in some freshwater prawns. /. Exp. Biol., 34: 417^23. PROSSER, C. L., AND S. J. F. WEINSTEIN, 1950. Comparison of blood volume in animals with open and with closed circulatory systems. Physiol. Zool., 23: 113-124. PROSSER, C. L., D. W. BISHOP, F. A. BROWN, JR., T. L. JAHN AND V. J. WULFI-, 1950. Com- parative Animal Physiology. W. B. Saunders, Philadelphia; pp. 6-102. PROSSER, C. L., J. W. GREEN AND T. J. CHOW, 1955. Ionic and osmotic concentrations in blood and urine of Pachygrapsus crassipcs acclimated to different salinities. Biol. Bull., 109: 99-107. RICHARDSON, H. E., 1905. A monograph on the isopods of North America. Bull. U. S. Nat. Mus., 54 : 1-727. RIEGEL, J. A., 1958. Osmoregulation and its ecological significance in certain sphaeromid isopod Crustacea. Ph.D. thesis, University of California, Berkeley. SCHLIEPER, C., 1929. t)ber die Einwirkung niederer Salzkonzentrationen auf marine Organ- ismen. Zeitschr. f. vergl. Physiol., 9: 478-514. WELSH, J. H., AND R. I. SMITH, 1953. Laboratory Exercises in Invertebrate Physiology. Burgess Publ. Co., Minneapolis ; 126 pp. \YIKGKKX, B., 1953. Osmotic regulation in some aquatic animals with special reference to the influence of temperature. Ada Zool. Fennica, 71 : 1-102. M< >TILITY AND POWER DISSIPATION IN FLAGELLATED CELLS, ESPECIALLY CHLAMYDOMONAS x R. R. RONKIN Department of Biological Sciences, University of Dclmvare, Newark, Dclaivarc The energetics of cellular motion have evoked much interest over the past few decades. Muscle, amoeboid cells, and ciliated or flagellated cells have all been studied, but skeletal muscle has received the most attention. This is true partly because the motion of muscle cells can be stopped and started at the will of the experimenter. This fortunate property, absent in amoeboid and ciliated cells, allows the muscle cell to be compared with itself during rest and exercise. Meta- bolic poisons can be used to stop movement in non-muscular cells, but chemical inhibition is seldom reversible or specific enough for experimental designs as elegant as those possible in studies on muscle. Recently, genetic mechanisms have been discovered for controlling the motility of certain flagellated cells: the bacterium Salmonella typhimurium (Stocker, Zinder and Lederberg, 1953) and the autotrophic green alga, Chlamydomonas (Lewin, 1952). Of the two organisms, Chlamydomonas has some advantages as an experi- mental object, since it is nonpathogenic and has simple, well-defined nutrient re- quirements. By using ultraviolet light, Lewin (1954) has produced several single- locus mutant strains with abnormal flagellar characters, including some which look- just like the wild-type strain but do not move their flagella. The paralysis must be related to an abnormality either of flagellar structure or of some other part of the cell. The failure of Mintz and Lewin (1954) to find serological differences between the flagella of normal and paralyzed strains suggests that these flagella may be structurally similar. If this is so, the loss of motility is probably related to a metabolic change elsewhere in the cell. It is now possible by using these algal strains to compare the metabolism of "normal" and "paralyzed" flagellated cells which are presumably alike in other respects. For this comparison it is necessary to assume that a large and definite proportion of the cells in the "normal" culture is motile. An estimate of this proportion, the motility index, will be developed primarily for use in later studies. Its use in this paper will be only to justify the above assumption. The energetic cost of flagellar motion will be estimated in two ways. One estimate is based on microscopic study of the motile cells, the other on measure- ments of respiration. The two estimates will be compared. It is a pleasure to acknowledge the technical assistance of Karl M. Buretz, L. \\ . Clem, Miss Mary C. Straughn, and Irwin D. Zimmerman ; the use of the facilities of the Marine Biological Laboratory, Woods Hole, Massachusetts, in 1 Aided by a contract between the Office of Naval Research, Department of the Navy, and the University of Delaware, NR 164-280. Technical Report 58-2. 285 286 R. R. RONKIN 1955 ; and the helpful comments of Drs. Paul Plesner and Erik Zeuthen, who read the manuscript. METHODS The marine organisms used in this study, Amphidinium Klebsi, Carteria (?) sp. and DunalicUa sp., were obtained from Dr. J. H. Ryther at the Woods Hole Oceanographic Institute. The three strains of Chlamydomonas came from the Department of Botany, Indiana University (I.U.) : they were C. Moewusii ( + ) (I.U. No. 97) herein called "CMW," C. Moewusii ( + ) (Lewin's paralyzed strain No. M 1001; I.U. No. 697) herein called "CMP," and C. Reinhardi ( + ) (I.U. No. 89, Sager and Granick, 1953) herein called "CRW." Marine organisms were studied in filtered, autoclavecl Woods Hole sea water and kept on agar slants made with sea water. Fresh-water Chlamydomonas was grown and studied in a liquid medium suggested by Fuller " which contained KNO 3 , 1 M 5.0 ml. K 2 HPO 4 , 1 M 0.5 ml. KH 2 PO 4 , 1 M 0.5 ml. MgSO 4 , 1 M 2.0 ml. Ca(NO 3 ) 2 , 1 M 0.25 ml. 'Trace element solution" 1.0 ml. "Iron solution" 1.0 ml. Iron-free water to make 1000 ml. 'Trace element solution" contained H 3 BO 3 1.43 g. MnSO 4 -H 2 O 1.05g. ZnCl 2 0.05 g. CuSO 4 -5H 2 O 0.04 g. H 2 MoO 4 -H 2 O 0.01 g. Distilled water to make 1000 ml. "Iron solution" contained Disoclium ethylenediaminetetra-acetate 0.5 g. FeSO 4 -7H 2 O 5.0 g. Distilled water to make 1000 ml. The culture vessels were 125-ml. Erlenmeyer flasks containing 50 ml. of medium and 2.5-liter, wide, flat-bottomed culture flasks (like A. H. Thomas No. 4372-F) containing one liter of medium. Air containing 5% CO 2 was bubbled through the larger cultures. The flasks were shaken mechanically to swirl the contents gently. Four fluorescent lamps (type F40T12W or SW) were mounted under a glass-bottomed water thermostat kept at 23 C., in which the larger flasks were immersed to the level of the medium inside. The illumination 2.5 cm. above the bottom of the flasks was about 500 foot-candles as estimated with a photographic exposure meter. The large flasks were inoculated either with 100 ml. of a previous one-liter culture, or with a 50-ml. culture, reared for the purpose in a 2 R. C. Fuller, personal communication (1955). POWER DISSIPATION IN FLAGELLATES 287 small flask. The small flasks were illuminated from above and shaken gently but were not otherwise aerated. One-liter cultures were ready for harvest (about 2 X 10 6 cells mlr 1 ) in two to four days, depending on the inoculum. The harvest was usually concentrated by gentle centrifugation, and the cells were re-suspended in fresh medium before use. Motility of whole populations of cells was studied by comparing photomicro- graphs of samples of cell suspensions. The film (Du Pont Microcopy) was ex- posed for 8 seconds and developed for maximal contrast with elon-hydroquinone contrast developer (Kodak formula No. D-ll). After being processed, the photo- graphs were projected onto a screen for counting those cells which were stationary long enough to form images. Images of moving cells failed to register because of the long photographic exposure. The use of a haemacytometer 3 and a phase- contrast microscope in photography made the counting easier. This method leads easily to the formulation of a motility index, M. A practical definition of M is J in which n\ - -- the number of cells counted in a defined area of the photograph of a cell suspension, made with a time exposure of 8 seconds; n>2 ---- the number of cells in the first photograph whose images fail to appear in the second, otherwise similar, photograph taken one minute later; //- = : the number of cells appearing in a photograph of a different drop of the same suspension, in which all the cells are immobilized (e.g. , with HCHO or I 2 vapor). The second measure of motility used here is based on the speed of locomotion of individual motile cells in a drop of a dilute suspension, placed on a slide and covered with a coverglass, at room temperature (21 to 23 C.). The individuals to be studied were selected at random by tracking every cell which crossed a line bisecting the field, for as long as it remained in the field. The image of the cell was projected on to a sheet of paper, using a camera lucida. The path of motion was described by pencil marks indicating the position of the cell every two seconds. A loudly ticking clock or mechanical sounder was found to be essential. The dis- tance travelled by the cell per second was calculated from a summation of the line segments connecting the pencil marks on the sheet, and from the time elapsed between the placement of the first and the last marks. The distances travelled per second by several cells in the same suspension were averaged to estimate the average speed of locomotion for the population. Oxygen consumption was measured at 23 C. by the Warburg method. Each 14-ml. reaction vessel was inclosed in a light-tight cloth bag and contained 2 ml. of a suspension of cells which had been washed by gentle centrifugation (700 X G, 30 seconds) and re- suspended in fresh medium. The manometers were read every 10 minutes. The respiratory rate was found to decline slowly with time, but 3 A haemacytometer chamber for phase-contrast microscopy is manufactured by the Ameri- can Optical Co. 288 R. R. RONKIN not appreciably during the first 90 minutes ; the readings during this period were fitted with a straight line by the method of least squares. Respiratory rates were then expressed in /x,l. of O 2 (S.T.P.) per mg. total nitrogen per hour (Qo 2 (N)). Total N was estimated by sulfuric-acid digestion of an aliquot, with three succes- sive additions of H 2 O 2 , followed by direct Nesslerization and reading of the samples in a Klett-Summerson photoelectric colorimeter (Miller and Miller, 1948). RESULTS Degrees of tnotility in a culture of C. Reinhardi When samples of a culture of C. Reinhardi were observed with the microscope, they were found to contain some stationary cells. Some of these became motile from time to time ; at the same time swimming individuals settled down to become members of the stationary group. In general the stationary group seemed to remain constant in size ; therefore, in any series of observations the number of originally stationary cells becoming active in any time interval may be expected to bear a constant relation to the number of originally stationary cells remaining. To test this supposition, a single drop of a culture was photographed repeatedly over a period of several minutes. The photographs were studied, and numbers of originally stationary cells remaining were plotted on a logarithmic scale against time. In one experiment (Fig. 1) the points fell on a straight line for the first I50i o u (0 0.125 o o> JC 'jc 1 100 u fc k. 0> .0 E 75 Nonmotility in Chlamydomonas Of 1,915 cells in this group, 7.4% (141) were nonmotile at time zero. = 3 min. 5 10 15 Time in minutes FIGURE 1. Degrees of motility in a culture of Chlamydomonas Reinhardi. For the first 5 minutes many of the originally stationary cells became motile as shown by the points fitted with a straight line. During the first 30 seconds a more active group of cells dominated ; a slower, possibly more heterogeneous group dominated after 5 minutes. POWER DISSIPATION IN FLAGELLATES 289 five minutes ; this supports the hypothesis that a constant proportion of the re- maining non-motile cells become active during each time interval. However, the graph also revealed that the entire original population of stationary cells was made up of three classes, according to their rates of decrease. The first two had half-times of one and three minutes, respectively. Cells in the third class, pos- sibly including dead individuals in the culture, failed to move in 16 minutes. For this population the first photograph showed 141 (,) stationary cells; in the second photo 23 (n 2 ) of these particular cells were missing. A photo of a killed sample showed 1915 ( 3 ) cells. Thus, J\l = 0.94. In general, samples from other cultures gave similar results, except that the "one-minute" class often could not be found. Locomotion of individual cells of several species A different quantitative concept of cellular motion results from the detailed observation of single motile cells selected at random from a culture. The path of motion of a flagellated algal cell is a series of straight lines or arcs of large radius. Cells may occasionally change direction abruptly or spin briefly in place as if held by a mucous attachment. In addition, cells which are swimming for- ward often revolve about an axis parallel to the direction of motion (Brown, 1945) and may oscillate as they swim. For studies of the velocity of motion, several kinds of elliptical or nearly spherical flagellates were selected. Table I shows the observations and calculations derived from them. The "average radius" is one fourth the sum of length and width. The minimal power dissipation, P, per cell was calculated from Stokes's Law relating to the force, /, needed to propel a sphere through a fluid : / : = 6-rrrrju, and from the relation P -- 10 17 /z<, where / = force needed to overcome fluid resistance (dyne), r = average radius of cell (cm.), 77 =: viscosity of fluid (poise = dyne sec. cm.~ 2 ), u = average speed of locomotion (cm. sec." 1 ), P = power (watt = 10 7 dyne cm. sec." 1 ). Oxygen consumed by normal and by paralysed Chlamydomonas A third aspect of flagellar motion concerns the intensity of metabolism of the flagellated cell. In this study, the oxygen consumption of a population of normal Chlamydomonas Moezvusii (CMW) was compared with that of the ultraviolet- induced, "paralyzed" mutant (CMP). The mutant cells have flagella but fail to use them ; these are held out rigidly almost perpendicular to the main axis of the cell. Occasionally a flagellum showed a little motion at its tip, but this hardly ever caused the whole cell to move. The figures in Table II are based on 12 reaction vessels for CMW and 13 for CMP. In preparation for each experiment the cells of the two strains were reared 290 R. R. RONKIN TABLE I Minimal power output of selected flagellated cells Size Species Medium Aver, velocity 10~ 2 cm. sec." 1 Min. power output/cell, 10~ 16 watt Aver, radius Length 10~ 4 cm. Width ^4 tnphidinium Klebsi 7.98 (11) 1.30 Sea water 0.739 (11) 7.5 Carteria (?) sp. 6.54 (9) 1.67 Sea water 1.25 (10) 17 Chlatnydomonas Moewnsii (CM\Y) 5.54 (11) 1.48 Fresh water 1.28 (16) 15 Dunaliella sp. 4.40 (9) 1.35 Sea water 2.26 (9) 38 Chlani. Reinhardi (CRW) 3.26 (100) 1.08 Fresh water 0.828 (100) 3.9 1 Numbers of individuals studied are in parentheses. Viscosities (corrected for density) used in calculations were: sea water, 0.965 cp (estimated from Miyake and Koizumi, 1948) ; fresh water, 0.931 cp. in one-liter cultures under identical conditions. In each experiment the oxygen consumption of the paralyzed cells was less than that of the normal cells when expressed in terms of total cellular nitrogen. DISCUSSION The quantitative description of cellular motility will be discussed before con- sidering the energy required for flagellar motion. This study presents two quanti- tative methods of studying locomotion in populations of flagellated cells. The motility index (photographic method) can be used for distinguishing the behavior of cell populations exposed to varying experimental treatments. It may prove helpful in pharmacologic and toxicologic studies on suspensions of algae, protozoa, bacteria, or sperm cells; these forms may offer the experimenter ad- vantages over larger and more expensive animal subjects. Compared with other proposed estimates of the proportion of non-motile cells in a microscopic field (Emmens, 1947; White, 1954) it would appear to avoid certain subjective errors in sampling and counting, and to minimize the error due to the inclusion of cells TABLE II Oxygen consumption (Qo (N)) of normal and paralyzed Chlamydomonas Moewusii (paired comparison) Experiment no. CMW CMP Difference (CMW-CMP) 6-27-57 1.24 1.14 0.10 7- 1-57 1.36 1.24 0.12 7-22-57 1.31 0.79 0.52 8- 3-57 1.19 1.14 0.05 8- 5-57 2.33 2.06 0.27 8- 7-57 2.00 1.72 0.28 Mean difference and its standard error (n = 6) 0.223 0.0706 POWER DISSIPATION IN FLAGELLATES 291 which may stop for momentary "rest" periods. It cannot distinguish degrees of impairment of locomotion. The average speed of locomotion appears to be valuable for distinguishing populations of cells which show normal speeds of locomotion from populations with impaired locomotion. It takes no notice of non-motile cells, and thus becomes most useful in estimating the degree of motility in cultures where the motility index is high. It is similar in principle to one devised by Baker, Cragle, Salis- bury and Van Demark (1957) who measured the time required for 100 free- swimming sperm cells to pass through a segment of a plane. Their method, which seems admirably suited to cells displaying the sperm type of locomotion, has the advantage of presenting the result of an experiment immediately without waiting for photographic processing. The decision to use a given method will rest partly upon the extent to which its assumptions are fulfilled by the swimming habits of the organism. The method described here is of special value, since from it can be derived an estimate of the external work done by the motile cells in the population. The estimates of power dissipation in Table I are certainly low, because the premises on which they are based all tend to reduce the estimates. It is supposed, for example, that the cell's internal energetic conversions are 100% efficient. The other assumptions, each known to be false to some extent, are : that there are no degrees of motion other than uniform in a straight line (contradicted by Brown, 1945, and others), that the cell is a sphere (contradicted in Table III), and that TABLE III Estimates of size of Chlamydomonas Strain CRW CMW CMP Length, M Width, M 6.49 0.14 5.24 0.17 7.64 0.17 5.62 0.14 7.41 0.14 4.92 0.10 The "" s ign is inserted between the mean and its standard error. Fifty cells of a single culture of each strain were measured. the frictional drag of the flagellum, apparently of major importance in the locomo- tion of sea urchin sperm (Gray and Hancock, 1955), is negligible in Chlamydo- monas. Excepting C. Reinhardi, the smaller flagellates travelled faster and dis- played a higher power output than the larger cells. Whether this difference is related to a greater metabolic rate of the smaller cells has not been determined. When normal and paralyzed C. Moe^vusi^ were reared and studied under the same conditions in several successive experiments, the paralyzed cells (CMP) always consumed less oxygen than did the wild-type, motile cells (CMW). The average difference in Qo 2 (N) was about 14% of that of the normal cells, and was found to be statistically significant (Mest, n -- 6) at the 5% level, but not at the 2% level. It must be assumed that the proportion of dead cells in the CMP culture is no greater than in the CMW culture. In interpreting this dif- ference, certain other features of the two strains should be borne in mind. Ocular micrometer measurements showed that although CMW and CMP are of equal length, the paralyzed cells are, on the average, a little more slender than 292 R. R. RONKIN the motile ones (Table III). Thus, a paralyzed cell's surface-to-volume ratio is slightly greater than that of a normal cell. From the size of this difference alone one would expect the Qo 2 (N) of the paralyzed strain to be a little greater than that of the normal strain ; it appears in fact to be less. The single mutation which resulted in paralysis of the flagella may have had other expressions, possibly in- volving alterations in the efficiency of biochemical pathways of metabolism. In summary, the physiologic differences between the two strains may be much greater than appeared at first. In ignorance of the magnitudes of these possible factors, it is tempting to suggest that the difference in oxygen consumption is actually related to the state of motility of the cell, but a cautious attitude seems desirable. As a partial test of this relationship, we may now compare the two available estimates of the energy required for motility. One of these (Table I) states that C. Moeu'usii dissipates at least 10~ 15 watt per cell in overcoming the frictional losses in water. The other estimate is derived from the difference in Qo 2 (N) between the normal and paralyzed strains, which is 0.22 p\. hr." 1 (mg. N)" 1 . If we suppose that the exclusion of light from the Warburg vessel does not affect motility (Lewin, 1953), the two figures are comparable; the latter figure can then be transformed to watts per cell by making the following reasonable assumptions : 1. The consumption of 1 fA. of (X releases about 4.8 X 10~ 3 calorie or 5.58 X 10- 6 watt hour. 2. A CMW cell contains 2.65 X 10~ 9 mg. N (estimated from cell counts and N determinations on a single culture at the time of harvest) . 3. The motility index in the CMW culture is high. The observed difference in the rate of oxygen consumption thus corresponds to a difference in power dissipation of 3.3 X 10~ 15 watt per cell. Rothschild's (1953) reworking of Taylor's figures gives estimates close to these for the minimal energy dissipated by bull sperm: for two kinds of assumptions, 3.74 X 10~ 14 watt and 2.04 X 10~ 15 watt per cell. In our comparison, the efficiency of conversion of chemical to mechanical energy is not taken into account. The closeness of the two estimates derived in this paper suggests that the lower rate of oxygen consumption of paralyzed cells may be correlated with their loss of motility. SUMMARY 1. The paper describes a method for estimating the minimal power output of individual, nearly spherical, flagellated cells. A comparison of 5 species of green flagellates suggests no relationship between size and power dissipation (Table I). 2. A simple photographic method for estimating the fraction of motile organisms in a culture is described. 3. Cultured populations of Chlamydomonas Reinhardi may contain two or more distinct groups of cells with different degrees of motility (Fig. 1). 4. The motile, "wild-type" C. Moeu'iisii consumed 1.57 /*!. O 2 (S.T.P.) per hour per mg. total N. 5. A paralyzed mutant strain of the same species consumed 14% less oxygen than the wild type. The extra oxygen consumed by the motile strain is com- mensurate with its estimated minimal power output. POWER DISSIPATION IN FLAGELLATES 293 LITERATURE CITED BAKER, F. N., R. G. CRAGLE, G. W. SALISBURY AND N. L. VAN DEMARK, 1957. Spermatozoan velocities in vitro, a simple method of measurement. 1-ertil. Steril., 8: 149-155. BROWN, H. P., 1945. On the structure and mechanics of the protozoan flagellum. Ohio J . Sci., 45: 247-301. EMMENS, C. W., 1947. The motility and viability of rabbit spermatozoa at different hydrogen- ion concentrations. J. Pliysioi., 106: 474-481. GRAY, J., AND G. J. HANCOCK, 1955. The propulsion of sea-urchin spermatozoa. /. E.\-p. Biol., 32 : 802-814. LEWIN, R. A., 1952. Ultraviolet-induced mutations in Chlamydomonas moczcnsii Gerloff. J. Gen. MicrobioL, 6 : 233-248. LE\VIN, R. A., 1953. Studies on the flagella of algae. II. Formation of flagella by Chlamydo- monas in light and in darkness. Ann. Nezv York Acad. Sci., 56: 1091-1093. LEWIN, R. A., 1954. Mutants of Chlainvdonwnas moeu'iisii with impaired motility. /. Gen. MicrobioL. 11: 358-363. MILLER, G. L., AND ELIZABETH E. MILLER, 1948. Determination of nitrogen in biological ma- terials. Anal. Chcm., 20: 481-488. MINTZ, RITA H., AND R. A. LEWIN, 1954. Studies on the flagella of algae. V. Serology of paralyzed mutants of Chlamydomonas. Canadian J. MicrobioL, 1 : 65-67. MIYAKE, Y., AND M. KOIZUMI, 1948. The measurement of the viscosity coefficient of sea water. /. Mar. Res., 7 : 63-66. ROTHSCHILD, LORD, 1953. The movements of spermatozoa. /;;: G. E. W. Wolstenholme (ed.), Mammalian Germ Cells, Little, Brown and Company, Boston, pp. 122-130. SAGER, RUTH, AND S. GRANICK, 1953. Nutritional studies with Chlamydomonas reinhardi. Ann. New York Acad. Sci., 56: 831-838. STOCKER, B. A. D., N. O. ZINDER AND J. LEDERBERG, 1953. Transduction of flagellar characters in Salmonella. J. Gen. MicrobioL, 9: 410-433. WHITE, 1. G., 1954. The effect of some seminal constituents and related substances on diluted mammalian spermatozoa. Austral. J. Biol. Sci., 7: 379-390. CONSEQUENCES OF UNILATERAL ULTRAVIOLET RADIATION OF SEA URCHIN EGGS 1 RONALD C. RUSTAD 2 Department of Zoology, University of California, Berkeley 4, California The suppression of the elevation of the fertilization membrane on the half of a sea urchin egg which directly receives high doses of ultraviolet light has been described by Reed (1943) and Spikes (1944). The experiments reported herein are an examination of the consequences of unilateral U.V. irradiation of the sea urchin egg in terms of changes in cell morphology with dose, the physical state of the cytoplasm, the effects of time and temperature, and the effects on subsequent cell division. Particular attention is directed toward observations on hyaline layer formation, local gelation, and excentric formation of the mitotic figure. MATERIALS AND METHODS Gametes were obtained from the sea urchin Strongylocentrotus purf>itratus by injection with 0.5 M KC1. The groups of eggs selected were more than 99% fertilizable, were free from visible abnormalities, yielded symmetrical fertilization membranes, and showed little distortion when the lifting of the fertilization mem- brane began. The pattern of morphological changes at different doses was con- firmed with suitable eggs obtained from a single female of the related species Strongylocentrotus franciscanus, which has larger eggs with less yolk. The ultraviolet source was an Electrotherapy Products Corp. low pressure mercury vapor lamp, which produces approximately 95% of its U.V. energy in a 2537 A band. The intensity was measured with a Hanoviameter. In some experiments the eggs were centrifuged in a Servall refrigerated angle- head centrifuge, either in sea water or in a sucrose gradient formed by layering sea water over 0.88 M sucrose. Unless otherwise noted, all experiments were carried out in 1 cm. deep, filtered sea water at 17.5 0.1 C. Artificial calcium-free sea water was prepared accord- ing to the formula of Moore (1956). Clarification of terminology In order to describe concisely and accurately the changes associated with uni- lateral irradiation of the strongly-absorbing egg certain special terms must be defined. The directly-irradiated hemisphere is the surface of the egg which faces 1 Supported by grants from the American Cancer Society and the Office of Naval Research awarded to Dr. Daniel Mazia. 2 This work was performed under the tenure of a Research Fellowship of the National Cancer Institute, United States Public Health Service. Present address: Department of Biological Sciences, Florida State University, Tallahassee, Florida. 294 CONSEQUENCES OF UNILATERAL U.V. 295 the U.V. lamp. The shaded hemisphere is the surface which does not face the lamp, and, hence, is shaded by the cytoplasm. The shaded-irradiated axis is an imaginary line drawn between the poles or centers of these two hemispheres. Uni- lateral membranes are fertilization membranes which lift off the egg on the shaded hemisphere only. All drawings and photographs except Figures 1 and 8 have been mounted with the shaded pole facing the top of the page. RESULTS When eggs were irradiated with large doses of U.V. and then fertilized, the height of the fertilization membrane and the hyaline layer on the directly-irradiated hemisphere was reduced. Sufficiently large doses unilaterally inhibited the forma- tion of these membranes entirely. The dose required to produce a definable level of effect varied by as much as a factor of three between the most sensitive and the most resistant groups of eggs. Nevertheless, the ratio of doses necessary to produce two definable effects on the majority of eggs in a population appeared to be constant even in the extreme cases. The data presented represent the most frequently encountered dose relations. Less than 1600 ergs/mm. 2 did not interfere with the normal membrane eleva- tion. When the dose was increased the fertilization membranes did not elevate to their normal height over the irradiated pole (Fig. 2). Doses of approxi- mately 2800 ergs/mm. 2 resulted in the almost complete suppression of the fertiliza- tion membrane over a small area, but the hyaline layer differentiated over the entire surface. When the dose was increased to 4800 ergs/mm. 2 the fertilization membrane covered only one hemisphere, while the hyaline layer appeared normal (Fig. 3). With slightly higher doses a reduction in the thickness of the hyaline layer was sometimes found (Fig. 4). With doses above 7200 ergs/mm. 2 the hyaline layer could be distinguished only slightly beyond the cell equator (Fig. 5). No further changes in the pattern of membrane elevation were noted at increased doses up to the range of 40,000 to 50,000 ergs/mm. 2 At this dose level partial cytolysis often occurred immediately on the directly-irradiated hemisphere, and complete cytolysis usually followed after standing or at fertilization. Identification of the inhibited surface A simple experimental procedure was devised to demonstrate that the irradiated surface was in fact the one that showed inhibition at fertilization. Stationary eggs were irradiated from above with 7200 ergs/mm.- in a large petri dish on a micro- scope stage and observed as sperm were carefully added. In four experiments there was no detectable net rotation of any of the eggs in the field of a low power objective. By careful focussing it was established that the fertilization membranes first encircled the lower hemispheres which had been shaded by cytoplasm. As the membranes raised further the eggs rolled over and came to rest on their sides revealing total suppression of membrane elevation on the irradiated hemispheres. Rclationsliif> to time and temperature Eggs were fertilized at regular intervals from a few seconds after irradiation to as much as twelve hours later without any visible changes in the unilateral 296 RONALD C. RUSTAD 2 3 5 6 FIGURES 1 to 6 Photomicrographs of sea urchin eggs showing different degrees of suppression of the fertilization reaction when irradiated with increasing doses of U.V. from the direction of the bottom of the page. FIGURE 1. Control. FIGURE 2. Reduction of the height of the fertilization membrane. FIGURE 3. Complete suppression of the elevation of the fertilization membrane on the directly-irradiated hemisphere. FIGURE 4. Reduction of the height of the hyaline layer. FIGURE 5. Complete suppression of both fertilization membrane elevation and the hyaline layer differentiation on one hemisphere. FIGURE 6. Later swelling of the initially flattened shaded hemisphere of an egg similar to Figure 4. fertilization reaction. In five separate experiments there was no increase or de- crease in the inhibited area with time. In general, the irradiated eggs cytolyzed sooner than the controls, but in most experiments both the irradiated and the control eggs became unfertilizable at approximately the same time, even with very high concentrations of sperm. Attempts were made to re-fertilize eggs which had been fertilized but did not completely differentiate the hyaline layer. The simple addition of viable sperm did not cause re-fertilization at any time up to 28 hours after irradiation. The sperm were observed to accumulate in the egg jelly which adhered to the irradiated hemisphere in each of these experiments. Irradiating eggs from the same females at 18 and 8 C. with various doses revealed that there were no differences in sensitivity at the two temperatures. CONSEQUENCES OF, UNILATERAL. U-V... 297 C /ian(/cs in morphology and physical state of the cytoplasm The progressive dose-dependent suppression on the elevation of the fertilization membrane and the differentiation of the hyaline layer have already been described. Sometimes at high doses the fertilization membrane was elevated to an abnormal height above the shaded pole and the cytoplasm under it was considerably flat- tened (Figs. 4 and 5). A large amount of participate matter, possibly cortical granule materials, was found in the perivitelline space under these conditions. The amount of this material was apparently greater at all doses than in the controls. After flattening, the cytoplasm under the unilateral membranes sometimes swelled and reduced the thickness of the perivitelline space (Fig. 6). In some cases the thickness was less than the controls. Under these conditions there was a constriction around the cell at the equator where the fertilization membrane met the hyaline layer (Fig. 6). There were no cases of membrane elevation activation by U.V. at any dose in any of the experiments. Unfertilized irradiated eggs were centrifuged for ten minutes at approximately 12,000 g in a sucrose gradient. In two such experiments 90% of the eggs stratified with the center of the light pole (identified by an oil cap over a clear region of cytoplasm) in the center of the shaded hemisphere (identified by subsequent fertili- zation) (Fig. 7). Almost all of the remaining 10% had an asymmetry of less than 30 between the light-heavy and the shaded-irradiated axis. A very small fraction of a per cent were 30 to 90 off center, and no cases were found in which the shaded pole appeared to have a greater density than the irradiated one. When unfertilized eggs were placed in 70% sea water after irradiation they swelled on one pole only, giving the eggs a somewhat pear-shaped appearance. Standing in this hypotonic medium for several hours did not result in any further changes in shape. The treated eggs were fertilized to establish that the shaded pole was the swollen one. Hence, while both unirradiated eggs and the shaded side of an irradiated one swell in 70% sea water, the directly irradiated surface does not. Irradiated eggs placed in 70 % sea water had a dense darkened area near the irradiated pole, a somewhat less dense region at the shaded pole, and a lighter less granular region near the equator. Occasionally this pattern appeared in eggs kept in normal sea water and seemed to be accompanied by a slight enlargement of the shaded hemisphere. With doses of the order of 40,000 ergs/mm. 2 a large blister of non-granular material formed on the irradiated pole when the eggs were placed in the hypotonic sea water. With slightly higher doses these blisters ap- peared spontaneously. Irradiated eggs were centrifuged at approximately 12,000 g in sufficiently dense suspensions that some of the eggs were confined in a random orientation with respect to their light-heavy axes. Some of these cells showed stratification only on the shaded side, which was identified by subsequent fertilization. When the direction of centrifugation was perpendicular to the shaded-irradiated axis there was a narrow region near the irradiated surface with a very high gel strength that resisted stratification when the central cytoplasm and the shaded side stratified (Fig. 8). FIGURES 7-12 298 CONSEQUENCES OF UNILATERAL U.V. 299 Eggs irradiated after equilibration in calcium-free artificial sea water and fertilized immediately when returned to normal sea water showed the same degree of inhibition as eggs irradiated in normal sea water. J\Iitotic abnormalities Cells irradiated at doses that inhibited the full differentiation of the hyaline layer seldom divided. At lower doses some or all of the eggs would divide several times and sometimes form apparently normal swimming blastulae. Gastrulation was usually abnormal. In some experiments even the first division was abnormal. A systematic group of abnormalities occurred as a result of the mitotic figure failing to migrate to the center of the egg. The nucleus of the unfertilized egg is excentrically located, and in normal division the mitotic apparatus is positioned approximately in the center of the cell. The position of the furrow is determined by the plane formerly occupied by the metaphase plate both in normal cells and these abnormal cells. When the mitotic figure located in either hemisphere was oriented perpendicular to the shaded-irradiated axis, the furrow formed along that axis and the egg cleaved into two equal-sized blastomeres (Figs. 9 to 12). When the mitotic figure was oriented parallel to the shaded-irradiated axis in either hemisphere, the furrow formed perpendicular to the axis and the sizes of the resulting blastomeres were quite different (Figs. 13 to 16). Variable results were observed when the mitotic figure was formed with other orientations with respect to the shaded-irradiated axis (Figs. 17 and 18). Excentric spindles were also found in eggs which were irradiated during the early part of the first mitotic cycle with comparatively low doses of U.V. The blastomeres in such experiments were always equal in size. Whenever the mitotic apparatus was excentric the furrow formed first on the surface that was closest to the spindle. At later stages of cytokinesis the furrow on the near side would always be deeper than the furrow on the far side. In some cases the furrow actually passed through the spindle before the first indentation occurred on the far side of the cell. DISCUSSION The progressive unilateral inhibition of the fertilization reaction has been de- scribed in terms of the U.V. doses required to produce different degrees of inhibi- FIGURES 7 to 18 Schematic drawings of eggs irradiated from the direction of the bottom of the page (except Fig. 8) ; refer to text for explanation. FIGURE 7. Egg centrifuged in a sucrose gradient and then fertilized. Stratification direc- tion indicates that the irradiated pole was heavier than the shaded pole. FIGURE 8. Egg irradiated from the left side of the page and centrifuged while confined with the shaded-irradiated axis perpendicular to the direction of centrifugation. A narrow region near the surface of the irradiated hemisphere resisted stratification indicating a local increase in gel strength. FIGURES 9 to 12. Division patterns of cells with spindles oriented perpendicular to the shaded-irradiated axis. 13 15 16 17 18 FIGURES 13-18 300 CONSEQUENCES OF UNILATERAL U.V. 301 tion of both the elevation of the fertilization membrane and the differentiation of the hyaline layer. Hyaline layer differentiation is less sensitive to U.V. than fertilization membrane elevation ; however, it may be suppressed completely on the directly-irradiated hemisphere with high doses. The inhibition of the elevation of the fertilization membrane has been described previously by Reed (1943) and Spikes (1944). By means of local dye experiments Spikes (1944) was able to demonstrate that the directly-irradiated hemisphere is the site of inhibition. His findings have been reconfirmed with the direct observations of undisturbed eggs reported herein. Giese (1947) has shown that the sea urchin egg strongly absorbs or scatters 2537 A U.V. light. Harvey and Lavin's ( 1944) U.V. photomicrographs also indicate that a considerable amount of the light is absorbed in sea urchin eggs of another genus. Since the shaded pole is not inhibited even at very high doses, it may be concluded that the transmission of the cytoplasm is too low to allow the necessary energy to reach the sensitive sites on the shaded side of the egg. The demonstration that there was no spreading of the damaged area with time indicates that the U.V. action is relatively direct, and, in particular, that there is no secondary effect of "diffusible poisons." There was no recovery with time ; hence, the damage seems to be irreversible by any metabolic mechanism. Since the degree of injury did not decrease with time, and a diffusible toxic product would be expected to decrease in local concentration, this observation provides additional evidence against the action of such substances. The sensitivity was the same at 8 and 18 C. Direct photochemical action has been shown repeatedly to have a O 10 of approxi- mately 1. Therefore, insofar as visually equivalent degrees of damage may be used as a measure of the rate of damage, it appears that the injury results from direct photochemical action. The time and temperature relations together offer evidence that the effect is localized and that there is a lack of intermediary toxic products. The observation that the inhibited surface could not be re-fertilized by the addition of fresh sperm could be interpreted in two ways : either the U.V. damage rendered it unfertilizable or some of the steps of the fertilization reaction occurred on this side when the egg was initially fertilized. If some substances necessary for the initial steps of the reaction had been used up the sperm could not initiate a response later. A pronounced green Becke line appears in the out-of-focus image of the damaged hemisphere of heavily irradiated eggs after fertilization. This change is probably similar to the dark-field changes which have been observed prior to membrane elevation (Runnstrom, 1928; Rothschild and Swann, 1949) and indi- cates that some step in the fertilization reaction has taken place. Two types of evidence for local gelation in the irradiated hemispheres were obtained : first, that swelling in 70% sea water was confined to the shaded pole, and, second, that a narrow band near the irradiated surface resisted stratification with centrifugation when the rest of the cytoplasm stratified. Reed (1948) found FIGURES 13 to 16. Division patterns of cells with spindles oriented parallel to the shaded- irradiated axis. FIGURES 17 and 18. An example of one of several division patterns obtained when the spindles have intermediate angular orientations. 302 RONALD C. RUSTAD that moderate doses of unilateral U.V. did not change the permeability of the egg to a large variety of solutions. Although no measurements were made, he discussed possible differences at higher doses and proposed that some sort of gela- tion occurred on the basis that vacuoles were formed in the irradiated pole. Spikes (1944) also proposed that gelation occurred, because he found that while normal eggs only swelled in 50% sea water, irradiated ones lysed on the irradiated side. Spikes' data might also be interpreted as indicating either that the surface of the shaded hemisphere was weakened or that the osmotically inert volume had been increased permitting greater than normal swelling followed by lysis. The obser- vation of the large amounts of granular material released into the perivitelline space at the shaded pole suggests the weakening either of the cell membrane or of some other surface structure. The flattening of the shaded pole at fertilization at high doses seems to fit either hypothesis, although an enhancement of the vigor of the fertilization reaction would yield the same pattern. It would not be un- reasonable to suppose that U.V. damage could affect both the surface strength and the osmotically inert volume, perhaps by a common mechanism. The observation that eggs irradiated in calcium-free sea water showed the same degree of damage as eggs in normal sea water cannot be interpreted directly in terms of the often demonstrated role of calcium in gelation (Heilbrunn, 1952). First, the eggs had to be fertilized in normal sea water since fertilization will not occur in the absence of external calcium ion; hence, new calcium may have been introduced before the damage was measured. Second, since Heilbrunn and his co-workers have shown that U.V. causes solation in low doses and gelation in high doses, it is quite possible that the calcium ion left in the egg after treatment with calcium-free sea water shifts between the less and more heavily damaged portions of the cytoplasm. The second possibility is quite attractive, since it would pro- vide a mechanism for an increase in osmotically inert volume in the less damaged hemisphere and introduces the possibility that the surface on the shaded side might be weakened by small amounts of U.V. penetrating the cytoplasm to cause solation. Spikes (1944) reported that in Lytechinus pie t us furrow formation almost always occurs along the shaded-irradiated axis. Clearly this is not the case in the Strongylocentrotus purpuratus used in these experiments; cleavage may take place with any orientation. Successful cleavage w r ith the furrow passing through the irradiated portion of the egg indicates either that the furrowing strength exceeds the resistance of the radiation-induced gel or that the gel is solated in the course of cytokinesis. Cleavage into equal or unequal sized blastomeres is determined by the orienta- tion of the spindle with respect to the shaded-irradiated axis. It occurs because the mitotic figure remains centered around the original location of the nucleus. The nucleus is excentrically located in unfertilized eggs of this species. When the axis of the mitotic figure is perpendicular to the shaded-irradiated axis the blastomeres are equal in size. Where the axes are parallel the blastomeres are unequally sized. In intermediate angular orientations the results are variable. While both parallel and perpendicular orientations can occur when the mitotic figure is located in either the shaded or irradiated hemisphere, mitotic figures near the equator seem to be restricted to intermediate angular orientations. It is clear that the migration of the nucleus to its normal central position is inhibited. An CONSEQUENCES OF UNILATERAL U.V. 303 increase in cytoplasmic viscosity would provide a plausible explanation for this failure of migration. &' It is a great pleasure to acknowledge my gratitude to Professor Daniel Muzia for his helpful advice and encouragement during the course of this work. I also wish to thank Professors J. E. Gullherg, L. V. Heilbrunn and C. B. Metz for their valuable comments about the results, and Mr. Fred Burnet for his skillful preparation of the drawings. SUMMARY 1. The progressive dose-dependent inhibition of the fertilization reaction on the directly-irradiated hemisphere of the unilaterally U.V. -irradiated sea urchin egg has been described in terms of changes in the ability to elevate the fertilization membrane and to differentiate the hyaline layer. 2. Membrane elevation was not activated by 2537 A U.V. light. 3. No spreading of the extent of injury or recovery was found with time ; and no temperature sensitivity differences were found ; hence, the injury appeared to be the result of direct photochemical action. 4. The irradiated hemisphere of the fertilized egg maintained its jelly for con- siderable periods of time. 5. Evidence was obtained showing partial gelation of the irradiated hemisphere and suggesting that the gelled cytoplasm had a higher density than the rest of the egg. Irradiation in calcium-free sea water did not change the degree of dam- age observed after fertilization in normal sea water. 6. The behavior of the cytoplasm of the shaded hemisphere at fertilization suggested either that the surface structure was damaged or that the osmotically inert volume had been increased. 7. Unilateral irradiation caused excentric spindle formation which resulted in equal sized blastomeres if the spindle axis was perpendicular to the axis of irradia- tion and unequal sized blastomeres if the axes were parallel. LITERATURE CITED GIESE, A. C., 1947. Radiations and cell division. Quart. Rcr. Biol., 22 : 253-282. HARVEY, E. B., AND G. I. LAVIX, 1944. The chromatin in the living Arbacia punctiilata egg and the cytoplasm of the centrifuged egg as photographed by ultraviolet light. Biol. Bull, 86:" 163-168. HEILBRUNN, L. V., 1952. An Outline of General Physiology. Third ed. W. B. Saunders Co., Philadelphia. MOORE, A. R., 1956. In: Formulae and Methods IV, Marine Biological Laboratory, Woods Hole, Massachusetts. REED, E. A., 1943. Unilateral membrane formation in the sea urchin egg treated with ultra- violet light. Anat. Rcc., 87 : 467. REED, E. A., 1948. Ultraviolet light and permeability of sea urchin eggs. /. Cell. Comp. Physiol., 31 : 261-280. ROTHSCHILD, LORD, AND M. M. SWANN, 1949. The fertilization reaction in the sea urchin egg. A propagated response to sperm attachment. /. E.vp. Biol., 26: 164-176. RUNNSTROM, J., 1928. Die Veranderungen der Plasmakolloide bei der Entwicklungserregung des Seeigeleies. Protoplasma, 4: 388-514. SPIKES, J. D., 1944. Membrane formation and cleavage in unilaterally irradiated sea urchin eggs. /. E.rp. Zoo!.. 95: 89-103. THE ROLE OF THE INITIATOR CELL IN SLIME MOLD AGGREGATION x MAURICE SUSSMAN - AND HERBERT L. ENNIS 3 Department of Biological Sciences, Northwestern ['nii'crsity, Evanston, Illinois Previous studies of slime mold aggregation (Sussman and Noel, 1952) had shown that the number of aggregative centers is linearly related to the number of cells present and, further, that centers are distributed in accord with the Poisson series among small, replicate population samples. These and supporting data were considered to dictate the existence of specially endowed individuals termed "initiator cells," each of which could evoke the aggregative response by its neighbors, the "responder cells." Recently a distinctive cell type was detected by morphological criteria in Dictyosteliuni discoideiun Raper and evidence was presented in support of the contention that cells of this type are in fact the initiators of aggregation (Ennis and Sussman, 1958a, 1958b ; Sussman, 1958). The distinctive individuals, termed I -cells, are much larger than the remainder of the population (R-cells), the difference amounting to 2-3-fold in diameter, 3-10-fold in area. They are much flatter and more heavily granulated and vacuolated. In contrast to the R-cells which move sluggishly, the I-cells are highly motile and extensive lobopodia and filopodia are seen to protrude constantly and explosively. Figure 1 presents histograms to illustrate the size differences. Two modes are apparent without overlap. The evidence (Ennis and Sussman, 1958b) supporting the candidacy of the I-cells for the appellation of "initiator" is summarized below : a) The ratio of I-cells to R-cells remained .constant during the pre-aggregative period at 1 : 1940. This figure agrees closely with the ratio of centers formed to cells present at optimal density (1:2200). b) A high correlation was encountered between the positions of I-cells and of subsequently formed aggregative centers. c) The appearance of centers among small, replicate population samples was correlated (perfectly in one experimental series and almost perfectly in another) with the previously determined incidence of I-cells. That is, centers appeared in samples containing I-cells ; none appeared in samples without I-cells. d) Removal of I-cells at an early enough time prevented subsequent center formation. 1 This work was supported by grants from the National Cancer Institute and the Office of Naval Research. - Present address : Department of Biology, Brandeis University, Waltham, Massachusetts. 3 Postdoctoral Fellow, N.I.H. Present address : Department of Bacteriology and Immunol- ogy, Harvard University School of Medicine, Boston, Massachusetts. 304 INITIATOR CELL 305 60 40 20 LARGEST SMALLEST RANDOM I -CELL MEAN=64.15 r =18.8 CV =29 21 I MEAN=299 re- incubation, R-cells were individually micro-manipulated to test areas. See text for details Pre-incubation period in hours Experimental Background Total No. with aggregates % Total No. with aggregates % 1 53 7 13.2 79 11 13.9 4-6 65 14 21.6 250 29 11.6 10-12 71 26 36.6 70 9 12.8 Washed myxamoebae were dispensed on washed agar at a density of 150-200 cells/mm. 2 . After 1, 4-6, and 10-12 hours, R-cells were picked up individually with a glass loop mounted in a deFonbrune micromanipulator and moved to test areas. The test areas had been prepared by dispensing washed myxamoebae on washed agar at a density of 250 cells/mm.-, one hour prior to use. After the excess fluid had been absorbed, an area, 1 mm. 2 , was delineated in the middle of each drop as described in the previous section. The outlying cells were brushed away leaving test squares containing 250 myxamoebae at a density of 250. The center: cell ratio being 1:2200, one would expect 11.3% of the squares to have aggregated spontaneously. The background controls shown in Tables II and III showed an incidence of 72 squares with aggregates out of a total of 578, or 12.4%. The extent to which addition of R-cells, pre-incubated for periods between 1 and 12 hours, affected the background incidence is shown in Table II. R-cells pre- TABLE III Initiative capacity of R-cells tested upon their developmental juniors A. Samples with I-cells Samples without I-cells No. No. with aggregates % No. No. with aggregates % 21 18 86 13 R-cells from samples with I-cells R-cells from samples without I-cells Background B . Experi- ment Total No. with aggregates % Total No. with aggregates % Total No. with aggregates % A 27 5 18.5 27 8 29.6 54 8 14.8 B 27 8 29.6 27 3 11.1 53 8 15.1 C 36 8 22.2 30 7 23.3 72 7 9.7 Total 90 21 23.4 84 18 21.4 179 23 12.8 Twenty-one which certainly con- The percentages of samples that A. Samples of 500 cells were dispensed on washed agar. tained I-cells and 13 which certainly did not were chosen, produced aggregates are shown. B. After 8 hours' pre-incubation, R-cells, taken from the samples with and without I-cells, were micromanipulated to test areas. See text for details. 314 MAURICE SUSSMAN AND HERBERT L. ENNIS incubated for one hour did not affect the background frequency but increases of 10 and 24% over background were obtained by adding R-cells pre-incubated for 4-6 and 10-12 hours, respectively. In other words, when pre-incubated for 10-12 hours and then moved to test areas, one out of four R-cells could induce the formation of a center among the test cells, 12 hours after its introduction. 2468 TIME IN HOURS 10 12 FIGURE 6. A kinetic comparison of : I. The capacity of small population samples to ag- gregate when isolated from their neighbors after varying periods of incubation. Ordinate : per cent of 250 cell samples that aggregated. Abscissa : time of incubation on washed agar prior to isolation. (Data from Table I.) II. The capacity of R-cells incubated for varying times on washed agar to initiate centers amongst their developmental juniors. Ordinate: per cent of R-cells capable of initiation. Abscissa time of incubation on washed agar prior to their micromanipulation to test areas. (Data from Table II.) Figure 6 is a graphic comparison of the kinetics of induction of centers in test squares (I) by progressively delayed removal of outlying I-cells (data from Table I) and (II) by addition of pre-incubated R-cells (data from Table II). The crude ki- netic similarity suggested that the outlying I-cell might not only 'be responsible for the subsequent aggregation of the R-cells but also for the concomitant increase in their capacity to themselves initiate centers. To test this possibility, replicate samples INITIATOR CELL 315 of 500 washed myxamoebae were dispensed on washed agar. In three experiments 21 samples were chosen which certainly contained I-cells and 13 which certainly did not. The data in Table III confirm the correctness of these choices since 86% of the samples said to contain I-cells aggregated while none of those said not to contain I-cells did so. After these samples had been incubated for 8 hours, R-cells were picked and moved to test squares as described in the preceding paragraph. Table III shows that R-cells, whether pre-incubated in the presence or absence of I- cells, were equally capable of inducing center formation. Thus, the rise of the initia- tive capacity of the R-cells during the pre-aggregative period is not dependent upon their contiguity with I-cells. Two points must be kept in mind here. First, it must be remembered that prior to their deposition on the washed agar, R-cells had all been in contact with I-cells and therefore could have been at this time the subject of interactions emanating from the latter. Second, even though the R-cells after 12 hours of incubation had attained a significant degree of initiative capacity, they fell far short of the level displayed by the I-cells after only 20 minutes of incubation. Therefore, the phenotypic difference between the two cell types in this respect remains clear. Finally, the results reveal a most puzzling paradox. When R-cells were pre- incubated for 8 hours in the absence of an I-cell and then placed in the presence of test cells for an additional 12 hours, at least one out of ten could induce center formation. Yet the samples from which these R-cells originally came, when in- cubated for a total of 20 or indeed 36 hours, had not aggregated. It is clear, there- fore, that the observed increase in the initiative capacity of R-cells during the pre-aggregative period in the development of a population is of no consequence to the ultimate aggregation of that population. In other words, the initiative capacity of such R-cells, demonstrated by movement to another population, is an experimental artifact bearing no relation to normal aggregation but which may possibly be used to understand the biochemical and genetic differences between the I-cell and R-cell Phcnotypes. DISCUSSION The data presented here and previously suggest a developmental program of slime mold aggregation that may serve as a useful working hypothesis. I-cells arise during the growth of an R-cell population (which in turn had originated from the spores of the preceding fruit), and attain a steady-state ratio of approximately 1:2000 early in the exponential phase (Sussman, 1956; unpublished data). Entrance into the stationary phase marks the beginning of the pre-aggrega- tive period. At the beginning of this period, the I-cells secrete material which, during the ensuing 12 hours, so conditions the neighboring R-cells as to induce them to aggregate. This interaction, as might be expected, affects the nearest neighbors first but its influence is progressively extended. Concomitant with, but unrelated to either the presence of the I-cell or the subsequent course of aggregation in the same population is a significant rise in the initiative capacity of the R-cells them- selves. Such cells upon extended incubation never do attain the degree of initiative capacity displayed by the I-cells nor can they act upon their developmental con- temporaries but only upon cells at an earlier developmental stage to which they have been added by the observer. 316 MAURICE SUSSMAN AND HERBERT L. ENNIS The first overt sign of aggregation is the formation of cell clumps concentrically about and usually at the I-cell. This is followed by excitation and elongation of the loose and clumped cells in response to the chemotactic complex (Sussman et al., 1956; Shaffer, 1956; Sussman, 1958). The appearance of oriented streams estab- lishes the position of the aggregative center. This is usually coincident with the final position of the I-cell but sometimes with the position of a particularly large clump nearby, and possibly reflects the point of greatest production of the chemo- tactic complex. In the latter case, the position of the center need bear no relation to the previous path of the I-cell. The picture as drawn raises many questions and offers a number of predictions under current study. The most important of the latter involves the hypothetical existence of an "initiator" substance. In view of the I-cell removal experiments, one ought under the same conditions to be able to induce test cells to aggregate by dispensing them in an area previously but no longer occupied by an I-cell. This is being tested. The I-cell addition experiments raise the question as to what is the minimum period of time after contact with the I-cell in which the induced R-cells can begin aggregation. Is the 12-hour period subsequent to contact manda- tory or does it involve preparations by the R-cells for aggregation, unconnected with the function of the I-cell? In the latter case, one ought to be able to pre- incubate the test cells for twelve hours, add I-cells, and observe the onset of aggregation very shortly thereafter. The fact that R-cells can also attain initiative capacity to a far smaller degree, albeit much later than do the I-cells and ineffectively so far as inducing their contemporaries to aggregate is concerned, still suggests that the metabolic path- ways involved in initiation are not unique to the I-cells. Indeed, one may imagine that the sole basis for the difference between I-cells and R-cells in this respect is the much greater size of the former. Perhaps, then, any of the diverse methods for producing giant cells may serve to create initiators just as does the normally occurring R-cell to I-cell transformation. This point is also under current study. SUMMARY Dictyostelium discoidcinn myxamoebae occur as two distinct morphological types, termed I-cells and R-cells. Data presented in a previous publication demon- strate that I-cells can initiate centers of aggregation and suggest compellingly that they are in fact the initiator cells for normal aggregation. The present communi- cation extends and amplifies these findings. A. Time lapse camera lucida drawings and photomicrographs illustrate the sequence of events dviring the onset of aggregation. B. Small population samples of myxamoebae, when isolated from their neigh- bors shortly after deposition on washed agar, showed a distribution of aggregative centers consistent with the distribution of I-cells within the samples. Longer periods of contact with neighboring cells (including other I-cells) that surrounded the samples prior to isolation permitted progressively greater proportions of the samples to aggregate. The possibility arises of an "initiator substance" whose effect may extend over relatively great distances. C. R-cells, incubated for long periods of time on washed agar, were found to have acquired initiative capacity. At best, only a small proportion did so and fur- INITIATOR CELL 317 thermore could only induce the formation of aggregative centers amongst their developmental juniors (by twelve hours) but not amongst their developmental contemporaries. LITERATURE CITED ENNIS, H. L., AND M. SUSSMAN, 1958a. The initiator cell for slime mold aggregation. Bacteriol. Proceedings, p. 32. ENNIS, H. L., AND M. SUSSMAN, 1958h. The initiator cell for slime mold aggregation. Proc. Nat. Acad. Set., 44: 401-411. SHAFFER, B. M., 1956. Properties of acrasin. Science, 123: 1172-1173. SUSSMAN, M., 1956. On the relation between growth and morphogenesis in the slime mold Dictyostettwm discoidcnm. Biol. Bull., 110: 91-95. SUSSMAN, M., 1958. A developmental analysis of slime mold aggregation. McCollum-Pratt Symposium on the chemical basis of development. (In press.) Johns Hopkins Uni- versity Press, Baltimore, Md. SUSSMAN, M., AND E. NOEL, 1952. An analysis of the aggregation stage in the development of the slime molds Dictyosteliaceae. I. The populational distribution of the capacity to initiate center formation. Biol. Bull.. 103 : 259-268. SUSSMAN, M., F. LEE AND N. S. KERR, 1956. Fractionation of acrasin. Science, 123 : 1171-1172. SHELL REPAIR IN CHITONS JOHN S. TUCKER AND ARTHUR C. GIESE Hopkins Marine Station of Stanford University, California l Cryptochiton stelleri (the "gumboot") is not only the largest member of the class Amphineura, but also one of the most specialized in that the girdle tissue has completely overgrown the skeletal plates (Heath, 1897). It therefore lacks the outer shell layer, the tegmentum. While preparing some of the skeletal plates for display, it was noticed that occasional plates were cracked and that many of these cracks were repaired by an amber-colored membrane resembling conchiolin. It seemed of interest to determine the frequency of damage, the stages of repair, the possible significance of this ability to the survival of the animal, and the relative incidence of breakage and repair in several other species of chitons (Katherina tunicata, Mopalia hindsii). Cryptochiton (Amicula) stelleri is a subtidal browsing herbivore, but it is also found in fair numbers up into the middle zone of the intertidal region. When found in the intertidal zone it is attached loosely to rock encrusted with coralline algae or to algal curtains, and occasionally it is found on a sandy bottom. Cryptochiton holds to its substrate only gently and can be removed easily by hand. It is also dislodged by w r ave action as evidenced by the large number (approximately 75) counted on three local beaches after a heavy storm in April, 1958. The plates of the storm-tossed animals were shattered and all but five of the animals were dead. It is possible that after seeking food in shallower tidepools and crevices during high water, the chiton is left by the subsequent receding tide and falls from its loosely-held position among the algae. Caught by wave action, it may be beaten against the rocks before it can re-establish its hold or before it can get back to deeper waters. The animals when strongly stimulated in the laboratory have been seen to contract with sufficient force to crack their plates ; perhaps some are also broken in this manner in nature. Of the 146 sets of plates (Fig. 1A) collected 2 87, or 59.5 per cent, had one or more plates broken (about 18 per cent had one, 17 per cent had two, 11 per cent had three, 6.2 per cent had four, 3.4 per cent had five, 2.7 per cent had six, 1.3 per cent had seven, but none had all eight). Two animals had seven of the eight plates broken. The middle plates were broken most often, these being the widest and flattest (6.2 per cent plate 1, 11.4 per cent plate 2, 14.1 per cent plate 3, 18 per cent plate 4, 21.2 per cent plate 5, 16.8 per cent plate 6, 8.4 per cent plate 7, and 3.9 per cent plate 8). Often two or three adjacent plates were found with similar breaks, suggesting a blow from a large surface. 1 Supported in part by U. S. Public Health Grant RG 4578 to A. C. Giese. 2 The chitons were being used in a study of the annual reproductive cycle and of the biochemistry of the blood and tissues ; hence the plates were available in numbers, from specimens collected for these purposes. 318 SHELL REPAIR 319 Plates that had been broken just prior to the animal's death, either by storms or in the laboratory, showed a clean cleavage with the parts fitting perfectly together. Depending upon the severity of the blow, the cleavage was in a single straight line or in an arborescent pattern. Repairs were seen in few plates that had been shattered into as many as seven pieces. The first step in repair is the formation of a strip of membrane overlapping the crack on both sides of the plate. The second stage seems to be the accumulation of fine granules of a calcium salt, presumably in the form of carbonate (Bevelander and Benzer, 1948), under the conchiolin strip with the concomitant erosion of the underlying crystalline shell. This erosion often extends for some distance laterally from the crack under an extension of the membrane strip (Fig. 1C). The last step in repair (Figs. 1D-G) is the invasion or growth of existing minute crystals (Bevelander and Benzer, 1948; Bevelander, 1953) of the surface of the membrane strip by crystals of calcium carbonate in the form of aragonite (Prenant, 1927). The crystals are imbedded in the surface of the conchiolin, leaving a ridge over the crack, and often an air space or a layer of membrane between the old shell and the new material (Fig. IE). This leaves the plate weakened so that a second blow usually splits the plate along the old crack. To determine the rate of repair of broken plates, five chitons subjected to hammer blows were kept in the laboratory with ample food and in running sea water, and sacrificed after varying lengths of time. The results are quite variable but they serve to illustrate the slowness of repair. For example, while one chiton developed membranes around the cracks in twenty days and granular calcium carbonate deposition in twenty-four, another showed no visible sign of repair in the same period of time. In still another chiton, dissected sixty days after breaking the plates, crystalline calcium carbonate was evident in the cracks. However, in two chitons examined 100 days after injury, only membranes had been laid down over the cracked edges of the plates. Energy for mobilization of the shell calcium is available only during active feeding and digestion in some mollusks (Wagge, 1951, 1952; Robertson, 1941). The effects of starvation were not tested here in view of the variability of results with well-fed specimens. Wilbur and Jodrey (1955) inhibited shell deposition in the oyster with car- bonic anhydrase inhibitor. However, no tests were made with such inhibitors on Cryptochiton in view of the variability of results and the long time required for repair of broken skeletal plates. Furthermore, it is not even known whether amphineurans possess carbonic anhydrase although Freeman and Wilbur (1948) found it in most, but not all, of the species of gastropods and pelecypods tested. Katherina tunicata Fifty-five sets of plates of Katherina were examined and only five plates were found to be broken, although many of them were eroded to some extent, possibly by a disease. Of these, two showed slight evidence of repair. One had a thin membrane with a few lime crystals, the other showed an old crack completely repaired. It is possible that the other cracked shells were broken when the eviscerated specimens were boiled to loosen the plates for examination. One valve broken experimentally showed a conchiolin membrane after a few weeks. 320 JOHN S. TUCKER AND ARTHUR C. GIESE B *** SHELL REPAIR 321 Katherina lives in the surf zone on exposed shores among the sea palms and between mussel beds where, at certain times of the day, it withstands an almost continual pounding by the waves. Even at low tides the animals hold fast to bare rock or crustose algae with such strength that a knife or screwdriver is needed to pry them loose. At that, an inexperienced collector will often get only the plates and girdle, the foot and viscera remaining on the substrate. The storms of 1958 which left so many specimens of Cryptochiton on the beaches presumably failed to dislodge specimens of Katherina; at least none was seen on the beaches with Cryptochiton. The infrequency of broken plates in Katherina suggests that its plates are proportionally stronger than those of Cryptochiton. The average weight of the eight plates (10 specimens; average wet weight 33 grams) was 19.6 per cent of the wet weight of the entire chiton, in comparison to the 7.4 per cent for Cryptochiton (45 specimens; average wet weight 850 grams). It is also possible that the shape of a skeletal plate has some bearing on its resistance to shock. Plates No. 2 to No. 7 of Cryptochiton are in the shape of butterflies (Fig. 1A) and are relatively flat. The skeletal plates in Katherina consist of a heavy, roughly circular, disc with one pair of thin lateral lobes (Fig. IB). Mopalia hindsii Twenty-six sets of skeletal plates of Mopalia were examined and, other than chipping along the edges of the thin membrane, eleven had broken plates (six had one plate broken, three had two and one each had three or four plates broken). The most common crack was from the lateral notch to the beak of the plate. Along this line the plate is porous. Although many of the cracks are clean and may have resulted from boiling eviscerated specimens to release the plates, Mopalia suffers a fairly high incidence of infection from an unidentified boring animal which weakens the prismatic layer of the plate with long tunnels. One of these weakened plates was broken and the shell was thickened along the cracked tunnel. A thin membrane and some lime crystals were also deposited after a lapse of several weeks along cracks in plates No. 2 to No. 7 broken by a blow from a hammer. One unbroken plate showed deposition of new material where an attached barnacle overlapped the edge of the plate (Fig. 1H). Mopalia therefore can to some extent repair its skeletal plates. The specimens of Mopalia used in this work were collected from concrete pilings in Monterey Harbor, a relatively protected habitat. While the skeletal plates are broad and flat (Fig. 1C) and in this respect resemble those of Cryptochiton, at the same time they constitute about 22.1 per cent of the wet weight of the animals. Apparently they are adequate for the conditions to which the animals are exposed. FIGURE 1. A. Shell plates of Cryptochiton stclleri. X %. Plate No. 1 (anterior) is toward the top of the page. B. Shell plates of Katherina tunicata. X %. Plate No. 1 (anterior) is toward the top of the page. C. Shell plates of Mopalia hindsii. X %. Plate No. 1 (anterior) is toward the top of the page. D. Representative cracks in shell plates of Cryptochiton undergoing repair. X ^o- E. A section along a crack in a plate of Cryptochiton showing the space between the old shell and the new material deposited during repair. X 1. F. Lateral extension of the conchiolin strip (dark material) over two breaks. X 1. G. Inva- sion of the conchiolin strip (dark) by crystalline calcium carbonate (light). X 1. H. Deposi- tion of new shell (above, right) along edge of barnacle attached to Mopalia plate. X 1. 322 JOHN S. TUCKER AND ARTHUR C. GIESE SUMMARY AND CONCLUSIONS The skeletal plates of Katherina tunicata and Mopalia hindsii are sturdy, con- stituting about a fifth of the wet weight of the animal. They were seldom found broken in the specimens examined, but some broken plates were undergoing re- pair. The skeletal plates of Cryptochiton stcllcri, on the other hand, are flat and thin and constitute only 7.4 per cent of the wet weight of the animal. The majority of cryptochitons examined showed breaks in one or more skeletal plates and in almost all of these, some degree of repair and deposition of membrane or mineral could be observed. The ability to repair its plates is probably of value to this species in view of the weakness in design of its skeleton. Irregularities of plates and variations in numbers of skeletal plates have been described for other species of chitons (Crozier, 1919; Berry, 1925, 1935; Taki, 1932). It is interesting that apart from an occasional asymmetrical terminal plate of a Cryptochiton, no such irregularities in number or shape were observed in the three species of chiton studied here. LITERATURE CITED BERRY, S. S., 1925. On an abnormal specimen of the chiton, Acanthoplcura qrannlata. Ann. and Mag. Nat. Hist., 16: 173-175. BERRY, S. S., 1935. A further record of a Chiton (Nuttalina) with nine valves. Nautilus, 48: 89-90. BEVELANDER, G., 1953. Interrelations between protein elaboration and calcification in molluscs. Anat. Rec., 117: 568-569. BEVELANDER, G., AND P. BENZER, 1948. Calcification in marine molluscs. Biol. Bull., 94 : 176-183. CROZIER, W. J., 1919. Coalescence of the shell plates in Chiton. Amcr. Nat., 53: 278-279. FREEMAN, J. A., AND K. M. WILBUR, 1948. Carbonic anhydrase in molluscs. Biol. Bull., 94 : 55-59. HEATH, H., 1897. External features of young Cryptochiton. Proc. Acad. Nat. Sci. Phil., 8 : 299-302. PRENANT, M., 1927. Les formes mineralogiques du calcaire chez les etres vivants, et le problem de leur determinisme. Biol. Rev., 2 : 365-393. ROBERTSON, J. D., 1941. The function and metabolism of calcium in the Invertebrata. Biol. Rev., 16: 106-133. TAKI, I., 1932. On some cases of abnormality of the shell plates in chitons. Mem. Coll. Sci. Kyoto Imp. Univ., 8: 27-64. WAGGE, L. E., 1951. Amoebocytic activity and alkaline phosphatase during shell regeneration in Helix. Quart. J. Micr. Sci., 92 : 307-321. WAGGE, L. E., 1952. Quantitative studies of calcium metabolism in Helix aspcrsa. J. Exp. Zool, 120: 311-342. WILBUR, K. M., AND L. H. JODREY, 1955. Studies on shell formation. V. The inhibition of shell formation by carbonic anhydrase inhibition. Biol. Bull., 108 : 359-365. THE JUVENILE HORMONE. I. ENDOCRINE ACTIVITY OF THE CORPORA ALLATA OF THE ADULT CECROPIA SILKWORM CARROLL M. WILLIAMS 1 The Biological Laboratories, Harvard University, Cambridge 38, Massachusetts The endocrine role of the corpora allata of insects was discovered by V. B. Wigglesworth (1934, 1936) over twenty years ago. In a series of simple and decisive experiments on Rhodnius he showed that the corpora allata secrete a "juvenile hormone" which opposes metamorphosis. In these early studies Wig- glesworth also recognized that the corpora allata undergo pronounced changes in endocrine activity during the course of metamorphosis ; namely, that they are active in the immature nymph, inactive in the mature nymph just prior to metamor- phosis, and active again in the adult insect after metamorphosis. Subsequently, the general validity of these conclusions has been confirmed repeatedly and found to apply to both hemi- and holometabolous insects (for review, see Wigglesworth, 1954, pages 56-64). During the past twelve years, in the course of studies of the metamorphosis of the Cecropia silkworm, the juvenile hormone has necessarily been an object of detailed attention. While confirming the essential elements in Wigglesworth's theory, the study has helped to resolve certain persistent mysteries and, more recently, has pointed the way to the successful extraction and purification of the hormone itself. This first of a series of communications is concerned with the endocrine activity of the corpora allata of the adult moth. MATERIALS AND METHODS 1. Experimental animals The experiments were performed on Cecropia, Cynthia, and Polyphemus silk- worms. Taxonomists continue to amuse themselves by changing the generic and specific names of these Saturniids. What began as Phalacna cecropia became Samia cecropia, then Platysamia cecropia, and now Hyalophora cecropia (Michener, 1952). The Cynthia silkworm, known throughout the world as Philosamia cynthia, was changed to Samia walkcri, and then back to Samia cynthia. Telea polyphemus is now Antheraea polyphemus. As in the analogous cases discussed by Wald (1952, page 339), the "common names" have escaped the attention of taxonomists and have remained firm and unchanging. Therefore, the common names will be used routinely in the present reports. 1 This study was aided by a grant from the National Institutes of Health of the U. S. Public Health Service. It is a pleasure to acknowledge the advice and counsel of Prof. Berta Scharrer. 323 324 CARROLL M. WILLIAMS Cecropia silkworms were reared under nylon nets on wild-cherry trees. Poly- phemus were reared on oak or maple ; Cynthia, on cherry or ailanthus or purchased from dealers. The cocoons were harvested and stored as previously described (Williams, 1946a; Shappirio and Williams, 1957). 2. Surgical procedures Experimental animals must be deeply anesthetized during surgical procedures. We use carbon dioxide for this purpose and with mixtures of air and carbon dioxide have maintained pupae anesthetized for as long as one month without injury. Groups of animals are placed in a capped, flat-bottom Buchner funnel and exposed for about twenty minutes to a slow stream of carbon dioxide from a compressed cylinder. The gas is bubbled through water en route to the funnel. The animals are flaccid when fully anesthetized, and one can no longer elicit any movements of the abdominal segments. Surgical procedures are performed in a second Buchner funnel (diameter 11 cm., height 3 cm.) \vhich is mounted flush on the top of the operating bench. A slow stream of carbon dioxide is bubbled through water and passed through the bottom of the uncovered funnel. Carbon dioxide, being heavier than air, fills the cavity of the funnel and maintains a continuous anesthesia during the surgical procedure (Williams, 1946b). Operations are carried out under the low magnification of the dissecting micro- scope, making use of 9 X oculars and 0.7, 1, or 2 X objectives. The foot of the microscope is removed and the vertical pillar permanently attached to the operating bench on the distal side of the funnel. A hinged-arm permits the microscope to scan the entire diameter of the funnel. In order to leave both hands free, the microscope is equipped with a foot-focusing device (designed and built by Mr. Robert Chapman of the Harvard Biological Laboratories). Illumination is pro- vided by a 6-volt microscope lamp (Zeiss "Osram") attached to and moving with the microscope. The lamp is equipped with an infra-red filter. Anesthetized animals are transferred to the carbon dioxide-filled funnel for the surgical procedure. They are then returned to air, placed in individual num- bered glass containers ("creamers"), and stored in a room having a controlled humidity of sixty per cent and a temperature of 25 C. Dissecting instruments consist of the following: watchmaker's forceps (Dumont "rustless"; two of No. 3 and two of No. 5) ; a scalpel (Bard-Parker No. 3 handle with a No. 11 detachable blade); stainless iris scissors curved on the flat and closing to the tip ; several forms of stainless steel iridectomy and micro-scissors ; a stainless steel dental probe ; a 5-ml. hypodermic syringe filled with insect Ringer and capped with a 25-gauge needle. Prior to each group of operations the instruments are briefly rinsed in seventy per cent ethanol and wiped dry. Rigorous asepsis is unnecessary because the blood of the silkworms apparently contains an anti-bacterial substance that protects it from the ordinary contaminants. However, it fails to protect from insect pathogens and no diseased insect should be operated upon with the same instruments or even in the same room. Healthy pupae can withstand almost any degree of surgery provided that a few crystals of the potent anti-tyrosinase, phenylthiourea, are placed in the operat- INSECT JUVENILE HORMONE 325 ing field. We routinely use an equal part mixture of phenylthiourea (twice recrystal- lized from hot 95 per cent ethanol) and streptomycin sulphate, the two having been ground together in a mortar and stored in a capped vial in the refrigerator. Small amounts of the powder are removed and discarded within two days after being placed at room temperature. Ephrussi-Beadle Ringer's solution is utilized containing 7.5 gm. NaCl, 0.35 gm. KG, and 0.21 gm. CaCL, per liter of distilled water. The stock solution is brought to a boil, capped, and stored in the dark under refrigeration. Fungal contamination of physiological solutions, especially those containing bicarbonate, is a common source of difficulty when solutions are stored at room temperature. Excised tissues and organs are transferred to small depression dishes made of black glass and filled with Ringer. Black plastic bottle-caps are also satisfactory for this purpose. Dissections of sacrificed animals are performed in a glass Petri dish which fits snugly into the cavity of the Biichner funnel. Plasticine is pressed into the bottom of the dish to receive short stainless steel pins. The dish is filled with Ringer and the dissection performed with the animal spread and pinned under the solution. After surgical procedures on surviving pupae. Ringer's solution is added from a hypodermic syringe so that the blood is flush with the surface of the cuticle. The area of excised cuticle is then capped by a plastic window of appropriate size. The latter is punched or cut with scissors from cellulose acetate cover slips ("Turtox," thickness 1 or 2). The window is sealed in place with paraffin wax which is melted in an alcohol lamp and transferred with a curved needle or drawing pen. The melted wax adheres to the cuticle and the underside of the rim of the plastic slip provided that both are dry. The operating field is thereby equipped with a transparent window which permits one to look inside the living animal. 3. Excision of pupal corpora allata and corpora cardiaca An anesthetized pupa is placed in a plasticine cradle in the bottom of the carbon dioxide-filled funnel. The cuticle of the facial region is first removed. For this purpose a scalpel incision is made through the integument on each side of the face. The two cuts are joined by a transverse cut and the rectangle of cuticle is grasped with forceps and pulled free from its attachment at the base of the legs. The insect's abdomen is then pressed forward with plasticine and held in this position so that the blood fills, but does not overflow, the operating field. The naked epidermis is grasped with forceps, split down the middle, and trimmed free with scissors. The brain is thereby exposed. This is pressed down in the field to reveal the tiny corpus allatum-corpus cardiacum complex on each side. The complexes are dorso-lateral to the brain and attached on each side to a large tracheal trunk at this position (see Figure 1). A pair of tiny nerves emerges from the posterior face of each brain hemisphere and passes to the corpus cardiacum on that side. These nerves are very delicate and difficult to see in a dissection of this type. By means of forceps the connections between glandular complex and the adjacent trachea are broken, and the complex transferred to Ringer's solution in a black- dish. Alternatively, the tracheal segment can be excised with iridectomy scissors and removed along with the glandular complex. 326 CARROLL M. WILLIAMS INSECT JUVENILE HORMONE 327 4. Excision of adult corpora allata The moth is anesthetized and its head dipped momentarily into seventy per cent ethanol to wet the scales and hairs. The head is then cut off with scissors and placed in Ringer's solution. (The headless moth will continue to live for approximately the normal life-span of 7 to 10 days at 25 C.) The antennae are excised at their bases. Then with fine scissors the head is cut along the dorsal midline from its posterior margin to the mouth parts. The head is then spread apart with foreceps and pinned under Ringer. The pair of corpora allata-corpora cardiaca complexes is attached to the aorta just behind the brain. The brain is split in the midline to expose the aorta. The glandular complexes can now be broken free from the rear of the brain and transferred to a black dish by grasping the aorta with forceps. Under the favorable conditions of illumination in the black dish, one can recognize the corpus cardiacum ; it is attached by short nerves to the much larger corpus allatum. The latter is ordinarily flattened or wedge-shaped and sub- divided into a number of lobes and lobules. If necessary, the glandular complex may now be subdivided into its constituent parts by breaking the nerves between corpus cardiacum and corpus allatum. 5. Isolation of pupal abdomens This procedure has already been described for the Cecropia silkworm (Williams, 1947). The principal difficulty is to isolate the terminal abdominal segments with- out puncturing the fluid-filled midgut. This difficulty is circumvented by the use of the Cynthia silkworm. In this species the midgut contains only a solid, rod-like mass. Therefore the perforation of the midgut is inconsequential. The pupa is transected just behind the metathorax with a single transverse cut of a sharp razor blade. The abdomen is then supported with the cut surface facing upward. Crystals of the phenylthiourea-streptomycin mixture are spread in the wound, and Ringer's solution is added to fill the cavity of the abdomen. The wound is then capped with a plastic slip in which a central hole has been punched. The plastic is sealed in place with melted wax. Ringer is finally added via the central hole to replace all air, and the hole itself sealed with wax. RESULTS 1. Role of the corpora allata in adult development and sexual maturation The pair of corpora allata-corpora cardiaca complexes was removed from each of a series of twenty chilled male or female Cecropia pupae via the facial approach. The integumentary defect was capped and sealed with a plastic window, and the animals placed at 25 C. Adult development was initiated after about two weeks and proceeded in syn- chrony with the time-table for the normal development of Cecropia at 25 C. (Schneiderman and Williams, 1954). The moths, emerging after three weeks FIGURE 1. Brain and corpora allata of the Cecropia silkworm are shown in cutaway views of the head of larva (top), pupa (middle), and adult (bottom). The corpora allata are the two small bodies attached by tiny nerves to the back of the brain. (This figure is used with the permission of Scientific American.) 328 CARROLL M. WILLIAMS FIGURE 2. After receiving implants of three pairs of corpora allata of adult Cecropia, the Polyphemus pupa, here illustrated, has transformed into a second pupal stage. (See right side of preparation where the old pupal cuticle has been trimmed away.) FIGURE 3. This Cecropia pupa received implants of two pairs of adult Cecropia corpora allata. Development has given rise to a mixture of pupa and adult. (The old pupal cuticle has been completely removed.) INSECT JUVENILE HORMONE 329 of adult development, could not be distinguished from un-operated individuals. The females deposited a normal complement of eggs and both sexes survived for the customary period of 7 to 10 days at 25 C. The absence of corpora allata was confirmed in dissections of many of these moths. All the internal organs, including the gonads, showed full and complete development. The abdomens of females were packed with ripe eggs, and the males showed normal spermatogenesis. The experiment was repeated on a series of six male and six female pupae to produce moths lacking corpora allata. The two sexes were cross-mated and each of the six females was allowed to oviposit in a paper bag. A normal number (150-225) of eggs was collected from each female. These were placed under large nylon nets and the larvae reared to maturity on wild-cherry leaves. No deviation from normal development could be detected. These experiments show that the corpora allata play no evident role in the transformation of the pupa into an adult Cecropia or in the gonadal function of the adult itself. 2. Endocrine activity of adult corpora allata In the absence of any obvious function of the corpora allata of adult Cecropia, it is paradoxical to find that the glands, when excised and tested for endocrine activity, are more active in the moth than at any other stage in the life history (Williams, unpublished data). This fact was discovered eleven years ago in the course of an experiment performed for other purposes. It happened by chance that a pair of adult corpora allata was implanted into a brainless diapausing Cecropia pupa. Ten days later, the host showed the termination of diapause and the initia- tion of development. This result would have been puzzling in a normal diapaus- ing pupa ; in a brainless diapausing pupa it was incomprehensible. Even more puzzling w T as the character of the development which then took place. Within two weeks the brainless pupa transformed, not into a moth, but into a bizarre creature in which large areas of pupal cuticle had been freshly formed (see Figs. 3 and 4). The animal, in short, was a mosaic of pupal and adult characteristics (Williams, 1952b). During the past eleven years this result has been duplicated on numerous occasions. The experimental series includes fifty-one brainless Cecropia pupae which received one to three pairs of corpora allata-corpora cardiaca complexes derived from male or female Cecropia moths. As shown in Table I a total of twelve individuals (23 per cent) showed the result just described. The residual 77 per cent showed no effect of the implantation and continued to diapause. But the twelve positive experiments were of sufficient interest in themselves. Not only FIGURE 4. Pupal-adult monstrosity after implantation of adult corpora allata. Note the pupal cuticle on head, palps, and antennae. However, the wings show scale-covered adult cuticle and the eyes show considerable adult development. FIGURE 5. This isolated pupal abdomen received implants of adult corpora allata, plus an injection of ecdysone. The tip of the old pupal cuticle has been torn away to reveal a second pupal abdomen that has formed. 330 CARROLL M. WILLIAMS TABLE I Tests of adult corpora allata-corpora cardiaca complexes* in brainless diapausing pupae Adult donors Brainless hosts Normal development Mixed development No development Cecropia Cecropia 12 39 Cecropia Cynthia 2 2 Cecropia Polyphemus 2 1 Cynthia Cynthia 5 Cynthia Cecropia 20 Polyphemus Polyphemus 2 Polyphemus Cecropia 4 10 Totals 22 77 * One to three pairs of complexes from male or female moths were implanted into each brainless pupa. had the implants caused the formation of mixtures of pupa and adult; seemingly, they also had substituted for the brain and provoked the termination of diapause. As shown in Table I, this result was duplicated when corpora allata of adult male or female Cecropia were implanted into brainless diapausing pupae of Cynthia or Polyphemus. Here again, a certain percentage of animals terminated diapause and developed into pupal-adult mixtures. The corpora allata-corpora cardiaca complexes of male and female Cynthia and Polyphemus moths were also tested. The three species seem to differ among them- selves in the endocrine activity of the adult corpora allata. For example, the corpora allata of adult Cynthia gave negative tests in all twenty-five preparations. By contrast, the glands of adult Polyphemus gave positive tests in six of sixteen preparations. Moreover, when used as recipients of implants, brainless Poly- phemus pupae seemed to have a lower developmental threshold than the other two species, for four of five individuals gave a positive reaction to the implantation of adult corpora allata. In retrospect, Polyphemus appears to be the animal of choice for experiments of this type. In the far more numerous tests of Cecropia corpora allata, the conditions of the experiment were subjected to minor variations in the hope of recruiting a positive response in a larger proportion of individuals. By increasing the number of implanted glands from one to two or three pairs, little additional effect was realized. However, the developmental response was markedly enhanced when the host animals w f ere placed at 15 or 20 C. rather than at 25 C. after the implantation of corpora allata. It was also observed that the experimental animals which developed at the lower temperature retained a far larger proportion of pupal characters than in similar animals developing at 25 C. 3. Inactivity of corpora cardiaca In the experiments just considered, the adult corpora allata were implanted together with the attached corpora cardiaca. However, in thirty-five additional preparations, the corpora allata were carefully dissected from the attached corpora cardiaca and then implanted into brainless diapausing pupae. INSECT JUVENILE HORMONE 331 TABLE II Tests of adult corpora allata* (minus corpora cardiaca) in brainless diapausing pupae Adult donors Brainless hosts Normal development Mixed development No development Cecropia Cecropia 4 23 Cvnthia Cvnthia 2 Cynthia Cecropia 1 3 Polyphemus Polyphemus 1 Polyphemus Cecropia 1 Totals 6 29 * One to three pairs of corpora allata from male or female moths were implanted into each brainless pupa. The results, recorded in Table II, were substantially the same as those observed in the previous experiments. Once again, a certain low percentage of brainless animals terminated diapause and transformed into pupal-adult monstrosities. The inactivity of implanted corpora cardiaca was further confirmed in fourteen experiments in which adult corpora cardiaca were freed from corpora allata and tested, as such, in brainless diapausing pupae. No developmental response was obtained even when as many as ten pairs of adult corpora cardiaca were implanted. Indeed, in the course of twelve years of experimentation, we have never detected any trace of developmental response after the implantation of corpora cardiaca of larvae, pupae, or adults. For present purposes it is necessary to conclude that the developmental reactions under consideration are attributable to the adult corpora allata per sc. This implies that in a certain proportion of individuals the adult corpora allata have two effects : they first promote the initiation of adult development ; they then prevent the transformation of the pupa into a normal adult moth. 4. Effects of brain implantation As noted in Tables I and II, the vast majority of brainless Cecropia pupae continued to diapause when implanted with adult corpora allata. In all of these preparations the implants gave the impression of being inert. The true state of affairs is suggested by the following experiment : Two pairs of adult corpora allata were implanted into each of five brainless Cecropia pupae. Six weeks later the pupae showed no change from their condition at the outset. Two brains of previously chilled Cecropia pupae were implanted at this time to cause the initiation of development. The latter gave rise to creatures showing large areas of pupal cuticle. In effect, the initiation of development un- masked the endocrine activity of the previously implanted corpora allata. Further information was provided by the following experiment : Two pairs of adult Cecropia corpora allata were implanted under a facial window in each of two brainless diapausing Cecropia pupae. One month later the implants were removed and the pupae caused to develop by the injection of 125 ^g. of a 332 CARROLL M. WILLIAMS purified extract of prothoracic gland hormone (ecdysone). 2 Both individuals transformed into moths which retained large areas of pupal cuticle. This experiment shows that the presence of the brain is not necessary for the secretion of juvenile hormone by adult corpora allata. In the absence of the initiation of adult development, the implants had built up a substantial titer of juvenile hormone. But the host could not signal this fact until its development was brought about by ecdysone. 5. Experiments on isolated pupal abdomens Eight abdomens were isolated from diapausing Cecropia pupae. Preparations of this type remain in permanent diapause unless provided with ecdysone by in- jection (Williams, 1954), or by the implantation of active prothoracic glands, or by the implantation of inactive prothoracic glands plus active brains (Williams, I952a). In the present experiment efforts w r ere made to evoke a developmental response of isolated abdomens by the implantation of adult corpora allata either alone, or in conjunction with brains, prothoracic glands, or injections of ecdysone. TABLE III Effects of implantations into isolated abdomens of diapausing cecropia Abdomen no. Implant Result 1415 1 pr. adult C.C. + C.A. No development 1447 1 pr. adult C.C. + C.A. No development 2123 3 pr. adult C.C. + C.A. No development 2090. . 5 pr. adult C.C. + C.A. No development 2212 2^ pr. adult C.C. + C.A. plus 2 chilled No development pupal brains 1515 1 pr. adult C.C. + C.A. plus 2 pr. No development prothoracic glands of diapausing pupae 2109 3J pr. adult C.C. + C.A. plus 4 pr. Molted to form second prothoracic glands of diapausing pupae pupal abdomen 9320 2 pr. adult C.A. (-C.C.) plus 25 M g. of Molted to form second crystalline ecdysone pupal abdomen Table III summarizes the several types of preparations. It is of particular interest and importance to note that no development took place when the abdomens received only adult corpora allata. We have checked this finding in twelve ad- ditional experiments performed on isolated Cynthia abdomens ; in this case the pupal abdomens were distributed at 15, 20, and 25 C. after the implantation of two to five pairs of corpora allata derived from adult Cecropia or Polyphemus. In short, no trace of development was ever observed in response to the implantation of adult corpora allata per se. The same negative result was also recorded in an experiment where adult corpora allata were implanted along with active brains. The preparation numbered 9320 in Table III is of particular interest. Here, two pairs of adult corpora allata were implanted into an isolated abdomen. A - I am indebted to Dr. Peter Karlson for supplying highly purified preparations of ecdysone. INSECT JUVENILE HORMONE 333 month later 25 /zg. of crystalline ecdysone were injected. Development began within two days. Within the following ten days the pupal abdomen transformed and molted into a second pupal abdomen (see Figure 5). This result was duplicated in two additional experiments utilizing Cynthia abdomens. It is clear that ecdysone is the prime-mover in the developmental response and that the juvenile hormone is inactive in the absence of ecdysone. Attention is now directed to preparation 2109 in Table III. This pupal abdomen received implants of adult corpora allata plus diapausing pupal prothoracic glands. Precisely the same result was observed as after the injection of ecdysone: the pupal abdomen molted and transformed into a second pupal abdomen. In this case it seems necessary to conclude that the corpora allata activated the diapausing prothoracic glands that, in this sense, a hormone from the corpora allata had substituted for the brain hormone. However, there is no indication in Table III that this corpus allatum hormone can substitute for ecdysone itself. 6. Tests of adult corpora allata hi previously chilled pupae The results considered to this point lead to the prediction that adult corpora allata should be uniformly active when tested in previously chilled pupae just prior to the initiation of adult development. During the past ten years this prediction has been confirmed on a large scale. The experimental series includes ninety-eight preparations in which corpora allata of male and female moths of Cecropia, Polyphemus, and Cynthia were tested in chilled pupae of each of the same three species. All except eight animals gave rise to adults retaining pupal characters. In the eight negative tests the implanted glands had been derived from elderly adults just prior to death. There was a rough correlation between the number of implanted glands and the degree to which pupal characters were preserved a finding which will be considered in further detail in the following paper. Moreover, as was true in the earlier experiments on brainless pupae, the effects of the implanted corpora allata were amplified when the host pupae were placed at 15 or 20 C., rather than at 25 C., immediately after the implantation. The retention of pupal characters was extreme in many of the test animals. As shown in Figure 2, the pupa transformed into a second pupa which showed only traces of adult characteristics. In several experiments performed on Polyphemus and Cecropia, the secondary pupa molted into a tertiary form. In this case, the pupal characteristics were less prominent after the second molt than after the first. None of these animals was viable for any prolonged period after transforming into mixed forms. Although the old pupal cuticle became thin and crisp and the ecdysial lines were eroded to the surface, spontaneous escape from the old pupal cuticle occurred only in individuals showing minimal retention of pupal character- istics. All other animals remained enveloped in the old pupal cuticle until they died or were sacrificed. In many of the individuals the molting process proceeded to a normal terminal phase accompanied by a complete breakdown of the old endocuticle and a partial or complete resorption of the molting fluid. Yet, for some unexplained reason, the insect failed to undertake the vigorous muscular efforts that accompany a normal ecdysis. It did not "try to molt" even though it possessed the nervous and 334 CARROLL M. WILLIAMS muscular equipment to do so. The use of forceps was therefore necessary to peel off the old pupal exuviae. In many individuals it was difficult or impossible to withdraw the lining of the old tracheal tubes through the spiracular openings. Indeed, in the case of Cecropia, the larger branches of this old system became stiff and melanized and therefore incapable of being shed. The net effect is that the juvenile hormone is a lethal agent for all these Saturniid pupae. 7. Inactivity of killed corpora allata The high activity recorded for implanted adult corpora allata suggested the possibility that substantial amounts of hormone might be stored within the glands themselves. This prospect was tested in five experiments. In one experiment eight adult Cecropia corpora allata were frozen and thawed twice at 40 C. and then implanted into a previously chilled pupa. Normal development ensued. In four other experiments adult corpora allata, in numbers ranging from 9 to 44, were homogenized in 0.1 ml. of insect Ringer and then introduced into four previously chilled pupae. All four animals developed into normal adult moths. Evidently, little or no hormone is stored in the living gland, for the activity of a single living adult corpus allatum was not duplicated by the implantation of up to forty-four dead glands. DISCUSSION 1. Secretion of the juvenile hormone by the adult corpora allata The experimental results demonstrate the endocrine activity of the corpora allata of Cecropia, Polyphemus, and Cynthia moths. As is amply evident in Wigglesworth's (1954) recent review, this finding is consistent with the picture presented in all other insects that have been studied in detail including several families of Lepidoptera. In the Cecropia silkworm the corpora allata, when re- moved and tested, are found to be more active in the adult moth than at any other stage in the life history (Williams, unpublished data). Moreover, there is general agreement that at least one of the secretory products of the adult corpora allata is the same juvenile hormone which is secreted weeks or months earlier by the corpora allata of the immature insect. This conclusion was first proposed by Pflugfelder (1938a, 1938b) and Pfeiffer (1945), and will be further documented in the subsequent papers in this series. 2. The role of the juvenile hormone in adult moths We have been unable to detect any function for the corpora allata in the pupal or adult stages of these Lepidoptera. Thus, as we have seen, the corpora allata can be removed from pupae of either sex without disturbing the development of normal, viable, sexually mature moths. These findings are the same as those re- ported for Bombyx mori by Bounhiol (1938) and Fukuda (1944). The present study enlarges the negative evidence by showing that the absence of corpora allata fails to interfere with the maturation of functional gametes and the production of normal offspring. INSECT JUVENILE HORMONE 335 The situation in the Lepidoptera therefore departs from that described for most other orders of insects where the corpora allata are necessary for the deposition of yolk in the adult female and for the secretory activity of the accessory glands in the adult male (for summary, see Wigglesworth, 1954, pages 77-80). In the Lepidoptera which have been studied, all these functions can go forward in the absence of corpora allata. For the sexual maturation of both males and females all that is required is the presence of prothoracic gland hormone (ecdysone). The brain hormone is also unnecessary for the sexual maturation of these silkworms. Pupae from which the brain, corpora cardiaca, and corpora allata have been removed develop into sexually mature moths after the injection of crystalline ecdysone (Williams, 1954). Adult Lepidoptera therefore present the paradoxical picture of the presence of highly active corpora allata for which there appears to be no apparent function. However, it is worth recalling that corpora allata have been tested only in species of adult Lepidoptera which are short-lived and unable to feed. In adults of the giant silkworms, as in the commercial silkworm, functional mouth-parts are absent. Consequently, the duration of the adult stage is greatly curtailed : ripe eggs must be ready for oviposition at the time of adult emergence. In short, the absence of mouth-parts has enforced on these short-lived moths a precocious maturation of the gonads during the course of pupal-adult development. Indeed, months before the development of the adult moth, the proteins which later appear in the yolk of the eggs are already present in high concentrations in the blood of the diapausing pupa (Telfer, 1954). It is among the feeding, long-lived species of adult Lepidoptera that one would anticipate a gonadotropic function for the corpora allata akin to that seen in most other orders of insects. This inference is in accord with the histological studies of Kaiser (1949) on long-lived butterflies of the genus Vanessa. Presumably, in the Ephemeroptera and other non-feeding adults one should find the same picture as presented by the Saturniidae. The absence of functional adult mouth-parts is clearly a secondary affair in the evolution of the Lepidoptera. Indeed, the very same moths contain digestive tracts of normal organization, but of no apparent function. Evidently, the presence of active corpora allata is a memento of a more primitive endocrinological situation. 3. Biological role of the juvenile hormone The juvenile hormone plays no role in the transformation of a pupa into an adult moth. All that is required is that the juvenile hormone be absent throughout the early phases of this transformation (Williams, 1952b). This conclusion is in line with the finding that the corpora allata are inactive throughout the entire pupal stage and during the first two-thirds of adult development (Williams, unpublished data). A pupa can be supplied with juvenile hormone by the implantation of living, active corpora allata obtained from larvae or adults. However, as demonstrated in the experiments on isolated pupal abdomens (Table III), the juvenile hormone has no effects in the absence of the prothoracic gland hormone, ecdysone. Only when the abdomen is provided with this hormone can one detect any action of 336 CARROLL M. WILLIAMS the implanted corpora allata. The outcome is that the pupal abdomen terminates diapause, molts, and transforms into a second pupal abdomen (Fig. 5). Substantially the same result is seen in experiments performed on brainless diapausing pupae. Here again the implantation of adult corpora allata is in- consequential unless ecdysone is supplied by injection or by the secretory activity of the animal's own prothoracic glands. The juvenile hormone then opposes the transformation of the pupa into an adult moth. The result (Figs. 3 and 4) is a creature showing to varying degrees a retention of pupal characters of the type previously described by Piepho (1952) and Williams (1952b). When the titer of juvenile hormone is high, then one may witness the formation of a bona fide second pupal instar a phenomenon hitherto unknown in any insect (Fig. 2). But, even in the presence of the highest concentrations of juvenile hormone, we have never observed in this material the reappearance of larval characters such as described in Rhodnius (Wigglesworth, 1954, 1957, 1958). 4. Mimicking of brain hormone In a certain proportion of brainless diapausing pupae the implantation of active corpora allata causes the termination of diapause and the initiation of adult development. This result is not seen in isolated pupal abdomens or other prepara- tions lacking prothoracic glands. But, as noted in Table III, the developmental re- action becomes possible if an isolated abdomen receives active corpora allata plus inactive prothoracic glands, or active corpora allata plus an injection of ecdysone (Fig. 5). Moreover, in numerous experiments to be described on a later occasion, the development of brainless diapausing pupae has been provoked by the injection of crude or purified extracts of juvenile hormone. Evidently, under certain un- defined conditions, a hormonal secretion of the corpora allata can activate the prothoracic glands and, in this sense, mimic the function of the brain hormone. Whether this hormone is the juvenile hormone or some further secretory product of the corpora allata is impossible to state at the present time. A decision on this point will become possible only when the juvenile hormone is isolated and tested in pure form. The finding that the corpora allata can turn on the prothoracic glands has an obvious bearing on the endocrine control of larval molting. If the corpora allata can activate the pupal prothoracic glands, there is no reason to suppose that they cannot do so in the immature larva. We begin to see a multiplicity of agencies which can promote the secretion of ecdysone by the prothoracic glands. The brain can turn on the prothoracic glands. Ecdysone can turn on the prothoracic glands (Williams, 1952a, 1954). And, evidently, under certain undefined conditions, so also can the corpora allata. Nature has apparently found it prudent to surround the prothoracic glands by a net-work of controls. The present study suggests that the corpora allata are a part of that net- work. SUMMARY 1. Juvenile hormone is secreted in high concentration by the corpora allata of the adult Cecropia moth. INSECT JUVENILE HORMONE 337 2. Notwithstanding this fact, the juvenile hormone has no apparent function in the adult moth. Extirpation of the corpora allata in the pupal stage fails to interfere with the production of normal moths whose gametes give rise to normal offspring. 3. The corpora allata are inactive during the entire pupal stage as well as during the first two-thirds of adult development. If active corpora allata are implanted into a pupa just prior to the initiation of adult development, the juvenile hormone acts to oppose the differentiation of the adult moth. Development gives rise to an insect showing a mixture of pupal and adult characters. In the presence of high con- centrations of juvenile hormone the pupa molts and transforms into a second pupa showing only traces of adult characters. 4. The biological action of juvenile hormone is seen only in the presence of active prothoracic glands or their secretory product, ecdysone. Isolated pupal abdomens fail to respond to juvenile hormone unless ecdysone is simultaneously present. When both hormones are present, the pupal abdomen terminates dia- pause, molts, and transforms into a second pupal abdomen. 5. Evidence is presented that the corpora allata secrete a factor which can mimic the brain hormone and activate the prothoracic glands. This finding is considered in relation to the endocrine control of larval molting. LITERATURE CITED BOUNHIOL, J. J., 1938. Recherches experimentales sur le determinisme de la metamorphose chez les Lepidopteres. Bull. Biol., SuppL, 24: 1-199. FUKUDA, S., 1944. The hormonal mechanism of larval molting and metamorphosis in the silkworm. /. Fac. Sci. Tokyo Univ. sec. IT, 6: 477-532. KAISER, P., 1949. Histologische Untersuchungen iiber die Corpora allata und Prothoraxdrusen der Lepidopteren in Bezug auf ihre Funktion. Arch. f. Ent^v., 144 : 99-131. MICHENER, C. D., 1952. The Saturniidae (Lepidoptera) of the Western Hemisphere. Bull. Amer. Museum Nat. Hist., 98 : Article 5. PFEIFFER, I. W., 1945. The influence of the corpora allata over the development of nymphal characters in the grasshopper Melanoplus differentialis. Trans. Conn. Acad. Arts Sci., 36: 489-515. PFLUGFELDER, O., 1938a. Untersuchungen iiber die histologischen Veranderungen und das Kernwachstum der Corpora allata von Termiten. Zcitschr. f. wiss. ZooL, 150: 451-467. PFLUGFELDER, O., 1938b. Weitere experimentelle Untersuchungen iiber die Funktion der Corpora allata von Dixippus morosus Br. Zeitschr. f. iviss. Zool., 151 : 149-191. PIEPHO, H., 1952. liber die Lenkung der Insektenmetamorphose durch Hormone. Verh. deutsch. Zool. Ges. (Leipzig), 1952: 62-75. SCHNEIDERMAN, H. A., AND C. M. WILLIAMS, 1954. Physiology of insect diapause. IX. The cytochrome oxidase system in relation to the diapause and development of the Cecropia silkworm. Biol. Bull, 106 : 238-252. SHAPPIRO, D. G., AND C. M. WILLIAMS, 1957. The cytochrome system of the Cecropia silk- worm. I. Spectroscopic studies of individual tissues. Proc. Roy. Soc. London, Ser. B, 147 : 218-232. TELFER, W. H., 1954. Immunological studies of insect metamorphosis. II. The role of a sex-limited blood protein in egg formation by the Cecropia silkworm. /. Gen. Physiol., 37 : 539-558. WALD, G., 1952. Biochemical evolution. In: Modern Trends in Physiol. and Biochem., E. S. G. Barren, Edit., Acad. Press, N. Y. : 337-376. WIGGLESWORTH, V. B., 1934. The physiology of ecdysis in Rhodnius prolixiis (Hemiptera). II. Factors controlling moulting and "metamorphosis." Quart. J. Micr. Sci., 77: 191-222. 338 CARROLL M. WILLIAMS WIGGLESWORTH, V. B., 1936. The function of the corpus allatum in the growth and reproduction of Rhodnins prolixus (Hemiptera). Quart. J. Micr. Sci., 79: 91-121. WIGGLESWORTH, V. B., 1954. The Physiology of Insect Metamorphosis. Monographs in Exp. Biol., No. 1. Cambridge Univ. Press. WIGGLESWORTH, V. B., 1957. The action of growth hormones in insects. Symp. Soc. Exp. Biol, 11 : 204-227. WIGGLESWORTH, V. B., 1958. Some methods for assaying extracts of juvenile hormone of insects. /. Insect Physiol., 2 : 73-84. WILLIAMS, C. M., 1946a. Physiology of insect diapause: the role of the brain in the production and termination of pupal dormancy in the giant silkworm, Platysamia cecropia. Biol. Bull., 90 : 234-243. WILLIAMS, C. M., 1946b. Continuous anesthesia for insects. Science, 103 : 57-59. WILLIAMS, C. M., 1947. Physiology of insect diapause. II. Interaction between the pupal brain and prothoracic glands in the metamorphosis of the giant silkworm, Platysamia cecropia. Biol. Bull., 93: 89-98. WILLIAMS, C. M., 1952a. Physiology of insect diapause. IV. The brain and prothoracic glands as an endocrine system in the Cecropia silkworm. Biol. Bull., 103 : 120-138. WILLIAMS, C. M., 1952b. Morphogenesis and the metamorphosis of insects. Harvey Lectures, 47: 126-155. WILLIAMS, C. M., 1954. Isolation and identification of the prothoracic gland hormone of insects. Anat. Rec.. 120 : 743. Vol. 116, No. 3 June, 1959 THE BIOLOGICAL BULLETIN PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY EMBRYOLOGICAL DEVELOPMENT OF THE POLYCHAETOUS ANNELID, DIOPATRA CUPREA (BOSC) 1 M. JEAN ALLEN - Marine Biological Laboratory, ]] 7 oods Hole, Massachusetts Insofar as the writer knows, the normal embryology of Diopatra cuf>rea has never been completely worked out. The main trouble seems to have been that investigators, with the exception of Just (1922), have found that it is difficult to activate the eggs of this species even when they appear ripe. Andrews had similar difficulty with the eggs of the closely related species, Diopatra magnet (since designated Onn[>his nia;/ua ). He made the statement (1891b, page 115) that "attempts at artificial fertilization were unsuccessful" although the eggs seemed ripe as indicated by their size and the large numbers present packing the coelom, as well as the occasional finding of similar eggs amongst the larvae in the egg masses which he found during the breeding season. However, Just (1922), in a paper concerned primarily with raising mature Platynercis nicyalops from eggs, noted (page 477), "Though it is usually stated that artificial insemination of Dio- patra eggs is not possible, every attempt made by the writer . . . was successful," and that he reared Diopatra citprea to a length of 4 cm. No record of development was given. The problem of activation has remained a significant one throughout the course of this investigation. \Yith perseverance (particularly initially ) larvae from many batches of eggs have been raised during the course of several summers to a stage where 6 sets of setae have been formed and, in the summer of 1958, a few were raised to a stage with 7 sets of setae. Thus far two abstracts have been published on this work (Allen, 1951, 1953) and more recently Costello ct al. (1957) have included some additional previously unpublished data (furnished by the present writer ) in their book on handling marine eggs and embryos. The study of the development of D. citprea is still incomplete but enough addi- tional material has recently been worked out so that it was thought advisable to publish a more detailed account of development than has thus far been done. There is little material in the literature on the development of the genus, Diopatra. As further observations on living material were made, the confusion in the litera- ture surrounding the development of the species, D. cuf>rca, became more apparent. 1 Supported in part by summer research grants from the University of New Hampshire and Wilson College. - Present address : Department of Biology, Wilson College, Chambersburg, Pennsylvania. 339 b Copyright 1959, by the Marine Biological Laboratory 340 M. JEAN ALLEN Observations made during the present study suggest that most, if not all, of the material which has been published on the development of Diopatra cuprca has been incorrectly attributed to this species, so that the investigations of the writer may represent the only material published on the development of this polychaete. MATERIAL AND METHODS The adult worm. The characteristics and habits of the adult worms of this species have been described by various investigators (Andrews, 1891a; Sumner, Osborn and Cole, 1911; Hartman, 1945, 1951; ct a!.}. The parchment-like tubes of these polychaetes, which are found in the intertidal zone, go down two to three feet into the substratum. When disturbed the worm retreats into the tube so that in digging for the adults one rarely obtains the whole worm. As a result, the posterior tip with its four anal cirri is rarely seen. The head bears five occipital tentacles and two shorter frontal tentacles. Larvae have been raised to a stage when the five occipital tentacles and two anal cirri are noticeable. Males which are sexually mature are cream to yellowish in color as a result of the sperm packed in the coelom. In males with fewer sperm only the parapodia are yellowish in color. Females when sexually mature are usually grey-green due to the color of the eggs (which have a green pigment) packed in the coelom. This species is plentiful in Woods Hole waters. Most of the collecting for this investigation was done at Northwest Gutter, Hadley Harbor, Massachusetts, and some of it was done in the harbor at North Falmouth and at Woods Hole, on the Buzzards Bay side. The adults for the most part were kept in aquaria in running sea water. The worms were fed every day or two with pieces of the mussel, Mytilus. Procuring and handling living developmental stages. The writer has raised larvae of Diopatra cuprca from mid-June through August following artificial ferti- lization. The problem of activation of apparently ripe eggs was present throughout this period but artificial insemination was more successful in June and July than in August. This is contrary to the remark of Bumpus (1898, page 855) that "the ova are nearly ripe in August." During the breeding period of a sexually mature worm, the coelom becomes packed with gametes. When eggs or sperm are needed, the posterior end of a worm is exposed by cutting the end of the tube with scissors. The exposed portion is then held lightly with forceps. This usually results in the worm's pinching off its posterior segments. Eggs were obtained from the isolated posterior sections by slitting the body wall along the bases of the parapodia with No. 5 watchmaker's forceps. Eggs thus obtained were washed in Syracuse dishes with sand-filtered sea water. In general, spermatozoa were obtained by making a small slit at the base of a parapodium with a No. 5 watchmaker's forceps and diluting the "dry" sperm with sand-filtered sea water. Under the dissecting microscope ripe sperm were observed to be active immediately. Polyspermy should be avoided. Within a few minutes after insemination the eggs were washed several times with sand-filtered sea water. Usually they were given fresh sea water one to two hours later. If development were normal, ciliated larvae developed at room temperature within three hours after insemination. At this stage larvae usually were transferred to stender dishes and placed on the sea water table in a moist DEVELOPMENT OF DIOPATRA CUPREA 341 chamber with 90 % sea water in the bottom. The water was changed at least once a day thereafter. Apparently egg laying in D. cuprca is a phenomenon rarely observed (Sumner et al., 1911). In only one instance did the writer observe natural egg laying in the laboratory. This was on the evening of June 23, 1949. A worm tube was picked up and eggs were immediately released in a transparent, only slightly viscous, jelly which dissolved readily in sea w^ater. The eggs w r ere fertilized arti- ficially and almost \00 c /c cleaved. Only a few other times in the experience of the writer has fertilization approached 100%, as the method of artificial insemina- tion described is frequently unsuccessful. To get a batch of eggs with 50% of the eggs cleaving is good. Observations were made on living stages with the dissecting and compound microscopes and, in the summer of 1958, additional observations w-ere made with the phase microscope. For study and for photomicrographs the ciliated stages were slowed down with a little dry MS-222 (tricain) added with a dissecting needle to a drop of filtered sea water containing the larvae (optimal concentrations for quieting various larval stages were not determined). For the setal studies the larvae were placed on a slide in a drop of filtered sea water and then a cover slip was applied. They were examined briefly under a magnification of X 430 and then left to dry a little. This treatment in many cases spread out the setae which were then studied in more detail under X 430. Handling of fixed material. Various stages were fixed, paraffin-embedded, and serially sectioned (usually at 7 or 10 micra). Whole mounts of stained and un- stained stages were also made. The fixatives used for the early stages were usually Allen's B-15 or Bouin's, and for later larval stages Schaudinn's or Bouin's heated to 60 C. A series was also fixed in Meves'. A variety of stains was tried includ- ing Heidenhain's hematoxylin, Harris' hematoxylin, acetocarmine, alum-cochineal, Giemsa's, toluidin blue, and Feulgen's. Sections and whole mounts usually were mounted in Permount or Canada balsam. It was considered important to use such whole mounts to make a cell lineage study through at least the early cleavage stages. However, the method which had given excellent results with cleavage stages of the gastropod, Crepidula, failed completely with Diopatra. Various other techniques have been tried, including pre-treatment to remove lipids or ribonucleic acid, either of which might take up the stain in the cytoplasm. To date a technique has not been developed that would stain the chromosomes and enable one to follow the orientation of the spindles without staining the cytoplasm. NORMAL DEVELOPMENT The writer has indicated already (1951 ) that the cleavage of Diopatra cnprea occurs with amazing rapidity, functional cilia being formed within three hours after insemination. Prior to this age, it is difficult to construct a time table of development because there is considerable variability among different batches of eggs and also among different eggs in the same batch, particularly in cases in which low percentages of fertilization occur. The following represents a slight elabora- tion of the schedule recorded in Costello ct al. ( 1957) which is based on the writer's data obtained over several summers. The times are recorded from insemination at temperatures of 21-24 C. 342 M. JEAN ALLEN Stage Time First polar body 15-20 minutes Second polar body 20-30 minutes Two- to four-cells 40-60 minutes Eight-cells 50-90 minutes Mid- to late cleavage 90-120 minutes Functional cilia 3 hours Apical tuft (apparent in some) 8-9 hours Apical tuft (present in all normal larvae) 12 hours Rotating trochophores 24 hours 2 to 3 sets of internal setae 36 hours 3 sets of external setae, no tentacles 2 days 4 sets of external setae, some with 3 tentacles 34 days 5 sets of external setae, 5 tentacles 4^-5 % days 6 sets of external setae, 5 tentacles 6^-8 days 7 sets of external setae, 5 tentacles 13-17 days (typical?) The various stages of normal development are described in more detail below. The unfertilized e*<< *i. .' ;*v. ^;-v 17 ,. ._ 18 19 PLATE III EXPLANATION OF FIGURES FIGURE 16. Section of two young eggs in the coelom, showing attached algal-like strings of cells (second string not in plane of section), the nucleus and prominent nucleolus in the egg and in each of the "nurse" cells. FIGURE 17. Fertilized egg in metaphase I, showing the DEVELOPMENT OF DIOPATRA CUPREA 349 Larvae of 2 days, 8-12 hours. These larvae, about the same width as the pre- ceding, have elongated by about 100 micra and measure approximately 325 X 200 micra (in measurements of larvae, widths indicate the broadest portion). The tendency of some larvae to settle on the bottom at this stage seems to be correlated with the secretion of mucus ; other larvae, however, are still actively rotating, positively phototactic swimmers. Their invariable swarming towards the light makes changing the water easy at this stage. The larvae usually have differentiated three sets of setae externally (sometimes only two), with a fourth set forming internally in some. The third set, though extending externally, may be incom- pletely formed ( see Table I ) . Larvae have two prominent red eyespots and several pigment spots anteriorly. The apical tuft, though reduced, is still prominent, being roughly 55 micra long. The anterior arms of the opaque Y-shaped mid-gut surround the colorless pharynx. Scattered black pigment spots can be seen in surface view. The prototroch is still present as is the telotroch of longer cilia, and between them are shorter cilia. Rarely seen, but very clear when observed with the phase microscope, is a little patch of cilia just posterior to each set of setae. The characteristic refractile drop- lets are still present at the widest part of the prototroch and this area appears con- tinuous with the mid-gut region. The hind-gut is not clearly defined. Larvae of 3 days, 8 to 12 hours. Larvae of this stage are slightly longer and usually somewhat narrower than those of the preceding stage (for example, one measured 400 X 180 micra). A few are still swimming and are positively photo- tactic, but most tend to crawl on the bottom, secreting mucus as they do so. They sometimes stick together in clumps in which case they should be separated before they die. Some have formed transparent slime tubes. Usually four functional sets of setae are visible externally (Figs. 11 and 12) and the parapodium of the first setigerous segment has two protrusions, a finger-like postsetal lobe and a shorter presetal lobe (Fig. 33). A tuft of cilia, rarely observed, is present at the base of each parapodium. An apical tuft is still prominent but is often missed even with the phase microscope, for it tends to bend backward when slowed with MS-222. The fairly broad prototroch extends from the anterior level of the eye- spots to just anterior to the first set of setae (compare Figures 11 and 12). The prominent telotroch lies just posterior to the last set of setae (Fig. 12). Incipient jaws have differentiated which have an extra toothed plate on one side of the otherwise symmetrical maxillae (similar to Fig. 29). This asymmetry of the jaws is characteristic of the adult. These larval jaws are movable indicating that pharyngeal muscle is differentiating. Peripheral vacuolated mucous cells are clearly defined. Two of the large posterior vacuoles may be visible externally (Fig. 11). The broad anterior region, with its bubbly cytoplasm, still appears contrast between yolky and non-yolky cytoplasm. FIGURE 18. Two-cell stage in metaphase of second cleavage showing that the CD blastomere is larger than the AB. Note the fertiliza- tion membrane. FIGURE 19. Three-cell stage showing that the CD blastomere sometimes cleaves before the AB. This may represent abnormal development. FIGURE 20. Blastomeres of late cleavage held firmly within the egg membrane. The peripheral vacuolated region is beginning to appear and one blastomere is in metaphase. FIGURE 21. Longitudinal section through an early ciliated stage, approximately three hours, showing central mound of cells at the animal pole, two of the four vacuolated plates of cells, and small round body (probably a polar body) beneath membrane at the right. 350 M. JEAN ALLEN 26 PLATE IV DEVELOPMENT OF DIOPATRA CUPREA 351 continuous with the droplet-filled darker mid-gut region (Fig. 11, droplets not in focus ) . The arms of the Y-shaped mid-gut surround the pharynx. The thick- \valled, rather transparent hind-gut, presumably ectodermal, is forming. In some batches, buds of the three more dorsal tentacles are obvious, as well as the rudi- ments of the two anal cirri. Further internal structure can be seen in serial sections. Figure 27 is a sagittal section of this stage, showing pharynx and incipient jaws, narrow esophageal por- tion, and the mid-gut which has no lumen as yet and contains some dark pigment spots. A coelom has appeared, two flattened nuclei of the ventral peritoneal cells being clearly visible. The ventral body wall is thick compared with the dorsal and a ventral nerve cord is differentiating just beneath the peritoneum. A cerebral ganglion is visible just anterior to the pharynx. At least four large posterior vacuoles are visible. Larvae of 4 days. By this stage four sets of setae are visible externally and a fifth is beginning to form internally. The apical tuft was not observed and proto- and telotrochs are reduced. A few superficial scattered dark pigment spots can be seen in living larvae, and the endodermal and mid-gut contains some pig- ment. The transparent hind-gut has a narrow lumen. In most larvae, three well developed tentacular protrusions have appeared (Fig. 13) and buds of the two more ventral tentacles, as well as two anal cirri. Also visible through the body wall are the developing jaws (Fig. 13). Larvae of 4 days, 8 to 12 hours. Larvae of this stage have settled on the bot- tom and some may be observed in transparent slime tubes. They have four sets of functional setae externally with a fifth beginning to protrude in some. The presetal and postsetal lobes on the parapodia of the first setigerous segment are retained in this stage and in the subsequent stages described (compare Fig. 33). Five occipital tentacles are present, one mid-dorsal, two dorso-lateral, and two ventro-lateral ones, the last two being shorter. Two anal cirri are represented by PLATE IV EXPLANATION OF FIGURES FIGURE 22. Frontal section of 24-hour trochophore (anterior at right) showing pharynx near center, light undifferentiated yolk mass just posterior to it, and mesodermal bands flanking the mid-gut. FIGURE 23. Transverse section through the central mound in a larva similar to that in Figure 21, showing the four plates of vacuolated cells surrounding the mound. FIGURE 24. Frontal section through a 36-hour larva (cut at 15 micra) showing pharynx (note anaphase), light undifferentiated yolk mass, and four prominent posterior vacuoles. FIGURE 25. Transverse section through the pharynx of a larva that is similar to Figure 24, showing peripheral vacuolated cells and the cilia penetrating the larval membrane. FIGURE 26. Frontal section through a 36-hour larva (cut at 10 micra) showing the pharynx (note anaphase), yolk mass, and two large posterior vacuoles. Two sets of internal setae are forming (tip of lower arrow) and two of the mucus-secreting cells with basal nuclei are visible (tip of upper arrow). FIGURE 27. Sagittal section through larva of 3% days, with four sets of setae. The jaws are beginning to form in the pharynx, the cerebral ganglion (light area) is anterior to them, and the mid-gut (without a lumen) is posterior to them. Note also the posterior vacuoles, the coelom around the gut, and the peritoneal cells (two nuclei clear) lying in contact with the ventral nerve cord. The ventral body wall is thicker than the dorsal. FIGURE 28. Sagittal section through larva of 5^/2 days, with five sets of setae. The same structures seen in Figure 27 may be noted, although they are more highly differentiated. The mid-gut region now has a lumen continuous with the intestine which opens by way of a ventral anus, and some of the mid-gut cells have black pigment. 352 M. JEAN ALLEN 32 33 PLATE V EXPLANATION OF FIGURES FIGURE 29. Differentiating jaws of a larva of 4% days showing toothed asymmetrical maxillary plates on the left (an extra toothed portion is present on the left side) and mandibles on the right. Note also the bundle of curved pointed setae from the first setigerous segment. FIGURE 30. Jaws from a larva of approximately eleven days, showing further differentiation DEVELOPMENT OF DIOPATRA CUPREA 353 buds in some larvae of this stage, but are more obvious in others. Tufts of cilia, visible at the eye level in some, probably represent the remains of the prototroch. A prominent telotroch is still present. Also visible externally are jaws consisting of asymmetrical maxillary plates with well defined teeth and differentiating man- dibles (Fig. 29). An esophagus is differentiating between pharynx and mid-gut, and the latter continues posteriorly into the hind-gut. The dark yolk mass and droplets are restricted to the mid-gut and black pigment is visible in its lining. Some of the larvae appeared to be feeding on microorganisms. Larvae of 5 l / 2 to 7 1 /. days. Larvae of 5 l / 2 days have 5 sets of functional setae although the last set is usually not completely formed ; in some cases a sixth set is differentiating internally. Some larvae may be observed in transparent slime tubes on the bottom, and in one instance a larva was observed turning around in its tube. Larvae which have not formed tubes often stick to the bottom at this stage and may constrict in two in attempting to free themselves. The five occipital tentacles are "knobby" and well developed (Fig. 15) : the three more dorsal ones are ap- proximately 1 50 micra in length and have two basal segments by 7 days ; the two more ventral ones are shorter and have one basal segment each. Two anal cirri are well developed (approximately 30 micra in length) and "knobby" (Fig. 15). A number of the differentiating internal structures of this stage can be illus- trated by Figure 28. This is a sagittal section through a larva with 5 sets of setae (SVs days old) and with well developed jaws associated with the pharynx. The mid-gut is patent throughout, its lumen being continuous with that of the hind-gut which, in turn, opens ventrally through the anus. The coelom has enlarged as compared with the preceding stage (Fig. 27). Nuclei of two of the flattened peri- toneal cells are visible ventrally (the peritoneum can also be seen in living larvae), and the cerebral ganglion and ventral nerve cord are clearly visible. Larvae of 8 days, 8 hours and older. By 8% days, 6 sets of setae have formed externally in most cases and are complete, or almost so. However, some larvae take one to three days longer to form the sixth set (a few take even longer). The black jaws are well differentiated and active at these stages. The asymmetrical maxillary plates have a medial toothed margin in each half (as well as the toothed as compared with Figure 29. The bundle of curved pointed setae from the first setigerous segment and an additional slender rod are also visible. FIGURE 31. Curved pointed setae on the first setigerous segment of a larva of 8 l /2 days, with six sets of setae. Characteristically, four such setae are present but here the curved tip of a fifth set is appearing (off tip of right- hand arrow). Note also the aciculum with a deeper origin than the external setae, and the slender rod (off tip of left-hand arrow). FIGURE 32. Two anterior parapodia in a larva of approximately 5 days, with four sets of setae. The curved, pointed, claw-like setae of the first setigerous segment are visible; contrast these with the short-tipped winged capillary type (one in focus) characteristic of the second, third, and fourth setigerous segments. FIGURE 33. Parapodia of first and second setigerous segments (anterior at right) in a larva of 5 l /s days, with a small fifth set of setae. The finger-like postsetal lobe and the smaller presetal lobe which are characteristic of the first parapodium are visible. FIGURE 34. Setal types from the fourth, fifth, and sixth setigerous segments ( anterior at left ) . Note the three short-tipped winged capillary setae ( and basal aciculum ) characteristic of the second, third, and fourth setigerous segments, the two bidentate acicular setae and one long-tipped winged capillary seta (and basal aciculum) characteristic of the fifth, sixth, and seventh setigerous segments. The two-pronged tip (off tip of arrow) of the second bidentate acicular seta developing in the sixth setigerous segment is also visible. 354 M. JEAN ALLEN additional piece ; see Figure 30) and work in scissors-like fashion with the man- dibles either held stationary or with both jaws working alternately in an antero- posterior direction. The maxillary plates move forward, open, and then close during their posterior movement. In a few cases a culture of algae was allowed to accumulate in the stender dishes. The larvae in these cases appeared to be feeding on the algae although the mid-gut was still dark with stored food material and contained large food vacuoles. The larvae upon occasion will eat their own kind as in one instance black jaws of another larva were observed in the mid-gut of an lli/^-day larva, and one larva appeared to be "gnawing" on another living larva stuck to it. An active rolling movement from side to side was noted in the esophageal region of a number of larvae, and in one food particles were noted in this region of the fore-gut which is very thick-walled. The five occipital tentacles are similar to those of the preceding stage except that they are longer, the dorsal ones measuring approximately 225 micra in 9-day larvae. Anal cirri in larvae of this age are approximately 50 micra long. Headless larvae, capable of moving about, were observed occasionally. Larvae of this age tend to stick to the bottom of the dish, often on their backs, in which case they may constrict in two in an attempt to become free. The larvae were not fed (except for any microorganisms which came through the sand-filtered sea water ) and may live as long as the yolk material lasts in the mid-gut (this area becomes transparent when the food supply is gone). Over several summers, 6 sets was the maximum number of setae observed in these larvae of D. euprea. However, in the summer of 1958, 7 sets were recorded for nine larvae, in two (from different batches) by 13Vi> days of development, in one by l4 l /2 clays, in two (from different batches ) by 171/2 days, and in one by IS 1 /-; days of development. One larva from this last batch did not develop a seventh set until the twenty-fifth day, and another from this batch until the thirtieth day of develop- ment. One from a different batch developed a seventh set by the twenty-sixth day. Among these larvae the oldest lived for 13 days after developing a seventh set of setae, dying at an age of 30 1 /o days. Most larvae died before developing a seventh set. The types of setae are described in more detail below. Types of larval setae and their order of appearance. By the time 5 sets of setae have formed in these larvae, four types of setae have differentiated. The type (or types) and distribution of each are characteristic for each segment. As indi- cated in Figures 29 to 34, those in the first setigerous segment are different from any of the others, those in segments two, three and four are similar, and those in segment five are new types which are retained in segments six and seven. One aciculum is associated with each setigerous sac at all levels. These internal basal setae have a deeper origin than the others (Figs. 31 and 34) and appear to direct the movements of the external ones. Once the direction of movement has been determined at any one level, the external setal complement seems to work against the aciculum which thus acts as a fulcrum. The following tables indicate the setigerous segments, the number and types of setae in each setigerous sac (omitting acicula which are present at all levels), the time of appearance at each level, and the setal complement of each segment at successive developmental stages. Photomicrographs are presented to help in the DEVELOPMENT OF DIOPATRA CUPREA 355 TABLE I Time of appearance of setal types in various segments Setigerous segment 1 Type of setae 3C 3C + tip of C 4C 2S 3S 2S 3S 2S 3S IB, 1L 2B, 1L IB, 1L 2B, 1L IB, 1L 2B, 1L Time of external appearance 2 days 3^ days 4^ days 2 days 1\ days 2\ days 3 days 1\ days 3^ days 4^ days 5J days 7 days 8 days 13 days (typical?) identification of these setal types. The key to the letters in the tables is as follows : C curved pointed type (Figs. 29 to 33), S short-tipped winged capillary type (Figs. 32 to 34), B bidentate acicular type (Fig. 34), L long-tipped winged capillary type (Fig. 34). The individual setae develop in a disto-proximal direction, the tip differentiating first. This was observed repeatedly in "dry" mounts. For example, in the first setigerous segment of a 4-day larva, three curved setae are complete and just the curved tip of the fourth is visible internally. In the fifth setigerous segment of 4- to 6-day larvae, one of the bidentate setae and the aciculum appear to develop simultaneously; then the long-tipped seta of this level develops and before it is completed the two-pronged tip of the second bidentate seta has developed inter- nally (Fig. 34). This sequence of setal development noted in setigerous segment number five is followed also in the sixth and seventh segments. In one larva (8V.> days old) the distal tip of a fifth seta of the curved type characteristic of segment 1 was noted (Fig. 31). This indicates that 4 curved setae may not be the full complement for this level ; however, this one case may not represent the typical condition. Also, in a number of larvae of 8 days, 8 hours TABLE II Distribution of setal types by segments at different stages Setigerous segment Larval stage 1 2 3 4 5 6 7 3 parapodia 3C 3S 3S 4 parapodia 3C 3S 3S 3S 5 parapodia 4C 3S 3S 3S 2B, 1L 6 parapodia 4C 3S 3S 3S 2B, 1L 2B, 1L 7 parapodia 4C 3S 3S 3S 2B, 1L 2B, 1L 2B, 1L 356 M. JEAN ALLEN and older, a tiny slender rod was noted in both of the first setigerous sacs (Figs. 30 and 31). Its presence was not observed consistently throughout this age group. As suggested by the tables, the setae once formed were retained throughout the period of observation. This is in contrast to Wilson's analysis of the succession of larval bristles in Nereis pclagica (1932) in which he found that as successive setae formed, the ones more anterior began falling out. DISCUSSION Certain aspects of the development of the egg and of the early larvae of Diopatra cuprca seem to be peculiar to this species, and in other instances to this genus or to the closely related genus, Onuphis. The curious process by which the eggs are formed in the ovary has been described by Andrews (1891b) and recently has been briefly reviewed by Costello ct al. (1957). Lieber (1931) has described this proc- ess for D. aniboinensis. Andrews (1891b) suggests that the algal-like strings of "nurse" cells attached to the developing egg may have a supportive function while the eggs are floating free in the coelom, rather than a nutritive one. However, Treadwell (1921, page 81) states that in the eggs of Diopatra cuprca at Woods Hole he was able to demonstrate a "definite communication pore between the ovum and the first cell of the chain, indicating that they are true 'nurse' cells." Lieber (1931 ) in a detailed study of oogenesis in Diopatra described and figured a cyto- plasmic connection between the developing egg of D. aniboinensis and its attached "nurse" cell and concluded that the cells were, in fact, nutritive in function and, therefore, properly termed nurse cells. The communication pore noted by Tread- well (1921) may conceivably represent the area where an amoeboid process of the egg could contact the cytoplasm of the "nurse" cell. Lieber ( 1931 ) has described a micropyle in the egg membrane of D. aniboincnsis. The defect observed near the vegetal pole in some eggs of D. cuprca in the present investigation may be a micropyle, although Andrews (1891b) makes no mention of it in either D. cuprca or D. uiagna. These defects may instead represent the remains of the communication pore noted by Treadwell (1921) in the developing oocyte. It has been noted that the ripe eggs of Diopatra cuprca appear to be perforated. The canalicular nature of the membrane has been demonstrated in stained eggs of Diopatra by Lieber (1931). A porous membrane is not restricted to the eggs of Diopatra but has been noted in other polychaete eggs, for example, those of Arcnicola cristata (Wilson, 1882). Retention of the egg membrane as a larval cuticle (noted in D. cuprea) ap- parently is not uncommon among polychaetes. Wilson (1882, page 295) states, : 'The persistence in some cases of the chorion as the larval cuticle is a remarkable occurrence entirely confined, so far as known, to the Chaetopods and Gephyrea, and by no means universal among them." Examples of species which retain the original egg membrane are Clyuienella torquata and Arcnicola cristata (Wilson, 1882), Nereis direr sic olor (Dales, 1950), and Thary.r inarioni (Dales, 1951). The four anterior vacuolated plates of cells which have formed by the time ciliation has been attained are peculiar to this form insofar as the writer knows, and appear to originate from the four groups of prototroch cells. DEVELOPMENT OF DIOPATRA CUPREA 357 The significance of the curious arrangement of yolk spheres into peripherally located yolk plates has not been determined, for the main mass of yolk remains in the central endodermal position (mid-gut region) of the trochophore. One pos- sibility is that these peripheral plates may serve as a more efficiently placed food supply for the rather precocious development of the setae and associated muscle strands which differentiate from the mesoderm just medial to them. As has been noted in the introduction there seems to be considerable confusion in the literature concerning the identification of larvae and earlier stages ascribed to Diopatra cuprea. It is well known that larval types are difficult to identify. Two important characteristics used for distinguishing between larvae are the jaws and setal types. The conspicuous asymmetry of the maxillary plates in Diopatra cuprea has been noted (Figs. 29 and 30). Monro (1924), in his description of the post-larval stage of D. cuprea, also pictures the unpaired, toothed plate associated with the otherwise symmetrical maxillae. This asymmetrical jaw type is charac- teristic of adult onuphids and eunicids. The functional significance of unpaired maxillary plates in otherwise symmetrical jaws, which appear to work in scissors- like fashion, is obscure. Comparing the diagram of the upper jaw pictured in Monro (1924, Fig. 6, page 197) with the writer's photomicrograph of the jaws of an 11 -day larva (Fig. 30), one may conclude that they are closely similar and in all probability could have come from larvae of the same species when one con- siders the difference in age. Monro (1924) includes a brief discussion of the possible evolution of jaws within the eunicids and closely related groups. Setae develop precociously in Diopatra cnprca. at least as compared with some of the nereids, such as Nereis pelagica (Wilson, 1932) and Nereis divcrsicolor (Dales, 1950). The importance of setal types in distinguishing between larvae is indicated by the work of Wilson (1932), Krishnan (1936), Dales (1950), et al. A comparison of the setae pictured here with the description and diagrams in Monro's post-larval stage (1924) suggests that the larvae described by Monro belong to a closely related species, if not to D. cuprea. Development of the first setigerous segment (Monro, 1924, Figure 2, and text, page 195) is in agreement with the findings described in the present study, but Monro indicates that from the second through the fifth set all setae are of the short-tipped winged capillary type. The view pictured is not clear (Fig. 3, page 195), and this setal type may or may not fit the type shown in the present investigation (Figs. 32, 33, and 34). In con- trast to Monro's larvae, the fifth set of setae observed in the present study has a new setal complement which includes a bidentate acicular type which is retained in segments 6 and 7 (Fig. 34). Beginning on the sixth segment of Monro's larvae a setigerous type (Fig. 4, page 196) appears which probably could be de- veloped from the bidentate acicular type described here ( Fig. 34 ) by the develop- ment of a hook. However, to be comparable to the larvae described by the writer, this hooked type should begin on the fifth parapodium instead of the sixth. Thus, the two species may not be identical. Wilson (1882) describes and figures some early stages in the development of a polychaete which he identifies as Diopatra cuprea. These larvae, however, were obtained from gelatinous egg masses, and Andrews (1891a, 1891b) states that these early stages and larvae described by Wilson do not belong to Diopatra cuprea but to Diopatra uiagna. Monro (1924) notes that Andrews does not give the 358 M. JEAN ALLEN basis for his statement and Monro, therefore, questions its validity. Treadwell (1921) has shown that the polychaetes described in the literature as D. magna in reality belong to another genus which he has designated as Onuphis. Both Diopatra and Onuphis are now accepted as distinct genera although they are closely related ones (Dr. Marian H. Pettibone, personal communication; also see Hartman, 1945, page 24, and Hartman, 1951, page 51, for keys separating these two genera). Treadwell (1921) further points out the possibility that the larvae de- scribed by Wilson are really those of Onuphis magna and seems inclined to agree with Andrew's interpretation. A comparison of the ciliated larva pictured by Treadwell from the gelatinous egg masses of Onuphis magna (1921, Plate 7, Fig- ure 5) with that figured by Wilson (1882, Plate XXIII, Fig. 10) shows more similarity between these two larvae than between Wilson's larvae and those of D. cuprca described in the present study. Comparing Wilson's larvae with the larvae pictured here, raised from the fer- tilized eggs of D. cuprca, certain differences are noted. No stages in the present study were observed that were as pear-shaped as Wilson's Figures 89 and 90 (Plate XXI), nor was any stage observed so markedly spotted with pigment as the larva in Wilson's Figure 89. Further, the rudimentary apical tuft shown is in marked contrast to the prominent apical tuft in the larvae here described. A comparison of larvae with five sets of setae shows that there are differences be- tween those of Wilson (1882, Plate XXIII, Fig. 10, and description on page 289) and those pictured and described by the writer. In Diopatra cuprca, in the present study, no dorsal cirri were observed, five occipital tentacles are present in normal larvae at this setal stage, and the mid-dorsal tentacle is almost the same size as the dorso-lateral (contrast Wilson's Fig. 10, Plate XXIII). Also a clearly defined pharynx and well developed jaws are visible at this stage (Figs. 14 and 15 of the present paper ; however, Wilson and Treadwell may have intentionally omitted internal structures from their drawings ) . Further, the enlarged tip of the one setal type shown in Wilson's larva (Plate XXI, Fig. 91) is different from any here described for D. cuprca (Figs. 31 and 34), although it is possible that this type might develop in a later stage. Distribution of the two species in question provides further evidence concerning the possibility of erroneous identification of their larvae. Both Diopatra cuprca and Onuphis magna are found intertidally in the Beaufort, North Carolina, area (Hartman, 1945 ) and in the Gulf of Mexico (Hartman, 1951 ) ; there is, therefore, a chance of confusing the egg cases of the two genera in these areas. Thus far, however, D. cuprca is the only onuphid found intertidally in the Woods Hole area (Dr. Marian H. Pettibone, personal communication), so to date there is no pos- sibility of confusion between these two onuphids (D. cuprca and 0. magna) in the intertidal zone at Woods Hole. The writer is led to the conclusion, there- fore, that the stages pictured by Wilson do not belong to Diopatra cuprca and probably belong to Onupliis magna (D. magna of Andrews) as Andrews has stated. If Andrews is correct and the evidence presented here indicates that he is then the gelatinous egg masses found by Wilson belong to Onuphis magna. Insofar as the writer knows, gelatinous egg masses of D. cuprca have never been found in the Woods Hole area where this species is common. She herself has never observed them and Mr. Milton B. Gray, who has collected D. cuprea for DEVELOPMENT OF DIOPATRA CUPREA 359 a number of summers in the Woods Hole area (both for investigators and for Course work), has never seen them (personal communication). Circumstantial evidence presented by Monro (1924) indicates that the eggs of D. cuprea are laid inside the tube (where the larvae develop) rather than in gelatinous egg capsules lying free on the sand. However, the possibility remains that Monro is not deal- ing with /). cuprea but with a closely related species. The one time normal spawn jelly was observed in the present study, it dissolved readily in sea water. This property of the jelly and the facts that cilia develop early and that the larva forms a prominent apical tuft suggest that D. cuprea may have a free-swimming stage. The writer, with the above observations in mind, would like to suggest that the egg masses with developing larvae which have been noted along the Gulf of Mexico (Hartman, 1951) as well as at Beaufort, North Carolina (Andrews, 1891b; Hartman, 1945; Wilson, 1882), belong to Onuphis inagna and not to Diopatra cuprea. Both species have been described as occurring together in these areas although their distribution along the Gulf of Mexico is somewhat different (Hart- man, 1951 ). With the confusion of these larval types apparent in the literature, the brief study of the setal types of D . cuprea included here may serve as at least one criterion for distinguishing between the species of onuphids in the future. The usefulness of setal types is apparent if one compares the table given by Krishnan ( 1936, page 521) for D. I'ariabills (Southern) with the tables included here for D. citprea. In summary, one is led to the conclusion that the early stages and larvae de- scribed by the several investigators cited probably do not belong to the species, Diopatra cuprea, but to a closely related genus or species, in two instances probably to Onuphis niagna which is the Diopatra inac/na of Andrews. Further, this would seem to indicate that the descriptions of the writer for Diopatra cuprea are the only ones which can be correctly attributed to this species, with the possible exception of Monro's post-larval description which may belong to D. cuprea. The possibility remains, however, that some investigation not here cited has escaped the writer's attention. The problem of activation of the egg of D. cuprea will have to be solved before this egg can be used to any extent either for experimental purposes or for class use. Some histochemical tests have been run on these stages (Allen, 1957) and it is hoped that in working further with the eggs of D. cuprea some of the problems noted will be solved. Further details of development may then be worked out to serve as a basis for experimental and histochemical studies. SUMMARY 1. Larvae of Diopatra cuprea (Bosc) have been raised, following artificial fer- tilization, to a stage with seven sets of setae. Observations on living stages and also on fixed and stained preparations have been described and photographed. 2. Cell lineage studies have not been made, but observations indicate that the early cleavages are typical of those for spiral cleavage and that the ciliated stage (age, three hours ) has a typical annelid cross and apical rosette. It, therefore, seems justifiable to conclude that the development of Diopatra cuprea follows the typical spiral pattern and mosaic development characteristic of other polychaetous annelids. 360 M. JEAN ALLEN 3. Peculiarities of the development of this polychaete, and possibly of closely related species, are the following : the peculiar algal-like nurse cells attached to the developing oocyte (also characteristic of Onuphis eggs) when floating free in the coelom, the amazing rapidity of development to the free-swimming stage (three hours ) , the four plates of cells which appear to develop from cells of the prototroch and their peculiar posterior extensions into at least four plates of yolk spheres, and the asymmetry of the maxillary plates. 4. Very little can be found in the literature on the embryology of the genus, Diopatra, and at least two authors have pointed out the possibility of error as to species in the identification of the developmental stages. Evidence is presented here which indicates that the early embryological and larval stages described by other investigators have been erroneously assigned to Diopatra cuprea. 5. If the above is correct and it would appear that Diopatra cuprea is the only onuphid found intertidally in the Woods Hole area one may conclude that the investigation presented by the writer is probably the only study recorded in the literature on the early developmental stages of Diopatra cuprea (Bosc). This is exclusive of Monro's description of the later (post-larval) stage which, if not be- longing to D. cuprea, is undoubtedly closely related to this species. LITERATURE CITED ALLEN, M. J., 1951. Observations on living developmental stages of the polychaete, Diopatra cuprea (Bosc). Anat. Rec., Ill: 550. ALLEN, M. J., 1953. Development of the polychaete, Diopatra cuprea (Bosc). Anat. Rec., 117: 572-573. ALLEN, M. J., 1957. Histochemical studies on developmental stages of polychaetous annelids. Anat. Rec.. 128: 515-516. ANDREWS, E. A., 1891a. Report upon the Annelida Polychaeta of Beaufort, North Carolina. Proc. U. S. Nat. Mus., 14 : 277-302. ANDREWS, E. A., 1891b. Reproductive organs of Diopatra. /. Morph., 5: 113-124. BORRADAILE, L. A., AND F. A. POTTS, 1935. The Invertebrata. Second edition. The Macmillan Co., New York. BUMPUS, H. C, 1898. The breeding of animals at Woods Holl during the months of June, July and August. Science, 8: 850-858. COSTELLO, D. P., M. E. DAVIDSON, A. EGGERS, M. H. Fox AND C. HENLEY, 1957. Methods for Obtaining and Handling Marine Eggs and Embryos. Marine Biological Labora- tory, Woods Hole. DALES, R. P., 1950. The reproduction and larval development of Nereis diversicolor O. F. Muller. /. Mar. Biol. Assoc., 29 : 321-360. DALES, R. P., 1951. Notes on the reproduction and early development of the cirratulid Thary.v marioni (St Joseph). /. Mar. Biol. Assoc., 30: 113-117. y HARTMAN, O., 1945. The marine annelids of North Carolina. Duke Univ. Mar. Station, Bull. no. 2. HARTMAN, O., 1951. The littoral marine annelids of the Gulf of Mexico. Publ. Inst. Mar. Sci., Univ. of Texas, 2 : 7-124. JUST, E. E., 1922. On rearing sexually mature Plat\nereis mcgalops from eggs. Amer. Nat., 56 : 471-478. KRISHNAN, G., 1936. The development of Diopatra variabilis (Southern). Zeitschr. wiss. Zoo/. Leipzig, 147: 513-525. LIBBER, A., 1931. Zur Oogenese einiger Diopatra-arten. Zeitschr. itnss. Zoo/. Leipzig, 138: 580-649. DEVELOPMENT OF DIOPATRA CUPREA 361 MEAD, A. D., 1897. The early development of marine annelids. /. Morpli.. 13: 227-326. MONRO, C. C. A., 1924. On the post-larval stage in Dia/mtni citprca. Bosc, a Polychaetous Annelid of the family Eunicidae. Aim. Mag. Nat. Hist., scr. 9, 14: 193-199. "SUMNER, F. B., R. C. OSBORN AND L. J. COLE, 1911. A biological survey of the waters of Woods Hole and vicinity. Part 2. Bull. U. S. Bur. Fisheries. 31 : 545-860. TREADWELL, A. L., 1921. Leodicidae of the West Indian region. Carnegie Inst. Wash., Pub., no. 293. WILSON, D. P., 1932. The development of Nereis pclagica Linnaeus. /. Mar. Biol. Assoc 18 : 203-217. WILSON, E. B., 1882. Observations on the early developmental stages of some polychaetous Annelides. Stud. Biol. Lab., Johns Hopkins Univ., 2: 271-299. A CONTRIBUTION TO THE BIOLOGY OF A DEEP SEA ECHINOID, ALLOCENTROTUS FRAGILIS (JACKSON) 1 R. A. BOOLOOTIAN, 2 A. C. GIESE, J. S. TUCKER AND A. FARMANFARMAIAN Hopkins Marine Station of Stanford University, California In February, 1957, a hydrographic team 3 from the Hopkins Marine Station accidentally discovered a bed of Allocentrotus fragilis (Swann, 1953) at a depth of 68 to 98 fathoms in Monterey Bay, California. This discovery was made during a routine hydrographic run. At the time a mid-water plankton haul with a stand- ard one-meter net was in progress. The Hopkins Marine Station research vessel, the "Tage," had apparently drifted with the onshore current. When the net was surfaced, to their surprise and delight, the team found approximately two dozen specimens of the deep sea urchin, Allocentrotus. This was the first time that the animal had been obtained alive and intact in large numbers. At this spot the fathometer indicated 80 fathoms and a radio "fix" recorded the position of the boat to be 3637'54" N and 12201'12" W. All subsequent hauls were started from this station. Since a project on the biology of the shore sea urchins, Strongyloccntrotus pur pit rat its and S. francisannts, was in progress at the Hopkins Marine Station, the chance finding of a bed of the deep sea urchins was of immediate comparative inter- est. Consequently, whenever possible, studies were made on the biology of Al- locentrotus for comparison with Strongylocentrotus. The oceanographic vessel, "Tage," was used for all work reported here. For dredging a four-meter beam trawl was employed. The average dredging time was twenty minutes. The entire sample, consisting of a variety of organisms, was brought into the laboratory in live condition in a tub of sea water. The animals were sorted and placed in separate tanks of running sea water. The species were identified and at times the number of individuals counted. The gonad index of the sea urchins, indicating the reproductive condition of the urchins, was determined as in previous studies, as were also the biochemical constituents of body fluid and tissues (Lasker and Giese, 1954; Bennett and Giese, 1955). Habitat of Allocentrotus Some of the physical features of the habitat of Allocentrotus should be con- sidered in order to gain an understanding of the conditions under which this species 1 This research was supported by USPH Grant 4578C to A. C. Giese. We are indebted to Dr. L. R. Blinks, Director of the Hopkins Marine Station, for making available the facilities of the laboratory, to Dr. R. L. Bolin for facilitating the use of the "Tage," to Dr. D. P. Abbott for sustained interest in the study, and to Mr. Joseph Balesteri, skipper of the "Tage," for his cooperation. - Now at the Department of Zoology, University of California at Los Angeles. 3 Under the direction of Professor R. L. Bolin of the Hopkins Marine Station and including Mr. Thomas Fast and Mr. Robert Aughtry operating with the financial assistance of Grant N60NR-26127 and Grant NSF-G-1780. 362 A DEEP SEA ECHINOID 363 lives in this area in Monterey Bay. By systematic grid dredging, the area of the sea urchin bed was estimated to be about one square mile. The depth of the area in which the urchins were taken varies between 55 to 90 fathoms, the shallow part of the bed lying on the continental shelf, the deeper part bordering the Monterey Canyon. Dredges at various depths indicate that the larger animals tend to inhabit the deeper regions near the Canyon, whereas the smaller animals are more frequently found in shallower areas. These results are summarized in Table I. The area nearest the Canyon is relatively flat and is composed of gravel and sand overlying gray silt (Galliher, 1932a, 1932b). From time to time, however, large boulders mainly of granite and shale, the largest of which weighed approxi- mately 15 kilograms, were brought up in the dredge. In the shale young urchins were frequently observed in their burrows, as illustrated in Figure IE. As the shoreline is approached the configuration of the bottom is somewhat changed, con- sisting mainly of granitic rock and coarse sand. TABLE I Sizes of Allocentrotus taken at various depths Bathymetrical range Range in size of test diameter* in fathoms in mm. 55-65 11.2- 21.3 60-65 11.2- 18.0 68 13.3- 29.4 65-90 55.0-103.3 * The measurement was made across the widest part of the test (the ambitus). Olga Hartman (1955) has published a photograph of Allocentrotus taken at. 350 to 400 fathoms in the San Pedro Basin 1 1 miles northeast of Avalon, Catalina Island, California. It was found in a sandy mud which appears to be relatively flat except for small mounds. As this species has been taken from 48 to 417 fathoms (Clark, 1912), the data considered in this paper represent only a limited aspect of the habitat of Allocentro- tus. It is possible that for the larger range over which it occurs, physical condi- tions other than those described above may obtain. Animals associated zvith Allocentrotus Since the organisms found in the same habitat as Allocentrotus may play a role in the ecology of the species, all of the organisms which came up in the beam trawl were identified when possible and counts of their numbers were made to ascertain their relative abundance. These organisms are listed in Table II. It is observed that protozoans, coelenterates, annelids, nematodes. mollusks, arthropods, echino- derms and fishes are found in the association. The interrelationships between the various forms have not been studied. Because of the random nature of the sampling it is difficult to say much about the relative abundance of the various species in the natural habitat. However, the crab, Mursia, is usually obtained, sometimes in large numbers as is the holothuroid, Stlchopus and an unknown tectibranch. The starfishes Mcdiaster, Pycnopodia, 364 BOOLOOTIAN, GIESE, TUCKER AND FARMANFARMAIAN FIGURE 1. A, An adult Allocentrotus fragilis 67 mm. in diameter. B, A test of Alloccn- t rot us fragilis 73 mm. in diameter. C, A photograph of the aboral half of the shell of Allo- centrotus showing the gonads. D, The Aristotle's lantern and the peripharyngeal coelom of Allocentrotus. E, A specimen of Allocentrotus fragilis (15 mm. in diameter) imbedded in its shale burrow. Henricia, Pterastcr and Astro[>cctcn are also rather likely to be among the speci- mens brought up in the trawl. From the numerous species and their relative abundance it seems likely that the habitat of Allocentrotus is one with relative abundance of food. Olga Hartman (1955) found AUoccntrotns in deep waters (350-400 fathoms) in association with a variety of animals (legend to plate 2A) : "A two-foot square A DEEP SEA ECHINOID 365 sample from the bottom yielded glass sponge, many foraminiferans, 20 or more species of annelids, many ophiuroids, and a large percentage of new or little known animals." In her photograph of the benthos a crinoid and a sea star are seen among the numerous Allocentrotus which appear to be spaced about a meter from one another. It is of interest to note that a rhabdocoel parasite similar to Syndesmus jrancls- canus commonly found in the shore urchin (Lehman, 1946) was observed in the gut of several specimens of Allocentrotus, and the specimens are of the same size as those found in Strongylocentrotus. One, two or three at most, were found in the gut and the incidence of infection was low. Protozoans Foraminiferans Coelenterates Psammogorgus Metridium senile TABLE II Animals taken in association with Allocentrotus fragilis Echinoderms Stylasterias sp. Astropecten californicus Luidia foliolata Annelids Three different species of polychaetes Nematodes A variety of specimens Mollusca Rosea pacifica (octopus) Numerous unidentified small gastropods Arthropods Crustaceans Munidopsis sp. Spirontocaris sp. Mursia quadichaudii Paguristes sp. Echinoderms Asteroids Mediaster aequalis Pycnopodia helianthoides Pteraster tessalatus Henricia aspera Orthasterias koehleri Ophiuroids Gorgonocephalus eucnemis Two other species of brittle stars Holothuroids Stichopus californicus Vertebrates Fishes representing the following families : Liparidae Agonidae Zoarcidae Ophidiidae Cottidae Batrachoididae Scorpaenidae Bothidae Pleuronectidae Petromyzontidae Entophenus tridentatus Rajidae Raja sp. Chimaeridae Hydrolagus colliei Nutrition of Allocentrotus Although the Allocentrotus bed occurs in the euphotic zone (down to 200 meters according to Sverdrup ct al., 1942), no conspicuous algae have ever come up in our numerous dredgings. The large algae serve as the main food of the shore urchins of the genus Strongylocentrotus (Lasker and Giese, 1954; Bennett and Giese, 1955). The sediments collected along with Allocentrotus in the dredge hauls con- sist of a variety of decomposing organic materials in which strands of algae, diatoms, 366 BOOLOOTIAN, GIESE, TUCKER AND FARMANFARMAIAN sponge spicules, nematodes, foraminiferan and other shells, as well as other protozo- ans are found among numerous bacteria. Sometimes live nematodes and protozo- ans were observed in the mud. The gut usually contains numerous olive-green pellets measuring 1.2 to 2.8 mm. in diameter, relatively compact but soft in texture. When these pellets are crushed and examined microscopically they are found to contain many small glassy rings (desmids?), foraminiferans, sponge spicules, a variety of diatoms, sand particles and unidentifiable organic particles. Acidification with HC1 indicates that most of the skeletal particles are silicious since they do not dissolve. Treatment with concentrated HNCX oxidized all the fluffy organic material leaving the silicious diatom skeletons, sponge spicules and glassy rings. In the collection of July 25, 1958 the intestines of all the animals sampled were more completely filled with pellets than in the other collections. The pellets were, in addition, a more vivid green than in all the other cases. Extracts indicated the presence of a brown pig- ment, fucoxanthin, plus a large amount of chlorophyll. The feeding was correlated with a rich plankton bloom in the surface waters nearby. In the collection made on August 14, 1958, some reddish pellets consisting entirely of organic debris and bacteria were found among the green ones. The constituents of the gut pellets are shown in Figure 2. Specimens of AUocentrotus which survive the hazards of the trip to the surface and arrive at the laboratory in good condition remain alive for many days. When the animals are kept out of water for even a brief time they lose body fluid and air is trapped inside the test, after which they float and die. Normal animals move about the aquaria like Strongylocentrotus purpitratus, though less actively, and they adhere less firmly so that they are more readily knocked off by even a small push. They right themselves much more slowly than the purple sea urchin. Attempts were made to feed AUocentrotus with boiled potatos, Phyllospadlx (eel grass) and the algae, Uh'a, Iridaea, and Gigartina, as well as with animal matter such as crushed mussel (Mytilus) and crushed deep sea crab (Mwrsia) after several days of fasting. The animals nibbled at some of the algae and at Mytilus and Mursia, dropping the material after a while, then going down to the bottom of the aquaria to nibble again. It would appear, therefore, that AUocentrotus is more selective than ,S\ pitrpuratits, which eats almost any organic material when hungry and shows sustained intake for hours. However, it must be remembered that the speci- mens are being tested at sea level and at about 15-16 C. whereas they come from a deep sea environment where they are subjected to about 15 atmospheres of pres- sure and temperatures of about 9 C. It is difficult to say what their behavior might be in their natural environment. It has been shown that the gonads of a purple sea urchin are probably the main storage organs of the animal, the gonads in a gravid animal increasing to a size which all but obliterates the body cavity left unoccupied by the gut and its contents. The relative mass of the gonads in gravid AUocentrotus is much less than that of a gravid Strongylocentrotus. At its peak the gonad of AUocentrotus is still a delicate structure, both in size and in color (pale creamy- white in the male and yellowish in the female). The gut of an AUocentrotus is generally well filled with pellets, but it does not appear to be as full as the gut of the two species of Strongylocentrotus studied. It appears, then, that food is generally less available in deeper waters A DEEP SEA ECHINOID 367 FIGURE 2. Food pellets of AUoccnirotus as seen under low and high powers. A, Food pellets as removed from the intestine (X 6). B, Crushed food pellets showing desmids (X 60). C, Diatoms and sponge spicules in crushed food pellets (X60). D and E, Foraminiferans in crushed food pellets (X 60). 368 BOOLOOTIAN, GIESE, TUCKER AND FARMANFARMAIAN than on the shore, except after an unusually rich bloom of plankton as in the col- lection of July 25, 1958. Like the gonad of the two species of Strongylocentrotus tested, the gonad of Allocentrotus contains a little stored glycogen (0.36 to 0.83 per cent or an average of 0.57 per cent of the dry weight), considerable protein (about 30 per cent of the dry weight), and a large store of lipid (an average of about 28 per cent of the dry weight). The chemical constitution of the gonad of Allocentrotus is much like that of the gonads of other species of sea urchins although it is smaller in proportion to body size. The perivisceral fluid, which is possibly one of the channels for distri- bution of the food from the gut, contains nutrients in solution much like the same fluid in the other species of sea urchins tested. Total nitrogen amounted to 3.78 to 4.98 milligrams per cent, non-protein nitrogen to 1.28 to 1.34 milligrams per cent, and a small amount of lipid is present. A variety of cells is present in the peri- visceral fluid, resembling those of the other species of sea urchin (Boolootian and Giese, 1958) and a clot forms much as in the other species of sea urchins tested (unpublished data). Healthy specimens of Allocentrotus kept in aquaria at about 15 C. in the laboratory defecate very slowly. This may be an indication of a rather slow rate of digestion but it may be the result of the abnormal conditions in the laboratory. When animals with the gut loaded with food were brought in on July 25, 1958, they defecated copiously. Defecation may therefore depend upon how full the gut is at the time of collection. All specimens collected sooner or later fall prey to a peculiar disorder. Small spots of dark red color begin to appear on the surface of the test. These spots then spread, covering the animal with large blotches of color. The tube feet degenerate and the spines fall off after which the animal dies. Microscopic ex- amination of the spots indicates that they are composed mainly of dead eleocytes, the pigmented cells of the perivisceral fluid. Reproduction The first collection of Allocentrotus in Februarv of 1957 contained individuals j in full reproductive condition, the gonads of many males and females containing mature gametes in large numbers. The eggs were readily fertilized and normal development to the pluteus followed. Development was best at temperatures between 9 14 C., cleavage being inhibited by higher temperatures. 4 The same was true for the second collection in March of 1957. However, the gonads of the animals collected in April no longer contained ripe gametes. Thereafter storms and other difficulties prevented collecting the urchins until September of 1957. The gonads of animals sampled in September, October, November and December of 1957 and in January of 1958 were well developed and of relatively large size until they spawned between January and the end of February, 1958, when the next collection was made. The gonads during the second breeding season were never as well developed as those of the first season, nor was as good a development of the embryonic stages observed. 4 4 The results on development of Allocentrotus are being published by Dr. A. R. Moore in a separate report. We are indebted to Dr. Moore for permitting us to quote here and in footnote 6 from his unpublished data. A DEEP SEA ECHINOID 369 The reproductive state of an animal can be ascertained by measuring the ratio of the volume of the gonad to the wet weight of the animal (Lasker and Giese, 1954). This ratio times 100 has been called the gonad index. The average gonad indices determined in this manner are plotted in Figure 3. The course of the FIGURE 3 A. o o X UJ Q O < z o I I I I I 1 I J I I I I I I i JFMAMJJASONDJFMAMJJASOND UJ LL) O Q. 50 40 30 20 10 B. "no data J L JFMAMJJ ASONDJFMAMJJASOND UJ OL $ a: LJ a. 5 UJ 10 C. J I I I I I I I I I I I I I J I I J I J FMAMJJ ASONDJFMAMJ JASOND FIGURE 3. A, Gonad index of Allocentrotus at different times from February, 1957 to July, 1958. B, Variations in phytoplankton during the years 1954 and 1955 as determined by Barham (1957). C, Variations of thermal monthly averages between 100-200 m. as reported by Skogsberg and Phelps (1946) for the years 1936 and 1937. Same locality as that used in the present study. 370 BOOLOOTIAN, GIESE, TUCKER AND FARMANFARMAIAN curve (dashed line) from April to September, 1957 is not known but since in 1958 the gonads of animals obtained in July were just beginning to enlarge, a period of reproductive quiescence may have occurred from April to the end of June, 1957 as happened in 1958. 3 All of the urchins used in determining the gonad index were mature, varying in wet weight from 45.5 grams to 264.0 grams and in test diameter G from 55.0 to 96 mm. Even a population of mature animals of similar size shows considerable variability in gonadal development at a given time. During the period when the gonads of some individuals are well developed and large, the gonads of other individuals are shrunken or undeveloped. The variability of gonad size is con- siderably smaller when the gonads are immature or spent. The great variability in the gonad index during the breeding season may indicate : 1 ) that some individuals do not have access to adequate food to ripen or to maintain their gonads, 2) that some individuals have just spawned while others are ready to do so, or 3 ) that some individuals may be immature when others are gravid. A histological study of the gravid and non-gravid gonads might make it possible to decide between these alternatives. DISCUSSION It is interesting to compare the biology of Allocentrotus jrai/ilis to that of the intertidal sea urchin, Strongylocentrotus purpuratns and to that of the subtidal urchin, S. franciscanus. Whereas the inshore urchins generally graze on algae, Allocentrotus appears to graze on whatever organic material occurs in the substrate, but chiefly on organic detritus, bacteria, and microscopic animals and plants of the organic "rain." 5". pnrpnratns is, on the other hand, omnivorous. When trapped in a burrow with an opening smaller than the test diameter it feeds largely on the detritus brought by sea water. In a sense, then, Allocentrotus represents an exten- sion of this special feeding habit of S. purpuratus. Allocentrotus lives in a community of invertebrates and fishes perhaps fewer in species and in numbers than the urchins of the intertidal and subtidal zone, although no decisive comparison can be made between the two communities because of the paucity of data for the deep sea community. It is also singularly interesting that a rhabdocoel containing hemoglobin should be present in the gut of the deep sea urchin as in the gut of shore forms. The data gathered in 1957-58 suggest that Allocentrotus has an annual breeding season although the span of the cycle cannot be defined precisely at the present time. During the fall and winter months from September, 1957 to January, 1958 the gonad index remained high. In both 1957 and 1958 the gonad index fell pre- cipitously between February and March. It is of interest to correlate 1) growth of gonads, and 2) spawning with physical conditions in Monterey Bay. Among the possible variables are 1) light, 2) temperature, 3) salinity and minerals and 4) planktonic bloom which may be correlated with up welling. 5 Only one Allocentrotus was obtained on August 14, 1957 but this male had a gonad index of 6.72 per cent, suggesting that the gonads were probably increasing in volume. Because of the general variability of size of gonads in any sample, the measurement is only indicative. 6 The largest test diameter observed in specimens from Monterey Bay is 103.3 mm. according to Dr. A. R. Moore. A DEEP SEA ECHINOID 371 Although day-length has been correlated with breeding cycles of some inverte- brates and vertebrates (Borthwick ct al., 1956), it does not seem likely that it is a controlling factor for Alloccntrotus because of the low intensity of light at the depths in which this animal lives. However, some photoperiodic animals are affected by very low light intensities and to them the span of illumination is of greater importance than the intensity of the light. The possible action of light in timing the reproductive cycle of Alloccntrotus is not excluded. Cyclic variations in temperature of the habitat of Alloccntrotus have been ob- served (Skogsberg, 1936; Skogsberg and Phelps, 1946). The data for the years 1936 and 1937 are given in Figure 3C at a depth between 100 and 200 meters. A seasonal rhythm is seen with low and fairly constant temperatures in spring and early summer. In May the temperature range at 150 meters was 8.2 to 8.5 C. in 1936, and 7.9 to 8.4 C. in 1937. In July the temperature at 150 meters began to rise, reaching a maximum by December at which time it ranged from 9.6 to 10.1 C. in 1936, and was 9.3 C. in 1937. The difference between highest and lowest tempera- tures is greater during upwelling of cold waters than during the period of warmer waters. The temperature variations may be correlated with three major water movements : the Oceanic period lasting from September to October, the Davidson current period lasting from November through February, and the Upwelling period occurring from late February through August. The Oceanic period and the Davidson Current generally coincide with the high thermal phase and the some- what lower chlorinity, although chlorinity variation is never large (Skogsberg, 1936). The onset of upwelling in late February coincides with the spawning of Alloccntrotus and may act as the trigger for initiation of the spawning. The sub- sequent warmer phase coincides with the period of growth of the gonads. As is to be expected, surface temperatures were found to be more variable than deep water temperatures according to Skogsberg and Phelps (1946) and the more recent CCOFI report of 1958. The upwelling in Monterey Bay is followed by a phytoplankton bloom (Bar- ham, 1956), as seen in Figure 3B. It is possible that the phytoplankton is used by the planktonic larvae of Alloccntrotus and by the metamorphosed young urchins themselves when they reach the sea bottom. In this way the timing of events in the breeding cycle may ultimately depend upon the food supply, the larvae ap- pearing at the most favorable time for their growth, namely, when phytoplankton is most abundant. All of these attempts to explain the breeding cycle of Allo- centrotus must be considered as tentative hypotheses for which substantiating data are still needed. SUMMARY 1. Following a chance collection of a deep sea urchin, Alloccntrotus jragilis, from a depth of 80 fathoms, it subsequently became possible to collect the urchins on numerous occasions from the same area. 2. The area of the bed was determined by grid dredging and the nature of the habitat determined to be relatively flat, gravel and sand underlaid with gray silt containing organic detritus and microscopic organisms. 3. The deep sea urchin appears to graze on the bottom since the organisms and organic debris of the bottom sediment appear in little pellets in its gut. 372 BOOLOOTIAN, GIESE, TUCKER AND FARMANFARMAIAN 4. Many types of invertebrates are associated with Allocentrotus f including various other echinoderms. A variety of fishes is found as well. 5. Individuals with mature gametes were obtained in February and March of 1957 and during the period of September. 1957 to January, 1958. Spawn-out ap- peared to occur between February and March during both years. 6. Attempts to correlate the life cycle of Allocentrotus with various environ- mental factors led to the suggestion that upwelling may trigger spawning. The planktonic larvae then presumably develop during the most effective time when the planktonic blooms occur. LITERATURE CITED BARHAM, E. G., 1956. The ecology of sonic scattering layers in the Monterey Bay Area, Cali- fornia. Ph.D. Thesis, Stanford. BENNETT, J., AND A. C. GIESE, 1955. The annual reproductive and nutritional cycles in two western sea urchins. Biol. Bull., 109: 226-237. BOOLOOTIAN, R. A., AND A. C. GIESE, 1958. Coelomic corpuscles of echinoderms. Biol. Bull., 115: 53-63. BORTHVVICK, H. A., S. B. HENDRICKS AND M. W. PARKER, 1956. Photoperiodism. In: Radia- tion Biology. A. Hollaender, ed., McGraw-Hill Book Co., N. Y. ///: Visible and Near- Visible Light, 479-517. CALIFORNIA COOPERATIVE OCEANIC FISHERIES INVESTIGATIONS (CCOFI) REPORT 1956-58, Progress Report. State of California, Department of Fish and Game. Marine Re- search Committee 7-56. CLARK, H. L., 1912. Hawaiian and other Pacific Echini. Mem. Mus. Comp. Zoo/., 34 : 209- ' 383. GALLIHER, E. W., 1932a. Sediments of Monterey Bay, California. Mining in California, 28: 42-79. GALLIHER, E. W., 1932b. Sediments of Monterey Bay, California. Ph.D. Thesis, Stanford. 135 pp. HARTMAN, O., 1955. Quantitative survey of the benthos of San Pedro Basin, Southern Cali- fornia. Part I, Preliminary Results. University of Southern California Publications. Alan Hancock Pacific Expeditions, 19: 1-185 (see especially Plate 2A, legend). LASKER, R., AND A. C. GIESE, 1954. Nutrition of the sea urchin, Strongylocentrotus purpuratus. Biol. Bull.. 106: 328-340. LEHMAN, H. E., 1946. A histological study of Syndisyrinx franciscanus, gen. et sp. nov., an endoparasitic rhabdocoel of the sea urchin, Strongylocentrotus frauciscanus. Biol. Bull., 91: 295-311. SKOGSBERG, T., 1936. Hydrography of Monterey Bay, California. Thermal conditions, 1929- 1933. Trans. Aincr. Philos. Soc., 29: 1-152. SKOGSBERG, T., AND A. PHELPS, 1946. Hydrography of Monterey Bay, California. Thermal conditions, Part II (1934-1937). Proc. Amer. Philos. Soc., 90: 350-386. SVERDRUP, H. U., M. JOHNSON AND R. FLEMING, 1942. The Oceans. Prentice-Hall Inc., N. Y. SWAN, E. F., 1953. The Strongylocentrotidae (Echinoidea) of the Northeast Pacific. Evolu- tion, 7 : 269-273. THE LARVAL DEVELOPMENT OF CALLINECTES SAPIDUS RATHBUN REARED IN THE LABORATORY 1 JOHN D. COSTLOW, JR. AND C. G. BOOKHOUT Duke University Marine Laboratory, Beaufort, North Carolina, and Department of Zoology, Duke Unii'ersity, Durliain. N. C. The crabs which comprise the family Portunidae include several commercially important species and studies on their life history have been in progress for the last 100 years. Of the British species only Port-units f>uber (L.) has been success- fully reared in the laboratory through all larval stages to the first crab (Lebour, 1928). Larvae of Carcinus inacnas Penn. have been described by a variety of workers but the complete development is not known from laboratory rearing. Of the American species Callinectcs sapidus Rathbun is the most important com- mercial crab in the Western Atlantic and Gulf of Mexico. Churchill (1942) described the larval development of C. sapidus by reconstructing the sequence of stages from planktonic material. Hopkins (1943, 1944), rearing the larvae through the third zoeal stage, found that not all of the stages fit the description given by Churchill (1942) and was of the opinion that the larvae described by Churchill (1942) represented several different species. The complete larval development of C. sapidus, from hatching to the first crab stage and beyond, was first reported from laboratory rearing by Costlow, Rees and Bookhout (1959). While a brief account is given of the number of stages, the duration of the intermolt periods, and the time required for complete development, the larval stages are not described nor is detailed information given on the various environmental factors under which complete development occurred. The present study has had two major objectives : one, to provide a detailed description of all the larval stages of Callinectcs sapidus Rathbun reared in the laboratory ; and two, to determine the effects of salinity and temperature on larval development. METHODS Ovigerous Callinectcs sapidus females were obtained from the Beaufort Inlet through the cooperation of Mr. David Beveridge, captain of the commercial trawler "Beveridge." Additional females were obtained from crab pots placed in waters of lower salinity. The crabs were placed in glass battery jars containing running, filtered sea water of a salinity of 23-26 p.p.t. The battery jars were tilted so that the slight overflow passed through a series of glass trays. When the eggs hatched the larvae were carried into the glass trays by the overflow, removed by large-bore pipettes as they collected on the light side, and segregated into cultures of 50-75 zoeae per finger bowl. These were further subdivided into 1 These studies were aided by a contract between the National Science Foundation and Duke University, G 4400. The authors wish to express their appreciation to Mrs. W. A. Chipman and Mrs. C. King for their assistance throughout the study. 373 374 J. D. COSTLOW, JR. AND C. G. BOOKHOUT plastic compartmented boxes with one zoea per compartment. Larvae which hatched from these crabs (Series a, c, and d) were maintained at 25 C., 26.7 p.p.t. with a photoperiod of approximately 12 hours light and 12 hours darkness. The larvae which would have been designated "b" did not hatch. To assure acclimation of the larvae to different salinities before hatching, other ovigerous crabs were placed in battery jars which did not incline but were partially filled with water of approximately the same salinity as the inlet water during the summer months (32 p.p.t.). Four salinities were obtained from the 32 p.p.t. sea water by the gradual addition of appropriate volumes of distilled water. The four salinities used were: 15 p.p.t., 20.1 p.p.t., 26.7 p.p.t. and 31.1 p.p.t. The water used for the adult crabs was aerated but not changed. The crabs were not fed and any fecal material which did appear was removed. Some larvae which hatched at 20.1 p.p.t. were gradually changed to water of 10 p.p.t. Additional zoeae were hatched and maintained through most of the larval period at 32 p.p.t. TABLE I Original number of Callinectes sapidus larvae maintained in 15 combinations of salinity and temperature. Because the larvae reared at 25 C., 26.7 p.p.t. were hatched from three different females at different times they are designated as a, c, and d. S~ per cent survival to first crab stage; * maintained on shaker, 120 'mi H. \p-p.t. c.\ 10.5 15.6 20.1 S 26.7 s 31.1 S 32.0 S 20 108 108 108 108 108 25 100 100 100 1.0 a) 18* 5.5 80 108* 108* 108* c) 150* 2.7 150* 1.3 lOOOf < 1 d) 100 8.0 30 108 108 108 108 t Diluted to 28 p.p.t. on day 41. When hatching occurred in the jars without any overflow the zoeae were removed with a large-bore pipette to finger bowls. The salinity of the water in the finger bowls was identical to the water in which hatching had occurred. Both plastic compartmented boxes and Syracuse watch glasses were used as rearing containers for larvae within each salinity. Ten zoeae were maintained in each Syracuse watch glass and 6 zoeae in each plastic compartment. Zoeae in each of the salinities were maintained at three different temperatures : 20 C., 25 C., and 30 C. Zoeae of all series were fed Arbacia eggs and Artcmia nauplii which were added each day when the larvae were changed to freshly filtered sea water and clean receptacles. Some plastic boxes were maintained on an Eberbach shaker (120/min.) at 25 C. but the majority of the containers were stationary (Table I). The megalops and crab stages were fed Artemia nauplii plus beef liver. The compartments containing the zoeae were observed daily for exuvia and, at this time, the number of molts and the mortality were recorded. Drawings of the zoeal stages and megalops stage were made from the exuvia of known molts and from larvae preserved at a known stage of development. All LARVAL DEVELOPMENT OF CALLINECTES 375 figures were made to scale on graph paper with the aid of a Whipple disc inserted in the ocular of a compound microscope. The detailed drawings of the appendages of each stage are also drawn to scale, different from that used for the whole larva, from appendages dissected out with glass needles. RESULTS Larval stages First zoea: The characteristics of the first stage zoeae agree closely with those given by Hopkins (1943). A small seta, described as between the dorsal and lat- eral spines of the cephalothorax (Hopkins, 1943) was not found. The abdomen has five segments plus a telson. As shown in Figure 1, A, B, the eyes are not stalked. The conical antennule (Fig. 1, C) bears a total of 5 terminal processes, the three aesthetes being longer and flatter than the two small setae. The proto- podite of the antenna (Fig. 1, D) is elongated, bears two rows of minute spines on the distal half, and the small exopodite terminates in two unequal setae. The mandibles are small, with a broad cutting surface (Fig. 1, E). The endopodite of the maxillule (Fig. 1, F) bears four terminal spines, equal in length, and two slightly subterminal spines. The basal and coxal endites of the protopodite have 5 and 6 spines, respectively, and show slight bifurcation. The unsegmented endop- odite of the maxilla (Fig. 1, G) also bears four terminal spines and two sub- terminal spines. The basal endite of the protopodite bears four spines on each bifurcation and three spines project from each lobe of the coxal endite. The scaphognathite has three setae on the outer margin of the distal portion plus two apical setae at the proximal tip. The first maxilliped (Fig. 1, H) has 4 natatory setae (cut short in the figures) on the exopodite and a spine arrangement of 2, 2, 0, 2, 5 on the 5 segments of the endopodite. The second maxilliped also has 4 swimming hairs and a 1, 1, 4 spine arrangement on the three segments of the endopodite (Fig. 1, I). The second segment of the abdomen bears a short lateral knob and the third segment has a short hook on each side. Segments 3 to 5 also have prominent lateral spines which project caudally, overlapping the adjacent segment. A pair of small setae project dorsally from all abdominal segments other than the first. Each furcus of the telson bears a small dorsal spine and a larger lateral spine (Fig. 1, A, B). The inner margin of each furcus has three spines. The pattern of the chromatophores was consistent for all zoeal stages. The location of those evident in Bourns-fixed larvae were : between the eyes ; posterior to the eye and dorso-lateral to anterior part of gut ; dorsal to gut in posterior region of cephalothorax ; below base of carapace spine ; mandible ; distal region of basop- odite of first maxilliped ; middle of first abdominal segment, dorsal to gut ; margin of third through last abdominal segments. Second zoea: Eyes stalked. Number of aesthetes of antennule identical to first stage. Endopodite of maxillule bears 4 terminal and 2 subterminal spines (Fig. 2, F) ; basal endite bears 7 spines and coxal endite has 7 spines; a small spine is now present on outer margin of protopodite. Basal endite of maxilla (Fig. 2, G) has 8 spines and coxal endite 6 spines. Five spines are present on distal margin of scaphognathite and two project from apical tip. On third segment of endopodite of first maxilliped, one spine is added (2, 2, 1, 2, 5) (Fig. 2, H). The exopodite 376 J. D. COSTLOW, JR. AND C. G. BOOKHOUT FIGURE 1. Side (A) and ventral view (B) of first zoeal stage of Callinectes sapidits with appendages. C, antennule ; D, antenna ; E, mandible ; F, maxillule ; G, maxilla ; H, first maxil- liped; I, second maxilliped. Whole zoea, X 65 ; appendages, X 290. LARVAL DEVELOPMENT OF CALLINECTES 377 FIGURE 2. Side (A) and ventral view (B) of second zoea of Callincctes sapidus with appendages. C, antennule ; D, antenna ; E, mandible ; F, maxillule ; G, maxilla ; H, first maxil- liped ; I, second maxilliped. Whole larvae, X 65 ; appendages, X 290. 378 J. D. COSTLOW, JR. AND C. G. BOOKHOUT FIGURE 3. Side (A) and ventral view (B) of third zoea of Callincctes sapidus with appendages. C, antennule ; D, antenna; E, mandible; F, maxillule ; G, maxilla; H, first maxil- liped; I, second maxilliped. Whole larvae, X 43 ; appendages, X 170. LARVAL DEVELOPMENT OF CALLINECTES 379 FIGURE 4. Side (A) and ventral view (B) of fourth zoea of Callincctes sapidus with appendages. C, antennule ; D, antenna ; E, mandible ; F, maxillule ; G, maxilla ; H, first maxil- liped; I, second maxilliped. Whole larva, X 43 ; appendages, X 170. 380 J. D. COSTLOW, JR. AND C. G. BOOKHOUT bears 6 plumose swimming setae. Endopodite of second maxilliped has one addi- tional subterminal spine (Fig. 2, I). This setation, 1, 1, 5, remains constant through the remaining larval stages. Exopodite of second maxilliped hears 6 plumose swimming setae. Inner margin of each furcus of telson now bears one additional spine without setules (Fig. 2, B). Third zoea: Setation of antennule and antenna unchanged from previous stage. The mandible (Fig. 3, E) has several small teeth in addition to the broad cutting surface. Basal endite of maxillule bears 8 spines and 7 spines project from coxal endite (Fig. 3, F). Basal and coxal endites of maxilla (Fig. 3, G) have 9 and 7 spines, respectively. Scaphognathite has 8 hairs on distal margin and 4 hairs at apical tip. A second, subterminal spine added to the fifth segment of the endop- odite of the first maxilliped gives a spine arrangement (2, 2, 1, 2, 6) which re- mains constant in the remaining larval stages (Fig. 3, H). The exopodites of both maxillipeds terminate in 8 swimming setae (Fig. 3, H, I). A sixth segment has been added to the abdomen. It bears the small dorsal setae but does not have lateral spines (Fig. 3, B). Fourtli zoea: A slight swelling in the basal region of the antenna indicates the beginning of the endopodite bud (Fig. 4, D). A small, unsegmented palp appears with the mandible (Fig. 4, E). The basal endite of the maxillule bears 10 ter- minal spines and one smaller subterminal spine (Fig. 4, F). Six spines project from the terminal portion of the coxal endite and two more appear at the margin. The basal endite of the maxilla bears 10 spines and 7 project terminally from the coxal endite (Fig. 4. G). The exopodites of both the first and the second maxilli- peds bear 9 swimming setae of unequal length (Fig. 4. H, I). The lateral edges of the cephalothorax have three small setae (Fig. 4, A). Fift/i zoea: The developing endopodite bud of the antenna (Fig. 5, D) is larger than in the previous stage. The maxillule remains as in the previous stage but setation of the maxilla is increased to 8 spines on the coxal endite (Fig. 5, F) and the soft hairs on the Scaphognathite are increased to 20. The number of swim- ming setae on the first maxilliped remains as in the previous stage (9) while the second maxilliped now bears a total of 11 setae. Buds of the third maxilliped, chela, and pereiopods are visible beneath the carapace. The number of setae pro- jecting from the edge of the carapace has increased. Si.vtli zoca: A fourth aesthete, subterminal to the original 3 aesthetes and 2 setae, is added to the antennule (Fig. 6, C). Hairs appear on the small, unseg- mented palp of the mandible (Fig. 6, E). A plumose spine is added to the basal segment of the endopodite of the maxillule (Fig. 6, F) and the coxal endite bears a total of 9 spines. Spines on the basal endite of the maxilla (Fig. 6, G) have increased to 13 and the marginal hairs of the Scaphognathite total approximately 25. There are 11 swimming setae on the first maxilliped and 12 on the second maxilliped. Pleopod buds appear for the first time on the abdominal segments 2 through 6 (Fig. 6, A, B). A small, non-plumose spine is added to the 8 spines within the inner margin of the telson. The number of setae on the margin of the carapace is also increased. Seventh zoca: The terminal aesthetes of the antennule increase to 7 and 5 subterminal aesthetes have been added (Fig. 7, C). The basal portion of the an- tennule is swollen and there is a slight indentation in the distal half. The devel- LARVAL DEVELOPMENT OF CALLINECTES 381 FIGURE 5. Side (A) and ventral view (B) of fifth zoea of Calliticctcs sapid us with appendages. C, antennule ; D, antenna; E, maxillule; F, maxilla; G, endopodite of first maxil- liped; H, endopodite of second maxilliped. Whole larva, X 43 ; appendages, X 170. 382 J. D. COSTLOW, JR. AND C. G. BOOKHOUT FIGURE 6. Side (A) and ventral view (B) of sixth zoea of Callincctcs sapidus with appendages. C, antennule ; D, antenna ; E, mandible ; F, maxillule ; G, maxilla ; H, endopodite of first maxilliped; I, endopodite of second maxilliped. Whole larva, X 43 ; appendages, X 170. LARVAL DEVELOPMENT OF CALLINECTES 383 FIGURE 7. Side (A) and ventral view (B) of seventh zoea of Callinectes sapidus with appendages. C, antennule ; D, antenna ; E, maxillule ; F, maxilla ; G, endopodite of first maxil- liped ; H, endopodite of second maxilliped ; I, third maxilliped. Whole larva, X 43 ; appendages, X170. 384 J. D. COSTLOW, JR. AND C. G. BOOKHOUT FIGURE 8. Side (A) and ventral view (B) of eighth zoea of Callincctcs sapidus and appendages. C, antennule ; D, antenna ; E, maxillule ; F, maxilla. Whole larva, X 32 ; ap- pendages, X 135. LARVAL DEVELOPMENT OF CALLINECTES 385 oping enclopodite bud of the antenna (Fig. 7, D) is approximately half the length of the antenna. The basal endite of the maxillule (Fig. 7, E) bears 17 spines and the coxal endite retains the 9 spines observed in the previous stage. The spines of the basal endite of the maxilla number 14 and 10 spines are present on the coxal endite (Fig. 7, F). On the scaphognathite approximately 29 soft, plumose hairs fringe the outer margin. The swimming setae have increased to 14 on the first maxilliped and to 13 on the second maxilliped (Fig. 7, A, B). The developing thoracic appendages have increased in size and project below the margin of the carapace. Eighlh zoca: The aesthetes of the antennule are arranged in three tiers: 7 ter- minal, 6 subterminal, and 5 in the most basal row (Fig. 8, C). Basal portion of the antennule is more inflated and the endopodite is visible as a small knob. Endop- odite of antenna (Fig. 8, D ) is now almost equal in length to protopodite and shows evidence of segmentation. Basal endite of maxillule (Fig. 8, E) bears 21 spines and coxal endite has 15 spines. A second spine is added below the endop- odite. Spines of the basal and coxal endites of the maxilla have increased to 15 and 10, respectively (Fig. 8, F). On the scaphognathite the plumose hairs have increased to approximately 36. Swimming setae on the first maxilliped have de- creased to 12 and 14 setae are found on the second maxilliped (Fig. 8, A, B ). On the first maxilliped an epipodite, partially developed, bears short setae and soft, non-plumose hairs (Fig. 9, A). Exopodite of the third maxilliped (Fig. 9, C) bears two short terminal spines and the epipodite terminates in one small, non- plumose spine. Chela and pereiopods are larger and project well beyond border of the carapace. Pleopod buds (Fig. 8, A, B) bear short non-plumose hairs. Spines on inner margin of telson total 10. Four small hairs project dorsally from posterior margin of first abdominal segment. Megalops: Rostrum pointed, longer than antennules but shorter than antennae ; eyes stalked (Fig. 9, D, E). Appendages, eyes, and margins of carapace pro- vided with small hairs. Antennule (Fig. 10, A) now divided into peduncle of three segments and two flagella. The unsegmented flagellum bears 6 non-plumose setae and the four seg- ments of the other flagellum bear numerous aesthetes. The longer, terminal seg- ment also bears two non-plumose setae. The antenna is composed of 1 1 segments, some of which bear setae as shown in Figure 10, B. The mandible (Fig. 10, C) has a palp of two segments with 11 bristles on distal segment. Endopodite of maxillule (Fig. 10, D) has 4 spines on terminal segment and 6 spines on first segment. The number of spines on the coxal and basal endites has increased to 17 and 25, respectively. Endopodite of maxilla (Fig. 10, E) reduced in size and bearing only three spines. There is an increase in the number of spines on endites of the protopodite and on the scaphognathite. First maxilliped (Fig. 11, A) is considerably modified from swimming ap- pendage of zoeal stages. Endopodite broader with 8 non-plumose setae on distal border. Exopodite of two segments, with 6 terminal setae on second segment. Epipodite well developed and fringed with long, non-plumose hairs. Second maxil- liped (Fig. 11, B) has endopodite of 4 segments with stout spines on terminal seg- ment. Exopodite is two-segmented with 6 terminal hairs. The epipodite is small. Third maxilliped (Fig. 11, C) with large endopodite bearing numerous spines on 386 J. D. COSTLOW, JR. AND C. G. BOOKHOUT FIGURE 9. Appendages of eighth zoea and side and dorsal view of megalops of Callinectes sapidus. A, first maxilliped; B, second maxilliped; C, third maxilliped; D, side view of megalops ; E, dorsal view of megalops ; F, ventral view of abdominal segments of megalops (setae removed on alternate pleopods for clarity). Whole megalops, X32; appendages, X 135. LARVAL DEVELOPMENT OF CALLINECTES 387 FIGURE 10. Appendages of megalops of Callinectes sapidus. A, antennule; B, antenna; C, mandible ; D, maxillule ; E, maxilla. X 135. 388 J. D. COSTLOW, JR. AND C. G. BOOKHOUT O.I FIGURE 11. Appendages of megalops of Callinectes sapidns. A, first maxilliped ; B, second maxilliped; C, third maxilliped; D, terminal segment of third maxilliped. X 135. LARVAL DEVELOPMENT OF CALLINECTES 389 all segments ; exopodite unsegmented and bearing 6 terminal setae ; epipodite fringed at distal portion by soft, non-plumose hairs. Spine on lateral surface of basi-ischiopodite of cheliped (Fig. 9, D, E), and dactylopodite of fifth pereiopod has 5 terminal spines. Cornua project from posterior edge of cephalothorax TABLE II Time of molting, expressed as days after hatching, for larvae of C. sapidus in salinity-temperature combinations in which development was complete or partially complete \p.p.t. "ex 20.1 26.7 31.1 Molt I Molt II Molt III Molt IV Molt V Molt VI Molt VII (to megalops) Molt VIII (to crab) 25 30 6-13 a) 7-9 c) 6-12 cl) 7-9 5-11 7-13 25 30 12-16 a) 10-12 c) 10-20 d) 10-12 11-16 11-19 25 30 17-27 a) 15 c) 17-26 d) 14-23 14-18 15-27 25 24-30 a) 19 c) 20-32 d) 18-26 20-29 25 28-34 a) 22 c) 24-39 d) 22-33 24-39 25 38 a) 27 c) 28-39 d) 26-38 29-43 25 43 a) 31 c) 35-49 d) 32-45 35-47 25 50 a) 37 c) 50-55 d) 39-53 45-55 (Fig. 9, E, F). Fifth abdominal segment retains lateral spines, projecting caudally past the smaller sixth abdominal segment (Fig. 9, D, F). Endopodites developed on all pleopods other than fifth pair. Exopodites of pleopods on segments 2 through 6 with 24, 23, 22, 21, and 12 long, non-plumose setae (Fig. 9, F). Four small, curled spines are found on inner surface of endopodite of the pleopod of 390 J. D. COSTLOW, JR. AND C. G. BOOKHOUT the second abdominal segment and three similar spines are present on endopodites of remaining pleopods. Telson with 6 to 8 short spines on posterior border. Larval development Hatching was observed at all experimental salinities except 15 p.p.t. In water of 20.1 p.p.t.-32 p.p.t. the zoeae hatched as first stage larvae and the so-called "pre- zoea" was never observed. Complete development to the first crab stage occurred in the four temperature-salinity combinations shown in Table I. As shown in Table II, the time of molting of the three series of larvae main- tained at 26.7 p.p.t., 25 C. (Series a, c and d) was similar. The first molt oc- curred within the same period of time for larvae at 20.1, 26.7, and 31.1 p.p.t. At these three salinities there was also little difference in the time of the later molts (Table II ) and in the range of time for complete larval development (Table III). The only difference in time required for total development was found in the series of larvae hatched and reared at 32 p.p.t. After dilution to 28 p.p.t. on day 41, at which time all the larvae had been either sixth or seventh stage zoea TABLE III Number of days observed for development of all zoeal stages (Z), duration of the megalops stage (M), and time for total development to the first crab stage (T) for larvae of Callinectes sapidus hatched and maintained at 25 C. in the salinities shown 20.1 26.7 31.1 32.0* Z M T Z M T z M T Z M T 43 7 50 a) 31 6 37 35-47 10-20 45-57 46 15 61 b) 35-49 7-9 44-56 d) 32-45 6-9 38-53 * Diluted to 28 p.p.t. on day 41. for some time, some molted to the megalops stage and eventually metamorphosed to the first crab on day 61. The one series in which zoeae completed the first three molts at 30 C., 26.7 p.p.t., shows no significant difference in the time of the molts in spite of the additional 5 C. in temperature (Table II). Mortality of C. sapid us larvae (Table IV) was highest during the first two zoeal stages in all temperature-salinity combinations. In all salinities larvae never went beyond the first zoeal stage when maintained at 20 C. At 10.5 and 15.6 p.p.t. mortality was also highest during the first stage at all three temperatures. Larvae maintained at one temperature-salinity combination, 25 C., 15.6 p.p.t., did molt to the second stage but died within a few days (Table IV). Once the second molt had been completed some of the remaining larvae usually lived to complete meta- morphosis to the crab. The number of zoeal stages of C. sapidus varied from 7 to 8. Most of the larvae which molted to the megalops did so following the seventh zoeal stage but one completed 8 zoeal stages and then metamorphosed to the megalops. The majority of the eighth stage zoeae died without additional molts. The variation LARVAL DEVELOPMENT OF CALLINECTES 391 TABLE IV Mortality of larvae of Callinectes sapidns at different stages, expressed as per cent of original number of zoeae, in those temperature-salinity combinations which permitted at least partial development. Vp.p.t. C.\ 15.6 20.1 26.7 31.1 Stage I 25 95 42 a) 72.2 c) 30.0 d) 11.0 53.3 30 95 58.3 60.1 Stage II 25 5 36 a) 16.7 c) 57.5 d) 42 22.8 30 5 37.0 37.0 Stage III 25 11 a) 5.5 c) 3.5 d) 10.0 12.0 30 2.7 2.8 Stage IV 25 8 a) 0.0 c) 2.0 d) 5.0 0.6 30 1.8 Stage V 25 1 a) 0.0 c) 0.0 d) 9.0 4.6 Stage VI 25 1 a) 0.0 c) 0.0 d) 9.0 0.6 Stage VII 25 0.0 a) 0.0 c) 0.0 d) 4.0 4.0 Megalops 25 0.0 a) 0.0 c) 4.3 d) 1.0 0.0 in number of stages occurred within one salinity-temperature combination (26.7 p.p.t., 25 C.) as well as in the other salinities. The megalops stage metamor- phosed directly to the first crab stage. DISCUSSION Larval stages The only existing description of all larval stages of Callinectes sap id us (Churchill, 1942) is based entirely on reconstruction from planktonic material. Hopkins 392 J. D. COSTLOW, JR. AND C. G. BOOKHOUT (1943, 1944) was able to rear C. sapldns through the first three zoeal stages and concluded that Churchill's (1942) description of the larvae included zoeae from several species. Reconstruction of the stages in larval development is always susceptible to this error in an area which includes more than one species. By rearing zoeae, liberated in the laboratory from the egg mass of an identified female, the species can definitely be known and confusion resulting from the mixing of larvae from several species is avoided. The larval development of many crabs has been reported to include a "pre- zoeal" stage. The "pre-zoea" is described for C. sapidus by Robertson (1938) and by Churchill (1942). In the present study the larvae, although varying con- siderably in size, always hatched as first zoeae in salinities of 20.1, 26.7, 31.1 and 32 p.p.t. Lochhead, Lochhead and Newcombe (1942) observed that 90 per cent of the eggs hatched as first zoeae under "favorable conditions" but that "pre-zoeae" were obtained if conditions were "unfavorable." Sandoz and Rogers (1944) found hatching to be associated with salinity : below 20 p.p.t. the per cent of larvae which emerged as "pre-zoeae" increased. The setation of the maxillipeds of C. sapidus larvae has been given by Churchill (1942) and, for the first three stages reared in the laboratory, by Hopkins (1943, 1944). The results of the present study agree with previous findings for the first two zoeal stages. Beginning with the third zoea, however, our description does not agree with that given by previous workers. Churchill ( 1942) gives 6 and 7 setae for the first and second maxillipeds, respectively, Hopkins (1944) found 8 and 9 setae, and we observed 8 swimming setae on each maxilliped. Hopkins (1944), describing a fourth stage zoea obtained from the plankton, gave the setation of the first and second maxillipeds as 8 and 10 while we found it to be 9 and 9. Robertson (1938) and Churchill (1942) put great emphasis on the cornua as a distinguishing feature of the C. sapidus megalops. Aikawa (1937) described the megalops of several species of Porhinus, obtained from the plankton, and included the cornua in the figures for these species. Aikawa (1937) also mentioned the hook on the basi-ischiopodite of the chela and the lateral spines on the fifth ab- dominal segment of the megalops. Lebour (1928), describing the megalops of Port unns pubcr reared from the egg in the laboratory and megalops of other species of Portunus obtained from the plankton, did not figure or describe these three characters for any species of Portunus. The present description of setation of the maxillule and maxilla agrees with Hopkins' (1943, 1944) findings for the first three stages. In many previous studies on larvae of the Brachyura the zoeae have been staged very largely by differences in the number of swimming hairs on the first and second maxillipeds. Aikawa (1937) compares setation of the maxillule and maxilla for a great variety of brachyuran larvae but includes only the first stage zoea. In each zoeal stage of C. sapidus examined in the present study it was found that there was always a progressive change in the setation of the maxilla. Setation of the maxillule was also different, except for the fourth and fifth zoeae. Hence these appendages, and others, may be important in staging larvae of different crabs. The significance of these appendages as diagnostic characters, however, will have to await a com- parative study of all stages in the larval development of other species of crabs. LARVAL DEVELOPMENT OF CALLINECTES 393 Larval development Although the effects of salinity and temperature on larval development of other crabs have been studied (Coffin, 1958; Costlow and Bookhout, unpublished results), Sandoz and Rogers (1944, 1948) give the only available data dealing specifically with the blue crab, Callincctcs sapidus. In the present study on larvae of this species the results agree closely with those reported for the first zoeal stage by Sandoz and Rogers (1944). If the salinity were reduced beyond 20.1 p.p.t. by dilution with distilled water, the zoeae did not usually live beyond the first molt. Sandoz and Rogers (1944) obtained some second zoeae at 20 p.p.t. and 25 p.p.t. (24-29 C.) but the few which molted to the third stage did not live. In the present study the time of molting (Table II) was quite variable, even within one salinity-temperature com- bination. Sandoz and Rogers (1944) reported an average of from 67 days for the first molt at 20 and 25 p.p.t., 24-29 C., although some larvae molted as late as the eleventh day. In the present study the first molt was completed in from 5 to 13 days in several salinity-temperature combinations (Table II). The later molts became more variable in time in all three salinities in which development was complete. In the present study isolated larvae did molt and successfully complete develop- ment to the crab stage. Sandoz and Rogers (1944) did not observe any molting among isolated larvae and all eventually died. One series of larvae, hatched and maintained for 41 days at 32 p.p.t., was of particular interest. The sixth and seventh stage zoeae were active but did not molt to the megalops. On day 41 the larvae were divided into three groups. The water containing one group of zoeae was reduced from 32 p.p.t. to 28 p.p.t. in approximately 4 hours. All zoeae of this group died within 24 hours. Water containing the second group of larvae was diluted to 28 p.p.t. over a period of approximately 24 hours. Five days later one zoea molted to the megalops and on day 61, metamorphosed to the crab. Larvae of the third group, retained at 32 p.p.t., died without any additional molting. While the number of larvae used should not be relied upon for any definite conclusions, it may be pointed out that the larvae hatched and reared at 31.1 p.p.t. completed metamorphosis to the crab without dilution to a lower salinity. Thus it would appear that the threshold which exists in the upper range of salinities is abrupt and well defined. At 25 C. the duration of the megalops stage (6-9 days) was similar for larvae maintained at 20.1 and 26.7 p.p.t. (Table III). In the higher salinity (31.1 p.p.t.) 10-20 days were required and in water diluted from 32 p.p.t. to 28 p.p.t., the megalops persisted for 15 days before molting to the crab. Sandoz and Rogers (1948) found little difference in the time required for the megalops to molt to the crab in 20 p.p.t. and 31 p.p.t. The 2.6-2.9 days which they record, however, were for stages obtained from the plankton and the exact age could not be known. If, as suggested by Sandoz and Rogers (1948), the megalops were approximately 2-3 days old when first obtained, the total period of 5-6 days would correspond closely with our results at 20.1 and 26.7 p.p.t. Churchill (1942) estimated that zoeal development of C. sapidus in the Chesa- peake Bay was completed in approximately one month. Zoeal development in 394 J. D. COSTLOW, JR. AND C. G. BOOKHOUT the laboratory required a minimum of 31 days and a maximum of 49 days, at various salinities. In the laboratory 7 zoeal stages and one megalops stage were observed whereas Churchill (1942) described 5 zoeal stages and one megalops from planktonic material. The use of Artemia nauplii has proven successful in rearing a variety of decapod larvae (Broad, 1957; Chamberlain, 1957; Knudsen, 1958; Coffin, 1958; Costlow and Bookhout, unpublished results) and Cirripedia larvae have been reared from hatching to settling and metamorphosis on Arbacia eggs (Costlow and Bookhout, 1957, 1958). The combination of Arbacia eggs and recently hatched Artemia nauplii used in the present study provides a source of motile food of different sizes. In our experience with other decapod larvae, also reared at different salinity-temperature combinations, the zoeae were vigorous and fed actively. C. sapidus larvae, even after completion of several molts, often appeared fragile and less vigorous than larvae of other species. Algae have been used unsuccessfully in attempts to rear the larvae of many decapods by previous workers. We have found that while C. sapidus zoeae will ingest many of the unicellular algae and live 10-13 days, the larvae never molt. Even though the gut is full of the cells, and fecal pellets are numerous, further development does not occur. In the present study algae were not used because zoeae which were provided algae have been observed to feed less actively on Artemia nauplii. Dean (1958) has suggested that what have been interpreted as differences in the nutritive quality of algae may represent "resistance" to digestion. The 7 zoeal stages described for C. sapidus may not represent the number of stages present in development under natural conditions. A main criticism of laboratory rearing has been that suboptimal conditions may produce "abnormal" stages and give a picture of larval development which is not consistent with that assumed to be found in the natural environment (Gurney, 1942). In the few existing examples of successful rearing of Brachyura in the laboratory no reference is made to "extra" or "abnormal" stages. Lebour (1930), dealing with larvae of the Anomura, noted that 5 larval stages usually represent the normal development of Galathca but that the fourth and fifth stages may be omitted. In the Macrura, Templeman (1936) found a stage in the larvae of Homarus americanus, inter- mediate in form between the recognized third and fourth stages, and attributed it to unfavorable rearing conditions. More recently, Broad (1957) has shown that the number of larval stages of Palacmonetes is directly associated with the availability of food. Lebour (1928), discussing the primitive nature of the Brachyrhyncha larvae, considers Portunus as the most primitive because of the many zoeal stages (5) and the spine structure of the telson. The 7 zoeal stages described for C. sapidus, a form closely allied to Portunus, may indicate a primitive adaptive quality which has, in part, accounted for the success of this species all along the Atlantic and Gulf coasts. If larval development is complete, and the post-larval stage is reached, it appears erroneous to refer to "abnormal" stages of development. Our present knowledge of the factors involved in the physiology of larval development of the Brachyura is too limited to predetermine the number of larval stages required for the develop- ment of any crab. LARVAL DEVELOPMENT OF CALLINECTES 395 SUMMARY AND CONCLUSIONS The larvae of Callinectes sapidus Rathbun were reared in the laboratory from hatching to the post-larval stages under conditions which combined 20 C, 25 C., 30 C, and 6 salinities (10.5, 15.6, 20.1, 26.7, 31.1 and 32 p.p.t.). Of the 3,014 zoeae maintained in 15 different combinations of salinity and temperature 1-8 per cent completed development at 25 C., in salinities of 20.1, 26.7, and 31.1 p.p.t. The zoeal stages and megalops stage are described and figured. From this study the following conclusions may be made : 1. Eggs hatched as first zoeae and the "pre-zoea" stage was not observed. 2. Seven zoeal stages and one megalops stage were observed in the complete development to the first crab in the laboratory. An eighth zoeal stage was some- times observed but usually did not complete metamorphosis to the megalops. 3. Setation of the maxillipeds and the maxillule showed a progressive increase with each larval stage and may be useful in the staging of species obtained from the plankton. 4. Development to the megalops required a minimum of 31 days and a maximum of 49 days. The megalops persisted from 6-20 days in the salinities used. 5. There is no significant difference in time of zoeal development in water with salinities of 20.1-31.1 p.p.t. 6. At a higher salinity (31.1 p.p.t.) a greater length of time is required for the megalops to complete metamorphosis to the first crab than when reared in lower salinities (20.1-26.7 p.p.t.). 7. Even though some zoeae completed development in salinities of 20.1-31.1 p.p.t. mortality was usually highest during the first two zoeal stages. Below 20.1 p.p.t. larvae rarely completed the first molt. 8. The large number of zoeal stages may not reflect development under natural conditions. The 7 zoeal stages may, however, indicate a primitive adaptive quality which has accounted for the success of Callinectes sapidus Rathbun along the Western Atlantic and Gulf of Mexico coasts. LITERATURE CITED AIKAWA, H., 1937. Further notes on Brachyuran larva. Rec. Oceanogr. Wks. Japan, IX: 87-162. BROAD, A. C., 1957. The relationship between diet and larval development of Palaemonetes. Biol. Bull., 112: 162-170. CHAMBERLAIN, N. A., 1957. Larval development of the mud crab Neopanope texana sayi (Smith). Biol. Bull., 113: 338. CHURCHILL, E. P., 1942. The zoeal stages of the blue crab, Callinectes sapidus Rathbun. Chesapeake Biol. Lab., Publ. No. 49, pp. 1-26. COFFIN, H. G., 1958. The laboratory culture of Pagurns samuelis (Stimpson) (Crustacea, Decapoda). Walla Walla College Publ. No. 22, pp. 1-5. COSTLOW, J. D., JR., AND C. G. BOOKHOUT, 1957. Larval development of Balanus eburneus in the laboratory. Biol. Bull., 112: 313-324. COSTLOW, J. D., JR., AND C. G. BOOKHOUT, 1958. Larval development of Balanus amphitrite var. denticulata Broch reared in the laboratory. Biol. Bull., 114: 284295. COSTLOW, J. D., JR., G. REES AND C. G. BOOKHOUT, 1959. A preliminary note on the complete larval development of Callinectes sapidus Rathbun reared in the laboratory. Limn. and Oceanography. In press. 396 J. D. COSTLOW, JR. AND C. G. BOOKHOUT DEAN, D., 1958. New property of the crystalline style of Crassostrea virginica. Science, 128 : 837. GURNEY, R., 1942. Larvae of Decapod Crustacea. Pp. 1-306, Ray Society, London. HOPKINS, S. H., 1943. The external morphology of the first and second zoeal stages of the blue crab, Callinectcs sapidus Rathbun. Trans. Amer. Micro. Soc., 62 : 85-90. HOPKINS, S. H., 1944. The external morphology of the third and fourth zoeal stages of the blue crab, Callinectes sapidtis Rathbun. Biol. Bull., 87: 145-152. KNUDSEN, J. W., 1958. Life cycle studies of the Brachyura of Western North America, I. General culture methods and the life cycle of Lophopanopcus Icucomanus leucomanus (Lockington). Bull. So. Calif. Acad. Sci., 57: 51-59. LEBOUR, M. V., 1928. The larval stages of the Plymouth Brachyura. Proc. Zool. Soc. London, 1928 : 473-560. LEBOUR, M. V., 1930. The larvae of the Plymouth Galatheidae. I. Munida banffica, Galathea strigosa and G. dispersa. J. Mar. Biol. Assoc., 17 : 175-186. LOCHHEAD, M. S., J. H. LOCHHEAD AND C. L. NfiwcoMBE, 1942. Hatching of the blue crab, Callinectcs sapidus Rathbun. Science, 95 : 382. ROBERTSON, R. L., 1938. Observations on the growth stages in the common blue crab, Cal- linectes sapidus Rathbun, with special reference to post-larval development. Thesis, Univ. of Maryland, 46 pp. SANDOZ, M., AND R. ROGERS, 1944. The effect of environmental factors on hatching, moulting, and survival of zoea larvae of the blue crab, Callinectcs sapidus Rathbun. Ecology, 25 : 216-228. SANDOZ, M., AND R. ROGERS, 1948. The effect of temperature and salinity on moulting and survival of megalops and post-larval stages of the blue crab, Callinectes sapidus. Va. Fish. Lab., unpubl. MS, 12 pp. TEMPLEMAN, W., 1936. Fourth stage larvae of Homarus americanus intermediate in form between normal third and fourth stages. /. Biol. Bd. Canada, 2: 349-354. STUDIES OX THE FORM OF THE AMPHIBIAN RED BLOOD CELL JOHN DAVISON Department of Biology, Princeton University, Princeton, N. 7., 1 and Department of Biological Sciences, Florida State University, Tallahassee, Florida - To a student of cell form the erythrocyte is an ideal subject for investigation. It is a free cell, not permanently involved in contact with other cells, and it has a definite and relatively simple form. I recently published an account of a model which was proposed as a partial explanation for the elliptical form of the amphibian red cell (Davison, 1957). Since the model has served as a guide to the present work, I will briefly describe its salient features as an introduction to these further observations. The blood cells of the newt Tritunis viridescens approximate thin elliptical discs in form. Viewed as plane elliptical figures, triploid cells have approximately 1.5 times greater area than diploid blood cells, but are apparently no greater in thickness, a relationship similar to that described by Fankhauser for 2n and 3n skin epidermal cells (Fankhauser, 1952). Not only are the 3n cells larger, they clearly have a different shape than 2n cells, being more eccentric regarded as elliptical figures. Using the ratio of the major to minor axes (a/b) as an index to cell form, 2n and 3n Tritunis red cells were found to have mean eccentricities of 1.55 and 1.82, respectively. It has long been recognized that liquid drops can, under the proper physical conditions, simulate many protoplasmic structures (Thompson, 1942). Reasoning that the blood cell exists in a system of cylinders, the blood vessels, I thought it might prove interesting to examine the form characteristics of a fluid drop in con- tact with a cylindrical surface. If one places a large (29 cm. in diameter) cylin- drical glass vessel with the axis horizontal, and pours mercury on the inside of the cylinder, the mercury will assume the form of a flat elliptical disc. Adding more mercury to the pool increases both the area and the eccentricity of the drop but does not appreciably increase its thickness. The model thus simulates the form differences observed between 2n and 3n blood cells. In the model the mercury is in contact with the cylindrical surface through the deforming force of gravity. In the animal it is clear that the blood cells are applied to the wall of the capillary but are not so oriented during their passage through larger vessels. No significant differences were found in the diameter of 2n and 3n capillaries, an essential point, since it is also clear from the model that the larger the cylinder the less eccentric the fluid drop. The latter observations from the model suggest that changes in capillary diameter should lead to alterations in red cell form, with an increase in 1 I would like to express my sincere appreciation to Dr. Gerhard Fankhauser of Princeton University who so generously offered me the use of his laboratory and supported this work through a grant from the Pfeiffer Foundation. - Permanent address : Department of Biological Sciences, Florida State University, Talla- hassee, Florida. 397 398 JOHN DAVISON cell eccentricity following a decrease in capillary diameter and a decrease in cell eccentricity following an increase in capillary diameter. With this background in mind, the further objectives of the study may be stated as follows : ( 1 ) To examine cell form when expressed as a continuous function of cell area, especially with reference to the cross-sectional area of the capillary. (2) To examine the effect of changes in capillary diameter on red cell form under conditions of constant cell area. (3) To quantitatively relate these variables. ANIMALS AND METHODS Since both diploid and triploid Spanish newts (Pleurodeles waltlii) were avail- able, this animal was selected to examine cell eccentricity as a function of cell area. Pleurodeles cells are less eccentric than those of Triturus, better permitting an analysis of the manner in which the blood cell approaches the circular form. The studies on adult Triturus followed the accidental discovery that cold-adapted (8.5 C.) animals have much more eccentric blood cells than the same animals maintained at room temperature (air conditioned 21 C.). Also one can con- veniently measure capillary diameter in the tail fin of adult Triturus, especially the males, while this is not possible in the heavily pigmented Pleur