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