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
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11
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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
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o
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Illllllllll
i
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2 3
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5 6
MINUTES
7-
/5.5
/5.5
T 3
6-
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C
k
o
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-
u O
I
i 4 - I
5 i
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|
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^
o o
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o
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*l 1
1 1
i
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^
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Illll
,
Illllllll
k 11
Illll mil
llllllllll II
..,., nil
1
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2 3
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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
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\
\
x
.
II
!
/55 *
\
\
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\
\
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-
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21
4-
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10 20 30
40 50
60 70
SECONDS
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SECONDS
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2 3
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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. On the feeding reactions and digestion of the coral polyp Astrangia
danac, with notes on its symbiosis with zooxanthellae. Biol. Bull., 49 : 407-439.
BOSCHMA, H., 1926. On the food of reef corals. Proc. Acad. Sci. Amsterdam, 29: 993-997.
BOSCHMA, H., 1929. On the food of reef corals and some other coelenterates. Xe Congres Int.
de Zool. Budapest, pp. 920-923.
BOSCHMA, H., 1936. Sur la croissance de quelques coraux des recifs de 1'Ile d'Edam (Baie de
Batavia). Mus. Roy. Hist. Nat. Belg., (2) 3: 101-114.
DUERDEN, J. E., 1902. West Indian madreporarian polyps. Mem. Nat. Acad. Sci., 8 : 401-648.
CHAVE, K. E., 1954. Aspects of the biogeochemistry of magnesium. I. Calcareous marine
organisms. /. Geol., 62 : 266-283.
EDMONDSON, C. H., 1928. The ecology of an Hawaiian coral reef. Bishop Mus. Bull., 45 : 1-64.
74 THOMAS F. GOREAU
EDMONDSON, C. H., 1929. Growth of Hawaiian corals. Bishop Mus. Bull., 58 : 1-38.
EMERY, K. O., J. I. TRACEY AND H. S. LADD, 1954. Geology of Bikini and nearby Atolls.
U. S. Geol. Surv. Profess. Pap., 260-A : 1-265.
GOREAU, T. F., 1956. A study of the biology and histochemistry of corals. Ph.D. thesis, Yale
University ; 227 pp.
GOREAU, T. F., 1957. Calcification in reef corals. Abstract, 1st Inter-Island Marine Biological
Conference, Puerto Rico.
GOREAU, T. F., 1958. The coral reefs of Jamaica. I. Species composition and zonation.
Ecology (in press).
GOREAU, T. F., AND V. T. BOWEN, 1955. Calcium uptake by coral. Science, 122: 1188-1189.
HAYASHI, K., 1937. On the detection of calcium in the calicoblast of some reef corals. Palao
Trap. Biol. Stat. Stud., 2: 168-176.
KAWAGUTI, S., 1937a. On the physiology of reef corals. II. The effect of light on color and
form of reefs. Palao Trap. Biol. Stat. Sttid., 2 : 199-208.
KAWAGUTI, S., 1937b. On the physiology of reef corals. III. Regeneration and phototropism
in reef corals. Palao Trop. Biol. Stat. Stud., 2 : 209-216.
KAWAGUTI, S., 1941. On the physiology of reef corals. IV. The growth of Goniastrea aspera
measured from numerical and areal increase of calyces. Palao Trop. Biol. Stat.
Stud., 2 : 309-317.
KAWAGUTI, S., 1944. Zooxanthellae as a factor of positive phototropism in those animals con-
taining them. Palao Trop. Biol. Stat. Stud., 2 : 681-682.
KAWAGUTI, S., AND D. SAKUMOTO, 1948. The effect of light on the calcium deposition of
corals. Bull. Oceanogr. hist. Taiivan, 4: 65-70.
MA, J. S., AND G. ZUAZAGA, 1942. Micrc-Kjeldahl determination of nitrogen. Ind. and Chem.
Eng., Anal Ed., 14 : 280-282.
MA, TING YING H., 1937. On the growth rate of reef building corals and its relation to sea
water temperatures. Nat. hist. Zool. Bot. (Academia Sinica) Mem. Zool., 1 : 1-226.
MATTHAI, G., 1918. Is the madreporarian skeleton an extraprotoplasmic secretion of the
polyp? Camb. Phil. Soc. Proc., 19: 160-163.
MAYOR, A. G., 1924. The growth rate of Samoan corals. Pap. Dept. Mar. Biol. Carnegie
Inst. Wash., 19: 1-25, 51-72.
MEIGEN, W., 1903. Beitrage zur Kenntniss des Kohlen-sauren Kalk. Nahtnmss. Gesellsch.
Freiburgh, Ber., 13 : 1-55.
MELDRUM, N. U., AND F. J. W. ROUGHTON, 1933. Carbonic anhydrase : its preparation and
properties. /. Physiol., 80: 113-170.
MOTODA, S., 1940. A study of the growth rate in the massive coral Goniastrea aspera Verrill.
Palao Trop. Biol' Stat. Stud., 2 : 1-6.
ODUM, H. T., AND E. P. ODUM, 1955. Trophic structure and productivity of a windward
coral reef community on Eniwetok Atoll. Ecol. Monogr., 25 : 291-320.
SARGENT, M. C., AND T. S. AUSTIN, 1954. Biologic economy of coral reefs. Geol. Survey
Profess. Pap., 260-E : 293-300.
STEPHENSON, T. A., AND ANNE STEPHENSON, 1933. Growth and asexual reproduction in
corals. Gt. Barrier Reef Exped. Set. Rep., 3(7) : 167-217.
STOLKOWSKY, J., 1950. Essai sur le determinisme des formes mineralogiques du calcaire chez
les formes vivantes. Theses presentees a la Faculte des Sciences de 1'Universite de
Paris; 113 pp. . .
TAMURA, T., AND Y. HADA, 1932. Growth of reef building corals inhabiting the South Sea
Islands. Tohoku Imp. Univ. Sci. Rep. (4},, 7 : 433-455. ,
VAUGHAN, T. W., 1919. Corals and the formation of coral reefs. Smithsonian hist., Ann.
Rep., 1917: 1.89-238.
VAUGHAN, T. W., AND J. W. WELLS, 1943. Revision of the suborders, families and genera
of the Scleractinia. Geol. Soc. Amer. Spec. Pap., 44 : 363 pp.
VERWEY, J., 1930. Depth of coral reefs and the penetration of light. Fourth Pacific Sci.
Congr. Proc. Java, II A : 277-299.
VINOGRADOV, A. P., 1953. The elementary composition of marine organisms. Sears Found.
Mar. Res. Mem., II: 647 pp. Yale University.
VOGEL, A. L., 1943. A Textbook of Quantitative Inorganic Analysis. Second Ed. London,
Longmans; 856 pp.
SKELETON FORMATION IN CORALS 75
WILBUR, K. M., AXD L. H. JODREY, 1955. Studies on shell formation. V. The inhibition of
shell formation by carbonic anhydrase inhibitors. Biol. Bull., 108 : 359-365.
YONGE, C. M., 1940. The biology of reef building corals. Gt. 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. They show that the chief sites of
regulation are the antennary glands and the gills with some regulation by the stomach
and possibly the mid-gut gland.
LITERATURE CITED
BURGER, J. W., 1957. The general form of excretion in the lobster, Homarus. Biol. Bull.,
113: 207-223.
GREEN, J. W., AND M. HARSCH, 1958. The influence of salinity concentration on Na 24 pene-
tration in fiddler crabs. Anat. Rec., 131 : 562.
GROSS, W. J., 1954. Osmotic responses in the sipunculid, Dendrostomum sostericolum. J.
Exp. Biol., 31 : 402-423.
IONIC REGULATION IN FIDDLER CRABS 87
GROSS, W. J., 1957. An analysis of response to osmotic stress in selected decapod Crustacea.
Biol Bull, 112: 43-62.
GROSS, W. J., 1958. Potassium and sodium regulation in an intertidal crab. Biol. Bull., 114:
334-347.
HUF, E., 1936. Der Einfluss des mechanischen Innendrucks auf die Fliissigkeitsausscheidung
bei gepanzerten Siisswasser und Meereskrebsen. Pflug. Arch. ges. Physiol, 237 :
240-250.
JONES, L. L., 1941. Osmotic regulation in several crabs of the Pacific coast of North America.
/. Cell. Comp. Physiol, 18: 79-92.
KOCH, H. J., 1954. Cholinesterase and active transport of sodium chloride through the isolated
gills of the crab Eriocheir sinensis. Proceedings of the Seventh Symposium of the
Colston Research Society, pp. 15-27.
KROGH, A., 1938. The active absorption of ions in some fresh-water animals. Zeitschr. f.
vcrgl. Physiol, 25 : 335-350.
NAGEL, H., 1934. Die Aufgaben der Exkretionsorgane und der Kiemen bei der Osmoregulation
von Carcinus maenus. Zeitschr. f. vergl Physiol., 21 : 468^91.
NALESFSKI, L. A., AND F. TAKANO, 1950. A photonephelometric method for the determination
of sulfates in biological fluids. /. Clin. Lab. Mcd., 36 : 468-470.
PARRY, G., 1954. Ionic regulation in the palaemonid prawn, Palaemon ( = Leander) servitus.
J. Exp. Biol, 31 : 601-613.
PROSSER, C. L., D. W. BISHOP, F. A. BROWN, JR., T. L. JAHN AND V. J. WULFF, 1950. Com-
parative Animal Physiology. W. B. 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
R
7 fi}
80
25
Medium change (mEq./l.)
I-
Blood change (mEq./l.)
S(S
4i
?0
1 00
70
24
Medium change (mEq./l.)
Pn
Blood change (mEq./l.)**
03
2 77
7 t
n 78
66
78
i^a
Medium change (mEq./l.)
~\l\rr
Blood change (mEq./l.)**
n QC
XO
71
fi-i
-10
1 ()
Mg
Medium change (mEq./l.)
* Change in medium for all ions is corrected to a volume equal to the weight of the crab.
** Blood change for calcium and magnesium equals the difference between mean of normal
crabs and the observed blood concentration after treatment for each crab. Medium change is the
observed concentration change in the medium after treatment for each crab.
Analyses of blood potassium and sodium were made before and after desiccation on
individual crabs.
RESULTS
Table I presents the urine and blood concentrations of sodium, potassium,
calcium, and magnesium after the following treatments: a) immersion in normal
sea water; b) immersion in 50% sea water; c) immersion in 150% sea water
and d) desiccation for a water loss of about 7% body weight. Comparing the
blood values after immersion in 100% sea water with those of Prosser et al. (1955),.
sodium and calcium appear in agreement. However, the potassium (7.43 mEq./l.)
and magnesium (20.0 mEq./l.) values are considerably less than those reported by
the above workers (12.1 mEq./l. and 58.4 mEq./l., respectively). On the other
hand Schlatter (1941) reported blood ion concentrations for this same species
which agree closely with the values of the present investigation.
It should be emphasized that the indicated stress media (Table I) represent
only the initial sea water concentrations, and that these necessarily were altered
by exchanges of salts with the animal. However, an accurate knowledge of the
sustained osmotic gradient and the final blood concentrations is of little meaning in
this investigation, since as described above, the animals were able to raise them-
IONIC EXCHANGES IN A CRAB 251
selves out of the water. The main objectives of this study are to demonstrate :
1) the degree to which a blood ion change is reflected in the external medium
and 2) the role of the antennary glands in controlling the ion content of the animal.
It also should be pointed out that in this crab alterations in the blood concentration
in aqueous media are effected by salt exchanges, not water (Gross, 1957).
Data in Table I, however, do reveal something of the ability of Pachygrapsus
to regulate ions in the different sea water concentrations. Thus blood sodium is
held above the sodium concentration of the dilute medium and normal sea water,
but below the concentration of the hypertonic medium. Blood potassium is held
above the concentration of the dilute medium, but below the concentration of normal
sea water or the concentrated medium. Gross (1958) reported that when Pachv-
grapsus was immersed in a small volume of 50% sea water, the blood potassium
remained less concentrated than the medium potassium. However, these animals
were immersed for longer periods than those reported in the present studies
(Table I) during which time the animal lost more potassium and the medium gained
potassium. Table I also show r s that the blood calcium remains more concentrated
than the medium calcium for all treatments. Blood magnesium, on the other hand,
is less concentrated than the medium magnesium for all aqueous conditions. All
four ions increase under conditions of desiccation.
The ratios, urine concentration/blood concentration (U/B ratio), for each
respective ion suggest the role of the antennary glands in the ion regulatory mech-
anism. Values in Table T are means of U/B ratios observed in individual speci-
mens, not ratios of means. Thus all the mean U/B ratios for sodium are less than
one, indicating that the antennary glands do not regulate sodium under this set
of conditions. That is, sodium is not eliminated effectively \vhen the gradient
between blood and medium favors a gain ; nor is it conserved effectively when the
gradient favors a loss to the medium (mean U/B ratio in 50% sea water == 0.96).
With respect to potassium the mean U/B ratio is less than one when the crab
is immersed in 100% or 150% sea water. Thus the antennary gland does not
regulate potassium for this set of conditions. In 50% sea water the mean U/B
ratio is 1.45 which means, if anything, potassium is being wasted when it is needed.
However, for conditions of desiccation the mean U/B ratio is 1 .34 which is signifi-
cantly greater than one. P < 0.01. If then there were sufficient production of
urine under conditions of desiccation, the antennary glands would tend to keep the
blood concentration of potassium at a normal level.
^'ith respect to calcium the mean U/B ratios for crabs immersed in 50% sea
water or subjected to desiccation are not significantly different from unity. Thus
the antennary glands are ineffective as regulators of calcium for these two con-
ditions. On the other hand, after immersion in 150% sea water the mean U/B
ratio is 1.32 which is significantly different from one, P < 0.01. In normal sea
water the U/B ratio is 1.17, again being significantly greater than one, P < 0.01.
Thus, the antennary glands might have a small role in regulating calcium, but in
no sense as large a role as they have for magnesium.
Data in Table I demonstrate that the mean U/B ratios for magnesium under all
conditions studied are much greater than unity. Even after immersion in 50%
sea water, the mean ratio is 5.62. However, it should be pointed out that even in
this diluted sea water the gradient betw r een blood and external medium favors the
252 WARREN J. GROSS
uptake of magnesium. Also, it will he noted that the mean ratio under conditions
of desiccation is 23.6 which suggests that the urine concentration depends on the
blood concentration, not entirely on the rate of influx from the external medium.
The data presented in Table I concerning the treatments in aqueous media are
qualitatively in general agreement with the findings of Prosser ct a I. (1955),
particularly with regard to the role of antennary glands in the regulation of
magnesium. Quantitatively the data presented in Table I differ somewhat from
those reported by Prosser ct al. ( 1955). However, precise comparison should not
be attempted because of differences in experimental procedure. For example,
crabs of the present investigation were immersed directly in small volumes of
stress media for a maximum of about 24 hours. The data presented by the above
workers were obtained on animals gradually acclimated to osmotic stresses for a
period of at least 5 days in relatively large volumes of media.
On the other hand there are certain differences which warrant attention.
Normal blood potassium and magnesium differences already have been mentioned
above. It will be observed that blood calcium after immersion of the animal in 50%
sea water ( 34.S mEq./l.) is higher than it is for animals from normal sea water
(29.6 mEq. /I.). These means are significantly different ; P =0.01. Prosser ct ul.
(1955) showed decreases in blood calcium in 50% sea water which, of course,
would be expected. It was thought that perhaps the increased blood calcium
resulting from immersion in dilute sea water was an effect of the small volume of
medium. Therefore, blood calcium of crabs immersed in large volumes (about
700 ml.) of 50% sea water for 24 hours was determined. The mean blood calcium
of 24 crabs thus treated was 30.9 mEq./l., S.D. = 9.0. This is not significantly
different from the mean (34.8) obtained by the other treatment; nor is it signfi-
cantly different from the average blood calcium of normal crabs. These workers
also called attention to the inverse relationship between the urine sodium con-
centration and the blood sodium concentration. That is, the urine sodium of
animals immersed in concentrated sea water was less concentrated than that of
animals immersed in normal sea water, which in turn was less concentrated than
that of animals immersed in dilute sea water. The means for urine sodium after
treatment in the three aqueous media (Table I) cannot be shown to be significantly
different, but the U/B ratios do suggest the same phenomenon. That is, the ratios
decrease as the animal is placed in increasing concentrations of sea water. These
ratios are all significantly different from each other; P < 0.01. The U/B ratio
for the desiccated crabs is not significantly different from the U/B ratio in crabs
exposed to concentrated media, but is significantly different from the ratios ob-
tained for crabs given the other treatments; P < 0.01.
Data in Table II demonstrate the ionic changes that occur in the medium when
a given change in the blood is effected. The measurement of calcium exchanges
with stress media was complicated by the fact that this ion is lost in significant
amounts when the animal is immersed in normal sea water. Such was not the
case for the other ions. It became necessary, therefore, to apply a correction to
the calcium exchanges, based on an average loss to normal sea water by 30 crabs.
This amounted to 0.5 mEq./l. per gram of crab for a 24-hour period in 50 ml. of
medium. It was thus necessary to assume that this normal loss is constant in
all concentrations of sea water, an assumption which subjects the values for calcium
change in the medium to considerable error.
IONIC EXCHANGES .IN A CRAB
253
The values for sodium and potassium have been reported previously (Gross,
1958) and represent means of the ratios, blood change (mEq./l.) /medium change
(mEq./l.), in individual crabs where the blood change is the difference between
the concentration before treatment and the concentration after treatment. For cal-
cium and magnesium the values in Table II also represent means of the ratios,
blood change (mEq./l.) /medium change (mEq./l.), in individual crabs, but since
only one sample of blood could be extracted from single specimens for calcium and
magnesium determinations, the blood change (mEq./l.) in the ratio for calcium
and magnesium equals the difference between the observed blood concentration
after treatment and the average blood concentration for crabs from normal sea
water.
With respect to sodium, the mean ratios are greater than 2.5 in both 50% and
150% sea water. The response to hypertonic stress and hypotonic stress seems
to be symmetrical. \Yith respect to potassium the ratio is unity or less ; while
it is 0.56 for crabs immersed in 50 r /c sea water, it is 1.00 for crabs immersed in
150% sea water. However when ion exchanges were measured in crabs transferred
from 50% to 150% sea water or vice versa, a symmetrical response for potassium
TABLE 1 1 1
Ion in c reuse in blood caused by desiccation
Mean change in concentra-
No. crabs
tion (% original) per
1% body weight loss
S.D.
by evaporation
\a
84
+ 2.20
0.71
K
50
+ 8.68
11.75
Ca
34
+5.47
4.23
Mg
35
+3.87
9.42
exchanges is observed, the mean ratio, change in blood (mEq./l.) /change in medium
(mEq./l.), being about unity in both extreme stresses (Gross, 1958).
The mean ratio for calcium and magnesium is less than one for all treatments.
Attention should be called to the large variance for the calcium ratio, following
immersion in 50% sea water. It also should be mentioned that the ratio, mean
+ 5.2
blood change (mEq./l.) /mean medium change (mEq./l.), is - -, ^ -- 2.87, the
signs of the numerator and denominator being opposite to expectation. Not only
does the average value for the blood calcium increase after treatment in dilute sea
water, but the medium apparently loses rather than gains calcium. The difference
between the mean of the ratios (0.93) and the ratio of the means (2.87) can be
explained on the basis of the large variance.
Table III reveals ionic changes that occur in the blood when Pachygrapsus is
desiccated for a loss of about 7% body weight. The sodium and potassium values,
again, have been reported previously (Gross, 1958) and represent averages of
changes in individual crabs, where the blood concentration change was determined
by before- and after-treatment readings on the same individual. The values for
calcium and magnesium are means of blood concentration changes for individual
254 WARREN J. GROSS
crabs, but since only after-treatment blood samples were taken, the blood change
for these two ions is represented by the difference between the observed concentra-
tion in an animal following desiccation and the mean blood concentration of the
respective ions in crabs from normal sea water. In Table III it can be seen that
the average change for sodium is less than the values for the other ions. While
the potassium and calcium changes are significantly greater than the sodium change,
P C 0.001, the mean magnesium change cannot be considered significantly different
from the sodium change. It will be explained below that blood ions which increase
more in concentration than blood sodium probably shift from a salt pool (perhaps
the intra-cellular space) into the blood when the animal is desiccated.
DISCUSSION
The ratios, blood change (mEq./l.) /medium change (mEq./L), presented in
Table II suggest that the principal exchanges of potassium, calcium, and mag-
nesium between animal and medium are not ultimately between blood and external
medium. A ratio of unity means that the concentration change in an external
medium which is equal in volume to the animal is identical to the concentration
change in the blood. Of course, much of the animal's volume is isolated from the
osmotic and ionic processes which occur in the blood. Thus for a ratio of unity,
the actual loss or gain of ions with the medium would be greater than the loss or
gain of ions in the blood. Therefore a source other than the blood must be con-
tributing to these exchanges. These ratios also can be expressed as "apparent
volume of distribution," using the equation F - M/P X 100 (Gross, 1958) where:
V "apparent volume of distribution" in % body weight;
weight of medium
M =
P =
weight of animal
change in blood ion concentration (mEq./l.)
change in medium ion concentration (mEq./l.)'
Thus, the "apparent volume of distribution" for sodium is 38.5% body weight
and for potassium, calcium and magnesium more than 100% body weight, which
only can be interpreted as an aggregation of these three ions in some sort of pool
where they are much more concentrated than they are in the blood. This also
means that the extra-vascular pools ultimately contribute more to potassium, calcium
and magnesium exchanges with the medium than does the original blood supply
(more than twice as much). At least, in the case of potassium, the pool probably
lies mainly in the intra-cellular space, because it is well known that intra-cellular
potassium concentrations are high. In the crab Carcinus the relative muscle con-
centrations of sodium, potassium, calcium and magnesium are 50, 120, 11 and 32
(mEq./kg. water), respectively (Shaw, 1955). If this were representative of
intra-cellular concentrations, it would seem unlikely that the intra-cellular space
harbors the pool for magnesium and calcium. Although the nature of the pools
is unknown, it becomes apparent that a change of a blood ion concentration can
occur without a loss or gain in the medium. Or exchanges between animal and
medium can occur without being reflected in the blood. The probable exception to
IONIC EXCHANGES IN A CRAB 255
this is sodium. The "apparent volume of distrihution" for sodium was calculated
to be 38.5% body weight for the moderate stresses of 50% and 150% sea water.
Webb (1940) estimates the blood volume of the crab Carcinus as 36% body
weight. Thus the calculated volume, 38.5% body weight, which seems close to
a reasonable value for blood space, means that the major sodium exchanges are
between the blood and external medium. Though there is evidence that a sodium
pool contributes to such exchanges when the animal is exposed to extreme osmotic
stress, its role is relatively small percentage-wise, compared with the other ions
(Gross, 1958). On the other hand sodium contributes about half the ions of the
blood ; thus the small percentage effect of a sodium pool would nevertheless affect
significantly the total osmotic pressure of the blood.
Burger (1957) immersed lobsters in media of abnormally high magnesium con-
centrations and noted that neither the blood nor the urine magnesium elevated.
On this evidence he concluded that the animal was impermeable to magnesium.
However, he did not consider the possibility that the magnesium could enter the
animal and be fixed outside of the vascular system, a phenomenon which obviously
occurs in Pachygrapsus.
The variance for the mean of the calcium ratios, blood change/medium change,
when the stress was 50% sea water is high. Nevertheless this ratio for calcium
(0.93) is significantly less than the mean ratio for sodium (2.56), F < .025. It
should be emphasized that the mean blood calcium after immersion in 50% sea
water was more concentrated than that for crabs from normal sea water. Also,
the corrected average change for calcium in the medium indicated a loss rather
than the expected gain. Now, it was revealed above that crabs in normal sea
water tend to lose calcium, and the average loss in normal sea water was applied
as a correction to the medium measurements, assuming that a loss of calcium
(probably by way of the gut) would be the same in a stress as in a normal medium,
but if there were a curtailment of normal calcium output in dilute sea w^ater, then
the correction would be too large and falsely could make the sign of the change in
the medium negative. It should be mentioned that the observed changes in the
medium without correction were all positive. If the sign of the corrected medium
change is in error, then the increase in the blood calcium concentration after im-
mersion in 50% sea water could be caused only by contributions from a calcium
reservoir.
Data in Table III demonstrate that for a given weight loss by evaporation the
average increase in the blood sodium concentration is less percentage-wise than the
increase for the other ions. It was concluded by Gross (1958) that such a differ-
ence in increase between sodium and potassium under conditions of desiccation
could not be explained on the basis of sodium exclusion from the blood. Rather,
it was concluded that it represented a shift of potassium ions from extra-vascular
spaces into the blood space. Data for calcium presented in Table III suggest that
the same phenomenon happens in the case of this ion ; values for magnesium are
questionable. No adaptive significance can be assigned to such a phenomenon ;
rather it is interpreted as a physiological failure which imposes a limitation on the
terrestrial habits of this crab.
The U/B ratios presented in Table I suggest the role of the antennary gland
as an, ion regulator. It has been established previously (Prosser ct ai, 1955)
256 WARREN J. GROSS
that this organ is ineffective as an osmotic regulator. Thus, it seems probable
that a principal function of the antennary gland is the regulation of magnesium.
That is, the U/B ratio with respect to magnesium is much greater than unity. Yet
the effectiveness of the antennary glands as magnesium regulators for each ex-
perimental condition cannot be known for certain until the volume of urine pro-
duction is known for each osmotic situation. Thus, even though the urine mag-
nesium is high when the animal is desiccated, it is possible that little or no urine
is produced when the animal is removed from an aqueous medium. Nevertheless,
the antennary glands may effectively remove magnesium ions from the blood, thus
tending to keep the blood levels normal, even though no ions are ejected from
the animal.
These studies were aided by a contract between the Office of Naval Research,
Department of the Navy and the University of California, NR 104-309.
I wish to thank Mr. David Allison for his able technical assistance. Also I
wish to express my gratitude to all those students who assisted in collecting the
experimental animals ; to Professor Theodore Holmes Bullock for reading the
manuscript ; to Professor Timothy Prout for his advice concerning the statistical
handling of the data and to Dr. Frank Bingham for suggesting the method for
the calcium and magnesium determinations.
SUMMARY
1. The effects of osmotic stress on the ion concentration in the blood of the
crab, Pachygrapsus crassipes, were investigated. Stresses imposed were 50% sea
water, 150% sea water and desiccation to a water loss of about 7% body weight.
2. The observed ratios, blood change (mEq./l.) /medium change (mEq./l.),
for sodium, potassium, calcium and magnesium after the crab was transferred from
normal sea water to 50% or 150% sea water yielded values for "apparent volume
of distribution." The average value for sodium was 38.5% body weight, but for
the other three ions w r as at least 100% body weight.
3. The large values for "apparent volume of distribution" in the cases of potas-
sium, calcium and magnesium indicate that these ions are contained in extra-
vascular pools in greater concentrations than they are in the blood and that these
pools participate in ion exchanges between animal and medium. Thus, a con-
centration change can occur in the blood without being reflected in the medium
or vice versa.
4. Calcium is lost to the medium by PacJiygrapsits when it is immersed in
normal sea water. Blood calcium increases when a crab is transferred from normal
sea water to dilute sea water.
5. When Pachygrapsus is desiccated, the blood concentrations of potassium,
calcium and magnesium average greater increases than does the sodium concentra-
tion. This suggests that potassium, calcium and possibly magnesium shift from
an extra- vascular pool into the blood space. The phenomenon is interpreted as a
physiological failure and a factor which may limit the terrestrial life of this species.
6. The ratio, urine concentration (mEq./l.) /blood concentration (mEq./L),
for the respective ions suggests the role of the antennary glands as ion regulators
IONIC EXCHANGES IN A CRAB 257
tinder the various stress conditions. Thus the antennary glands were found to
he relatively ineffective as regulators of sodium, potassium and calcium for all
conditions studied. The U/B ratio for magnesium averaged 5.62 when the crab
was immersed in 50% sea water; 13.6 for normal sea water; 15.4 for 150% sea
water and 23.6 when the crab was desiccated. These high ratios suggest that a
principal role of the antennary glands is magnesium regulation.
7 '. The volumes of urine production which have not been measured must be
known before the effectiveness of the antennary glands as magnesium regulators
can be determined.
LITERATURE CITED
BURGER, I. YY., 1957. The general form of excretion in the lobster Homanis. Biol. Bull.,
113: 207-223.
GROSS, W. J., 1957. An analysis of response to osmotic stress in selected decapod Crustacea.
Biol. Bull., 112: 43-62.
GROSS, W. J., 1958. Potassium and sodium regulation in an intertidal crab. Biol. Bull.. 114:
334-347.
KNIGHT, A. G., 1951. Estimation of calcium in water. Chemistry and Industry, 1951, 1141.
KROGH, A., 1939. Osmotic Regulation in Aquatic Animals. Cambridge at the University Press.
PROSSER, C. L., D. W. BISHOP, F. A. BROWN, JR., T. L. JAHN AND V. WULFF, 1950. Com-
parative Animal Physiology. W. B. Saunders Co., Philadelphia.
PROSSER, C. L., J. W. GREEN AND T. S. CHOW, 1955. Ionic and osmotic concentrations in
blood and urine of Pachygrapsus crassipes acclimated to different salinities. Biol.
Bull.. 109: 99-107.
ROBERTSON, J. D., 1949. Ionic regulation in some marine invertebrates. /. .r/>. Biol., 26:
182-200.
ROBERTSON, J. D., 1953. Further studies on the ionic regulation in marine invertebrates. /.
Ex p. Biol., 30: 277-296.
ROBERTSON, J. D., 1957. Osmotic and ionic regulation in aquatic invertebrates. Recent Ad-
vances in Invertebrate Physiology. University of Oregon Publications, pp. 229-246.
SCHLATTER, M. J., 1941. Analyses of the blood serum of Cambants clarkii, Pachygrapsus
crassipes and Paiiulinis interruptus. J . Cell. Coinf*. Physiol., 17 : 259-261.
SCHWARZENBACH, G., W. BlEDERMANN AND F. BANGERTER, 1946. KompleXOllC VI. NeUC
einfache Titriermethoden zur Bestimmung der Wasserharte. Hch'. Chini. Acta. 29:
811-818.
SHAW, J., 1955. Ionic regulation in the muscle fibres of Carciuus niacnas. II. The effect
of reduced blood concentration. /. Ex p. Biol.. 32 : 664-680.
WEBB, D. A., 1940. Ionic regulation in Carcimts inaenas. Proc. Roy. Soc. London, Scries B.
129: 107-136.
HISTOLOGY AND METABOLISM OF FROZEN INTERTIDAL
ANIMALS x
JOHN KANWISHER
Woods Hole Occanografihic Institution, Woods Hole, Massachusetts
Many invertebrate animals are normally exposed to environmental temperatures
far below the freezing point of their body fluids. Although supercooling may
sometimes be a factor in survival (Salt, 1950; Ditman et al., 1942 ; Scholander et al.,
1953), freezing occurs in nature among insects (Asahina ct al., 1954; Scholander
et al., 1953), shore animals (Kanwisher, 1955), and other groups (Luyet and
Gehenio, 1940.) On the shore during winter, for example, freezing and thawing
occurs twice a day when the animals are exposed to the cold air by the tide.
Intertidal animals in the Arctic may be frozen for as long as 6 months (Kanwisher,
1955). The survival of these animals depends on their being able to have most
of their body water turned to ice. It is remarkable that no injury is produced in a
living system when more than half of its bulk is changed to a crystalline solid.
I am reporting here some investigations on the histology and metabolism of these
intertidal animals.
HISTOLOGY
In the freeze-drying histological technique, tissue is cooled very quickly with
liquid nitrogen. Freezing occurs so fast that ice crystals do not have time to
grow very large and cellular organization is very little disturbed. The water is
removed by vacuum while the sample is kept cold. The resulting dehydrated
tissue matrix is imbedded, sectioned, and stained in a conventional manner. I
have used the method here to capture the situation in tissue from shore animals
frozen to relatively mild natural temperatures. Comparison with material from
unfrozen animals has shown the distortions caused by the freezing.
Animals were collected from the shore at Woods Hole in January and moved
to a --10 cold room without thawing. Sections of tissue about 1 mm. thick were
cut with a cold knife, held with cold tweezers, and plunged into a vial of isopentane
suspended in a container of liquid nitrogen. The isopentane allows a faster heat
transfer because it does not boil and form an insulating gas layer. The hard frozen
samples were quickly transferred to the already cold dehydrating chamber and
vacuum applied for 24 hours at about -45. The dehydrated tissue was then
imbedded in de-gassed paraffin already in the chamber with the vacuum still applied.
Photomicrographs of 10-micron sections are shown in Figure 1. The unfrozen
controls were tissue taken from identical animals that had thawed at room tem-
perature for an hour.
1 Contribution Number 1013 from the Woods Hole Oceanographic Institution. This study
was aided by a contract between the Office of Naval Research and the Arctic Institute of
North America.
258
FROZEN INTERTIDAL ANIMALS
259
B
D
FIGURE 1. Photomicrographs of unfrozen and frozen tissue.
Figure 1, A is the unfrozen foot of the shore snail Littorina littorea. The
purpose of the randomly arranged muscle fihers is related to the snail's type of
locomotion. In the frozen tissue in Figure 1, B. the ice forms in large pockets
with a resulting shrinkage and distortion of the cells.
The extreme distortions indicated in the initial results were surprising enough
260 JOHN KANWISHER
to warrant the following procedure. The frozen muscle slice was cut in two
pieces. One was used as the frozen specimen. The other was warmed for less
than a minute on the palm of my hand and then hard frozen in the liquid nitrogen.
When sectioned it appeared nearly the same as tissue from an unfrozen animal.
Figures 1, A and 1, B are actually sections from this run.
A transverse section of the unfrozen adductor muscle in the oyster, Crassostrea
virgimcus, is shown in Figure 1, C. The parallel muscle fibers are viewed end-on.
In its frozen counterpart in Figure 1. D, the fibers are clumped into groups to
make room for the intervening ice. The prominent elements that resulted are all
about the same size. There may be membranes not visible in the unfrozen muscle
to account for this regularity. The same regular clumping was seen in the ad-
ductor muscle of two mussels, Modiolus modiolus and Mytilus cdulis.
Figure 1, E is of the eggs in the unfrozen ovary of the blue mussel Mytilus
ednlis. When frozen as in Figure 1, F, the detail is much less distinct but the
eggs clearly have shrunken during the formation of the large amounts of inter-
cellular ice. Comparable distortions were seen in other tissues from these and other
species.
METABOLISM OF FROZEN ANIMALS
Scholander ct al. (1953) measured respiration at freezing temperatures by
following the decrease in oxygen concentration in a closed volume containing the
animal. The same method has been used here. Manometric and volumetric
techniques can not be used because of the volume change when water turns to ice.
The snails to be used were frozen in 20-nil. syringes in a cold bath. Only those
in which the snail froze while fully extended from its shell were used. A short
section of tubing on the tip of the syringe extended above the surface of the
liquid and was closed with a pinch clamp. A sample of gas could be withdrawn
without removing the syringe from the bath. The plunger was free to move up
and replace the volume lost in sampling. Allowance was made for the decreased
volume in calculating the rate of oxygen removal.
Duplicate oxygen analyses good to 0.02 per cent were made with the half-cc.
analyzer of Scholander (1947). Serial samples were plotted against time and
the slope was used in computing the oxygen consumption. The concentration
was never allowed to go below 18 per cent in any run. Respiration was assumed
to be independent of tension over this small range.
After the snails were placed in the cold bath, at least 6 hours were allowed for
phase equilibration between ice and water in the tissues. Previous experience
(Kamvisher, 1955) had shown that there was no appreciable increase in ice after
this length of time. The syringe was then flushed with cold outside air. A series
of oxygen determinations showed that such air did not vary appreciably from
20.94 per cent so this was considered the starting concentration. At intervals
ranging from 2 to 120 hours samples were withdrawn with a mercury gas sampler.
Volumetric respirometers (Scholander ct a!., 1952) were used above 0. One
ml. of sea water was included in the vial with the animals. At such values
w r ere in good agreement with those made by gas analysis which is specific for
oxygen. The often used and rarely proven hypothesis is thus confirmed that the
volume decrease is due to oxygen being consumed.
The respiration temperature data from 10 to +30 C. are plotted in Figure 2.
Between and +20, oxygen consumption shows the usual logarithmic increase
FROZEN INTERTIDAL ANIMALS
261
\vith a Q 10 of 2 to 3. Above this respiration decreases, probably due to thermal
injury. Below the metabolic activity drops sharply with an apparent Q 10 of
about 50.
At -10, respiration was so low it took 6 days for the snails to consume a
measureable amount of oxygen. Even in this length of time the concentration
change was smaller than desired for accurate determination. This may account
for the greater spread of values at this low temperature. At -15 the empty
syringes gave blank values of one-third the oxygen decrease in those containing
snails. This may be due to oxidation of grease used on the syringe plunger. It
100
- I
o
10
o
8
o
o
9
8
1
8
8
o
8
o
8
o
o
o
o
RESPIRATION
vs.
TEMPERATURE
LITTORINA LITTOREA
FIGURE 2
TEMP. C.
-10 -5 5 10 15 20
FIGURE 2. Variation of oxygen uptake with temperature.
25
30
did not seem that this technique could be trusted on the slower rates to be expected
at still lower temperatures.
SALINITY EFFECT ON RESPIRATION
Scholander et al. (1953) have given several reasons why the respiratory gas
exchange of a frozen animal drops so much more rapidly with temperature than it
does above when no ice is present. The ice may act as a diffusion barrier to
the gases. The increased viscosity of the body fluids may slow the reaction rates.
Finally the increased salinity may directly inhibit the animal's metabolism. No
way could be devised to test the first two hypotheses. The respiratory response
to increased salinity above can be determined independently of any ice effects.
262
JOHN KANWISHER
Higher than normal salinities were made by freezing sea water and using
the brine. Dilution with fresh water gave lower than normal salinities. Freshly
collected snails were placed in jars containing the different salinities for a minimum
of 6 hours before being used. At very high and low values the snails withdrew
into their shells. Experience had shown that the operculum blocks respiratory
gas exchange so these could not be used.
For the respiration measurements single snails were placed in 20-ml. syringes
filled with the desired salinity. The syringes were kept in a constant temperature
bath except when sampling. One-mi, samples were removed at convenient intervals
and analyzed gasometrically for oxygen by the method of Scholander et al. (1955).
200r
100
50
20
10
o
o.
o
CM
O
/
O
% SAL
\
o \ o
\
RESPIRATION
vs.
SALINITY
LITTORINA
\ LITTOREA
\
\o
\
\
FIGURE 3
INITY
2.5 5.0 7.5
FIGURE 3. Response of oxygen uptake to different salinities.
Since this is a physical extraction of the gases it could be relied on in spite of the
water sometimes becoming cloudy with waste products.
As in the low temperature gas analysis method several serial readings were
used to indicate the rate of oxygen removal by the snails. Low oxygen tensions
were avoided by working in the range of 2.5 to 6 mm. 3 of oxygen per ml. The
curves showed that respiration was independent of tension over this range.
The variations of oxygen consumption with changes in the external salinity are
shown in Figure 3. High salinity depresses the respiration of Littorina littorca.
This is a reversible effect since the rate increases again when the snail is returned
to normal salinity. When the snails withdrew into their shells at higher salinities
than shown, no oxygen consumption could be detected. They are apparently able
to subsist for long periods anaerobically.
FROZEN INTERTIDAL ANIMALS 263
Since freezing occurs throughout the animal, the remaining body fluids in all parts
of the animal are concentrated. If any effect of external salinity above is to be
related to freezing, it must be shown that the animal is not osmotically regulating.
Increased salinity could conceivably be effective in only altering the absorption of
oxygen at the surface. Tissues of snails from water of different salinities were
analyzed for chlorides. By carefully cracking the shell, the animal could be re-
moved whole. Excess water was mopped off and the weight quickly taken. The
water was removed by drying for several hours in a 100 oven. The dry weight
then gave the total water by difference. The dry tissue was then digested and
titrated for the amount of total chlorides present. This amount was considered
dispersed in all the animal water in order to calculate the concentration in the live
animal. From the resulting normalities at different external salinities it was
clear that internal chloride concentration was proportional to that outside the
animal. Any result of externally varied salinity can reasonably be viewed as
arising from a corresponding change throughout the animal.
DISCUSSION
Chambers and Hale (1932) observed plant and animal cells freezing under
the microscope. They found that ice formation inside the cellular membrane always
resulted in the death of the cell. The detailed studies of Asahina and his colleagues
(1954) have described the freezing process in the blood and isolated organs of
insect pre-pupae and in the intact insects themselves. They also found that
intra-cellular freezing is lethal to the cell or tissue. Such results are responsible
for the general belief that all freezing in animals and plants takes place outside
the cells.
The impressive liquid air freezing experiments of nematodes by Luyet and
Gehenio (1940) have almost certainly been an instance of ice within cells. Lack
of injury here has been attributed to the very rapid rate of freezing. This vitrifica-
tion does not allow time enough for ice crystals to grow to a size where they can
damage protoplasmic structures. It forms the basis of the histology used here.
This phenomenon probably has little to do with the normal ecology of these animals
in nature.
Shore animals that are exposed to freezing are in shells. This impedes heat
transfer and gives ice crystals time to grow. One can readily see them in an
opened animal. The tissues of such an animal are similar in texture and appearance
to a frozen piece of meat. It is not surprising when one considers that water makes
up three-fourths of the bulk of the animal and four-fifths or more of it may be ice.
The photomicrographs presented here show the large amount of distortion necessary
at the cellular scale to make room for this ice. Yet this can change back to a more
or less normal appearing tissue in 30 seconds as shown in Figures 1, A and B.
Siminovitch and Briggs (1949) have related frost hardiness in plants to the ability
of water to migrate rapidly in and out of the cells. Unfortunately no equivalent
test could be devised to apply this to shore animals.
Littorina littorea was found to remain out of its shell and behave normally in
salinities of 2 to 7 per cent. It sharply decreased its metabolism in response to a
salinity increase. In Figure 3. the data indicate that doubling the salinity above
the optimum decreases the oxygen demand to about a third. As the salinity of
264 JOHN KANWISHER
the body fluids is increased by the freezing out of water, oxygen uptake must
drop in the same fashion. From the freezing curves in a previous paper (Kan-
wisher, 1955) 70 to 80 per cent of the water in this species is frozen at 10.
This would result in a Q 10 from the salinity of about 10. Above the Q 10 due
to the usual temperature effect on reaction rates is between 2 and 3. Combining
these one would expect a Q lf , in the range of 20 to 30 below 0. The actually
observed one is closer to 50. The effect of ice as a gaseous diffusion barrier and
that from the loss of water itself may account for the difference. It is felt that
the present data do not warrant a more vigorous interpretation. Similar work with
intertidal algae (Kan wisher, 1957) has shown that the drying effect of freezing
was chiefly responsible for a similar large decrease in respiration. A three-times
increase in salinity had little effect on the oxygen uptake of these plants.
Freezing in shore animals to the extent shown here is a normal occurrence
twice daily in the winter with no obvious injury to the animal. This freezing
hardiness is probably connected with the ability to stand the internal distortions and
high salinities that result. The greatly lowered metabolism may be of adaptive
significance in severe locations where shore animals are frozen into the ice for
months at a time. As such it could represent a considerable saving in food reserves.
SUMMARY
1. Histology of frozen shore animals has shown large pockets of intercellular
ice with consequent shrinkage and distortion of the surrounding cells.
2. The Q 10 drops precipitously in the region of ice formation and may be as
high as 50.
3. High tissue salinity without freezing decreases oxygen uptake. Thus the
salinity increase that results from freezing is responsible for a large part of the
high Qio-
LITERATURE CITED
ASAHINA, E., K. AOKI AND J. SniNOZAKi, 1954. The freezing process of frost-hardy cater-
pillars. Bui. Entomological Research, 45: 329-339.
CHAMBERS, R., AND H. P. HALE, 1932. The formation of ice in protoplasm. Proc. Roy. Soc.
London, Ser. B, 110: 336-352.
DITMAN, L. P., G. B. VOGT AND D. R. SMITH, 1942. The relation of unfreezable water to
cold hardiness in insects. /. Economic Entomology, 35: 265-272.
KANWISHER, J. W., 1955. Freezing in intertidal animals. Biol. Bull., 109: 56-63.
KANWISHER, J. W., 1957. Freezing and drying in intertidal algae. Biol. Bull.. 113: 275-285.
LUYET, B. J., AND P. M. GEHENIO, 1940. Life and Death at Low Temperatures. Biodynamica,
Normandy, Missouri.
SALT, R. W., 1950. Time as a factor in the freezing of undercooled insects. Canadian J . Res.,
28: sect. D: 285-291.
SCHOLANDER, P. F., 1947. Analyzer for accurate estimation of respiratory gases in one-half
cubic centimeter samples. /. Biol. Chcin.. 167 : 235-250.
SCHOLANDER, P. F., C. LLOYD CLAFF, J. R. ANDREWS AND D. F. WALLACH, 1952. Micro-
volumetric respirometry. /. Gen. Physio!., 35 : 375395.
SCHOLANDER, P. F., W. FLAGG, R. J. HOCK AND L. IRVING, 1953. Studies on the physiology of
frozen plants and animals in the Arctic. /. Cell. Coinp. Physio!., 42: supplement 1,
1-56.
SCHOLANDER, P. F., L. VAN DAM, C. L. CLAFF AND J. W. KANWISHER, 1955. Micro-gasometric
determination of dissolved oxygen and nitrogen. Biol. Bull., 109 : 328-334.
SIMINOVITCH, D., AND D. R. BRiGGS, 1949. The chemistry of the living bark of the black
locust tree in relation to frost-hardiness. Arch. Biochcm., 23 : 8-17.
CHROMATOGRAPHIC ANALYSES OF AMINO ACIDS IN THE
DEVELOPING SLIME MOLD, DICTYOSTELIUM
DISCOIDEUM RAPER l
JEROME O. KRIVANEK AND ROBIN C. KRIVANEK
Department of Zoology, Neivcomb College of Tulane University, New Orleans 18, Louisiana
The slime mold, Dictyostelium discoideum Raper, is a relatively simple biological
system in which to study the processes of differentiation and morphogenesis.
From a seemingly homogeneous mass of cells (the aggregation mass), there are
eventually formed in the mature sorocarp two basic cell types the stalk cell and
the spore cell. The developmental cycle of D. discoideum has been described in
detail by Bonner (1944) and Raper (1935, 1940) and will not be repeated here.
In the recent literature, studies have been reported that suggest correlations
between nitrogen metabolism and the processes of differentiation and morphogenesis
in this slime mold. Gregg, Hackney and Krivanek (1954) detected the evolution
of ammonia and described changes in several nitrogenous fractions during the life
cycle of this organism. In this same study, they suggested that the cellulose of
the mature sorocarp was synthesized at the expense of a protein precursor and
pointed out that the major nitrogen changes took place while the spore and stalk
cells were being formed, i.e., during the culmination process. In addition, Krivanek
and Krivanek (1958), using the histochemical technique devised by Francis (1953),
demonstrated the occurrence of amine oxidase activity in various regions of the
slime mold undergoing differentiative changes. The simultaneous occurrence of
changes in nitrogen metabolism and of differentiative and morphogenetic phe-
nomena prompted the present study.
MATERIALS AND METHODS
The method as outlined by Block, Durrum and Zweig (1955) was used for
ascending paper chromatographic determinations of amino acids in the slime mold.
Chromatograms, using hydrolyzed and unhydrolyzed tissues, were made of four
representative stages of development migrating pseudoplasmodium, pre-culmina-
tion, culmination, and mature sorocarp. In the case of hydrolyzed tissue, in-
dividuals in the desired stage of development were isolated and homogenized in
6 N HC1, hydrolyzed for 18 hours, and evaporated over a boiling water bath.
The residue was placed in a soda lime desiccator for 48 hours and then taken up
in 2 cc. of warm glass-distilled water and filtered. After evaporating the water
filtrate, the residue therefrom was taken up in 1 cc. of iso-propanol, the vehicle
used in the application of the spot. In the case of the unhydrolyzed tissue,
1 This research was supported in part by Research Grant E 1453 from the National Insti-
tute of Allergy and Infectious Diseases, National Institutes of Health, U. S. Public Health
Service.
265
266
JEROME O. KRIVANEK AND ROBIN C. KRIVANEK
homogenates were made with water and evaporated. The residue was taken up
in 1 cc. of iso-propanol and applied to the paper.
The microhomogenizer described by Gregg, Hackney and Krivanek (1954)
was used for the preparation of the tissue homogenates. All homogenization took
place at room temperature (22 C.). Depending upon the stage of development to
be analyzed, the homogenization procedure lasted from thirty minutes to an hour.
All evaporation took place over a boiling water bath with the evaporation lasting
ALL STAGES
(HYDROLYZED)
o
PHENOL
FIGURE 1. Diagram of the results of two-dimensional chromatography on hydrolyzed
tissue of D. discoideum. Spots are identified as the leucines (1), phenylalanine (2), methionine
(3), proline (4), tyrosine (5), alanine (6), threonine (7), histidine (8), glycine (9), glutamic
acid (10), serine (11), asparagine (12), unknown (13), cystine (14), and aspartic acid (15).
no more than five minutes in any case. Rupture of virtually all cells was insured
by means of periodic microscopic examination of the homogenate.
For both types of analyses, i.e., hydrolyzed and unhydrolyzed, two-dimensional
chromatograms were made on Whatman No. 1 filter paper. For the first dimension,
n-butanol, acetic acid and water (250, 60, 250 v/v/v) were used as the solvent
mixture. For the second dimension, an 80% solution of phenol in water was
used as solvent. Development of the spots was accomplished by means of spraying
the chromatograms with a solution of 0.3% ninhydrin in 95% ethanol. After
AMINO ACIDS IN DICTYOSTELIUM
267
spraying, the chromatograms were allowed to dry in complete darkness for 18
hours. No less than 6 and no more than 10 runs were made for each analysis.
In the majority of cases, consistent spot patterns were achieved and only 6 runs
were made. However, in those few cases where slight inconsistencies in the
patterns were evident, additional runs were made to achieve reproducibility.
Identification of the spots was achieved in two ways. Firstly, Rf values were
calculated and compared with the R f values of known amino acids. Secondly, one-
dimensional as well as two-dimensional "control" runs were made using solutions
of known amino acids, both singly and grouped, and the loci of spots were com-
pared between the control and experimental series.
RESULTS
Hydrolyzcd tissue. Results of the chromatographic studies of amino acids in
hydrolyzed tissues of D. discoidcinn are shown in Figure 1. With the exception
MIGRATING PSEUOOPL ASMODIUM
(UNHYDROLYZED)
FIGURE 2. Diagram of the results of two-dimensional chromatography on unhydrolyzed
tissue of D. discoidcwn in the migrating pseudoplasmodium stage. Identified spots are the
leucines (1), methionine (2), tyrosine (4), alanine (5), threonine (6), glycine (9), serine (10),
glutamic acid (11), aspartic acid (13), and cystine (15). Spots 7, 14, 16, and 17 are unknowns.
of one spot (no. 13), all spots were identified. The identified spots included the
leucines (1), phenylalanine (2), methionine (3), proline (4), tyrosine (5), alanine
(6), threonine (7), histidine (8), glycine (9), glutamic acid (10), serine (11),
asparagine (12), cystine (14), and aspartic acid (15).
The same spot pattern persisted throughout the four analyzed stages of develop-
ment. Although no quantitative determinations of the amino acids were made,
comparisons of the relative spot intensities afforded some degree of quantification.
Glutamic acid presented the most intense color in each stage. Also quite intense,
but not to the degree of glutamic acid, were the spots of the leucines, methionine,
alanine, threonine, serine, and asparagine. Medium light spots resulted from
268
JEROME O. KRIVANEK AND ROBIN C. KRIVANEK
O
PRE-CULMINATION STAGE
(UNHYDROLYZEO I
O
PHENOL
FIGURE 3. Diagram of the results of two-dimensional chromatography on unhydrolyzed
tissue of D. discoideum in the pre-culmination stage. Spots as in Figure 2, plus spot 12, an
unknown.
phenylalanine, tyrosine, glycine, and histidine. The faintest spots were those of
proline, cystine and aspartic acid.
In addition to these well-formed spots, a very faint, vaguely-defined spot was
occasionally found in the approximate locus of cysteine. Because of its vagueness
O
CULMINATION STAGE
(UNHYDROLYZED)
PHENOL
FIGURE 4. Diagram of the results of two-dimensional chromatography on unhydrolyzed
tissue of D. discoidcum in the culmination stage. Spots as in Figure 2, plus spots 3 and 12,
unknowns.
AMINO ACIDS IN DICTYOSTELIUM
269
and the failure of our controls to show a clear cysteine spot, we cannot state
positively either the presence or absence of cysteine.
Unhydrolyzed tissue. Results of the chromatographic studies of amino acids
in unhydrolyzed tissue of D. discoidewn are shown in Figures 2, 3, 4 and 5.
Whereas a consistent spot pattern occurred throughout the developmental cycle
in the case of hydrolyzed tissue, considerable variability in the spot patterns oc-
curred between the several stages in the case of unhydrolyzed tissue. A total of
17 spots appeared in all or nearly all of the stages of development. However, only
ten were identified. They were the spots of the leucines (1), methionine (2),
tyrosine (4), alanine (5), threonine (6), glycine (9), serine (10), glutamic acid
(11), aspartic acid (13), and cystine (15). The remaining seven spots 3, 7, 8,
12, 14. 16, and 17- were not identified. Presumably these ninhydrin-positive
o
MATURE SOROCARP
(UNHYDROLYZED)
03 o ar
FIGURE 5. Diagram of the results of two-dimensional chromatography on unhydrolyzed
tissue of D. discoidewn in the mature sorocarp stage. Spots as in Figure 2, plus spots 3, 8,
and 12, unknowns.
spots were simple peptides. It is possible that these spots were the products of
partial hydrolysis by enzymes derived from the cells. However, in view of the
rapidity with which the tissues were prepared, this would seem unlikely. Those
spots which were evident in all stages of development were 1, 2, 5, 6, 7, 9, 10, 11,
13. 14, 15, and 16. Although spot 12, an unknown, did not appear in the migrating
pseudoplasmodium. it did appear in the succeeding three stages of development.
Spot 3, also unknown, appeared only in the culmination and mature sorocarp
stages, while spot 8, a third unknown, appeared only in the mature sorocarp stage.
Spot 4, identified as tyrosine, was present in all stages except the mature sorocarp,
and spot 17 appeared erratically being present in all but the pre-culmination stage.
As in the case of hydrolyzed tissue, cysteine could not be definitely ascertained as
either being present or absent.
270 JEROME O. KRIVANEK AND ROBIN C. KRIVANEK
DISCUSSION
In their quantitative studies of the nitrogen metabolism in the slime mold, D.
discoideum, Gregg, Hackney and Krivanek (1954) demonstrated a decrease in the
total nitrogen/dry weight during the transition from the migrating pseudoplas-
modiuni to the mature sorocarp. They attributed this decrease to a decrease in
the total extractable protein nitrogen and total unextractable nitrogen components
of the slime mold. In addition, they found that ammonia was being given off
by the slime mold during its life cycle. In a subsequent study, Gregg and
Bronsweig (1956) found a steady increase in the total amount of reducing sub-
stances (presumably carbohydrates) as the life cycle progressed. On the basis of
these data, it was suggested that the protein fraction of the slime mold served
as a precursor for the carbohydrate of the mature sorocarp. However, no indica-
tion was made of the possible pathway (s) involved in this conversion. The present
study may be suggestive in this respect.
Glutamic acid invariably presented the most intense spot of any of the deter-
mined amino acids. This was evident in both hydrolyzed and unhydrolyzed
tissues. The deamination of glutamic acid to a-ketoglutaric acid with the cor-
responding release of ammonia is known. Because of the reversibility of this
reaction, it is considered to be one of the prime mechanisms responsible for the
interconversion of ammonia and a-amino group nitrogen. The reaction is catalyzed
by glutamic acid dehydrogenase, requiring either DPN or TPN as a coenzyme
(Meister, 1957). The importance of this reaction, as it relates to D. discoideum,
lies in the fact that not only has glutamic acid been detected to a high degree in
the slime mold, but, also, the liberation of ammonia during the life cycle suggests
such a deamination reaction. Further, Krivanek and Krivanek (1958) demon-
strated non-specific dehydrogenase activity in the pre-stalk area of the pre-culmina--
tion and culmination stages stages in which the future sorophore sheath (con-
sisting primarily of polysaccharides) is being secreted by the stalk cells as they
move apically to become eventually enclosed within the sorophore sheath. This
non-specific dehydrogenase activity could logically be attributed to glutamic acid
dehydrogenase. By virtue of the relationship between glutamic acid, a-ketoglutarate,
areas of dehydrogenase activity, and sites of carbohydrate secretion, there thus can
be postulated this link between carbohydrate metabolism and protein metabolism in
the slime mold.
The glutamic acid-ketoglutarate relationship, if actually operative in the slime
mold, need not be the only link between carbohydrate metabolism and protein
metabolism. Aspartic acid, also demonstrated in hydrolyzed and unhydrolyzed
tissues of the slime mold, can be deaminated to fumarate, another intermediate in
the citric acid cycle (Meister, 1957), thus creating a second possible link between the
two types of metabolism. Further, there is the possibility that alanine can undergo
deamination forming the Krebs cycle intermediate pyruvate as has been sug-
gested by Meister (1957), and serine, as well as cysteine, can undergo the same
process yielding ammonia and pyruvate.
The suggested relationships already discussed do not preclude the possibility
of other mechanisms relating carbohydrate metabolism to protein metabolism, such
as clecarboxylation and transamination. There is as yet, however, no evidence
to indicate the presence of these mechanisms in the slime mold.
AMINO ACIDS IN DICTYOSTELIUM 271
Several of the amino acids of the hydrolyzed tissues appear as well in un-
hydrolyzed tissue. Consequently, it is not possible to determine whether these
amino acids occur as free amino acids only, or also as bound amino acids. How-
ever, four amino acids appear only in the hydrolyzed tissue (phenylalanine, proline,
histidine, and asparagine). They are considered therefore to exist only in the
bound form. The significance of these amino acids with respect to the differentia-
tive process in Dictyosteliuni is at present not apparent.
Studies have recently been initiated to test the validity of the above postulates.
These correlative studies will embrace the use of the analogs of the amino acids
shown to be present in D. discoideum.
SUMMARY
1. The amino acids in hydrolyzed and unhydrolyzed tissue of the slime mold,
Dictyosteliuni discoideum Raper, have been determined by means of two-dimen-
sional ascending paper chromatography. Analyses were made on four stages of
development migrating pseudoplasmodium, pre-culmination, culmination, and
mature sorocarp.
2. Unhydrolyzed tissue contained the leucines, methionine, tyrosine, alanine,
threonine, glycine, serine, glutamic acid, aspartic acid, cystine, and seven unidentified
spots, presumably simple peptides. Not all these spots were present in all tested
stages.
3. Hydrolyzed tissue contained in addition to the amino acids identified above,
phenylalanine, proline, histidine, asparagine, and one unknown spot. All tested
stages were identical.
4. The postulate is presented that glutamic acid (and possibly also to a lesser
degree aspartic acid, alanine, serine, and cysteine) through deamination may enter
the Krebs cycle and form a link between protein and carbohydrate metabolism,
the change in balance between protein and carbohydrate being one of the most
prominent features of differentiation in this organism.
LITERATURE CITED
BLOCK, R. J., E. L. DURRUM AND G. ZWEIG, 1955. A Manual of Paper Chromatography and
Paper Electrophoresis. Academic Press, Inc., New York.
BONNEK, J. T., 1944. A descriptive study of the slime mold, Dictyostelium discoideum. Anier.
J. Bot., 31 : 175-182.
FRANCIS, C. M., 1953. Histochemical demonstration of amine oxidase in liver. Nature, 171 :
701-702.
GREGG, J. H., A. L. HACKNEY AND J. O. KRIVANEK, 1954. Nitrogen metabolism of the slime
mold Distvostcliiim discoideum during growth and morphogenesis. Biol. Bull., 107 :
226-235.
GREGG, J. H., AND R. D. BRONSWEIG, 1956. Biochemical events accompanying stalk formation
in the slime mold, Distyostelium discoideum. J. Cell. Comp. Physiol., 48: 293-300.
KRIVANEK, J. O., AND R. C. KRIVANEK, 1958. The histochemical localization of certain bio-
chemical intermediates and enzymes in the developing slime mold, Dictyostelium
discoideum Raper. /. Exp. Zool, 37: 89-116.
MEISTER, A., 1957. Biochemistry of the Amino Acids. Academic Press, Inc., New York.
RAPER, K. B., 1935. Dictyostelium discoideum, a new species of slime mold from decaying forest
leaves. /. Agric. Res., 50: 135-147.
RAPER, K. B., 1940. Pseudoplasmodium formation and organization in Dictyostelium discoideum.
J. Elisha Mitchell Sci. Soc., 56 : 241-282.
SOME ASPECTS OF OSMOREGULATION IN TWO SPECIES OF
SPHAEROMID ISOPOD CRUSTACEA
J. A. RIEGEL 1
Department of Zoology, University of California, Davis, California
The internal effects of osmoregulation were studied in two euryhaline species
of isopod crustaceans, Gnorhnophaeroma orcgonensis (Dana) and Sphaeroma
pcntodon Richardson. Although a large literature exists on the subject of osmoreg-
ulation in Crustacea, only a little of it concerns isopods. Therefore, the present
study was undertaken to gain more information in this relatively unexplored area.
Bogucki (1932) studied the ionic composition of the body fluid in Mesidotea
cntomon, which according to Ekman (1953) is an inhabitant of the Baltic and
Arctic Seas and several fresh-water lakes in the land area of the northwest Pacific,
Siberia, and northern Europe. Bogucki found the body fluid concentration to be
hypertonic to the medium in lower salinities, becoming isotonic as the medium
approached sea water. Lockwood and Croghan (1957), studying the brackish- and
fresh-water races of the same species, found the osmotic behavior to be similar in
both races, except that the brackish-water animals could not survive in fresh water.
They concluded that the fresh-water race has developed a more effective osmoreg-
ulatory mechanism that enables it to maintain the high haemolymph concentrations
of the brackish-water race in fresh water. Bateman (1933) found that Ligia
oceanica maintained its body fluid hypertonic to a medium of about 80 per cent
sea water, but swelled and died in 50 per cent sea water. However, Parry (1953),
working with Ligia exotica, found that in well-aerated sea water, specimens of
the species could survive 17 to 30 days in salinities ranging from 50 to 125 per
cent sea water. In very concentrated media (A = 3.46 C.), the body fluid was
maintained hypotonic to the medium. Menzies (1954), in addition to splitting
Gnorimosphacroma orcgonensis into two subspecies, lute a and oregonensis, per-
formed preliminary experiments to test the ability of the two subspecies to survive
in various salinities. Specimens of G. o. oregonensis taken from 25 per cent sea
water and placed in tap water were all dead after one day. Specimens of G. o.
lutea taken from 1.6 per cent sea water and placed in tap water died slowly over
a period of three days. Menzies concluded that G. o. oregonensis is probably re-
stricted to sea water, but he was puzzled as to why G. o. lutea could survive in
sea water, but not in tap water (salts equivalent to 0.3% sea water), which was not
very much less saline than the normal habitat water (1.6% sea water). He
postulated that G. o. lutea required a slight concentration of salts, greater than the
tap water used, or that there were toxins present in that medium.
1 Present address : Department of Zoology, State College of Washington, Pullman,
Washington.
272
OSMOREGULATION IN SPHAEROMID ISOPODS 273
MATERIALS AND METHODS
Experimental animals
Gnorimosphaeroma oregonensis is widely distributed over the west coast of
North America from Alaska to central California (Menzies, 1954). It also occurs
in Hawaii (M. A. Miller, unpublished report). It may be collected intertidally in
bays, in estuarine conditions and occasionally populations of the species are found in
fresh-water creeks and ponds. Because of its ability to inhabit such a wide eco-
logical range, it was considered to be a suitable experimental type for the further
elucidation of osmoregulatory adaptations which enable a marine animal to live
in brackish and fresh water.
The following designations will be used for the three habitat groups of
Gnorimosphaeroma oregonensis. Animals taken from fresh water will be called
G. oregonensis (FW) ; those taken from estuarine populations will be called G.
oregonensis (EF = estuarine form), and those animals taken from intertidal bay
populations will be designated G. oregonensis (BF = bay form). G. oregonensis
(FW and EF) equal the subspecies G. o. lute a of Menzies. G. oregonensis (BF)
equals the subspecies G. o. oregonensis of Menzies.
Sphaeroma pentodon is known only from San Francisco Bay (Richardson,
1905), Tomales Bay (new locality), and Bolinas Lagoon (new locality), California.
It lives intertidally in burrows, which it constructs in mud, wooden logs and pilings,
and sandstone. The salinity of the habitats from which it was collected ranged
from about 11 per cent sea water to about 96 per cent sea water. 6\ pentodon
was included in the study because it is related to Gnorimosphaeroma oregonensis,
and its range overlaps that of the latter species in parts of its distribution.
Methods
Four principal types of studies were made : ( 1 ) Changes in the total osmotic
pressure of the body fluid after three, 24, and 48 hours exposure to the experimental
salinities were made in order to determine the relative degree and pattern of
osmoregulation exhibited by the animals. (2) The animals were weighed before
and after exposure to the experimental salinities for 24 hours in order to detect
possible changes in weight indicating water gain or loss. (3) Survival tests were
run to determine the length of time the experimental animals could live in the
experimental salinities. (4) Field checks were made by measuring changes in
the body fluid of all but Gnorimosphaeroma oregonensis (FW) during a portion of
a tidal cycle.
The laboratory experiments were conducted at 16 C., a temperature to which
all forms were accustomed. The animals were placed in 60 per cent sea water for
24 hours prior to the start of the experiments. The 60 per cent sea water permitted
a common starting salinity for all experimental series, facilitating comparisons.
E.vperimen tal salinities
The experimental salinities used in this study were 125, 100, 75, 50, and 25
per cent sea water, and fresh water (salts equivalent to 0.25% sea water by chloride
determination). The 100 per cent sea water (salinity = 34.44%o) was collected off
274 J. A. RIEGEL
the Marin County coast away from the influence of fresh-water streams. The 25,
50, and 75 per cent sea water solutions were made by diluting normal sea water
with distilled \vater. The 125 per cent sea water solution was prepared by boiling
normal sea water, taking care not to precipitate salts. The pH was checked before
and after boiling to ascertain that any loss of carbon dioxide was regained by
exposure to air. The fresh water was soft creek water collected at Pilarcitos
Creek, San Mateo County, California.
Salinity determinations
Salinity determinations on sea water concentrations greater than 25 per cent
sea water were made by a short method described by Welsh and Smith (1953).
The salinity of sea water diluted to less than 25 per cent normal sea water and fresh
water was determined by the standard silver nitrate titration method using the
Knudsen Tables (1901).
Melting point determinations
A method devised by Gross (1954) was used for determining the melting point
of body fluids. From repeated runs on standard samples, it was found that the
concentration of the body fluids could be obtained within an error of about two
per cent sea water (0.04 C).
Body fluid samples (ca. 1-2 mm 3 .) were collected into prepared melting point
capillaries (ca. 1 mm. ID X 3 cm. length) which were previously marked with a
coded series of dots corresponding to the experimental salinities to which the
animals had been exposed. Collection of the body fluid was facilitated by the use
of a hand control. After collection, both ends of the capillary were sealed with
petroleum jelly and the sample quick-frozen on dry ice.
Survival tests
The ability of the experimental animals to survive for extended periods of time
in the experimental salinities was tested as follows : Seventy animals of each experi-
mental group were placed, ten each, in six jars containing the experimental salini-
ties, and one jar containing filtered habitat water. The jars were checked daily
for 21 days, and the number of survivors recorded.
Field tests
Changes in the body fluid concentration of Gnorinwsphaeroma oregonensis
(EF), G. oregonensis (BF), and Sphacronm pcntodon during a 7 1 /o-hour period
from low to high tide in the field were measured as follows : In the case of G.
oregonensis (EF), which remains immersed in water during low tide, five body
fluid samples and one sea water sample were taken at 114 -hour intervals. In the
case of G. oregonensis (BF) and 5". pcntodon, which remain out of the water
during low tide, five body fluid samples and five samples of water around the
pleopods were collected. The body fluid and pleopod water samples were frozen
on dry ice and returned to Davis for determination.
OSMOREGULATION IN SPHAEROMID ISOPODS
275
RESULTS
The term "gradient" will be used in the following pages to indicate the differ-
ence in concentration (expressed in percentage sea water) between the body fluid
and the medium.
Melting point determination of body fluid concentrations
The results of melting point determination of body fluid concentrations are
shown in Figure 1. In general, changes in the body fluid concentrations seemed
Exposure Time (hours)
Exposure Time (hours)
co
O
c
CD
C
O
O
O
rruo
Exposure Time (hours)
Exposure Time (hours)
FIGURE 1. Body fluid concentration changes with time in the experimental salinities.
The dotted line represents the body fluid concentration changes of animals kept in habitat
salinities (controls) indicated.
to be rapid the major changes occurred within the first three hours of exposure
to the experimental salinities.
After 48 hours' exposure, the fresh-water and estuarine forms of Gnorimosphae-
roma oregoncnsis maintained their body fluids hypertonic to the medium in 50 per
276 J. A. RIEGEL
cent sea water and less, and hypotonic in 75 per cent sea water and above. How-
ever, in 75 per cent sea water after 24 hours' exposure, the body fluid concentration
values of G. oregonensis (FW) were quite variable, ranging between hypotonicity
and hypertonicity. Possibly that salinity is close to the medium concentration
where the "switch" from hyper- to hypo-osmotic regulation occurs. G. oregonensis
(BF) maintained its body fluid hypotonic to the medium in 75 per cent sea water
and above, and hypertonic to 50 and 25 per cent sea water. Apparently, there was
no maintenance of the body fluid concentration in fresh water. In that medium,
the body fluid concentration steadily dropped, and after 48 hours, all of the animals
were dead.
Comparing the above results with those of Menzies (1954) above it can
be seen that in both studies, Gnorimosphaeroma oregonensis (BF) (= G. o.
oregonensis of Menzies) could not survive in fresh water. However, in Men-
zies' study, G. oregonensis (EF) (= G. o. lutca of Menzies) were not surviving
after three days in tap water, while in the present study, that form lived
for several days in fresh water. It is possible that the tap water used by Menzies
(unchlorinated well water) contained some unknown toxic substance or had an
imbalance of ions. Its ion analysis is as follows : HCOs, 0.241%c ; SC>4, 0.037%c ;
Cl, 0.029% ; Ca, 0.01 \% \ Mg, 0.020^-,; and Na. 0.078% .
After 48 hours, Sphaeroma pentodon maintained its body fluid hypotonic to
the medium in 100 and 125 per cent sea water and hypertonic in the lower salinities.
It is interesting to note that 6". pentodon and Gnorimosphaeroma oregonensis (BF)
have extremely wide viability limits in terms of the concentration and dilution of
their body fluids surviving within a concentration range (of their body fluids)
of over 70 per cent sea water !
Weight changes in the experimental media
No weight changes were detected in any of the experimental animal groups,
except Gnorinwspliacronia oregonensis (BF) in fresh water. In that salinity, the
majority of the animals were very close to death at the end of the 24-hour period,
and the weight changes were considered to be subnormal. Those animals that
were still active at the end of the 24 hours did not lose weight. It was possible
to weigh the animals within an average error of one per cent of their body weight.
Survival tests
The survival experiment was terminated after 21 days. At termination, the
estuarine and fresh-water forms of Gnorhnosphaeronia oregonensis were surviving
in all salinities. G. oregonensis (BF) was surviving in all salinities except fresh
water, where the LD ; - )0 value (average survival time) was less than two days.
Sphaeroma pentodon was surviving in all experimental salinities, except fresh water,
where the LD 50 value was 1 1 clays. No unusual mortality was noted among the
controls.
Field tests
The results of the field test of body fluid concentration changes during a tidal
cycle showed that no significant changes in concentration of the body fluid or water
OSMOREGULATION IN SPHAEROMID ISOPODS 277
surrounding the pleopods were detected in Gnorimosphaeroma oregonensis (BF)
or Sphaeroma pentodon. In G. orcgonensis (EF), however, changes were rather
characteristic. Starting at low tide, when the animals were exposed to fresh water,
the body fluid concentration was 50 per cent sea water. This concentration did
not change until over five hours later, when the salinity of the habitat had reached
42 per cent sea water, at which time the body fluid concentration was 58 per cent
sea water. Then, by the time of the extreme high tide, 1^4 hours later, the body
fluid concentration had changed again to 70 per cent sea water, while the medium
concentration had changed to 65 per cent sea water.
DISCUSSION
Comparative osmoregulatory abilities
Figure 2 shows the 48-hour body fluid concentrations of the experimental
animals in the experimental media. It was assumed that all major changes in body
fluid concentration had occurred by 48 hours. In hypotonic media, Sphaeroma
pentodon appears to be a strong regulator, at least in 50 and 75 per cent sea water.
There is no apparent reason for the animals to maintain such high body fluid con-
centration in those salinities when they can live, at least for several days, in fresh
water and 25 per cent sea water with (presumably) much lower body fluid con-
centrations. Gnorimosphaeroma oregonensis (BF) has only limited regulation
in all media and appears to be the greatest conformer of the group, maintaining a
relatively small gradient between its body fluid and the medium in all salinities.
G. oregonensis (EF) and G. orcgonensis (FW) are the most able regulators in
terms of the ability to maintain their body fluid concentrations relatively constant
in hypotonic media. The body fluid concentration differences between the two
forms seen in fresh water, 25 per cent sea water, and 50 per cent sea water, are
statistically significant (t 6.15, 3.87, and 12.3, respectively, with 11, 10, and 9
degrees of freedom). The ability of G. orcgonensis (FW) to maintain its body
fluid more concentrated in the hypotonic media perhaps represents the major
osmoregulatory difference between the two forms. The estuarine form is inter-
mediate between the bay and fresh-water forms in osmoregulatory ability.
Comparing the osmoregulatory abilities of the isopods in this study with those
of other crustaceans, a similarity can be seen to species inhabiting similar salinity
ranges. From the results of Lockwood and Croghan (1957), it appears that
Mesidotca entomon is similar in its osmotic regulation to Gnorimosphaeroma
oregonensis. The former species consists of two "races" which have adapted to
brackish- and fresh-water. As in G. oregonensis (FW), the fresh-water M.
entotnon is able to live in salinities up to normal sea water. However, unlike
G. oregonensis (EF), the brackish-water "race" of M. cntomon cannot live in fresh
water. The brackish-water M. cntomon is thus closer to G. oregonensis (BF) and
Sphaeroma pentodon in its osmoregulatory abilities. However, M. entomon does
not show the high degree of hypo-osmotic regulation seen in the isopods in the
present study. Beadle and Cragg (1940) reported a difference in the ability to
retain chloride between the brackish- and fresh-water forms of the amphipod,
Gammarus duebeni, when placed in distilled water. The fresh-water form retained
sufficient chloride to survive for several days in distilled water, whereas the brackish-
J. A. RIEGEL
GNORIMOSPHAEROMA OREGONENSIS (EF) I I
GNORIMOSPHAEROMA OREGONENSIS (FW) 2-- ---2
SPHAEROMA PENTODON 3--- 3
GNORIMOSPHAEROMA OREGONENSIS (BF) 4 4
50 75
Medium Concentration (%SW)
125
FIGURE 2. Relation of the body fluid concentration to the medium concentration of
animals exposed for 48 hours to the experimental salinities.
water form lost chloride and died rapidly in that medium. It appears that the
osmoregulatory abilities of the isopods in this study are intermediate between those
of one group of crustaceans which can hyper-regulate in dilute sea water, but become
isosmotic, or nearly so, in salinities approaching normal sea water {e.g., Carcinus
maenasj Schlieper, 1929; Hemigrapsus oregonensis and H. nudus, Jones, 1941) and
a second group of crustaceans, which hyper-regulate in dilute sea water and hypo-
regulate in salinities approaching normal sea water (e.g., Heloecius cordiformis,
Edmonds, 1935 ; Uca crenulata and Pachygrapsus crassipes, Jones, 1941 ; Palae-
monetes varians, Panikkar, 1941; and Palaemon serratus, Parry, 1954). All but
the last two members of the latter group are primarily semi-terrestrial, which has
led Prosser ct al. (1950a, 1955) to suggest that hypo-osmotic regulation may be
associated with the semi-terrestrial habit. The isopods in this study are able to
survive for extended periods out of water, but they cannot be classified as semi-
terrestrial.
OSMOREGULATION IN SPHAEROMID ISOPODS 279
The mechanism of osmoregulation
Although there is little direct evidence elucidating the actual mechanisms of
osmotic regulation of the body fluid of the experimental animals, it is possible to
make certain hypotheses concerning that phenomenon based on data obtained in
the present study and in studies (unpublished) which were made prior to the
present study.
a. Evidence for water movement
There were no detectable weight changes in the experiments conducted at
16 C., which indicates that there was no net gain or loss of water from the
experimental animals' bodies. It is probable that the maintenance of a zero net
water flux (that is, no imbalance of the water gain/loss ratio) is dependent upon
the ability of the animal to maintain its metabolic rate at a normal level. Duplicate
experiments done at 5 C. (see Riegel, 1958) resulted in weight gains by the
experimental animals in the dilute salinities and weight losses in the more con-
centrated salinities. These results may be interpreted as being due to an inter-
ference by the low temperature with the normal metabolism of the animals.
b. Evidence for salt movement
Since there were no weight changes in the experiments conducted at 16 C.,
it must be assumed that body fluid concentration changes were due to salt move-
ment. In dilute media (fresh water to 50 per cent sea water), the salt concentra-
tion of the body fluid was actively maintained against a gradient. In more con-
centrated media (75 to 125 per cent sea water), salts were prevented from entering
the body (or were eliminated as fast as they came in), since after the initial
concentration of the body fluids (generally by 24 hours) the body fluid was
maintained hypotonic to the medium. This mechanism could possibly involve, at
least in part, an arrest of the mechanism for active salt absorption.
Except for Gnorimosphaeroma orcgonensis (FW) there was a rapid loss of
salts (within three hours) in the more dilute salinities. Whether this loss was
due to an active elimination of salts by the animal, thus reducing the concentration
gradient between their body fluids and the medium, or a passive loss from the
body is not known. There is some evidence suggesting an active elimination of
salts in the more dilute salinities, shown especially by G. orcgonensis (EF) and
Sphaeroma pentodon after three hours' exposure to fresh water. In those two
forms, the body fluid concentrations dropped more rapidly at 16 C. than at 5 C.
(see Riegel, 1958).
Whatever the mechanism for the maintenance of the body fluid concentrations
in lower salinities, low temperatures interfere with the metabolism of the animals,
causing variations in osmoregulation not seen at the higher temperature. In all
cases, except Gnorimosphaeroma oregonensis (BF) in fresh water, the animals were
able to maintain their body fluid concentrations within viable limits after 48 hours'
exposure at 16 C. But at 5 C., G. oregonensis (BF) was dead after 24 hours'
exposure to fresh water and 48 hours' exposure to 25 per cent sea water, and
Sphaeroma pentodon died after 48 hours' exposure to fresh water. Further, the
body fluid concentration of G. oregonensis (FW) and G. oregonensis (EF) dropped
280 J. A. RIEGEL
to subnormal values in fresh water at the lower temperature, but remained within
normal limits at the higher temperature.
Wikgren (1953) studied the effect of low temperature on various poikilotherm-
ons animals (a crayfish, a lamprey, and a bony fish) and concluded that low
temperatures have their chief effect in interfering with the ion-absorbing mechanism
of the animals. In the lamprey, urine production was decreased by low tem-
perature, which may have resulted in a weight gain, although Wikgren did not
indicate that such was the case. David (1925) performed experiments on the
living kidney of the frog, which indicated that that organ's urine diluting and con-
centrating activity was not affected by temperature. However, Wikgren (1953)
recalculated David's data and stated that the diluting capacity of the frog's kidney
was reduced by low temperature. Thus, evidence may be inferred from the review
by Wikgren (1953) that low temperature adversely affects the ability of cold-
blooded animals (at least, cold-blooded vertebrates) to rid the body of water.
The changes in body fluid concentration seen in the present study at 16 C.
were undoubtedly due to salt movement. Since there were demonstrated water
losses and gains at 5 C., the question arose as to whether the body fluid concentra-
tion changes which occurred at that low temperature were due entirely to water
movement or were partly due to salt movement.
Because the usual procedures for determining body fluid volume were hardly
applicable to animals of such small size as used in this study, that component
was estimated in the following manner. Ten animals of each experimental group
were weighed, and all the body fluid removed from their bodies that could be
collected into capillaries of 1-mm. bore. The animals were then re-weighed.
Average collectable body fluid weights as a percentage of total body weight were
9.5, 9.7, 11.1, and 6. 8, respectively, for Gnorimosphaeroma oregonensis (FW),
G. oregonensis (EF), G. oregonensis (BF), and Sphaeroma pentodon. These
values established the minimum possible weight of the body fluid. Ten animals of
each experimental group were weighed and dried to constant weight in a calcium
chloride desiccator. The average values for total body water as a percentage of
the total body weight were 56.5, 55.6, 56.4, and 53.8, respectively, for G. oregonensis
(FW), G. oregonensis (EF), G. oregonensis (BF), and 5". pentodon. These
values established the maximum possible weight of the body fluid as a percentage of
the total body weight.
Table I compares the calculated and actual dilution and concentration of the
body fluids in fresh water and 125 per cent sea water [using a 40-milligram speci-
men of Gnorimosphaeroma oregonensis (EF) as an example] based on estimates
of the body fluid weight ranging from ten to 50 per cent of the total body weight.
A sample calculation follows : Referring to Table I, it can be seen that a 40-milligram
animal, with a body fluid concentration of 50 per cent sea water (column 5), when
placed in fresh water would gain 11.3 per cent of its body weight (column 3) after
24 hours. If the weight gain is due entirely to water entry into the body, the
incoming water would dilute the body fluids by a factor, X, given by the relation :
wt (= original body fluid weight) . . a ., . CA
X = - If the body fluid comprises 50 per
wt 2 4 (= 24-hour body fluid weight)
cent of the total body weight (column 1), its dilution by the gain of 4.5 milligrams
OSMOREGULATION IN SPHAEROMID ISOPODS
281
v^ j.
(
of water (column 4) would result in a body fluid concentration of X-SQ
20 \
^T-I 50 I, or 40.8 per cent sea water (column 6) .
When a 40-milligram animal whose initial body fluid concentration is 50 per
cent sea water is placed in 125 per cent sea water, if the body fluid comprises 50
per cent of the total weight, the body fluid would be concentrated by the factor
X I - _ 1. Thus the body fluid will be concentrated to 57.8 per cent sea water
\ 1 / . 5 /
(column 10).
TABLE I
Comparison of actual and calculated concentration and dilution of the body fluids* (BF) at 5 C.
based on several estimates of the body fluid weight (BF Wt.) as a percentage of total
body weight (B W) and assuming the concentration and dilution to be due
entirely to water movement
1
2
3
4
5
6
7
8
9
10
11
Est. BF
Wt.
(% BW)
Est. BF
Wt.
(mg.)
% BW
gain
FW
BF Wt.
after
24 hrs.
FW
Start.
BF cone.
(% SW)
Calc.
BF cone.
FW
Actual
BF cone.
24 hrs.
FW
% BW
loss
125%
SW
BF Wt.
24 hrs.
125%
SW
Calc.
BF cone.
24 hrs.
in 125%
Actual
BF cone.
24 hrs.
in 125%
SW
SW
50
20
11.3
24.5
50
40.8
42
6.7
17.3
57.8
112
40
16
11.3
20.5
50
39.0
42
6.7
13.3
60.2
112
30
12
11.3
16.5
50
36.4
42
6.7
9.3
64.5
112
20
8
11.3
12.5
50
32.0
42
6.7
5.3
75.4
112
18
7.2
11.3
11.7
50
30.8
42
6.7
4.5
80.0
112
16
6.4
11.3
10.9
50
29.4
42
6.7
3.7
86.5
112
14
5.6
11.3
10.1
50
27.7
42
6.7
2.9
96.6
112
13
5.2
11.3
9.7
50
26.8
42
6.7
2.5
104.0
112
12
4.8
11.3
9.3
50
25.8
42
6.7
2.1
114.3
112
11
4.4
11.3
8.9
50
24.7
42
6.7
1.7
129.4
112
10
4.0
11.3
8.5
50
23.5
42
6.7
1.3
153.8
112
* A 40-mg. specimen of Gnorimosphaeroma oregonensis (EF) was used as an example.
From Table I it can be seen that the calculated body fluid concentrations and
dilutions in 125 per cent sea water and fresh water, based on estimates of the body
fluid weight percentage, do not completely match the actual results. If the
estimated body fluid weight of 50 per cent total weight is correct, the calculated
dilution in fresh water is close to the actual value. However, the calculated con-
centration in 125 per cent sea water is much lower than the actual value. If the
estimated body fluid weight of 12 per cent total body weight is correct, the calcu-
lated body fluid concentration in 125 per cent sea water is close to the actual value,
but the calculated body fluid concentration in fresh water is much lower than the
actual value. Therefore, it is likely that the actual body fluid weight lies somewhere
between 10 and 50 per cent of the total body weight. If a reasonable estimate of
20 to 30 per cent is close to the actual value for the body fluid component of the
total body weight, it appears that the actual body fluid concentrations in fresh water
and 125 per cent sea water at 5 C. are not due entirely to water movement. That
is. it is probable that there is a retention or reabsorption of salts in fresh water and
an absorption of salts in 125 per cent sea water.
J. A. RIEGEL
These results are in general agreement with those of Hukuda (1932) who
compared the theoretical and actual change in weight with the observed change in
osmotic pressure of the blood in P or tunas puber when that marine animal was.
immersed in % normal sea water. Gross (1957) found in Emerita analoga that
a weight change of less than two per cent of the body weight resulted in a body
fluid concentration change equivalent to 25 per cent sea water. Based on the
assumption that osmotically active water comprised 40 per cent of the body weight,,
he calculated that the weight change, if due entirely to water movement, would
have changed the body fluid concentration by less than six per cent.
The estimate of 20 to 30 per cent as the haemolymph component of the body
weight in Gnorimosphaeroma oregonensis (EF) only partially agrees with similar
estimates of that value in other crustaceans. A body fluid value of 50 per cent
of body weight was assumed by Lockwood and Croghan (1957) for Mesidotea
cntomon. Similarly, a body fluid of % body weight was assumed for Palaemonetcs
antennarius by Parry (1957). Gross (1957) made actual calculations of the
"solute space" in Pachygrapsus crassipcs and Emerita analoga which were, re-
spectively, 56 and 40 per cent of body weight. However, solute space would be
expected to be greater than the body fluid volume and less than the total body water.
Approximate measurements of blood volume of various crustaceans have been
made using sodium thiocyanate. Nagel (1934) found a blood volume of 37 per
cent of body weight in Carcinus maenas. Krogh (1939) measured a blood volume
of 33 per cent of body weight in Eriocheir sinensis. Prosser and Weinstein (1950)
measured the body fluid volume of the crayfish. Orconectes virilis, obtaining values
of 25.6 per cent and 25.1 per cent, respectively, with sodium ferrocyanide and a dye,
T-1824. The isopods in the present study seemed to have large amount of
exoskeleton relative to soft tissue. This fact was further borne out by the relatively
low total water values, and in the writer's opinion, supports the estimate of 20 to
30 per cent of total body weight as the body fluid component.
To summarize, it is probable that the osmoregulatory abilities of the experimental
animals include a mechanism for active salt uptake and retention. In the experi-
ments conducted at 16 C., the body fluid concentrations and dilutions were not
accompanied by detectable weight losses or gains, sviggesting that the concentration
and dilution are due to salt movement. Since concentrations and dilutions of the
body fluids could not be explained purely on the basis of water movement (weight
losses or gains), in experiments conducted at 5 C., there is evidence that con-
centration changes, especially in the higher salinities (75 to 125 per cent sea water)
were also due to salt movement at the low temperature. There is some evidence
that the experimental animals actively maintain the normal water content of the
body fluid. Though body fluid concentrations were well-marked at 16 C., no
weight changes were detected. Rather than propose that no water enters or leaves
the bodies of the experimental animals upon exposure to the experimental salinities,
it might be more reasonable to assume that the normal body water component is
actively maintained by pumping water out as fast as it comes in in hypotonic media
and by active water uptake and/or salt elimination in hypertonic media. The fact
that weight changes were well-marked in experiments conducted at 5 C. and
non-existent in experiments conducted at 16 C. indicates that the mechanism for
active maintenance of the water balance of the body is depressed or inactivated by-
low temperature.
OSMOREGULATION IN SPHAEROMID ISOPODS 283
The writer wishes to express his gratitude to Professor Milton A. Miller of the
University of California, Davis, for his guidance during the writer's period of
graduate study. Appreciation is expressed to Dr. Ralph I. Smith, of the Uni-
versity of California, Berkeley, for suggestions and helpful criticism during the
balance of the research embodied in this paper. Sincere thanks go to Dr. A. H.
Smith, of the University of California, Davis, for technical aid and advice and
critical review of the manuscript, and to the Committee on Research of the Uni-
versity of California for a Graduate Student Research Grant (DG-6) which made
a greater part of this work possible. Finally, a special note of thanks to Professor
C. Ladd Prosser, of the University of Illinois, who contributed much to the form
of the paper presented here by his generous comments and criticism.
SUMMARY
1. Osmoregulatory requirements were analyzed and compared in Menzies' two
subspecies of Gnoriinosphacroma orcgoncnsis (G. o. orcgoncnsis and G. o. lutca)
and Sphaeroma pcntodon Richardson.
2. The mechanism of osmoregulation was studied by measuring changes in
the total osmotic concentration of the body fluid after three to 48 hours' exposure
to various experimental salinities ranging from fresh water to 125 per cent sea
water. Changes were also measured in the field during a partial tidal cycle. The
principal findings and conclusions are as follows :
a.) The body fluids of the experimental animals became either diluted or con-
centrated in the experimental salinities. Generally, in more dilute media
(50% sea water or less), the body fluids were maintained hypertonic to the
medium, while in more concentrated media (75 to 125% sea water), they were
usually maintained hypotonic to the medium.
b.) The lack of weight changes in experimental salinities in experiments conducted
at 16 C. indicates that dilution and concentration of the body fluid at normal
temperatures are caused primarily by salt movement.
c.) Pronounced weight changes that occurred in experiments conducted at 5 C.
suggest that the normal water component of the body fluid is actively main-
tained and that low temperature interferes with this active maintenance, which
normally permits excess water to leave the body in diluted media and to enter
in more concentrated salinities. However, the fact that the degree of concentra-
tion and dilution of the body fluids at the low temperature could not be explained
solely on the basis of water movement suggests concurrent salt gains or losses.
LITERATURE CITED
BATEMAN, J. B., 1933. Osmotic and ionic regulation in the shore crab, Carcinus maenas, with
notes on the blood concentration of Gammanis locusta and Ligia oceanica. J. E.rp.
Biol, 10 : 355-372.
BEADLE, L. C., AND J. B. CRAGG, 1940. Osmotic regulation in fresh-water animals. Nature,
146 : 588.
BOGUCKI, M., 1932. Recherches sur la regulation osmotique chez 1'isopod marin, Mesidotea
entomon (L.). Arch. Int. Physiol, 35: 197-213.
DAVID, E., 1925. Ueber die Harnbildung in der Froschniere. VI Mitteilung. Ueber den Ein-
fluss der Temperature auf die Funktion der iiberlebenden Froschniere. Pfliig. Arch.
gcs. Physiol., 208: 146-176.
284 j. A. RIEGEL
EDMONDS, E., 1935. The relations between the internal fluid of marine invertebrates and the
water of the environment, with special reference to Australian Crustacea. Proc. Linn.
Soc. N. S. W., 60: 233-247.
EKMAN, S., 1953. Zoogeography of the Sea. Sidgwick and Jackson, London. 417 pp.
GROSS, W. J., 1954. Osmotic responses in the sipunculid, Dendrostomum zostericolum. J.
Exp. Biol, 31 : 402-423.
GROSS, W. J., 1957. An analysis of response to osmotic stress in selected decapod Crustacea.
Biol. Bull, 112: 43-62.
HUKUDA, K., 1932. Change of weight of marine animals in diluted media. /. Exp. Biol., 9 :
61-68.
JONES, L. L., 1941. Osmotic regulation in several crabs of the Pacific Coast of North
America. /. Cell. Comp. Physiol., 18: 79-91.
KNUDSEN, S., 1901. Hydrographical Tables. Copenhagen: G. E. C. Gad; 63 pp.
LOCKWOOD, A. P. M., AND P. C. CROGHAN, 1957. The chloride regulation of the brackish and
freshwater races of Mesidotea cntomon (L.). /. Exp. Biol., 34: 253-258.
MENZIES, R. J., 1954. A review of the systematics and ecology. of the genus "Exosphaeroma,"'
with the description of a new genus, a new species, and a new subspecies (Crustacea;
Isopoda, Sphaeromidae). Amcr. Mus. Nov., 1683: 1-24.
NAGEL, H., 1934. Die Aufgaben der Exkretionsorgane und der Kiemen bei der Osmoregulation
von Carcinus maenas. Zcitschr. f. vcrgl. Physiol., 21 : 468491.
PANIKKAR, N. K., 1941. Osmoregulation in some palaemonid prawns. J. Mar. Biol. Assoc.,
25 : 317-359.
PARRY, G., 1953. Osmotic and ionic regulation in the isopod crustacean Ligea occanica. J.
Exp. Biol., 30 : 567-574.
PARRY, G., 1954. Ionic regulation in the palaemonid prawn Palaemon ( Leandcr) scrratus.
J. Exp. Biol., 31 : 601-613.
PARRY, G., 1957. Osmoregulation in some freshwater prawns. /. Exp. Biol., 34: 417^23.
PROSSER, C. L., AND S. J. F. WEINSTEIN, 1950. Comparison of blood volume in animals with
open and with closed circulatory systems. Physiol. Zool., 23: 113-124.
PROSSER, C. L., D. W. BISHOP, F. A. BROWN, JR., T. L. JAHN AND V. J. WULFI-, 1950. Com-
parative Animal Physiology. W. B. Saunders, Philadelphia; pp. 6-102.
PROSSER, C. L., J. W. GREEN AND T. J. CHOW, 1955. Ionic and osmotic concentrations in blood
and urine of Pachygrapsus crassipcs acclimated to different salinities. Biol. Bull.,
109: 99-107.
RICHARDSON, H. E., 1905. A monograph on the isopods of North America. Bull. U. S. Nat.
Mus., 54 : 1-727.
RIEGEL, J. A., 1958. Osmoregulation and its ecological significance in certain sphaeromid isopod
Crustacea. Ph.D. thesis, University of California, Berkeley.
SCHLIEPER, C., 1929. t)ber die Einwirkung niederer Salzkonzentrationen auf marine Organ-
ismen. Zeitschr. f. vergl. Physiol., 9: 478-514.
WELSH, J. H., AND R. I. SMITH, 1953. Laboratory Exercises in Invertebrate Physiology.
Burgess Publ. Co., Minneapolis ; 126 pp.
\YIKGKKX, B., 1953. Osmotic regulation in some aquatic animals with special reference to the
influence of temperature. Ada Zool. Fennica, 71 : 1-102.
M< >TILITY AND POWER DISSIPATION IN FLAGELLATED
CELLS, ESPECIALLY CHLAMYDOMONAS x
R. R. RONKIN
Department of Biological Sciences, University of Dclmvare, Newark, Dclaivarc
The energetics of cellular motion have evoked much interest over the past
few decades. Muscle, amoeboid cells, and ciliated or flagellated cells have all been
studied, but skeletal muscle has received the most attention. This is true partly
because the motion of muscle cells can be stopped and started at the will of the
experimenter. This fortunate property, absent in amoeboid and ciliated cells,
allows the muscle cell to be compared with itself during rest and exercise. Meta-
bolic poisons can be used to stop movement in non-muscular cells, but chemical
inhibition is seldom reversible or specific enough for experimental designs as elegant
as those possible in studies on muscle.
Recently, genetic mechanisms have been discovered for controlling the motility
of certain flagellated cells: the bacterium Salmonella typhimurium (Stocker, Zinder
and Lederberg, 1953) and the autotrophic green alga, Chlamydomonas (Lewin,
1952). Of the two organisms, Chlamydomonas has some advantages as an experi-
mental object, since it is nonpathogenic and has simple, well-defined nutrient re-
quirements. By using ultraviolet light, Lewin (1954) has produced several single-
locus mutant strains with abnormal flagellar characters, including some which look-
just like the wild-type strain but do not move their flagella. The paralysis must
be related to an abnormality either of flagellar structure or of some other part of
the cell. The failure of Mintz and Lewin (1954) to find serological differences
between the flagella of normal and paralyzed strains suggests that these flagella
may be structurally similar. If this is so, the loss of motility is probably related
to a metabolic change elsewhere in the cell. It is now possible by using these
algal strains to compare the metabolism of "normal" and "paralyzed" flagellated
cells which are presumably alike in other respects. For this comparison it is
necessary to assume that a large and definite proportion of the cells in the "normal"
culture is motile. An estimate of this proportion, the motility index, will be
developed primarily for use in later studies. Its use in this paper will be only
to justify the above assumption.
The energetic cost of flagellar motion will be estimated in two ways. One
estimate is based on microscopic study of the motile cells, the other on measure-
ments of respiration. The two estimates will be compared.
It is a pleasure to acknowledge the technical assistance of Karl M. Buretz,
L. \\ . Clem, Miss Mary C. Straughn, and Irwin D. Zimmerman ; the use of the
facilities of the Marine Biological Laboratory, Woods Hole, Massachusetts, in
1 Aided by a contract between the Office of Naval Research, Department of the Navy, and
the University of Delaware, NR 164-280. Technical Report 58-2.
285
286 R. R. RONKIN
1955 ; and the helpful comments of Drs. Paul Plesner and Erik Zeuthen, who
read the manuscript.
METHODS
The marine organisms used in this study, Amphidinium Klebsi, Carteria (?)
sp. and DunalicUa sp., were obtained from Dr. J. H. Ryther at the Woods Hole
Oceanographic Institute. The three strains of Chlamydomonas came from the
Department of Botany, Indiana University (I.U.) : they were C. Moewusii ( + )
(I.U. No. 97) herein called "CMW," C. Moewusii ( + ) (Lewin's paralyzed strain
No. M 1001; I.U. No. 697) herein called "CMP," and C. Reinhardi ( + ) (I.U.
No. 89, Sager and Granick, 1953) herein called "CRW." Marine organisms were
studied in filtered, autoclavecl Woods Hole sea water and kept on agar slants
made with sea water. Fresh-water Chlamydomonas was grown and studied in
a liquid medium suggested by Fuller " which contained
KNO 3 , 1 M 5.0 ml.
K 2 HPO 4 , 1 M 0.5 ml.
KH 2 PO 4 , 1 M 0.5 ml.
MgSO 4 , 1 M 2.0 ml.
Ca(NO 3 ) 2 , 1 M 0.25 ml.
'Trace element solution" 1.0 ml.
"Iron solution" 1.0 ml.
Iron-free water to make 1000 ml.
'Trace element solution" contained
H 3 BO 3 1.43 g.
MnSO 4 -H 2 O 1.05g.
ZnCl 2 0.05 g.
CuSO 4 -5H 2 O 0.04 g.
H 2 MoO 4 -H 2 O 0.01 g.
Distilled water to make 1000 ml.
"Iron solution" contained
Disoclium ethylenediaminetetra-acetate 0.5 g.
FeSO 4 -7H 2 O 5.0 g.
Distilled water to make 1000 ml.
The culture vessels were 125-ml. Erlenmeyer flasks containing 50 ml. of medium
and 2.5-liter, wide, flat-bottomed culture flasks (like A. H. Thomas No. 4372-F)
containing one liter of medium. Air containing 5% CO 2 was bubbled through
the larger cultures. The flasks were shaken mechanically to swirl the contents
gently. Four fluorescent lamps (type F40T12W or SW) were mounted under
a glass-bottomed water thermostat kept at 23 C., in which the larger flasks were
immersed to the level of the medium inside. The illumination 2.5 cm. above
the bottom of the flasks was about 500 foot-candles as estimated with a photographic
exposure meter. The large flasks were inoculated either with 100 ml. of a
previous one-liter culture, or with a 50-ml. culture, reared for the purpose in a
2 R. C. Fuller, personal communication (1955).
POWER DISSIPATION IN FLAGELLATES 287
small flask. The small flasks were illuminated from above and shaken gently
but were not otherwise aerated. One-liter cultures were ready for harvest (about
2 X 10 6 cells mlr 1 ) in two to four days, depending on the inoculum. The harvest
was usually concentrated by gentle centrifugation, and the cells were re-suspended
in fresh medium before use.
Motility of whole populations of cells was studied by comparing photomicro-
graphs of samples of cell suspensions. The film (Du Pont Microcopy) was ex-
posed for 8 seconds and developed for maximal contrast with elon-hydroquinone
contrast developer (Kodak formula No. D-ll). After being processed, the photo-
graphs were projected onto a screen for counting those cells which were stationary
long enough to form images. Images of moving cells failed to register because
of the long photographic exposure. The use of a haemacytometer 3 and a phase-
contrast microscope in photography made the counting easier.
This method leads easily to the formulation of a motility index, M. A practical
definition of M is
J
in which n\ - -- the number of cells counted in a defined area of the photograph of
a cell suspension, made with a time exposure of 8 seconds;
n>2 ---- the number of cells in the first photograph whose images fail to
appear in the second, otherwise similar, photograph taken one
minute later;
//- = : the number of cells appearing in a photograph of a different drop
of the same suspension, in which all the cells are immobilized (e.g. ,
with HCHO or I 2 vapor).
The second measure of motility used here is based on the speed of locomotion
of individual motile cells in a drop of a dilute suspension, placed on a slide and
covered with a coverglass, at room temperature (21 to 23 C.). The individuals
to be studied were selected at random by tracking every cell which crossed a line
bisecting the field, for as long as it remained in the field. The image of the cell
was projected on to a sheet of paper, using a camera lucida. The path of motion
was described by pencil marks indicating the position of the cell every two seconds.
A loudly ticking clock or mechanical sounder was found to be essential. The dis-
tance travelled by the cell per second was calculated from a summation of the
line segments connecting the pencil marks on the sheet, and from the time elapsed
between the placement of the first and the last marks. The distances travelled
per second by several cells in the same suspension were averaged to estimate the
average speed of locomotion for the population.
Oxygen consumption was measured at 23 C. by the Warburg method. Each
14-ml. reaction vessel was inclosed in a light-tight cloth bag and contained 2 ml.
of a suspension of cells which had been washed by gentle centrifugation (700 X G,
30 seconds) and re- suspended in fresh medium. The manometers were read every
10 minutes. The respiratory rate was found to decline slowly with time, but
3 A haemacytometer chamber for phase-contrast microscopy is manufactured by the Ameri-
can Optical Co.
288 R. R. RONKIN
not appreciably during the first 90 minutes ; the readings during this period were
fitted with a straight line by the method of least squares. Respiratory rates were
then expressed in /x,l. of O 2 (S.T.P.) per mg. total nitrogen per hour (Qo 2 (N)).
Total N was estimated by sulfuric-acid digestion of an aliquot, with three succes-
sive additions of H 2 O 2 , followed by direct Nesslerization and reading of the
samples in a Klett-Summerson photoelectric colorimeter (Miller and Miller, 1948).
RESULTS
Degrees of tnotility in a culture of C. Reinhardi
When samples of a culture of C. Reinhardi were observed with the microscope,
they were found to contain some stationary cells. Some of these became motile
from time to time ; at the same time swimming individuals settled down to become
members of the stationary group. In general the stationary group seemed to
remain constant in size ; therefore, in any series of observations the number of
originally stationary cells becoming active in any time interval may be expected
to bear a constant relation to the number of originally stationary cells remaining.
To test this supposition, a single drop of a culture was photographed repeatedly
over a period of several minutes. The photographs were studied, and numbers
of originally stationary cells remaining were plotted on a logarithmic scale against
time. In one experiment (Fig. 1) the points fell on a straight line for the first
I50i
o
u
(0
0.125
o
o>
JC
'jc
1 100
u
fc
k.
0>
.0
E
75
Nonmotility in Chlamydomonas
Of 1,915 cells in this group,
7.4% (141) were nonmotile at
time zero.
= 3 min.
5 10 15
Time in minutes
FIGURE 1. Degrees of motility in a culture of Chlamydomonas Reinhardi. For the first
5 minutes many of the originally stationary cells became motile as shown by the points fitted
with a straight line. During the first 30 seconds a more active group of cells dominated ; a
slower, possibly more heterogeneous group dominated after 5 minutes.
POWER DISSIPATION IN FLAGELLATES 289
five minutes ; this supports the hypothesis that a constant proportion of the re-
maining non-motile cells become active during each time interval. However, the
graph also revealed that the entire original population of stationary cells was
made up of three classes, according to their rates of decrease. The first two had
half-times of one and three minutes, respectively. Cells in the third class, pos-
sibly including dead individuals in the culture, failed to move in 16 minutes. For
this population the first photograph showed 141 (,) stationary cells; in the
second photo 23 (n 2 ) of these particular cells were missing. A photo of a killed
sample showed 1915 ( 3 ) cells. Thus, J\l = 0.94. In general, samples from other
cultures gave similar results, except that the "one-minute" class often could not
be found.
Locomotion of individual cells of several species
A different quantitative concept of cellular motion results from the detailed
observation of single motile cells selected at random from a culture. The path
of motion of a flagellated algal cell is a series of straight lines or arcs of large
radius. Cells may occasionally change direction abruptly or spin briefly in place
as if held by a mucous attachment. In addition, cells which are swimming for-
ward often revolve about an axis parallel to the direction of motion (Brown, 1945)
and may oscillate as they swim.
For studies of the velocity of motion, several kinds of elliptical or nearly spherical
flagellates were selected. Table I shows the observations and calculations derived
from them. The "average radius" is one fourth the sum of length and width.
The minimal power dissipation, P, per cell was calculated from Stokes's Law
relating to the force, /, needed to propel a sphere through a fluid :
/ : = 6-rrrrju,
and from the relation
P -- 10 17 /z<,
where
/ = force needed to overcome fluid resistance (dyne),
r = average radius of cell (cm.),
77 =: viscosity of fluid (poise = dyne sec. cm.~ 2 ),
u = average speed of locomotion (cm. sec." 1 ),
P = power (watt = 10 7 dyne cm. sec." 1 ).
Oxygen consumed by normal and by paralysed Chlamydomonas
A third aspect of flagellar motion concerns the intensity of metabolism of the
flagellated cell. In this study, the oxygen consumption of a population of normal
Chlamydomonas Moezvusii (CMW) was compared with that of the ultraviolet-
induced, "paralyzed" mutant (CMP). The mutant cells have flagella but fail
to use them ; these are held out rigidly almost perpendicular to the main axis of the
cell. Occasionally a flagellum showed a little motion at its tip, but this hardly
ever caused the whole cell to move.
The figures in Table II are based on 12 reaction vessels for CMW and 13 for
CMP. In preparation for each experiment the cells of the two strains were reared
290
R. R. RONKIN
TABLE I
Minimal power output of selected flagellated cells
Size
Species
Medium
Aver, velocity
10~ 2 cm. sec." 1
Min. power
output/cell,
10~ 16 watt
Aver, radius
Length
10~ 4 cm.
Width
^4 tnphidinium Klebsi
7.98 (11)
1.30
Sea water
0.739 (11)
7.5
Carteria (?) sp.
6.54 (9)
1.67
Sea water
1.25 (10)
17
Chlatnydomonas Moewnsii (CM\Y)
5.54 (11)
1.48
Fresh water
1.28 (16)
15
Dunaliella sp.
4.40 (9)
1.35
Sea water
2.26 (9)
38
Chlani. Reinhardi (CRW)
3.26 (100)
1.08
Fresh water
0.828 (100)
3.9
1
Numbers of individuals studied are in parentheses. Viscosities (corrected for density) used
in calculations were: sea water, 0.965 cp (estimated from Miyake and Koizumi, 1948) ; fresh water,
0.931 cp.
in one-liter cultures under identical conditions. In each experiment the oxygen
consumption of the paralyzed cells was less than that of the normal cells when
expressed in terms of total cellular nitrogen.
DISCUSSION
The quantitative description of cellular motility will be discussed before con-
sidering the energy required for flagellar motion. This study presents two quanti-
tative methods of studying locomotion in populations of flagellated cells.
The motility index (photographic method) can be used for distinguishing the
behavior of cell populations exposed to varying experimental treatments. It may
prove helpful in pharmacologic and toxicologic studies on suspensions of algae,
protozoa, bacteria, or sperm cells; these forms may offer the experimenter ad-
vantages over larger and more expensive animal subjects. Compared with other
proposed estimates of the proportion of non-motile cells in a microscopic field
(Emmens, 1947; White, 1954) it would appear to avoid certain subjective errors
in sampling and counting, and to minimize the error due to the inclusion of cells
TABLE II
Oxygen consumption (Qo (N)) of normal and paralyzed Chlamydomonas Moewusii
(paired comparison)
Experiment no.
CMW
CMP
Difference
(CMW-CMP)
6-27-57
1.24
1.14
0.10
7- 1-57
1.36
1.24
0.12
7-22-57
1.31
0.79
0.52
8- 3-57
1.19
1.14
0.05
8- 5-57
2.33
2.06
0.27
8- 7-57
2.00
1.72
0.28
Mean difference and its standard error (n = 6)
0.223 0.0706
POWER DISSIPATION IN FLAGELLATES 291
which may stop for momentary "rest" periods. It cannot distinguish degrees of
impairment of locomotion.
The average speed of locomotion appears to be valuable for distinguishing
populations of cells which show normal speeds of locomotion from populations with
impaired locomotion. It takes no notice of non-motile cells, and thus becomes
most useful in estimating the degree of motility in cultures where the motility
index is high. It is similar in principle to one devised by Baker, Cragle, Salis-
bury and Van Demark (1957) who measured the time required for 100 free-
swimming sperm cells to pass through a segment of a plane. Their method, which
seems admirably suited to cells displaying the sperm type of locomotion, has the
advantage of presenting the result of an experiment immediately without waiting
for photographic processing. The decision to use a given method will rest partly
upon the extent to which its assumptions are fulfilled by the swimming habits
of the organism. The method described here is of special value, since from it can
be derived an estimate of the external work done by the motile cells in the
population.
The estimates of power dissipation in Table I are certainly low, because the
premises on which they are based all tend to reduce the estimates. It is supposed,
for example, that the cell's internal energetic conversions are 100% efficient. The
other assumptions, each known to be false to some extent, are : that there are no
degrees of motion other than uniform in a straight line (contradicted by Brown,
1945, and others), that the cell is a sphere (contradicted in Table III), and that
TABLE III
Estimates of size of Chlamydomonas
Strain
CRW
CMW
CMP
Length, M
Width, M
6.49 0.14
5.24 0.17
7.64 0.17
5.62 0.14
7.41 0.14
4.92 0.10
The "" s ign is inserted between the mean and its standard error. Fifty cells of a single
culture of each strain were measured.
the frictional drag of the flagellum, apparently of major importance in the locomo-
tion of sea urchin sperm (Gray and Hancock, 1955), is negligible in Chlamydo-
monas. Excepting C. Reinhardi, the smaller flagellates travelled faster and dis-
played a higher power output than the larger cells. Whether this difference is
related to a greater metabolic rate of the smaller cells has not been determined.
When normal and paralyzed C. Moe^vusi^ were reared and studied under the
same conditions in several successive experiments, the paralyzed cells (CMP)
always consumed less oxygen than did the wild-type, motile cells (CMW). The
average difference in Qo 2 (N) was about 14% of that of the normal cells, and
was found to be statistically significant (Mest, n -- 6) at the 5% level, but not
at the 2% level. It must be assumed that the proportion of dead cells in the
CMP culture is no greater than in the CMW culture. In interpreting this dif-
ference, certain other features of the two strains should be borne in mind.
Ocular micrometer measurements showed that although CMW and CMP are
of equal length, the paralyzed cells are, on the average, a little more slender than
292 R. R. RONKIN
the motile ones (Table III). Thus, a paralyzed cell's surface-to-volume ratio is
slightly greater than that of a normal cell. From the size of this difference alone
one would expect the Qo 2 (N) of the paralyzed strain to be a little greater than
that of the normal strain ; it appears in fact to be less. The single mutation which
resulted in paralysis of the flagella may have had other expressions, possibly in-
volving alterations in the efficiency of biochemical pathways of metabolism. In
summary, the physiologic differences between the two strains may be much greater
than appeared at first. In ignorance of the magnitudes of these possible factors,
it is tempting to suggest that the difference in oxygen consumption is actually
related to the state of motility of the cell, but a cautious attitude seems desirable.
As a partial test of this relationship, we may now compare the two available
estimates of the energy required for motility. One of these (Table I) states that
C. Moeu'usii dissipates at least 10~ 15 watt per cell in overcoming the frictional
losses in water. The other estimate is derived from the difference in Qo 2 (N)
between the normal and paralyzed strains, which is 0.22 p\. hr." 1 (mg. N)" 1 . If
we suppose that the exclusion of light from the Warburg vessel does not affect
motility (Lewin, 1953), the two figures are comparable; the latter figure can then
be transformed to watts per cell by making the following reasonable assumptions :
1. The consumption of 1 fA. of (X releases about 4.8 X 10~ 3 calorie or
5.58 X 10- 6 watt hour.
2. A CMW cell contains 2.65 X 10~ 9 mg. N (estimated from cell counts and
N determinations on a single culture at the time of harvest) .
3. The motility index in the CMW culture is high.
The observed difference in the rate of oxygen consumption thus corresponds
to a difference in power dissipation of 3.3 X 10~ 15 watt per cell. Rothschild's
(1953) reworking of Taylor's figures gives estimates close to these for the minimal
energy dissipated by bull sperm: for two kinds of assumptions, 3.74 X 10~ 14 watt
and 2.04 X 10~ 15 watt per cell. In our comparison, the efficiency of conversion of
chemical to mechanical energy is not taken into account. The closeness of the two
estimates derived in this paper suggests that the lower rate of oxygen consumption
of paralyzed cells may be correlated with their loss of motility.
SUMMARY
1. The paper describes a method for estimating the minimal power output of
individual, nearly spherical, flagellated cells. A comparison of 5 species of green
flagellates suggests no relationship between size and power dissipation (Table I).
2. A simple photographic method for estimating the fraction of motile organisms
in a culture is described.
3. Cultured populations of Chlamydomonas Reinhardi may contain two or more
distinct groups of cells with different degrees of motility (Fig. 1).
4. The motile, "wild-type" C. Moeu'iisii consumed 1.57 /*!. O 2 (S.T.P.) per
hour per mg. total N.
5. A paralyzed mutant strain of the same species consumed 14% less oxygen
than the wild type. The extra oxygen consumed by the motile strain is com-
mensurate with its estimated minimal power output.
POWER DISSIPATION IN FLAGELLATES 293
LITERATURE CITED
BAKER, F. N., R. G. CRAGLE, G. W. SALISBURY AND N. L. VAN DEMARK, 1957. Spermatozoan
velocities in vitro, a simple method of measurement. 1-ertil. Steril., 8: 149-155.
BROWN, H. P., 1945. On the structure and mechanics of the protozoan flagellum. Ohio J . Sci.,
45: 247-301.
EMMENS, C. W., 1947. The motility and viability of rabbit spermatozoa at different hydrogen-
ion concentrations. J. Pliysioi., 106: 474-481.
GRAY, J., AND G. J. HANCOCK, 1955. The propulsion of sea-urchin spermatozoa. /. E.\-p. Biol.,
32 : 802-814.
LEWIN, R. A., 1952. Ultraviolet-induced mutations in Chlamydomonas moczcnsii Gerloff. J.
Gen. MicrobioL, 6 : 233-248.
LE\VIN, R. A., 1953. Studies on the flagella of algae. II. Formation of flagella by Chlamydo-
monas in light and in darkness. Ann. Nezv York Acad. Sci., 56: 1091-1093.
LEWIN, R. A., 1954. Mutants of Chlainvdonwnas moeu'iisii with impaired motility. /. Gen.
MicrobioL. 11: 358-363.
MILLER, G. L., AND ELIZABETH E. MILLER, 1948. Determination of nitrogen in biological ma-
terials. Anal. Chcm., 20: 481-488.
MINTZ, RITA H., AND R. A. LEWIN, 1954. Studies on the flagella of algae. V. Serology of
paralyzed mutants of Chlamydomonas. Canadian J. MicrobioL, 1 : 65-67.
MIYAKE, Y., AND M. KOIZUMI, 1948. The measurement of the viscosity coefficient of sea
water. /. Mar. Res., 7 : 63-66.
ROTHSCHILD, LORD, 1953. The movements of spermatozoa. /;;: G. E. W. Wolstenholme (ed.),
Mammalian Germ Cells, Little, Brown and Company, Boston, pp. 122-130.
SAGER, RUTH, AND S. GRANICK, 1953. Nutritional studies with Chlamydomonas reinhardi.
Ann. New York Acad. Sci., 56: 831-838.
STOCKER, B. A. D., N. O. ZINDER AND J. LEDERBERG, 1953. Transduction of flagellar characters
in Salmonella. J. Gen. MicrobioL, 9: 410-433.
WHITE, 1. G., 1954. The effect of some seminal constituents and related substances on diluted
mammalian spermatozoa. Austral. J. Biol. Sci., 7: 379-390.
CONSEQUENCES OF UNILATERAL ULTRAVIOLET RADIATION
OF SEA URCHIN EGGS 1
RONALD C. RUSTAD 2
Department of Zoology, University of California, Berkeley 4, California
The suppression of the elevation of the fertilization membrane on the half of
a sea urchin egg which directly receives high doses of ultraviolet light has been
described by Reed (1943) and Spikes (1944). The experiments reported herein
are an examination of the consequences of unilateral U.V. irradiation of the sea
urchin egg in terms of changes in cell morphology with dose, the physical state of
the cytoplasm, the effects of time and temperature, and the effects on subsequent
cell division. Particular attention is directed toward observations on hyaline layer
formation, local gelation, and excentric formation of the mitotic figure.
MATERIALS AND METHODS
Gametes were obtained from the sea urchin Strongylocentrotus purf>itratus by
injection with 0.5 M KC1. The groups of eggs selected were more than 99%
fertilizable, were free from visible abnormalities, yielded symmetrical fertilization
membranes, and showed little distortion when the lifting of the fertilization mem-
brane began. The pattern of morphological changes at different doses was con-
firmed with suitable eggs obtained from a single female of the related species
Strongylocentrotus franciscanus, which has larger eggs with less yolk.
The ultraviolet source was an Electrotherapy Products Corp. low pressure
mercury vapor lamp, which produces approximately 95% of its U.V. energy in a
2537 A band. The intensity was measured with a Hanoviameter.
In some experiments the eggs were centrifuged in a Servall refrigerated angle-
head centrifuge, either in sea water or in a sucrose gradient formed by layering
sea water over 0.88 M sucrose.
Unless otherwise noted, all experiments were carried out in 1 cm. deep, filtered
sea water at 17.5 0.1 C. Artificial calcium-free sea water was prepared accord-
ing to the formula of Moore (1956).
Clarification of terminology
In order to describe concisely and accurately the changes associated with uni-
lateral irradiation of the strongly-absorbing egg certain special terms must be
defined. The directly-irradiated hemisphere is the surface of the egg which faces
1 Supported by grants from the American Cancer Society and the Office of Naval Research
awarded to Dr. Daniel Mazia.
2 This work was performed under the tenure of a Research Fellowship of the National
Cancer Institute, United States Public Health Service. Present address: Department of
Biological Sciences, Florida State University, Tallahassee, Florida.
294
CONSEQUENCES OF UNILATERAL U.V. 295
the U.V. lamp. The shaded hemisphere is the surface which does not face the
lamp, and, hence, is shaded by the cytoplasm. The shaded-irradiated axis is an
imaginary line drawn between the poles or centers of these two hemispheres. Uni-
lateral membranes are fertilization membranes which lift off the egg on the shaded
hemisphere only. All drawings and photographs except Figures 1 and 8 have
been mounted with the shaded pole facing the top of the page.
RESULTS
When eggs were irradiated with large doses of U.V. and then fertilized, the
height of the fertilization membrane and the hyaline layer on the directly-irradiated
hemisphere was reduced. Sufficiently large doses unilaterally inhibited the forma-
tion of these membranes entirely.
The dose required to produce a definable level of effect varied by as much as
a factor of three between the most sensitive and the most resistant groups of eggs.
Nevertheless, the ratio of doses necessary to produce two definable effects on the
majority of eggs in a population appeared to be constant even in the extreme
cases. The data presented represent the most frequently encountered dose relations.
Less than 1600 ergs/mm. 2 did not interfere with the normal membrane eleva-
tion. When the dose was increased the fertilization membranes did not elevate
to their normal height over the irradiated pole (Fig. 2). Doses of approxi-
mately 2800 ergs/mm. 2 resulted in the almost complete suppression of the fertiliza-
tion membrane over a small area, but the hyaline layer differentiated over the
entire surface. When the dose was increased to 4800 ergs/mm. 2 the fertilization
membrane covered only one hemisphere, while the hyaline layer appeared normal
(Fig. 3). With slightly higher doses a reduction in the thickness of the hyaline
layer was sometimes found (Fig. 4). With doses above 7200 ergs/mm. 2 the
hyaline layer could be distinguished only slightly beyond the cell equator (Fig. 5).
No further changes in the pattern of membrane elevation were noted at increased
doses up to the range of 40,000 to 50,000 ergs/mm. 2 At this dose level partial
cytolysis often occurred immediately on the directly-irradiated hemisphere, and
complete cytolysis usually followed after standing or at fertilization.
Identification of the inhibited surface
A simple experimental procedure was devised to demonstrate that the irradiated
surface was in fact the one that showed inhibition at fertilization. Stationary eggs
were irradiated from above with 7200 ergs/mm.- in a large petri dish on a micro-
scope stage and observed as sperm were carefully added. In four experiments
there was no detectable net rotation of any of the eggs in the field of a low power
objective. By careful focussing it was established that the fertilization membranes
first encircled the lower hemispheres which had been shaded by cytoplasm. As
the membranes raised further the eggs rolled over and came to rest on their sides
revealing total suppression of membrane elevation on the irradiated hemispheres.
Rclationsliif> to time and temperature
Eggs were fertilized at regular intervals from a few seconds after irradiation
to as much as twelve hours later without any visible changes in the unilateral
296
RONALD C. RUSTAD
2
3
5
6
FIGURES 1 to 6
Photomicrographs of sea urchin eggs showing different degrees of suppression of the
fertilization reaction when irradiated with increasing doses of U.V. from the direction of the
bottom of the page.
FIGURE 1. Control.
FIGURE 2. Reduction of the height of the fertilization membrane.
FIGURE 3. Complete suppression of the elevation of the fertilization membrane on the
directly-irradiated hemisphere.
FIGURE 4. Reduction of the height of the hyaline layer.
FIGURE 5. Complete suppression of both fertilization membrane elevation and the hyaline
layer differentiation on one hemisphere.
FIGURE 6. Later swelling of the initially flattened shaded hemisphere of an egg similar
to Figure 4.
fertilization reaction. In five separate experiments there was no increase or de-
crease in the inhibited area with time. In general, the irradiated eggs cytolyzed
sooner than the controls, but in most experiments both the irradiated and the
control eggs became unfertilizable at approximately the same time, even with
very high concentrations of sperm.
Attempts were made to re-fertilize eggs which had been fertilized but did not
completely differentiate the hyaline layer. The simple addition of viable sperm
did not cause re-fertilization at any time up to 28 hours after irradiation. The
sperm were observed to accumulate in the egg jelly which adhered to the irradiated
hemisphere in each of these experiments.
Irradiating eggs from the same females at 18 and 8 C. with various doses
revealed that there were no differences in sensitivity at the two temperatures.
CONSEQUENCES OF, UNILATERAL. U-V... 297
C /ian(/cs in morphology and physical state of the cytoplasm
The progressive dose-dependent suppression on the elevation of the fertilization
membrane and the differentiation of the hyaline layer have already been described.
Sometimes at high doses the fertilization membrane was elevated to an abnormal
height above the shaded pole and the cytoplasm under it was considerably flat-
tened (Figs. 4 and 5). A large amount of participate matter, possibly cortical
granule materials, was found in the perivitelline space under these conditions.
The amount of this material was apparently greater at all doses than in the controls.
After flattening, the cytoplasm under the unilateral membranes sometimes
swelled and reduced the thickness of the perivitelline space (Fig. 6). In some
cases the thickness was less than the controls. Under these conditions there was
a constriction around the cell at the equator where the fertilization membrane met
the hyaline layer (Fig. 6).
There were no cases of membrane elevation activation by U.V. at any dose
in any of the experiments.
Unfertilized irradiated eggs were centrifuged for ten minutes at approximately
12,000 g in a sucrose gradient. In two such experiments 90% of the eggs stratified
with the center of the light pole (identified by an oil cap over a clear region of
cytoplasm) in the center of the shaded hemisphere (identified by subsequent fertili-
zation) (Fig. 7). Almost all of the remaining 10% had an asymmetry of less
than 30 between the light-heavy and the shaded-irradiated axis. A very small
fraction of a per cent were 30 to 90 off center, and no cases were found in which
the shaded pole appeared to have a greater density than the irradiated one.
When unfertilized eggs were placed in 70% sea water after irradiation they
swelled on one pole only, giving the eggs a somewhat pear-shaped appearance.
Standing in this hypotonic medium for several hours did not result in any further
changes in shape. The treated eggs were fertilized to establish that the shaded
pole was the swollen one. Hence, while both unirradiated eggs and the shaded
side of an irradiated one swell in 70% sea water, the directly irradiated surface
does not.
Irradiated eggs placed in 70 % sea water had a dense darkened area near the
irradiated pole, a somewhat less dense region at the shaded pole, and a lighter
less granular region near the equator. Occasionally this pattern appeared in eggs
kept in normal sea water and seemed to be accompanied by a slight enlargement
of the shaded hemisphere. With doses of the order of 40,000 ergs/mm. 2 a large
blister of non-granular material formed on the irradiated pole when the eggs were
placed in the hypotonic sea water. With slightly higher doses these blisters ap-
peared spontaneously.
Irradiated eggs were centrifuged at approximately 12,000 g in sufficiently
dense suspensions that some of the eggs were confined in a random orientation
with respect to their light-heavy axes. Some of these cells showed stratification
only on the shaded side, which was identified by subsequent fertilization. When
the direction of centrifugation was perpendicular to the shaded-irradiated axis there
was a narrow region near the irradiated surface with a very high gel strength
that resisted stratification when the central cytoplasm and the shaded side stratified
(Fig. 8).
FIGURES 7-12
298
CONSEQUENCES OF UNILATERAL U.V. 299
Eggs irradiated after equilibration in calcium-free artificial sea water and
fertilized immediately when returned to normal sea water showed the same degree
of inhibition as eggs irradiated in normal sea water.
J\Iitotic abnormalities
Cells irradiated at doses that inhibited the full differentiation of the hyaline
layer seldom divided. At lower doses some or all of the eggs would divide several
times and sometimes form apparently normal swimming blastulae. Gastrulation
was usually abnormal. In some experiments even the first division was abnormal.
A systematic group of abnormalities occurred as a result of the mitotic figure
failing to migrate to the center of the egg. The nucleus of the unfertilized egg
is excentrically located, and in normal division the mitotic apparatus is positioned
approximately in the center of the cell. The position of the furrow is determined
by the plane formerly occupied by the metaphase plate both in normal cells and
these abnormal cells.
When the mitotic figure located in either hemisphere was oriented perpendicular
to the shaded-irradiated axis, the furrow formed along that axis and the egg
cleaved into two equal-sized blastomeres (Figs. 9 to 12).
When the mitotic figure was oriented parallel to the shaded-irradiated axis in
either hemisphere, the furrow formed perpendicular to the axis and the sizes of
the resulting blastomeres were quite different (Figs. 13 to 16).
Variable results were observed when the mitotic figure was formed with other
orientations with respect to the shaded-irradiated axis (Figs. 17 and 18).
Excentric spindles were also found in eggs which were irradiated during the
early part of the first mitotic cycle with comparatively low doses of U.V. The
blastomeres in such experiments were always equal in size.
Whenever the mitotic apparatus was excentric the furrow formed first on the
surface that was closest to the spindle. At later stages of cytokinesis the furrow
on the near side would always be deeper than the furrow on the far side. In some
cases the furrow actually passed through the spindle before the first indentation
occurred on the far side of the cell.
DISCUSSION
The progressive unilateral inhibition of the fertilization reaction has been de-
scribed in terms of the U.V. doses required to produce different degrees of inhibi-
FIGURES 7 to 18
Schematic drawings of eggs irradiated from the direction of the bottom of the page
(except Fig. 8) ; refer to text for explanation.
FIGURE 7. Egg centrifuged in a sucrose gradient and then fertilized. Stratification direc-
tion indicates that the irradiated pole was heavier than the shaded pole.
FIGURE 8. Egg irradiated from the left side of the page and centrifuged while confined
with the shaded-irradiated axis perpendicular to the direction of centrifugation. A narrow
region near the surface of the irradiated hemisphere resisted stratification indicating a local
increase in gel strength.
FIGURES 9 to 12. Division patterns of cells with spindles oriented perpendicular to the
shaded-irradiated axis.
13
15
16
17
18
FIGURES 13-18
300
CONSEQUENCES OF UNILATERAL U.V. 301
tion of both the elevation of the fertilization membrane and the differentiation of
the hyaline layer. Hyaline layer differentiation is less sensitive to U.V. than
fertilization membrane elevation ; however, it may be suppressed completely on
the directly-irradiated hemisphere with high doses. The inhibition of the elevation
of the fertilization membrane has been described previously by Reed (1943) and
Spikes (1944).
By means of local dye experiments Spikes (1944) was able to demonstrate that
the directly-irradiated hemisphere is the site of inhibition. His findings have been
reconfirmed with the direct observations of undisturbed eggs reported herein.
Giese (1947) has shown that the sea urchin egg strongly absorbs or scatters 2537 A
U.V. light. Harvey and Lavin's ( 1944) U.V. photomicrographs also indicate that
a considerable amount of the light is absorbed in sea urchin eggs of another genus.
Since the shaded pole is not inhibited even at very high doses, it may be concluded
that the transmission of the cytoplasm is too low to allow the necessary energy to
reach the sensitive sites on the shaded side of the egg.
The demonstration that there was no spreading of the damaged area with time
indicates that the U.V. action is relatively direct, and, in particular, that there is
no secondary effect of "diffusible poisons." There was no recovery with time ;
hence, the damage seems to be irreversible by any metabolic mechanism. Since
the degree of injury did not decrease with time, and a diffusible toxic product would
be expected to decrease in local concentration, this observation provides additional
evidence against the action of such substances.
The sensitivity was the same at 8 and 18 C.
Direct photochemical action has been shown repeatedly to have a O 10 of approxi-
mately 1. Therefore, insofar as visually equivalent degrees of damage may be
used as a measure of the rate of damage, it appears that the injury results from
direct photochemical action. The time and temperature relations together offer
evidence that the effect is localized and that there is a lack of intermediary toxic
products.
The observation that the inhibited surface could not be re-fertilized by the
addition of fresh sperm could be interpreted in two ways : either the U.V. damage
rendered it unfertilizable or some of the steps of the fertilization reaction occurred
on this side when the egg was initially fertilized. If some substances necessary for
the initial steps of the reaction had been used up the sperm could not initiate a
response later. A pronounced green Becke line appears in the out-of-focus image
of the damaged hemisphere of heavily irradiated eggs after fertilization. This
change is probably similar to the dark-field changes which have been observed prior
to membrane elevation (Runnstrom, 1928; Rothschild and Swann, 1949) and indi-
cates that some step in the fertilization reaction has taken place.
Two types of evidence for local gelation in the irradiated hemispheres were
obtained : first, that swelling in 70% sea water was confined to the shaded pole,
and, second, that a narrow band near the irradiated surface resisted stratification
with centrifugation when the rest of the cytoplasm stratified. Reed (1948) found
FIGURES 13 to 16. Division patterns of cells with spindles oriented parallel to the shaded-
irradiated axis.
FIGURES 17 and 18. An example of one of several division patterns obtained when the
spindles have intermediate angular orientations.
302 RONALD C. RUSTAD
that moderate doses of unilateral U.V. did not change the permeability of the
egg to a large variety of solutions. Although no measurements were made, he
discussed possible differences at higher doses and proposed that some sort of gela-
tion occurred on the basis that vacuoles were formed in the irradiated pole. Spikes
(1944) also proposed that gelation occurred, because he found that while normal
eggs only swelled in 50% sea water, irradiated ones lysed on the irradiated side.
Spikes' data might also be interpreted as indicating either that the surface of
the shaded hemisphere was weakened or that the osmotically inert volume had been
increased permitting greater than normal swelling followed by lysis. The obser-
vation of the large amounts of granular material released into the perivitelline
space at the shaded pole suggests the weakening either of the cell membrane or
of some other surface structure. The flattening of the shaded pole at fertilization
at high doses seems to fit either hypothesis, although an enhancement of the vigor
of the fertilization reaction would yield the same pattern. It would not be un-
reasonable to suppose that U.V. damage could affect both the surface strength
and the osmotically inert volume, perhaps by a common mechanism.
The observation that eggs irradiated in calcium-free sea water showed the
same degree of damage as eggs in normal sea water cannot be interpreted directly
in terms of the often demonstrated role of calcium in gelation (Heilbrunn, 1952).
First, the eggs had to be fertilized in normal sea water since fertilization will not
occur in the absence of external calcium ion; hence, new calcium may have been
introduced before the damage was measured. Second, since Heilbrunn and his
co-workers have shown that U.V. causes solation in low doses and gelation in high
doses, it is quite possible that the calcium ion left in the egg after treatment with
calcium-free sea water shifts between the less and more heavily damaged portions
of the cytoplasm. The second possibility is quite attractive, since it would pro-
vide a mechanism for an increase in osmotically inert volume in the less damaged
hemisphere and introduces the possibility that the surface on the shaded side might
be weakened by small amounts of U.V. penetrating the cytoplasm to cause solation.
Spikes (1944) reported that in Lytechinus pie t us furrow formation almost
always occurs along the shaded-irradiated axis. Clearly this is not the case in
the Strongylocentrotus purpuratus used in these experiments; cleavage may take
place with any orientation. Successful cleavage w r ith the furrow passing through
the irradiated portion of the egg indicates either that the furrowing strength exceeds
the resistance of the radiation-induced gel or that the gel is solated in the course
of cytokinesis.
Cleavage into equal or unequal sized blastomeres is determined by the orienta-
tion of the spindle with respect to the shaded-irradiated axis. It occurs because
the mitotic figure remains centered around the original location of the nucleus.
The nucleus is excentrically located in unfertilized eggs of this species. When
the axis of the mitotic figure is perpendicular to the shaded-irradiated axis the
blastomeres are equal in size. Where the axes are parallel the blastomeres are
unequally sized. In intermediate angular orientations the results are variable.
While both parallel and perpendicular orientations can occur when the mitotic
figure is located in either the shaded or irradiated hemisphere, mitotic figures near
the equator seem to be restricted to intermediate angular orientations. It is clear
that the migration of the nucleus to its normal central position is inhibited. An
CONSEQUENCES OF UNILATERAL U.V. 303
increase in cytoplasmic viscosity would provide a plausible explanation for this
failure of migration.
&'
It is a great pleasure to acknowledge my gratitude to Professor Daniel Muzia
for his helpful advice and encouragement during the course of this work. I also
wish to thank Professors J. E. Gullherg, L. V. Heilbrunn and C. B. Metz for
their valuable comments about the results, and Mr. Fred Burnet for his skillful
preparation of the drawings.
SUMMARY
1. The progressive dose-dependent inhibition of the fertilization reaction on
the directly-irradiated hemisphere of the unilaterally U.V. -irradiated sea urchin egg
has been described in terms of changes in the ability to elevate the fertilization
membrane and to differentiate the hyaline layer.
2. Membrane elevation was not activated by 2537 A U.V. light.
3. No spreading of the extent of injury or recovery was found with time ; and
no temperature sensitivity differences were found ; hence, the injury appeared to
be the result of direct photochemical action.
4. The irradiated hemisphere of the fertilized egg maintained its jelly for con-
siderable periods of time.
5. Evidence was obtained showing partial gelation of the irradiated hemisphere
and suggesting that the gelled cytoplasm had a higher density than the rest of
the egg. Irradiation in calcium-free sea water did not change the degree of dam-
age observed after fertilization in normal sea water.
6. The behavior of the cytoplasm of the shaded hemisphere at fertilization
suggested either that the surface structure was damaged or that the osmotically
inert volume had been increased.
7. Unilateral irradiation caused excentric spindle formation which resulted in
equal sized blastomeres if the spindle axis was perpendicular to the axis of irradia-
tion and unequal sized blastomeres if the axes were parallel.
LITERATURE CITED
GIESE, A. C., 1947. Radiations and cell division. Quart. Rcr. Biol., 22 : 253-282.
HARVEY, E. B., AND G. I. LAVIX, 1944. The chromatin in the living Arbacia punctiilata egg
and the cytoplasm of the centrifuged egg as photographed by ultraviolet light. Biol.
Bull, 86:" 163-168.
HEILBRUNN, L. V., 1952. An Outline of General Physiology. Third ed. W. B. Saunders Co.,
Philadelphia.
MOORE, A. R., 1956. In: Formulae and Methods IV, Marine Biological Laboratory, Woods
Hole, Massachusetts.
REED, E. A., 1943. Unilateral membrane formation in the sea urchin egg treated with ultra-
violet light. Anat. Rcc., 87 : 467.
REED, E. A., 1948. Ultraviolet light and permeability of sea urchin eggs. /. Cell. Comp.
Physiol., 31 : 261-280.
ROTHSCHILD, LORD, AND M. M. SWANN, 1949. The fertilization reaction in the sea urchin egg.
A propagated response to sperm attachment. /. E.vp. Biol., 26: 164-176.
RUNNSTROM, J., 1928. Die Veranderungen der Plasmakolloide bei der Entwicklungserregung
des Seeigeleies. Protoplasma, 4: 388-514.
SPIKES, J. D., 1944. Membrane formation and cleavage in unilaterally irradiated sea urchin
eggs. /. E.rp. Zoo!.. 95: 89-103.
THE ROLE OF THE INITIATOR CELL IN SLIME MOLD
AGGREGATION x
MAURICE SUSSMAN - AND HERBERT L. ENNIS 3
Department of Biological Sciences, Northwestern ['nii'crsity, Evanston, Illinois
Previous studies of slime mold aggregation (Sussman and Noel, 1952) had
shown that the number of aggregative centers is linearly related to the number
of cells present and, further, that centers are distributed in accord with the
Poisson series among small, replicate population samples. These and supporting
data were considered to dictate the existence of specially endowed individuals
termed "initiator cells," each of which could evoke the aggregative response by
its neighbors, the "responder cells." Recently a distinctive cell type was detected
by morphological criteria in Dictyosteliuni discoideiun Raper and evidence was
presented in support of the contention that cells of this type are in fact the
initiators of aggregation (Ennis and Sussman, 1958a, 1958b ; Sussman, 1958). The
distinctive individuals, termed I -cells, are much larger than the remainder of the
population (R-cells), the difference amounting to 2-3-fold in diameter, 3-10-fold
in area. They are much flatter and more heavily granulated and vacuolated. In
contrast to the R-cells which move sluggishly, the I-cells are highly motile and
extensive lobopodia and filopodia are seen to protrude constantly and explosively.
Figure 1 presents histograms to illustrate the size differences. Two modes are
apparent without overlap.
The evidence (Ennis and Sussman, 1958b) supporting the candidacy of the
I-cells for the appellation of "initiator" is summarized below :
a) The ratio of I-cells to R-cells remained .constant during the pre-aggregative
period at 1 : 1940. This figure agrees closely with the ratio of centers
formed to cells present at optimal density (1:2200).
b) A high correlation was encountered between the positions of I-cells and
of subsequently formed aggregative centers.
c) The appearance of centers among small, replicate population samples was
correlated (perfectly in one experimental series and almost perfectly in
another) with the previously determined incidence of I-cells. That is,
centers appeared in samples containing I-cells ; none appeared in samples
without I-cells.
d) Removal of I-cells at an early enough time prevented subsequent center
formation.
1 This work was supported by grants from the National Cancer Institute and the Office
of Naval Research.
- Present address : Department of Biology, Brandeis University, Waltham, Massachusetts.
3 Postdoctoral Fellow, N.I.H. Present address : Department of Bacteriology and Immunol-
ogy, Harvard University School of Medicine, Boston, Massachusetts.
304
INITIATOR CELL
305
60
40
20
LARGEST SMALLEST
RANDOM I -CELL
MEAN=64.15
r =18.8
CV =29 21
I
MEAN=299
re- incubation, R-cells were individually micro-manipulated
to test areas. See text for details
Pre-incubation
period in hours
Experimental
Background
Total
No. with
aggregates
%
Total
No. with
aggregates
%
1
53
7
13.2
79
11
13.9
4-6
65
14
21.6
250
29
11.6
10-12
71
26
36.6
70
9
12.8
Washed myxamoebae were dispensed on washed agar at a density of 150-200
cells/mm. 2 . After 1, 4-6, and 10-12 hours, R-cells were picked up individually with
a glass loop mounted in a deFonbrune micromanipulator and moved to test areas.
The test areas had been prepared by dispensing washed myxamoebae on washed
agar at a density of 250 cells/mm.-, one hour prior to use. After the excess fluid
had been absorbed, an area, 1 mm. 2 , was delineated in the middle of each drop as
described in the previous section. The outlying cells were brushed away leaving
test squares containing 250 myxamoebae at a density of 250. The center: cell
ratio being 1:2200, one would expect 11.3% of the squares to have aggregated
spontaneously. The background controls shown in Tables II and III showed an
incidence of 72 squares with aggregates out of a total of 578, or 12.4%. The
extent to which addition of R-cells, pre-incubated for periods between 1 and 12
hours, affected the background incidence is shown in Table II. R-cells pre-
TABLE III
Initiative capacity of R-cells tested upon their developmental juniors
A. Samples with I-cells
Samples without I-cells
No.
No. with
aggregates
%
No.
No. with
aggregates
%
21
18
86
13
R-cells from samples with
I-cells
R-cells from samples without
I-cells
Background
B . Experi-
ment
Total
No. with
aggregates
%
Total
No. with
aggregates
%
Total
No. with
aggregates
%
A
27
5
18.5
27
8
29.6
54
8
14.8
B
27
8
29.6
27
3
11.1
53
8
15.1
C
36
8
22.2
30
7
23.3
72
7
9.7
Total
90
21
23.4
84
18
21.4
179
23
12.8
Twenty-one which certainly con-
The percentages of samples that
A. Samples of 500 cells were dispensed on washed agar.
tained I-cells and 13 which certainly did not were chosen,
produced aggregates are shown.
B. After 8 hours' pre-incubation, R-cells, taken from the samples with and without I-cells,
were micromanipulated to test areas. See text for details.
314
MAURICE SUSSMAN AND HERBERT L. ENNIS
incubated for one hour did not affect the background frequency but increases of
10 and 24% over background were obtained by adding R-cells pre-incubated for
4-6 and 10-12 hours, respectively. In other words, when pre-incubated for 10-12
hours and then moved to test areas, one out of four R-cells could induce the
formation of a center among the test cells, 12 hours after its introduction.
2468
TIME IN HOURS
10
12
FIGURE 6. A kinetic comparison of : I. The capacity of small population samples to ag-
gregate when isolated from their neighbors after varying periods of incubation. Ordinate :
per cent of 250 cell samples that aggregated. Abscissa : time of incubation on washed agar
prior to isolation. (Data from Table I.) II. The capacity of R-cells incubated for varying
times on washed agar to initiate centers amongst their developmental juniors. Ordinate: per
cent of R-cells capable of initiation. Abscissa time of incubation on washed agar prior to
their micromanipulation to test areas. (Data from Table II.)
Figure 6 is a graphic comparison of the kinetics of induction of centers in test
squares (I) by progressively delayed removal of outlying I-cells (data from Table I)
and (II) by addition of pre-incubated R-cells (data from Table II). The crude ki-
netic similarity suggested that the outlying I-cell might not only 'be responsible for the
subsequent aggregation of the R-cells but also for the concomitant increase in their
capacity to themselves initiate centers. To test this possibility, replicate samples
INITIATOR CELL 315
of 500 washed myxamoebae were dispensed on washed agar. In three experiments
21 samples were chosen which certainly contained I-cells and 13 which certainly
did not. The data in Table III confirm the correctness of these choices since 86%
of the samples said to contain I-cells aggregated while none of those said not to
contain I-cells did so. After these samples had been incubated for 8 hours, R-cells
were picked and moved to test squares as described in the preceding paragraph.
Table III shows that R-cells, whether pre-incubated in the presence or absence of I-
cells, were equally capable of inducing center formation. Thus, the rise of the initia-
tive capacity of the R-cells during the pre-aggregative period is not dependent upon
their contiguity with I-cells. Two points must be kept in mind here. First, it
must be remembered that prior to their deposition on the washed agar, R-cells had
all been in contact with I-cells and therefore could have been at this time the
subject of interactions emanating from the latter. Second, even though the R-cells
after 12 hours of incubation had attained a significant degree of initiative capacity,
they fell far short of the level displayed by the I-cells after only 20 minutes of
incubation. Therefore, the phenotypic difference between the two cell types in this
respect remains clear.
Finally, the results reveal a most puzzling paradox. When R-cells were pre-
incubated for 8 hours in the absence of an I-cell and then placed in the presence
of test cells for an additional 12 hours, at least one out of ten could induce center
formation. Yet the samples from which these R-cells originally came, when in-
cubated for a total of 20 or indeed 36 hours, had not aggregated. It is clear, there-
fore, that the observed increase in the initiative capacity of R-cells during the
pre-aggregative period in the development of a population is of no consequence to
the ultimate aggregation of that population. In other words, the initiative capacity
of such R-cells, demonstrated by movement to another population, is an experimental
artifact bearing no relation to normal aggregation but which may possibly be used to
understand the biochemical and genetic differences between the I-cell and R-cell
Phcnotypes.
DISCUSSION
The data presented here and previously suggest a developmental program of
slime mold aggregation that may serve as a useful working hypothesis.
I-cells arise during the growth of an R-cell population (which in turn had
originated from the spores of the preceding fruit), and attain a steady-state ratio of
approximately 1:2000 early in the exponential phase (Sussman, 1956; unpublished
data). Entrance into the stationary phase marks the beginning of the pre-aggrega-
tive period. At the beginning of this period, the I-cells secrete material which,
during the ensuing 12 hours, so conditions the neighboring R-cells as to induce them
to aggregate. This interaction, as might be expected, affects the nearest neighbors
first but its influence is progressively extended. Concomitant with, but unrelated
to either the presence of the I-cell or the subsequent course of aggregation in the
same population is a significant rise in the initiative capacity of the R-cells them-
selves. Such cells upon extended incubation never do attain the degree of initiative
capacity displayed by the I-cells nor can they act upon their developmental con-
temporaries but only upon cells at an earlier developmental stage to which they
have been added by the observer.
316 MAURICE SUSSMAN AND HERBERT L. ENNIS
The first overt sign of aggregation is the formation of cell clumps concentrically
about and usually at the I-cell. This is followed by excitation and elongation of the
loose and clumped cells in response to the chemotactic complex (Sussman et al.,
1956; Shaffer, 1956; Sussman, 1958). The appearance of oriented streams estab-
lishes the position of the aggregative center. This is usually coincident with the
final position of the I-cell but sometimes with the position of a particularly large
clump nearby, and possibly reflects the point of greatest production of the chemo-
tactic complex. In the latter case, the position of the center need bear no relation
to the previous path of the I-cell.
The picture as drawn raises many questions and offers a number of predictions
under current study. The most important of the latter involves the hypothetical
existence of an "initiator" substance. In view of the I-cell removal experiments,
one ought under the same conditions to be able to induce test cells to aggregate
by dispensing them in an area previously but no longer occupied by an I-cell. This
is being tested. The I-cell addition experiments raise the question as to what is
the minimum period of time after contact with the I-cell in which the induced
R-cells can begin aggregation. Is the 12-hour period subsequent to contact manda-
tory or does it involve preparations by the R-cells for aggregation, unconnected
with the function of the I-cell? In the latter case, one ought to be able to pre-
incubate the test cells for twelve hours, add I-cells, and observe the onset of
aggregation very shortly thereafter.
The fact that R-cells can also attain initiative capacity to a far smaller degree,
albeit much later than do the I-cells and ineffectively so far as inducing their
contemporaries to aggregate is concerned, still suggests that the metabolic path-
ways involved in initiation are not unique to the I-cells. Indeed, one may imagine
that the sole basis for the difference between I-cells and R-cells in this respect is
the much greater size of the former. Perhaps, then, any of the diverse methods
for producing giant cells may serve to create initiators just as does the normally
occurring R-cell to I-cell transformation. This point is also under current study.
SUMMARY
Dictyostelium discoidcinn myxamoebae occur as two distinct morphological
types, termed I-cells and R-cells. Data presented in a previous publication demon-
strate that I-cells can initiate centers of aggregation and suggest compellingly that
they are in fact the initiator cells for normal aggregation. The present communi-
cation extends and amplifies these findings.
A. Time lapse camera lucida drawings and photomicrographs illustrate the
sequence of events dviring the onset of aggregation.
B. Small population samples of myxamoebae, when isolated from their neigh-
bors shortly after deposition on washed agar, showed a distribution of aggregative
centers consistent with the distribution of I-cells within the samples. Longer
periods of contact with neighboring cells (including other I-cells) that surrounded
the samples prior to isolation permitted progressively greater proportions of the
samples to aggregate. The possibility arises of an "initiator substance" whose
effect may extend over relatively great distances.
C. R-cells, incubated for long periods of time on washed agar, were found to
have acquired initiative capacity. At best, only a small proportion did so and fur-
INITIATOR CELL 317
thermore could only induce the formation of aggregative centers amongst their
developmental juniors (by twelve hours) but not amongst their developmental
contemporaries.
LITERATURE CITED
ENNIS, H. L., AND M. SUSSMAN, 1958a. The initiator cell for slime mold aggregation.
Bacteriol. Proceedings, p. 32.
ENNIS, H. L., AND M. SUSSMAN, 1958h. The initiator cell for slime mold aggregation. Proc.
Nat. Acad. Set., 44: 401-411.
SHAFFER, B. M., 1956. Properties of acrasin. Science, 123: 1172-1173.
SUSSMAN, M., 1956. On the relation between growth and morphogenesis in the slime mold
Dictyostettwm discoidcnm. Biol. Bull., 110: 91-95.
SUSSMAN, M., 1958. A developmental analysis of slime mold aggregation. McCollum-Pratt
Symposium on the chemical basis of development. (In press.) Johns Hopkins Uni-
versity Press, Baltimore, Md.
SUSSMAN, M., AND E. NOEL, 1952. An analysis of the aggregation stage in the development
of the slime molds Dictyosteliaceae. I. The populational distribution of the capacity
to initiate center formation. Biol. Bull.. 103 : 259-268.
SUSSMAN, M., F. LEE AND N. S. KERR, 1956. Fractionation of acrasin. Science, 123 :
1171-1172.
SHELL REPAIR IN CHITONS
JOHN S. TUCKER AND ARTHUR C. GIESE
Hopkins Marine Station of Stanford University, California l
Cryptochiton stelleri (the "gumboot") is not only the largest member of the
class Amphineura, but also one of the most specialized in that the girdle tissue
has completely overgrown the skeletal plates (Heath, 1897). It therefore lacks
the outer shell layer, the tegmentum. While preparing some of the skeletal
plates for display, it was noticed that occasional plates were cracked and that
many of these cracks were repaired by an amber-colored membrane resembling
conchiolin. It seemed of interest to determine the frequency of damage, the stages
of repair, the possible significance of this ability to the survival of the animal, and
the relative incidence of breakage and repair in several other species of chitons
(Katherina tunicata, Mopalia hindsii).
Cryptochiton (Amicula) stelleri is a subtidal browsing herbivore, but it is also
found in fair numbers up into the middle zone of the intertidal region. When
found in the intertidal zone it is attached loosely to rock encrusted with coralline
algae or to algal curtains, and occasionally it is found on a sandy bottom.
Cryptochiton holds to its substrate only gently and can be removed easily by hand.
It is also dislodged by w r ave action as evidenced by the large number (approximately
75) counted on three local beaches after a heavy storm in April, 1958. The plates
of the storm-tossed animals were shattered and all but five of the animals were
dead. It is possible that after seeking food in shallower tidepools and crevices
during high water, the chiton is left by the subsequent receding tide and falls from
its loosely-held position among the algae. Caught by wave action, it may be beaten
against the rocks before it can re-establish its hold or before it can get back to
deeper waters. The animals when strongly stimulated in the laboratory have been
seen to contract with sufficient force to crack their plates ; perhaps some are also
broken in this manner in nature.
Of the 146 sets of plates (Fig. 1A) collected 2 87, or 59.5 per cent, had one or
more plates broken (about 18 per cent had one, 17 per cent had two, 11 per cent
had three, 6.2 per cent had four, 3.4 per cent had five, 2.7 per cent had six, 1.3
per cent had seven, but none had all eight). Two animals had seven of the eight
plates broken. The middle plates were broken most often, these being the widest
and flattest (6.2 per cent plate 1, 11.4 per cent plate 2, 14.1 per cent plate 3, 18 per
cent plate 4, 21.2 per cent plate 5, 16.8 per cent plate 6, 8.4 per cent plate 7, and
3.9 per cent plate 8). Often two or three adjacent plates were found with similar
breaks, suggesting a blow from a large surface.
1 Supported in part by U. S. Public Health Grant RG 4578 to A. C. Giese.
2 The chitons were being used in a study of the annual reproductive cycle and of the
biochemistry of the blood and tissues ; hence the plates were available in numbers, from
specimens collected for these purposes.
318
SHELL REPAIR 319
Plates that had been broken just prior to the animal's death, either by storms
or in the laboratory, showed a clean cleavage with the parts fitting perfectly together.
Depending upon the severity of the blow, the cleavage was in a single straight line or
in an arborescent pattern. Repairs were seen in few plates that had been shattered
into as many as seven pieces.
The first step in repair is the formation of a strip of membrane overlapping the
crack on both sides of the plate. The second stage seems to be the accumulation of
fine granules of a calcium salt, presumably in the form of carbonate (Bevelander
and Benzer, 1948), under the conchiolin strip with the concomitant erosion of the
underlying crystalline shell. This erosion often extends for some distance laterally
from the crack under an extension of the membrane strip (Fig. 1C). The last
step in repair (Figs. 1D-G) is the invasion or growth of existing minute crystals
(Bevelander and Benzer, 1948; Bevelander, 1953) of the surface of the membrane
strip by crystals of calcium carbonate in the form of aragonite (Prenant, 1927).
The crystals are imbedded in the surface of the conchiolin, leaving a ridge over the
crack, and often an air space or a layer of membrane between the old shell and the
new material (Fig. IE). This leaves the plate weakened so that a second blow
usually splits the plate along the old crack.
To determine the rate of repair of broken plates, five chitons subjected to
hammer blows were kept in the laboratory with ample food and in running sea
water, and sacrificed after varying lengths of time. The results are quite variable
but they serve to illustrate the slowness of repair. For example, while one chiton
developed membranes around the cracks in twenty days and granular calcium
carbonate deposition in twenty-four, another showed no visible sign of repair in
the same period of time. In still another chiton, dissected sixty days after breaking
the plates, crystalline calcium carbonate was evident in the cracks. However,
in two chitons examined 100 days after injury, only membranes had been laid
down over the cracked edges of the plates.
Energy for mobilization of the shell calcium is available only during active
feeding and digestion in some mollusks (Wagge, 1951, 1952; Robertson, 1941).
The effects of starvation were not tested here in view of the variability of results
with well-fed specimens.
Wilbur and Jodrey (1955) inhibited shell deposition in the oyster with car-
bonic anhydrase inhibitor. However, no tests were made with such inhibitors on
Cryptochiton in view of the variability of results and the long time required for
repair of broken skeletal plates. Furthermore, it is not even known whether
amphineurans possess carbonic anhydrase although Freeman and Wilbur (1948)
found it in most, but not all, of the species of gastropods and pelecypods tested.
Katherina tunicata
Fifty-five sets of plates of Katherina were examined and only five plates were
found to be broken, although many of them were eroded to some extent, possibly
by a disease. Of these, two showed slight evidence of repair. One had a thin
membrane with a few lime crystals, the other showed an old crack completely
repaired. It is possible that the other cracked shells were broken when the
eviscerated specimens were boiled to loosen the plates for examination. One valve
broken experimentally showed a conchiolin membrane after a few weeks.
320
JOHN S. TUCKER AND ARTHUR C. GIESE
B
***
SHELL REPAIR 321
Katherina lives in the surf zone on exposed shores among the sea palms and
between mussel beds where, at certain times of the day, it withstands an almost
continual pounding by the waves. Even at low tides the animals hold fast to bare
rock or crustose algae with such strength that a knife or screwdriver is needed to
pry them loose. At that, an inexperienced collector will often get only the plates
and girdle, the foot and viscera remaining on the substrate. The storms of 1958
which left so many specimens of Cryptochiton on the beaches presumably failed
to dislodge specimens of Katherina; at least none was seen on the beaches with
Cryptochiton.
The infrequency of broken plates in Katherina suggests that its plates are
proportionally stronger than those of Cryptochiton. The average weight of the
eight plates (10 specimens; average wet weight 33 grams) was 19.6 per cent of
the wet weight of the entire chiton, in comparison to the 7.4 per cent for Cryptochiton
(45 specimens; average wet weight 850 grams).
It is also possible that the shape of a skeletal plate has some bearing on its
resistance to shock. Plates No. 2 to No. 7 of Cryptochiton are in the shape of
butterflies (Fig. 1A) and are relatively flat. The skeletal plates in Katherina
consist of a heavy, roughly circular, disc with one pair of thin lateral lobes (Fig. IB).
Mopalia hindsii
Twenty-six sets of skeletal plates of Mopalia were examined and, other than
chipping along the edges of the thin membrane, eleven had broken plates (six had
one plate broken, three had two and one each had three or four plates broken).
The most common crack was from the lateral notch to the beak of the plate. Along
this line the plate is porous. Although many of the cracks are clean and may
have resulted from boiling eviscerated specimens to release the plates, Mopalia
suffers a fairly high incidence of infection from an unidentified boring animal which
weakens the prismatic layer of the plate with long tunnels. One of these weakened
plates was broken and the shell was thickened along the cracked tunnel. A thin
membrane and some lime crystals were also deposited after a lapse of several
weeks along cracks in plates No. 2 to No. 7 broken by a blow from a hammer.
One unbroken plate showed deposition of new material where an attached barnacle
overlapped the edge of the plate (Fig. 1H). Mopalia therefore can to some extent
repair its skeletal plates.
The specimens of Mopalia used in this work were collected from concrete pilings
in Monterey Harbor, a relatively protected habitat. While the skeletal plates are
broad and flat (Fig. 1C) and in this respect resemble those of Cryptochiton, at
the same time they constitute about 22.1 per cent of the wet weight of the animals.
Apparently they are adequate for the conditions to which the animals are exposed.
FIGURE 1. A. Shell plates of Cryptochiton stclleri. X %. Plate No. 1 (anterior) is
toward the top of the page. B. Shell plates of Katherina tunicata. X %. Plate No. 1
(anterior) is toward the top of the page. C. Shell plates of Mopalia hindsii. X %. Plate
No. 1 (anterior) is toward the top of the page. D. Representative cracks in shell plates of
Cryptochiton undergoing repair. X ^o- E. A section along a crack in a plate of Cryptochiton
showing the space between the old shell and the new material deposited during repair. X 1.
F. Lateral extension of the conchiolin strip (dark material) over two breaks. X 1. G. Inva-
sion of the conchiolin strip (dark) by crystalline calcium carbonate (light). X 1. H. Deposi-
tion of new shell (above, right) along edge of barnacle attached to Mopalia plate. X 1.
322 JOHN S. TUCKER AND ARTHUR C. GIESE
SUMMARY AND CONCLUSIONS
The skeletal plates of Katherina tunicata and Mopalia hindsii are sturdy, con-
stituting about a fifth of the wet weight of the animal. They were seldom found
broken in the specimens examined, but some broken plates were undergoing re-
pair. The skeletal plates of Cryptochiton stcllcri, on the other hand, are flat and
thin and constitute only 7.4 per cent of the wet weight of the animal. The majority
of cryptochitons examined showed breaks in one or more skeletal plates and in
almost all of these, some degree of repair and deposition of membrane or mineral
could be observed. The ability to repair its plates is probably of value to this
species in view of the weakness in design of its skeleton.
Irregularities of plates and variations in numbers of skeletal plates have been
described for other species of chitons (Crozier, 1919; Berry, 1925, 1935; Taki,
1932). It is interesting that apart from an occasional asymmetrical terminal
plate of a Cryptochiton, no such irregularities in number or shape were observed
in the three species of chiton studied here.
LITERATURE CITED
BERRY, S. S., 1925. On an abnormal specimen of the chiton, Acanthoplcura qrannlata. Ann.
and Mag. Nat. Hist., 16: 173-175.
BERRY, S. S., 1935. A further record of a Chiton (Nuttalina) with nine valves. Nautilus,
48: 89-90.
BEVELANDER, G., 1953. Interrelations between protein elaboration and calcification in molluscs.
Anat. Rec., 117: 568-569.
BEVELANDER, G., AND P. BENZER, 1948. Calcification in marine molluscs. Biol. Bull., 94 :
176-183.
CROZIER, W. J., 1919. Coalescence of the shell plates in Chiton. Amcr. Nat., 53: 278-279.
FREEMAN, J. A., AND K. M. WILBUR, 1948. Carbonic anhydrase in molluscs. Biol. Bull., 94 :
55-59.
HEATH, H., 1897. External features of young Cryptochiton. Proc. Acad. Nat. Sci. Phil., 8 :
299-302.
PRENANT, M., 1927. Les formes mineralogiques du calcaire chez les etres vivants, et le problem
de leur determinisme. Biol. Rev., 2 : 365-393.
ROBERTSON, J. D., 1941. The function and metabolism of calcium in the Invertebrata. Biol.
Rev., 16: 106-133.
TAKI, I., 1932. On some cases of abnormality of the shell plates in chitons. Mem. Coll. Sci.
Kyoto Imp. Univ., 8: 27-64.
WAGGE, L. E., 1951. Amoebocytic activity and alkaline phosphatase during shell regeneration
in Helix. Quart. J. Micr. Sci., 92 : 307-321.
WAGGE, L. E., 1952. Quantitative studies of calcium metabolism in Helix aspcrsa. J. Exp.
Zool, 120: 311-342.
WILBUR, K. M., AND L. H. JODREY, 1955. Studies on shell formation. V. The inhibition of
shell formation by carbonic anhydrase inhibition. Biol. Bull., 108 : 359-365.
THE JUVENILE HORMONE. I. ENDOCRINE ACTIVITY OF
THE CORPORA ALLATA OF THE ADULT CECROPIA
SILKWORM
CARROLL M. WILLIAMS 1
The Biological Laboratories, Harvard University, Cambridge 38, Massachusetts
The endocrine role of the corpora allata of insects was discovered by V. B.
Wigglesworth (1934, 1936) over twenty years ago. In a series of simple and
decisive experiments on Rhodnius he showed that the corpora allata secrete a
"juvenile hormone" which opposes metamorphosis. In these early studies Wig-
glesworth also recognized that the corpora allata undergo pronounced changes in
endocrine activity during the course of metamorphosis ; namely, that they are
active in the immature nymph, inactive in the mature nymph just prior to metamor-
phosis, and active again in the adult insect after metamorphosis. Subsequently,
the general validity of these conclusions has been confirmed repeatedly and found
to apply to both hemi- and holometabolous insects (for review, see Wigglesworth,
1954, pages 56-64).
During the past twelve years, in the course of studies of the metamorphosis
of the Cecropia silkworm, the juvenile hormone has necessarily been an object of
detailed attention. While confirming the essential elements in Wigglesworth's
theory, the study has helped to resolve certain persistent mysteries and, more
recently, has pointed the way to the successful extraction and purification of the
hormone itself. This first of a series of communications is concerned with the
endocrine activity of the corpora allata of the adult moth.
MATERIALS AND METHODS
1. Experimental animals
The experiments were performed on Cecropia, Cynthia, and Polyphemus silk-
worms. Taxonomists continue to amuse themselves by changing the generic and
specific names of these Saturniids. What began as Phalacna cecropia became
Samia cecropia, then Platysamia cecropia, and now Hyalophora cecropia (Michener,
1952). The Cynthia silkworm, known throughout the world as Philosamia
cynthia, was changed to Samia walkcri, and then back to Samia cynthia. Telea
polyphemus is now Antheraea polyphemus. As in the analogous cases discussed by
Wald (1952, page 339), the "common names" have escaped the attention of
taxonomists and have remained firm and unchanging. Therefore, the common
names will be used routinely in the present reports.
1 This study was aided by a grant from the National Institutes of Health of the U. S.
Public Health Service. It is a pleasure to acknowledge the advice and counsel of Prof. Berta
Scharrer.
323
324 CARROLL M. WILLIAMS
Cecropia silkworms were reared under nylon nets on wild-cherry trees. Poly-
phemus were reared on oak or maple ; Cynthia, on cherry or ailanthus or purchased
from dealers. The cocoons were harvested and stored as previously described
(Williams, 1946a; Shappirio and Williams, 1957).
2. Surgical procedures
Experimental animals must be deeply anesthetized during surgical procedures.
We use carbon dioxide for this purpose and with mixtures of air and carbon
dioxide have maintained pupae anesthetized for as long as one month without
injury. Groups of animals are placed in a capped, flat-bottom Buchner funnel
and exposed for about twenty minutes to a slow stream of carbon dioxide from a
compressed cylinder. The gas is bubbled through water en route to the funnel.
The animals are flaccid when fully anesthetized, and one can no longer elicit any
movements of the abdominal segments.
Surgical procedures are performed in a second Buchner funnel (diameter 11
cm., height 3 cm.) \vhich is mounted flush on the top of the operating bench. A
slow stream of carbon dioxide is bubbled through water and passed through the
bottom of the uncovered funnel. Carbon dioxide, being heavier than air, fills the
cavity of the funnel and maintains a continuous anesthesia during the surgical
procedure (Williams, 1946b).
Operations are carried out under the low magnification of the dissecting micro-
scope, making use of 9 X oculars and 0.7, 1, or 2 X objectives. The foot of the
microscope is removed and the vertical pillar permanently attached to the operating
bench on the distal side of the funnel. A hinged-arm permits the microscope to
scan the entire diameter of the funnel. In order to leave both hands free, the
microscope is equipped with a foot-focusing device (designed and built by Mr.
Robert Chapman of the Harvard Biological Laboratories). Illumination is pro-
vided by a 6-volt microscope lamp (Zeiss "Osram") attached to and moving with
the microscope. The lamp is equipped with an infra-red filter.
Anesthetized animals are transferred to the carbon dioxide-filled funnel for
the surgical procedure. They are then returned to air, placed in individual num-
bered glass containers ("creamers"), and stored in a room having a controlled
humidity of sixty per cent and a temperature of 25 C.
Dissecting instruments consist of the following: watchmaker's forceps (Dumont
"rustless"; two of No. 3 and two of No. 5) ; a scalpel (Bard-Parker No. 3 handle
with a No. 11 detachable blade); stainless iris scissors curved on the flat and
closing to the tip ; several forms of stainless steel iridectomy and micro-scissors ; a
stainless steel dental probe ; a 5-ml. hypodermic syringe filled with insect Ringer
and capped with a 25-gauge needle.
Prior to each group of operations the instruments are briefly rinsed in seventy
per cent ethanol and wiped dry. Rigorous asepsis is unnecessary because the blood
of the silkworms apparently contains an anti-bacterial substance that protects it
from the ordinary contaminants. However, it fails to protect from insect pathogens
and no diseased insect should be operated upon with the same instruments or
even in the same room.
Healthy pupae can withstand almost any degree of surgery provided that a
few crystals of the potent anti-tyrosinase, phenylthiourea, are placed in the operat-
INSECT JUVENILE HORMONE 325
ing field. We routinely use an equal part mixture of phenylthiourea (twice recrystal-
lized from hot 95 per cent ethanol) and streptomycin sulphate, the two having
been ground together in a mortar and stored in a capped vial in the refrigerator.
Small amounts of the powder are removed and discarded within two days after
being placed at room temperature.
Ephrussi-Beadle Ringer's solution is utilized containing 7.5 gm. NaCl, 0.35 gm.
KG, and 0.21 gm. CaCL, per liter of distilled water. The stock solution is brought
to a boil, capped, and stored in the dark under refrigeration. Fungal contamination
of physiological solutions, especially those containing bicarbonate, is a common
source of difficulty when solutions are stored at room temperature.
Excised tissues and organs are transferred to small depression dishes made of
black glass and filled with Ringer. Black plastic bottle-caps are also satisfactory for
this purpose. Dissections of sacrificed animals are performed in a glass Petri dish
which fits snugly into the cavity of the Biichner funnel. Plasticine is pressed into
the bottom of the dish to receive short stainless steel pins. The dish is filled with
Ringer and the dissection performed with the animal spread and pinned under
the solution.
After surgical procedures on surviving pupae. Ringer's solution is added from
a hypodermic syringe so that the blood is flush with the surface of the cuticle. The
area of excised cuticle is then capped by a plastic window of appropriate size.
The latter is punched or cut with scissors from cellulose acetate cover slips
("Turtox," thickness 1 or 2). The window is sealed in place with paraffin wax
which is melted in an alcohol lamp and transferred with a curved needle or drawing
pen. The melted wax adheres to the cuticle and the underside of the rim of the
plastic slip provided that both are dry. The operating field is thereby equipped
with a transparent window which permits one to look inside the living animal.
3. Excision of pupal corpora allata and corpora cardiaca
An anesthetized pupa is placed in a plasticine cradle in the bottom of the
carbon dioxide-filled funnel. The cuticle of the facial region is first removed.
For this purpose a scalpel incision is made through the integument on each side
of the face. The two cuts are joined by a transverse cut and the rectangle of
cuticle is grasped with forceps and pulled free from its attachment at the base of
the legs. The insect's abdomen is then pressed forward with plasticine and held
in this position so that the blood fills, but does not overflow, the operating field.
The naked epidermis is grasped with forceps, split down the middle, and trimmed
free with scissors. The brain is thereby exposed. This is pressed down in the
field to reveal the tiny corpus allatum-corpus cardiacum complex on each side.
The complexes are dorso-lateral to the brain and attached on each side to a large
tracheal trunk at this position (see Figure 1). A pair of tiny nerves emerges from
the posterior face of each brain hemisphere and passes to the corpus cardiacum
on that side. These nerves are very delicate and difficult to see in a dissection of
this type.
By means of forceps the connections between glandular complex and the adjacent
trachea are broken, and the complex transferred to Ringer's solution in a black-
dish. Alternatively, the tracheal segment can be excised with iridectomy scissors
and removed along with the glandular complex.
326
CARROLL M. WILLIAMS
INSECT JUVENILE HORMONE 327
4. Excision of adult corpora allata
The moth is anesthetized and its head dipped momentarily into seventy per
cent ethanol to wet the scales and hairs. The head is then cut off with scissors
and placed in Ringer's solution. (The headless moth will continue to live for
approximately the normal life-span of 7 to 10 days at 25 C.)
The antennae are excised at their bases. Then with fine scissors the head is cut
along the dorsal midline from its posterior margin to the mouth parts. The head is
then spread apart with foreceps and pinned under Ringer. The pair of corpora
allata-corpora cardiaca complexes is attached to the aorta just behind the brain.
The brain is split in the midline to expose the aorta. The glandular complexes can
now be broken free from the rear of the brain and transferred to a black dish by
grasping the aorta with forceps.
Under the favorable conditions of illumination in the black dish, one can
recognize the corpus cardiacum ; it is attached by short nerves to the much larger
corpus allatum. The latter is ordinarily flattened or wedge-shaped and sub-
divided into a number of lobes and lobules. If necessary, the glandular complex
may now be subdivided into its constituent parts by breaking the nerves between
corpus cardiacum and corpus allatum.
5. Isolation of pupal abdomens
This procedure has already been described for the Cecropia silkworm (Williams,
1947). The principal difficulty is to isolate the terminal abdominal segments with-
out puncturing the fluid-filled midgut. This difficulty is circumvented by the use
of the Cynthia silkworm. In this species the midgut contains only a solid, rod-like
mass. Therefore the perforation of the midgut is inconsequential. The pupa is
transected just behind the metathorax with a single transverse cut of a sharp
razor blade. The abdomen is then supported with the cut surface facing upward.
Crystals of the phenylthiourea-streptomycin mixture are spread in the wound, and
Ringer's solution is added to fill the cavity of the abdomen. The wound is then
capped with a plastic slip in which a central hole has been punched. The plastic
is sealed in place with melted wax. Ringer is finally added via the central hole to
replace all air, and the hole itself sealed with wax.
RESULTS
1. Role of the corpora allata in adult development and sexual maturation
The pair of corpora allata-corpora cardiaca complexes was removed from each
of a series of twenty chilled male or female Cecropia pupae via the facial approach.
The integumentary defect was capped and sealed with a plastic window, and the
animals placed at 25 C.
Adult development was initiated after about two weeks and proceeded in syn-
chrony with the time-table for the normal development of Cecropia at 25 C.
(Schneiderman and Williams, 1954). The moths, emerging after three weeks
FIGURE 1. Brain and corpora allata of the Cecropia silkworm are shown in cutaway
views of the head of larva (top), pupa (middle), and adult (bottom). The corpora allata are
the two small bodies attached by tiny nerves to the back of the brain. (This figure is used
with the permission of Scientific American.)
328
CARROLL M. WILLIAMS
FIGURE 2. After receiving implants of three pairs of corpora allata of adult Cecropia,
the Polyphemus pupa, here illustrated, has transformed into a second pupal stage. (See right
side of preparation where the old pupal cuticle has been trimmed away.)
FIGURE 3. This Cecropia pupa received implants of two pairs of adult Cecropia corpora
allata. Development has given rise to a mixture of pupa and adult. (The old pupal cuticle
has been completely removed.)
INSECT JUVENILE HORMONE 329
of adult development, could not be distinguished from un-operated individuals. The
females deposited a normal complement of eggs and both sexes survived for the
customary period of 7 to 10 days at 25 C.
The absence of corpora allata was confirmed in dissections of many of these
moths. All the internal organs, including the gonads, showed full and complete
development. The abdomens of females were packed with ripe eggs, and the
males showed normal spermatogenesis.
The experiment was repeated on a series of six male and six female pupae to
produce moths lacking corpora allata. The two sexes were cross-mated and each
of the six females was allowed to oviposit in a paper bag. A normal number
(150-225) of eggs was collected from each female. These were placed under
large nylon nets and the larvae reared to maturity on wild-cherry leaves. No
deviation from normal development could be detected.
These experiments show that the corpora allata play no evident role in the
transformation of the pupa into an adult Cecropia or in the gonadal function of
the adult itself.
2. Endocrine activity of adult corpora allata
In the absence of any obvious function of the corpora allata of adult Cecropia,
it is paradoxical to find that the glands, when excised and tested for endocrine
activity, are more active in the moth than at any other stage in the life history
(Williams, unpublished data). This fact was discovered eleven years ago in the
course of an experiment performed for other purposes. It happened by chance that
a pair of adult corpora allata was implanted into a brainless diapausing Cecropia
pupa. Ten days later, the host showed the termination of diapause and the initia-
tion of development. This result would have been puzzling in a normal diapaus-
ing pupa ; in a brainless diapausing pupa it was incomprehensible.
Even more puzzling w T as the character of the development which then took
place. Within two weeks the brainless pupa transformed, not into a moth, but
into a bizarre creature in which large areas of pupal cuticle had been freshly
formed (see Figs. 3 and 4). The animal, in short, was a mosaic of pupal and
adult characteristics (Williams, 1952b).
During the past eleven years this result has been duplicated on numerous
occasions. The experimental series includes fifty-one brainless Cecropia pupae
which received one to three pairs of corpora allata-corpora cardiaca complexes
derived from male or female Cecropia moths. As shown in Table I a total of
twelve individuals (23 per cent) showed the result just described. The residual
77 per cent showed no effect of the implantation and continued to diapause. But
the twelve positive experiments were of sufficient interest in themselves. Not only
FIGURE 4. Pupal-adult monstrosity after implantation of adult corpora allata. Note the
pupal cuticle on head, palps, and antennae. However, the wings show scale-covered adult
cuticle and the eyes show considerable adult development.
FIGURE 5. This isolated pupal abdomen received implants of adult corpora allata, plus
an injection of ecdysone. The tip of the old pupal cuticle has been torn away to reveal a second
pupal abdomen that has formed.
330
CARROLL M. WILLIAMS
TABLE I
Tests of adult corpora allata-corpora cardiaca complexes* in brainless diapausing pupae
Adult donors
Brainless hosts
Normal
development
Mixed
development
No
development
Cecropia
Cecropia
12
39
Cecropia
Cynthia
2
2
Cecropia
Polyphemus
2
1
Cynthia
Cynthia
5
Cynthia
Cecropia
20
Polyphemus
Polyphemus
2
Polyphemus
Cecropia
4
10
Totals
22
77
* One to three pairs of complexes from male or female moths were implanted into each
brainless pupa.
had the implants caused the formation of mixtures of pupa and adult; seemingly,
they also had substituted for the brain and provoked the termination of diapause.
As shown in Table I, this result was duplicated when corpora allata of adult
male or female Cecropia were implanted into brainless diapausing pupae of Cynthia
or Polyphemus. Here again, a certain percentage of animals terminated diapause
and developed into pupal-adult mixtures.
The corpora allata-corpora cardiaca complexes of male and female Cynthia and
Polyphemus moths were also tested. The three species seem to differ among them-
selves in the endocrine activity of the adult corpora allata. For example, the
corpora allata of adult Cynthia gave negative tests in all twenty-five preparations.
By contrast, the glands of adult Polyphemus gave positive tests in six of sixteen
preparations. Moreover, when used as recipients of implants, brainless Poly-
phemus pupae seemed to have a lower developmental threshold than the other two
species, for four of five individuals gave a positive reaction to the implantation of
adult corpora allata. In retrospect, Polyphemus appears to be the animal of choice
for experiments of this type.
In the far more numerous tests of Cecropia corpora allata, the conditions of
the experiment were subjected to minor variations in the hope of recruiting a
positive response in a larger proportion of individuals. By increasing the number
of implanted glands from one to two or three pairs, little additional effect was
realized. However, the developmental response was markedly enhanced when the
host animals w f ere placed at 15 or 20 C. rather than at 25 C. after the implantation
of corpora allata. It was also observed that the experimental animals which
developed at the lower temperature retained a far larger proportion of pupal
characters than in similar animals developing at 25 C.
3. Inactivity of corpora cardiaca
In the experiments just considered, the adult corpora allata were implanted
together with the attached corpora cardiaca. However, in thirty-five additional
preparations, the corpora allata were carefully dissected from the attached corpora
cardiaca and then implanted into brainless diapausing pupae.
INSECT JUVENILE HORMONE
331
TABLE II
Tests of adult corpora allata* (minus corpora cardiaca) in brainless diapausing pupae
Adult donors
Brainless hosts
Normal
development
Mixed
development
No
development
Cecropia
Cecropia
4
23
Cvnthia
Cvnthia
2
Cynthia
Cecropia
1
3
Polyphemus
Polyphemus
1
Polyphemus
Cecropia
1
Totals
6
29
* One to three pairs of corpora allata from male or female moths were implanted into each
brainless pupa.
The results, recorded in Table II, were substantially the same as those observed
in the previous experiments. Once again, a certain low percentage of brainless
animals terminated diapause and transformed into pupal-adult monstrosities.
The inactivity of implanted corpora cardiaca was further confirmed in fourteen
experiments in which adult corpora cardiaca were freed from corpora allata and
tested, as such, in brainless diapausing pupae. No developmental response was
obtained even when as many as ten pairs of adult corpora cardiaca were implanted.
Indeed, in the course of twelve years of experimentation, we have never detected
any trace of developmental response after the implantation of corpora cardiaca of
larvae, pupae, or adults.
For present purposes it is necessary to conclude that the developmental reactions
under consideration are attributable to the adult corpora allata per sc. This
implies that in a certain proportion of individuals the adult corpora allata have two
effects : they first promote the initiation of adult development ; they then prevent
the transformation of the pupa into a normal adult moth.
4. Effects of brain implantation
As noted in Tables I and II, the vast majority of brainless Cecropia pupae
continued to diapause when implanted with adult corpora allata. In all of these
preparations the implants gave the impression of being inert. The true state of
affairs is suggested by the following experiment :
Two pairs of adult corpora allata were implanted into each of five brainless
Cecropia pupae. Six weeks later the pupae showed no change from their condition
at the outset. Two brains of previously chilled Cecropia pupae were implanted at
this time to cause the initiation of development. The latter gave rise to creatures
showing large areas of pupal cuticle. In effect, the initiation of development un-
masked the endocrine activity of the previously implanted corpora allata. Further
information was provided by the following experiment :
Two pairs of adult Cecropia corpora allata were implanted under a facial window
in each of two brainless diapausing Cecropia pupae. One month later the implants
were removed and the pupae caused to develop by the injection of 125 ^g. of a
332 CARROLL M. WILLIAMS
purified extract of prothoracic gland hormone (ecdysone). 2 Both individuals
transformed into moths which retained large areas of pupal cuticle.
This experiment shows that the presence of the brain is not necessary for the
secretion of juvenile hormone by adult corpora allata. In the absence of the
initiation of adult development, the implants had built up a substantial titer of
juvenile hormone. But the host could not signal this fact until its development
was brought about by ecdysone.
5. Experiments on isolated pupal abdomens
Eight abdomens were isolated from diapausing Cecropia pupae. Preparations
of this type remain in permanent diapause unless provided with ecdysone by in-
jection (Williams, 1954), or by the implantation of active prothoracic glands, or
by the implantation of inactive prothoracic glands plus active brains (Williams,
I952a). In the present experiment efforts w r ere made to evoke a developmental
response of isolated abdomens by the implantation of adult corpora allata either
alone, or in conjunction with brains, prothoracic glands, or injections of ecdysone.
TABLE III
Effects of implantations into isolated abdomens of diapausing cecropia
Abdomen no. Implant Result
1415 1 pr. adult C.C. + C.A. No development
1447 1 pr. adult C.C. + C.A. No development
2123 3 pr. adult C.C. + C.A. No development
2090. . 5 pr. adult C.C. + C.A. No development
2212 2^ pr. adult C.C. + C.A. plus 2 chilled No development
pupal brains
1515 1 pr. adult C.C. + C.A. plus 2 pr. No development
prothoracic glands of diapausing pupae
2109 3J pr. adult C.C. + C.A. plus 4 pr. Molted to form second
prothoracic glands of diapausing pupae pupal abdomen
9320 2 pr. adult C.A. (-C.C.) plus 25 M g. of Molted to form second
crystalline ecdysone pupal abdomen
Table III summarizes the several types of preparations. It is of particular
interest and importance to note that no development took place when the abdomens
received only adult corpora allata. We have checked this finding in twelve ad-
ditional experiments performed on isolated Cynthia abdomens ; in this case the
pupal abdomens were distributed at 15, 20, and 25 C. after the implantation of
two to five pairs of corpora allata derived from adult Cecropia or Polyphemus. In
short, no trace of development was ever observed in response to the implantation
of adult corpora allata per se. The same negative result was also recorded in an
experiment where adult corpora allata were implanted along with active brains.
The preparation numbered 9320 in Table III is of particular interest. Here,
two pairs of adult corpora allata were implanted into an isolated abdomen. A
- I am indebted to Dr. Peter Karlson for supplying highly purified preparations of ecdysone.
INSECT JUVENILE HORMONE 333
month later 25 /zg. of crystalline ecdysone were injected. Development began
within two days. Within the following ten days the pupal abdomen transformed
and molted into a second pupal abdomen (see Figure 5). This result was
duplicated in two additional experiments utilizing Cynthia abdomens. It is clear
that ecdysone is the prime-mover in the developmental response and that the
juvenile hormone is inactive in the absence of ecdysone.
Attention is now directed to preparation 2109 in Table III. This pupal
abdomen received implants of adult corpora allata plus diapausing pupal prothoracic
glands. Precisely the same result was observed as after the injection of ecdysone:
the pupal abdomen molted and transformed into a second pupal abdomen. In this
case it seems necessary to conclude that the corpora allata activated the diapausing
prothoracic glands that, in this sense, a hormone from the corpora allata had
substituted for the brain hormone. However, there is no indication in Table III
that this corpus allatum hormone can substitute for ecdysone itself.
6. Tests of adult corpora allata hi previously chilled pupae
The results considered to this point lead to the prediction that adult corpora
allata should be uniformly active when tested in previously chilled pupae just prior
to the initiation of adult development.
During the past ten years this prediction has been confirmed on a large scale.
The experimental series includes ninety-eight preparations in which corpora allata
of male and female moths of Cecropia, Polyphemus, and Cynthia were tested in
chilled pupae of each of the same three species. All except eight animals gave
rise to adults retaining pupal characters. In the eight negative tests the implanted
glands had been derived from elderly adults just prior to death.
There was a rough correlation between the number of implanted glands and
the degree to which pupal characters were preserved a finding which will be
considered in further detail in the following paper. Moreover, as was true in the
earlier experiments on brainless pupae, the effects of the implanted corpora allata
were amplified when the host pupae were placed at 15 or 20 C., rather than at
25 C., immediately after the implantation.
The retention of pupal characters was extreme in many of the test animals.
As shown in Figure 2, the pupa transformed into a second pupa which showed only
traces of adult characteristics. In several experiments performed on Polyphemus
and Cecropia, the secondary pupa molted into a tertiary form. In this case, the
pupal characteristics were less prominent after the second molt than after the first.
None of these animals was viable for any prolonged period after transforming
into mixed forms. Although the old pupal cuticle became thin and crisp and the
ecdysial lines were eroded to the surface, spontaneous escape from the old pupal
cuticle occurred only in individuals showing minimal retention of pupal character-
istics. All other animals remained enveloped in the old pupal cuticle until they
died or were sacrificed.
In many of the individuals the molting process proceeded to a normal terminal
phase accompanied by a complete breakdown of the old endocuticle and a partial
or complete resorption of the molting fluid. Yet, for some unexplained reason, the
insect failed to undertake the vigorous muscular efforts that accompany a normal
ecdysis. It did not "try to molt" even though it possessed the nervous and
334 CARROLL M. WILLIAMS
muscular equipment to do so. The use of forceps was therefore necessary to
peel off the old pupal exuviae.
In many individuals it was difficult or impossible to withdraw the lining of the
old tracheal tubes through the spiracular openings. Indeed, in the case of Cecropia,
the larger branches of this old system became stiff and melanized and therefore
incapable of being shed. The net effect is that the juvenile hormone is a lethal
agent for all these Saturniid pupae.
7. Inactivity of killed corpora allata
The high activity recorded for implanted adult corpora allata suggested the
possibility that substantial amounts of hormone might be stored within the glands
themselves. This prospect was tested in five experiments. In one experiment
eight adult Cecropia corpora allata were frozen and thawed twice at 40 C. and
then implanted into a previously chilled pupa. Normal development ensued.
In four other experiments adult corpora allata, in numbers ranging from 9 to
44, were homogenized in 0.1 ml. of insect Ringer and then introduced into four
previously chilled pupae. All four animals developed into normal adult moths.
Evidently, little or no hormone is stored in the living gland, for the activity of
a single living adult corpus allatum was not duplicated by the implantation of up
to forty-four dead glands.
DISCUSSION
1. Secretion of the juvenile hormone by the adult corpora allata
The experimental results demonstrate the endocrine activity of the corpora
allata of Cecropia, Polyphemus, and Cynthia moths. As is amply evident in
Wigglesworth's (1954) recent review, this finding is consistent with the picture
presented in all other insects that have been studied in detail including several
families of Lepidoptera. In the Cecropia silkworm the corpora allata, when re-
moved and tested, are found to be more active in the adult moth than at any other
stage in the life history (Williams, unpublished data). Moreover, there is general
agreement that at least one of the secretory products of the adult corpora allata is
the same juvenile hormone which is secreted weeks or months earlier by the
corpora allata of the immature insect. This conclusion was first proposed by
Pflugfelder (1938a, 1938b) and Pfeiffer (1945), and will be further documented
in the subsequent papers in this series.
2. The role of the juvenile hormone in adult moths
We have been unable to detect any function for the corpora allata in the pupal
or adult stages of these Lepidoptera. Thus, as we have seen, the corpora allata
can be removed from pupae of either sex without disturbing the development of
normal, viable, sexually mature moths. These findings are the same as those re-
ported for Bombyx mori by Bounhiol (1938) and Fukuda (1944). The present
study enlarges the negative evidence by showing that the absence of corpora allata
fails to interfere with the maturation of functional gametes and the production of
normal offspring.
INSECT JUVENILE HORMONE 335
The situation in the Lepidoptera therefore departs from that described for most
other orders of insects where the corpora allata are necessary for the deposition of
yolk in the adult female and for the secretory activity of the accessory glands in
the adult male (for summary, see Wigglesworth, 1954, pages 77-80). In the
Lepidoptera which have been studied, all these functions can go forward in the
absence of corpora allata. For the sexual maturation of both males and females
all that is required is the presence of prothoracic gland hormone (ecdysone). The
brain hormone is also unnecessary for the sexual maturation of these silkworms.
Pupae from which the brain, corpora cardiaca, and corpora allata have been
removed develop into sexually mature moths after the injection of crystalline
ecdysone (Williams, 1954).
Adult Lepidoptera therefore present the paradoxical picture of the presence of
highly active corpora allata for which there appears to be no apparent function.
However, it is worth recalling that corpora allata have been tested only in species
of adult Lepidoptera which are short-lived and unable to feed. In adults of the
giant silkworms, as in the commercial silkworm, functional mouth-parts are absent.
Consequently, the duration of the adult stage is greatly curtailed : ripe eggs must
be ready for oviposition at the time of adult emergence. In short, the absence of
mouth-parts has enforced on these short-lived moths a precocious maturation of
the gonads during the course of pupal-adult development. Indeed, months before
the development of the adult moth, the proteins which later appear in the yolk of
the eggs are already present in high concentrations in the blood of the diapausing
pupa (Telfer, 1954).
It is among the feeding, long-lived species of adult Lepidoptera that one would
anticipate a gonadotropic function for the corpora allata akin to that seen in most
other orders of insects. This inference is in accord with the histological studies
of Kaiser (1949) on long-lived butterflies of the genus Vanessa. Presumably, in
the Ephemeroptera and other non-feeding adults one should find the same picture
as presented by the Saturniidae.
The absence of functional adult mouth-parts is clearly a secondary affair in the
evolution of the Lepidoptera. Indeed, the very same moths contain digestive
tracts of normal organization, but of no apparent function. Evidently, the presence
of active corpora allata is a memento of a more primitive endocrinological situation.
3. Biological role of the juvenile hormone
The juvenile hormone plays no role in the transformation of a pupa into an adult
moth. All that is required is that the juvenile hormone be absent throughout the
early phases of this transformation (Williams, 1952b). This conclusion is in line
with the finding that the corpora allata are inactive throughout the entire pupal
stage and during the first two-thirds of adult development (Williams, unpublished
data).
A pupa can be supplied with juvenile hormone by the implantation of living,
active corpora allata obtained from larvae or adults. However, as demonstrated
in the experiments on isolated pupal abdomens (Table III), the juvenile hormone
has no effects in the absence of the prothoracic gland hormone, ecdysone. Only
when the abdomen is provided with this hormone can one detect any action of
336 CARROLL M. WILLIAMS
the implanted corpora allata. The outcome is that the pupal abdomen terminates
diapause, molts, and transforms into a second pupal abdomen (Fig. 5).
Substantially the same result is seen in experiments performed on brainless
diapausing pupae. Here again the implantation of adult corpora allata is in-
consequential unless ecdysone is supplied by injection or by the secretory activity
of the animal's own prothoracic glands. The juvenile hormone then opposes
the transformation of the pupa into an adult moth. The result (Figs. 3 and 4)
is a creature showing to varying degrees a retention of pupal characters of the
type previously described by Piepho (1952) and Williams (1952b). When
the titer of juvenile hormone is high, then one may witness the formation of a
bona fide second pupal instar a phenomenon hitherto unknown in any insect
(Fig. 2). But, even in the presence of the highest concentrations of juvenile
hormone, we have never observed in this material the reappearance of larval
characters such as described in Rhodnius (Wigglesworth, 1954, 1957, 1958).
4. Mimicking of brain hormone
In a certain proportion of brainless diapausing pupae the implantation of
active corpora allata causes the termination of diapause and the initiation of adult
development. This result is not seen in isolated pupal abdomens or other prepara-
tions lacking prothoracic glands. But, as noted in Table III, the developmental re-
action becomes possible if an isolated abdomen receives active corpora allata plus
inactive prothoracic glands, or active corpora allata plus an injection of ecdysone
(Fig. 5). Moreover, in numerous experiments to be described on a later occasion,
the development of brainless diapausing pupae has been provoked by the injection of
crude or purified extracts of juvenile hormone. Evidently, under certain un-
defined conditions, a hormonal secretion of the corpora allata can activate the
prothoracic glands and, in this sense, mimic the function of the brain hormone.
Whether this hormone is the juvenile hormone or some further secretory product
of the corpora allata is impossible to state at the present time. A decision on this
point will become possible only when the juvenile hormone is isolated and tested
in pure form.
The finding that the corpora allata can turn on the prothoracic glands has an
obvious bearing on the endocrine control of larval molting. If the corpora allata
can activate the pupal prothoracic glands, there is no reason to suppose that they
cannot do so in the immature larva.
We begin to see a multiplicity of agencies which can promote the secretion of
ecdysone by the prothoracic glands. The brain can turn on the prothoracic glands.
Ecdysone can turn on the prothoracic glands (Williams, 1952a, 1954). And,
evidently, under certain undefined conditions, so also can the corpora allata.
Nature has apparently found it prudent to surround the prothoracic glands by a
net-work of controls. The present study suggests that the corpora allata are a
part of that net- work.
SUMMARY
1. Juvenile hormone is secreted in high concentration by the corpora allata
of the adult Cecropia moth.
INSECT JUVENILE HORMONE 337
2. Notwithstanding this fact, the juvenile hormone has no apparent function in
the adult moth. Extirpation of the corpora allata in the pupal stage fails to
interfere with the production of normal moths whose gametes give rise to normal
offspring.
3. The corpora allata are inactive during the entire pupal stage as well as during
the first two-thirds of adult development. If active corpora allata are implanted into
a pupa just prior to the initiation of adult development, the juvenile hormone acts
to oppose the differentiation of the adult moth. Development gives rise to an insect
showing a mixture of pupal and adult characters. In the presence of high con-
centrations of juvenile hormone the pupa molts and transforms into a second pupa
showing only traces of adult characters.
4. The biological action of juvenile hormone is seen only in the presence of
active prothoracic glands or their secretory product, ecdysone. Isolated pupal
abdomens fail to respond to juvenile hormone unless ecdysone is simultaneously
present. When both hormones are present, the pupal abdomen terminates dia-
pause, molts, and transforms into a second pupal abdomen.
5. Evidence is presented that the corpora allata secrete a factor which can
mimic the brain hormone and activate the prothoracic glands. This finding is
considered in relation to the endocrine control of larval molting.
LITERATURE CITED
BOUNHIOL, J. J., 1938. Recherches experimentales sur le determinisme de la metamorphose
chez les Lepidopteres. Bull. Biol., SuppL, 24: 1-199.
FUKUDA, S., 1944. The hormonal mechanism of larval molting and metamorphosis in the
silkworm. /. Fac. Sci. Tokyo Univ. sec. IT, 6: 477-532.
KAISER, P., 1949. Histologische Untersuchungen iiber die Corpora allata und Prothoraxdrusen
der Lepidopteren in Bezug auf ihre Funktion. Arch. f. Ent^v., 144 : 99-131.
MICHENER, C. D., 1952. The Saturniidae (Lepidoptera) of the Western Hemisphere. Bull.
Amer. Museum Nat. Hist., 98 : Article 5.
PFEIFFER, I. W., 1945. The influence of the corpora allata over the development of nymphal
characters in the grasshopper Melanoplus differentialis. Trans. Conn. Acad. Arts
Sci., 36: 489-515.
PFLUGFELDER, O., 1938a. Untersuchungen iiber die histologischen Veranderungen und das
Kernwachstum der Corpora allata von Termiten. Zcitschr. f. wiss. ZooL, 150: 451-467.
PFLUGFELDER, O., 1938b. Weitere experimentelle Untersuchungen iiber die Funktion der Corpora
allata von Dixippus morosus Br. Zeitschr. f. iviss. Zool., 151 : 149-191.
PIEPHO, H., 1952. liber die Lenkung der Insektenmetamorphose durch Hormone. Verh.
deutsch. Zool. Ges. (Leipzig), 1952: 62-75.
SCHNEIDERMAN, H. A., AND C. M. WILLIAMS, 1954. Physiology of insect diapause. IX. The
cytochrome oxidase system in relation to the diapause and development of the Cecropia
silkworm. Biol. Bull, 106 : 238-252.
SHAPPIRO, D. G., AND C. M. WILLIAMS, 1957. The cytochrome system of the Cecropia silk-
worm. I. Spectroscopic studies of individual tissues. Proc. Roy. Soc. London, Ser. B,
147 : 218-232.
TELFER, W. H., 1954. Immunological studies of insect metamorphosis. II. The role of a
sex-limited blood protein in egg formation by the Cecropia silkworm. /. Gen. Physiol.,
37 : 539-558.
WALD, G., 1952. Biochemical evolution. In: Modern Trends in Physiol. and Biochem., E. S.
G. Barren, Edit., Acad. Press, N. Y. : 337-376.
WIGGLESWORTH, V. B., 1934. The physiology of ecdysis in Rhodnius prolixiis (Hemiptera).
II. Factors controlling moulting and "metamorphosis." Quart. J. Micr. Sci., 77:
191-222.
338 CARROLL M. WILLIAMS
WIGGLESWORTH, V. B., 1936. The function of the corpus allatum in the growth and reproduction
of Rhodnins prolixus (Hemiptera). Quart. J. Micr. Sci., 79: 91-121.
WIGGLESWORTH, V. B., 1954. The Physiology of Insect Metamorphosis. Monographs in Exp.
Biol., No. 1. Cambridge Univ. Press.
WIGGLESWORTH, V. B., 1957. The action of growth hormones in insects. Symp. Soc. Exp.
Biol, 11 : 204-227.
WIGGLESWORTH, V. B., 1958. Some methods for assaying extracts of juvenile hormone of
insects. /. Insect Physiol., 2 : 73-84.
WILLIAMS, C. M., 1946a. Physiology of insect diapause: the role of the brain in the production
and termination of pupal dormancy in the giant silkworm, Platysamia cecropia. Biol.
Bull., 90 : 234-243.
WILLIAMS, C. M., 1946b. Continuous anesthesia for insects. Science, 103 : 57-59.
WILLIAMS, C. M., 1947. Physiology of insect diapause. II. Interaction between the pupal
brain and prothoracic glands in the metamorphosis of the giant silkworm, Platysamia
cecropia. Biol. Bull., 93: 89-98.
WILLIAMS, C. M., 1952a. Physiology of insect diapause. IV. The brain and prothoracic
glands as an endocrine system in the Cecropia silkworm. Biol. Bull., 103 : 120-138.
WILLIAMS, C. M., 1952b. Morphogenesis and the metamorphosis of insects. Harvey Lectures,
47: 126-155.
WILLIAMS, C. M., 1954. Isolation and identification of the prothoracic gland hormone of insects.
Anat. Rec.. 120 : 743.
Vol. 116, No. 3 June, 1959
THE
BIOLOGICAL BULLETIN
PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY
EMBRYOLOGICAL DEVELOPMENT OF THE POLYCHAETOUS
ANNELID, DIOPATRA CUPREA (BOSC) 1
M. JEAN ALLEN -
Marine Biological Laboratory, ]] 7 oods Hole, Massachusetts
Insofar as the writer knows, the normal embryology of Diopatra cuf>rea has
never been completely worked out. The main trouble seems to have been that
investigators, with the exception of Just (1922), have found that it is difficult
to activate the eggs of this species even when they appear ripe. Andrews had
similar difficulty with the eggs of the closely related species, Diopatra magnet
(since designated Onn[>his nia;/ua ). He made the statement (1891b, page 115) that
"attempts at artificial fertilization were unsuccessful" although the eggs seemed
ripe as indicated by their size and the large numbers present packing the coelom,
as well as the occasional finding of similar eggs amongst the larvae in the egg
masses which he found during the breeding season. However, Just (1922), in
a paper concerned primarily with raising mature Platynercis nicyalops from eggs,
noted (page 477), "Though it is usually stated that artificial insemination of Dio-
patra eggs is not possible, every attempt made by the writer . . . was successful,"
and that he reared Diopatra citprea to a length of 4 cm. No record of development
was given.
The problem of activation has remained a significant one throughout the course
of this investigation. \Yith perseverance (particularly initially ) larvae from many
batches of eggs have been raised during the course of several summers to a stage
where 6 sets of setae have been formed and, in the summer of 1958, a few were
raised to a stage with 7 sets of setae. Thus far two abstracts have been published
on this work (Allen, 1951, 1953) and more recently Costello ct al. (1957) have
included some additional previously unpublished data (furnished by the present
writer ) in their book on handling marine eggs and embryos.
The study of the development of D. citprea is still incomplete but enough addi-
tional material has recently been worked out so that it was thought advisable to
publish a more detailed account of development than has thus far been done.
There is little material in the literature on the development of the genus, Diopatra.
As further observations on living material were made, the confusion in the litera-
ture surrounding the development of the species, D. cuf>rca, became more apparent.
1 Supported in part by summer research grants from the University of New Hampshire
and Wilson College.
- Present address : Department of Biology, Wilson College, Chambersburg, Pennsylvania.
339
b
Copyright 1959, by the Marine Biological Laboratory
340 M. JEAN ALLEN
Observations made during the present study suggest that most, if not all, of the
material which has been published on the development of Diopatra cuprca has
been incorrectly attributed to this species, so that the investigations of the writer
may represent the only material published on the development of this polychaete.
MATERIAL AND METHODS
The adult worm. The characteristics and habits of the adult worms of this
species have been described by various investigators (Andrews, 1891a; Sumner,
Osborn and Cole, 1911; Hartman, 1945, 1951; ct a!.}. The parchment-like tubes
of these polychaetes, which are found in the intertidal zone, go down two to three
feet into the substratum. When disturbed the worm retreats into the tube so that
in digging for the adults one rarely obtains the whole worm. As a result, the
posterior tip with its four anal cirri is rarely seen. The head bears five occipital
tentacles and two shorter frontal tentacles. Larvae have been raised to a stage
when the five occipital tentacles and two anal cirri are noticeable. Males which are
sexually mature are cream to yellowish in color as a result of the sperm packed in
the coelom. In males with fewer sperm only the parapodia are yellowish in color.
Females when sexually mature are usually grey-green due to the color of the
eggs (which have a green pigment) packed in the coelom. This species is plentiful
in Woods Hole waters. Most of the collecting for this investigation was done at
Northwest Gutter, Hadley Harbor, Massachusetts, and some of it was done in
the harbor at North Falmouth and at Woods Hole, on the Buzzards Bay side.
The adults for the most part were kept in aquaria in running sea water. The
worms were fed every day or two with pieces of the mussel, Mytilus.
Procuring and handling living developmental stages. The writer has raised
larvae of Diopatra cuprca from mid-June through August following artificial ferti-
lization. The problem of activation of apparently ripe eggs was present throughout
this period but artificial insemination was more successful in June and July than
in August. This is contrary to the remark of Bumpus (1898, page 855) that
"the ova are nearly ripe in August."
During the breeding period of a sexually mature worm, the coelom becomes
packed with gametes. When eggs or sperm are needed, the posterior end of a
worm is exposed by cutting the end of the tube with scissors. The exposed portion
is then held lightly with forceps. This usually results in the worm's pinching
off its posterior segments. Eggs were obtained from the isolated posterior sections
by slitting the body wall along the bases of the parapodia with No. 5 watchmaker's
forceps. Eggs thus obtained were washed in Syracuse dishes with sand-filtered
sea water. In general, spermatozoa were obtained by making a small slit at the
base of a parapodium with a No. 5 watchmaker's forceps and diluting the "dry"
sperm with sand-filtered sea water. Under the dissecting microscope ripe sperm
were observed to be active immediately. Polyspermy should be avoided.
Within a few minutes after insemination the eggs were washed several times
with sand-filtered sea water. Usually they were given fresh sea water one to
two hours later. If development were normal, ciliated larvae developed at room
temperature within three hours after insemination. At this stage larvae usually
were transferred to stender dishes and placed on the sea water table in a moist
DEVELOPMENT OF DIOPATRA CUPREA 341
chamber with 90 % sea water in the bottom. The water was changed at least once
a day thereafter.
Apparently egg laying in D. cuprca is a phenomenon rarely observed (Sumner
et al., 1911). In only one instance did the writer observe natural egg laying in
the laboratory. This was on the evening of June 23, 1949. A worm tube was
picked up and eggs were immediately released in a transparent, only slightly
viscous, jelly which dissolved readily in sea w^ater. The eggs w r ere fertilized arti-
ficially and almost \00 c /c cleaved. Only a few other times in the experience of
the writer has fertilization approached 100%, as the method of artificial insemina-
tion described is frequently unsuccessful. To get a batch of eggs with 50% of
the eggs cleaving is good.
Observations were made on living stages with the dissecting and compound
microscopes and, in the summer of 1958, additional observations w-ere made with
the phase microscope. For study and for photomicrographs the ciliated stages
were slowed down with a little dry MS-222 (tricain) added with a dissecting needle
to a drop of filtered sea water containing the larvae (optimal concentrations for
quieting various larval stages were not determined).
For the setal studies the larvae were placed on a slide in a drop of filtered sea
water and then a cover slip was applied. They were examined briefly under a
magnification of X 430 and then left to dry a little. This treatment in many cases
spread out the setae which were then studied in more detail under X 430.
Handling of fixed material. Various stages were fixed, paraffin-embedded, and
serially sectioned (usually at 7 or 10 micra). Whole mounts of stained and un-
stained stages were also made. The fixatives used for the early stages were usually
Allen's B-15 or Bouin's, and for later larval stages Schaudinn's or Bouin's heated
to 60 C. A series was also fixed in Meves'. A variety of stains was tried includ-
ing Heidenhain's hematoxylin, Harris' hematoxylin, acetocarmine, alum-cochineal,
Giemsa's, toluidin blue, and Feulgen's. Sections and whole mounts usually were
mounted in Permount or Canada balsam. It was considered important to use
such whole mounts to make a cell lineage study through at least the early cleavage
stages. However, the method which had given excellent results with cleavage
stages of the gastropod, Crepidula, failed completely with Diopatra. Various other
techniques have been tried, including pre-treatment to remove lipids or ribonucleic
acid, either of which might take up the stain in the cytoplasm. To date a technique
has not been developed that would stain the chromosomes and enable one to follow
the orientation of the spindles without staining the cytoplasm.
NORMAL DEVELOPMENT
The writer has indicated already (1951 ) that the cleavage of Diopatra cnprea
occurs with amazing rapidity, functional cilia being formed within three hours
after insemination. Prior to this age, it is difficult to construct a time table of
development because there is considerable variability among different batches of
eggs and also among different eggs in the same batch, particularly in cases in which
low percentages of fertilization occur. The following represents a slight elabora-
tion of the schedule recorded in Costello ct al. ( 1957) which is based on the writer's
data obtained over several summers. The times are recorded from insemination
at temperatures of 21-24 C.
342 M. JEAN ALLEN
Stage Time
First polar body 15-20 minutes
Second polar body 20-30 minutes
Two- to four-cells 40-60 minutes
Eight-cells 50-90 minutes
Mid- to late cleavage 90-120 minutes
Functional cilia 3 hours
Apical tuft (apparent in some) 8-9 hours
Apical tuft (present in all normal larvae) 12 hours
Rotating trochophores 24 hours
2 to 3 sets of internal setae 36 hours
3 sets of external setae, no tentacles 2 days
4 sets of external setae, some with 3 tentacles 34 days
5 sets of external setae, 5 tentacles 4^-5 % days
6 sets of external setae, 5 tentacles 6^-8 days
7 sets of external setae, 5 tentacles 13-17 days (typical?)
The various stages of normal development are described in more detail below.
The unfertilized e*<<
*i. .' ;*v.
^;-v
17
,. ._
18
19
PLATE III
EXPLANATION OF FIGURES
FIGURE 16. Section of two young eggs in the coelom, showing attached algal-like strings
of cells (second string not in plane of section), the nucleus and prominent nucleolus in the egg
and in each of the "nurse" cells. FIGURE 17. Fertilized egg in metaphase I, showing the
DEVELOPMENT OF DIOPATRA CUPREA 349
Larvae of 2 days, 8-12 hours. These larvae, about the same width as the pre-
ceding, have elongated by about 100 micra and measure approximately 325 X 200
micra (in measurements of larvae, widths indicate the broadest portion). The
tendency of some larvae to settle on the bottom at this stage seems to be correlated
with the secretion of mucus ; other larvae, however, are still actively rotating,
positively phototactic swimmers. Their invariable swarming towards the light
makes changing the water easy at this stage. The larvae usually have differentiated
three sets of setae externally (sometimes only two), with a fourth set forming
internally in some. The third set, though extending externally, may be incom-
pletely formed ( see Table I ) .
Larvae have two prominent red eyespots and several pigment spots anteriorly.
The apical tuft, though reduced, is still prominent, being roughly 55 micra long.
The anterior arms of the opaque Y-shaped mid-gut surround the colorless pharynx.
Scattered black pigment spots can be seen in surface view. The prototroch is still
present as is the telotroch of longer cilia, and between them are shorter cilia.
Rarely seen, but very clear when observed with the phase microscope, is a little
patch of cilia just posterior to each set of setae. The characteristic refractile drop-
lets are still present at the widest part of the prototroch and this area appears con-
tinuous with the mid-gut region. The hind-gut is not clearly defined.
Larvae of 3 days, 8 to 12 hours. Larvae of this stage are slightly longer and
usually somewhat narrower than those of the preceding stage (for example, one
measured 400 X 180 micra). A few are still swimming and are positively photo-
tactic, but most tend to crawl on the bottom, secreting mucus as they do so. They
sometimes stick together in clumps in which case they should be separated before
they die. Some have formed transparent slime tubes. Usually four functional
sets of setae are visible externally (Figs. 11 and 12) and the parapodium of the
first setigerous segment has two protrusions, a finger-like postsetal lobe and a
shorter presetal lobe (Fig. 33). A tuft of cilia, rarely observed, is present at the
base of each parapodium. An apical tuft is still prominent but is often missed
even with the phase microscope, for it tends to bend backward when slowed with
MS-222. The fairly broad prototroch extends from the anterior level of the eye-
spots to just anterior to the first set of setae (compare Figures 11 and 12). The
prominent telotroch lies just posterior to the last set of setae (Fig. 12). Incipient
jaws have differentiated which have an extra toothed plate on one side of the
otherwise symmetrical maxillae (similar to Fig. 29). This asymmetry of the
jaws is characteristic of the adult. These larval jaws are movable indicating that
pharyngeal muscle is differentiating. Peripheral vacuolated mucous cells are
clearly defined. Two of the large posterior vacuoles may be visible externally
(Fig. 11). The broad anterior region, with its bubbly cytoplasm, still appears
contrast between yolky and non-yolky cytoplasm. FIGURE 18. Two-cell stage in metaphase
of second cleavage showing that the CD blastomere is larger than the AB. Note the fertiliza-
tion membrane. FIGURE 19. Three-cell stage showing that the CD blastomere sometimes
cleaves before the AB. This may represent abnormal development. FIGURE 20. Blastomeres
of late cleavage held firmly within the egg membrane. The peripheral vacuolated region is
beginning to appear and one blastomere is in metaphase. FIGURE 21. Longitudinal section
through an early ciliated stage, approximately three hours, showing central mound of cells at
the animal pole, two of the four vacuolated plates of cells, and small round body (probably a
polar body) beneath membrane at the right.
350
M. JEAN ALLEN
26
PLATE IV
DEVELOPMENT OF DIOPATRA CUPREA 351
continuous with the droplet-filled darker mid-gut region (Fig. 11, droplets not
in focus ) . The arms of the Y-shaped mid-gut surround the pharynx. The thick-
\valled, rather transparent hind-gut, presumably ectodermal, is forming. In some
batches, buds of the three more dorsal tentacles are obvious, as well as the rudi-
ments of the two anal cirri.
Further internal structure can be seen in serial sections. Figure 27 is a sagittal
section of this stage, showing pharynx and incipient jaws, narrow esophageal por-
tion, and the mid-gut which has no lumen as yet and contains some dark pigment
spots. A coelom has appeared, two flattened nuclei of the ventral peritoneal cells
being clearly visible. The ventral body wall is thick compared with the dorsal
and a ventral nerve cord is differentiating just beneath the peritoneum. A cerebral
ganglion is visible just anterior to the pharynx. At least four large posterior
vacuoles are visible.
Larvae of 4 days. By this stage four sets of setae are visible externally and
a fifth is beginning to form internally. The apical tuft was not observed and
proto- and telotrochs are reduced. A few superficial scattered dark pigment spots
can be seen in living larvae, and the endodermal and mid-gut contains some pig-
ment. The transparent hind-gut has a narrow lumen. In most larvae, three well
developed tentacular protrusions have appeared (Fig. 13) and buds of the two more
ventral tentacles, as well as two anal cirri. Also visible through the body wall
are the developing jaws (Fig. 13).
Larvae of 4 days, 8 to 12 hours. Larvae of this stage have settled on the bot-
tom and some may be observed in transparent slime tubes. They have four sets
of functional setae externally with a fifth beginning to protrude in some. The
presetal and postsetal lobes on the parapodia of the first setigerous segment are
retained in this stage and in the subsequent stages described (compare Fig. 33).
Five occipital tentacles are present, one mid-dorsal, two dorso-lateral, and two
ventro-lateral ones, the last two being shorter. Two anal cirri are represented by
PLATE IV
EXPLANATION OF FIGURES
FIGURE 22. Frontal section of 24-hour trochophore (anterior at right) showing pharynx
near center, light undifferentiated yolk mass just posterior to it, and mesodermal bands flanking
the mid-gut. FIGURE 23. Transverse section through the central mound in a larva similar to
that in Figure 21, showing the four plates of vacuolated cells surrounding the mound. FIGURE
24. Frontal section through a 36-hour larva (cut at 15 micra) showing pharynx (note
anaphase), light undifferentiated yolk mass, and four prominent posterior vacuoles. FIGURE 25.
Transverse section through the pharynx of a larva that is similar to Figure 24, showing
peripheral vacuolated cells and the cilia penetrating the larval membrane. FIGURE 26. Frontal
section through a 36-hour larva (cut at 10 micra) showing the pharynx (note anaphase),
yolk mass, and two large posterior vacuoles. Two sets of internal setae are forming (tip of
lower arrow) and two of the mucus-secreting cells with basal nuclei are visible (tip of upper
arrow). FIGURE 27. Sagittal section through larva of 3% days, with four sets of setae. The
jaws are beginning to form in the pharynx, the cerebral ganglion (light area) is anterior to
them, and the mid-gut (without a lumen) is posterior to them. Note also the posterior vacuoles,
the coelom around the gut, and the peritoneal cells (two nuclei clear) lying in contact with the
ventral nerve cord. The ventral body wall is thicker than the dorsal. FIGURE 28. Sagittal
section through larva of 5^/2 days, with five sets of setae. The same structures seen in Figure 27
may be noted, although they are more highly differentiated. The mid-gut region now has
a lumen continuous with the intestine which opens by way of a ventral anus, and some of the
mid-gut cells have black pigment.
352
M. JEAN ALLEN
32
33
PLATE V
EXPLANATION OF FIGURES
FIGURE 29. Differentiating jaws of a larva of 4% days showing toothed asymmetrical
maxillary plates on the left (an extra toothed portion is present on the left side) and mandibles
on the right. Note also the bundle of curved pointed setae from the first setigerous segment.
FIGURE 30. Jaws from a larva of approximately eleven days, showing further differentiation
DEVELOPMENT OF DIOPATRA CUPREA 353
buds in some larvae of this stage, but are more obvious in others. Tufts of cilia,
visible at the eye level in some, probably represent the remains of the prototroch.
A prominent telotroch is still present. Also visible externally are jaws consisting
of asymmetrical maxillary plates with well defined teeth and differentiating man-
dibles (Fig. 29). An esophagus is differentiating between pharynx and mid-gut,
and the latter continues posteriorly into the hind-gut. The dark yolk mass and
droplets are restricted to the mid-gut and black pigment is visible in its lining.
Some of the larvae appeared to be feeding on microorganisms.
Larvae of 5 l / 2 to 7 1 /. days. Larvae of 5 l / 2 days have 5 sets of functional setae
although the last set is usually not completely formed ; in some cases a sixth set is
differentiating internally. Some larvae may be observed in transparent slime tubes
on the bottom, and in one instance a larva was observed turning around in its tube.
Larvae which have not formed tubes often stick to the bottom at this stage and
may constrict in two in attempting to free themselves. The five occipital tentacles
are "knobby" and well developed (Fig. 15) : the three more dorsal ones are ap-
proximately 1 50 micra in length and have two basal segments by 7 days ; the two
more ventral ones are shorter and have one basal segment each. Two anal cirri
are well developed (approximately 30 micra in length) and "knobby" (Fig. 15).
A number of the differentiating internal structures of this stage can be illus-
trated by Figure 28. This is a sagittal section through a larva with 5 sets of setae
(SVs days old) and with well developed jaws associated with the pharynx. The
mid-gut is patent throughout, its lumen being continuous with that of the hind-gut
which, in turn, opens ventrally through the anus. The coelom has enlarged as
compared with the preceding stage (Fig. 27). Nuclei of two of the flattened peri-
toneal cells are visible ventrally (the peritoneum can also be seen in living larvae),
and the cerebral ganglion and ventral nerve cord are clearly visible.
Larvae of 8 days, 8 hours and older. By 8% days, 6 sets of setae have formed
externally in most cases and are complete, or almost so. However, some larvae
take one to three days longer to form the sixth set (a few take even longer). The
black jaws are well differentiated and active at these stages. The asymmetrical
maxillary plates have a medial toothed margin in each half (as well as the toothed
as compared with Figure 29. The bundle of curved pointed setae from the first setigerous
segment and an additional slender rod are also visible. FIGURE 31. Curved pointed setae on
the first setigerous segment of a larva of 8 l /2 days, with six sets of setae. Characteristically,
four such setae are present but here the curved tip of a fifth set is appearing (off tip of right-
hand arrow). Note also the aciculum with a deeper origin than the external setae, and the
slender rod (off tip of left-hand arrow). FIGURE 32. Two anterior parapodia in a larva of
approximately 5 days, with four sets of setae. The curved, pointed, claw-like setae of the first
setigerous segment are visible; contrast these with the short-tipped winged capillary type (one
in focus) characteristic of the second, third, and fourth setigerous segments. FIGURE 33.
Parapodia of first and second setigerous segments (anterior at right) in a larva of 5 l /s days,
with a small fifth set of setae. The finger-like postsetal lobe and the smaller presetal lobe
which are characteristic of the first parapodium are visible. FIGURE 34. Setal types from the
fourth, fifth, and sixth setigerous segments ( anterior at left ) . Note the three short-tipped
winged capillary setae ( and basal aciculum ) characteristic of the second, third, and fourth
setigerous segments, the two bidentate acicular setae and one long-tipped winged capillary seta
(and basal aciculum) characteristic of the fifth, sixth, and seventh setigerous segments. The
two-pronged tip (off tip of arrow) of the second bidentate acicular seta developing in the sixth
setigerous segment is also visible.
354 M. JEAN ALLEN
additional piece ; see Figure 30) and work in scissors-like fashion with the man-
dibles either held stationary or with both jaws working alternately in an antero-
posterior direction. The maxillary plates move forward, open, and then close
during their posterior movement.
In a few cases a culture of algae was allowed to accumulate in the stender
dishes. The larvae in these cases appeared to be feeding on the algae although the
mid-gut was still dark with stored food material and contained large food vacuoles.
The larvae upon occasion will eat their own kind as in one instance black jaws of
another larva were observed in the mid-gut of an lli/^-day larva, and one larva
appeared to be "gnawing" on another living larva stuck to it. An active rolling
movement from side to side was noted in the esophageal region of a number of
larvae, and in one food particles were noted in this region of the fore-gut which is
very thick-walled.
The five occipital tentacles are similar to those of the preceding stage except
that they are longer, the dorsal ones measuring approximately 225 micra in 9-day
larvae. Anal cirri in larvae of this age are approximately 50 micra long.
Headless larvae, capable of moving about, were observed occasionally. Larvae
of this age tend to stick to the bottom of the dish, often on their backs, in which
case they may constrict in two in an attempt to become free.
The larvae were not fed (except for any microorganisms which came through
the sand-filtered sea water ) and may live as long as the yolk material lasts in the
mid-gut (this area becomes transparent when the food supply is gone). Over
several summers, 6 sets was the maximum number of setae observed in these larvae
of D. euprea. However, in the summer of 1958, 7 sets were recorded for nine
larvae, in two (from different batches) by 13Vi> days of development, in one by
l4 l /2 clays, in two (from different batches ) by 171/2 days, and in one by IS 1 /-; days
of development. One larva from this last batch did not develop a seventh set until
the twenty-fifth day, and another from this batch until the thirtieth day of develop-
ment. One from a different batch developed a seventh set by the twenty-sixth
day. Among these larvae the oldest lived for 13 days after developing a seventh
set of setae, dying at an age of 30 1 /o days. Most larvae died before developing a
seventh set. The types of setae are described in more detail below.
Types of larval setae and their order of appearance. By the time 5 sets of
setae have formed in these larvae, four types of setae have differentiated. The type
(or types) and distribution of each are characteristic for each segment. As indi-
cated in Figures 29 to 34, those in the first setigerous segment are different from
any of the others, those in segments two, three and four are similar, and those in
segment five are new types which are retained in segments six and seven. One
aciculum is associated with each setigerous sac at all levels. These internal basal
setae have a deeper origin than the others (Figs. 31 and 34) and appear to direct
the movements of the external ones. Once the direction of movement has been
determined at any one level, the external setal complement seems to work against
the aciculum which thus acts as a fulcrum.
The following tables indicate the setigerous segments, the number and types
of setae in each setigerous sac (omitting acicula which are present at all levels),
the time of appearance at each level, and the setal complement of each segment at
successive developmental stages. Photomicrographs are presented to help in the
DEVELOPMENT OF DIOPATRA CUPREA
355
TABLE I
Time of appearance of setal types in various segments
Setigerous segment
1
Type of setae
3C
3C + tip of C
4C
2S
3S
2S
3S
2S
3S
IB, 1L
2B, 1L
IB, 1L
2B, 1L
IB, 1L
2B, 1L
Time of external appearance
2 days
3^ days
4^ days
2 days
1\ days
2\ days
3 days
1\ days
3^ days
4^ days
5J days
7 days
8 days
13 days (typical?)
identification of these setal types. The key to the letters in the tables is as follows :
C curved pointed type (Figs. 29 to 33), S short-tipped winged capillary type
(Figs. 32 to 34), B bidentate acicular type (Fig. 34), L long-tipped winged
capillary type (Fig. 34).
The individual setae develop in a disto-proximal direction, the tip differentiating
first. This was observed repeatedly in "dry" mounts. For example, in the first
setigerous segment of a 4-day larva, three curved setae are complete and just the
curved tip of the fourth is visible internally. In the fifth setigerous segment of
4- to 6-day larvae, one of the bidentate setae and the aciculum appear to develop
simultaneously; then the long-tipped seta of this level develops and before it is
completed the two-pronged tip of the second bidentate seta has developed inter-
nally (Fig. 34). This sequence of setal development noted in setigerous segment
number five is followed also in the sixth and seventh segments.
In one larva (8V.> days old) the distal tip of a fifth seta of the curved type
characteristic of segment 1 was noted (Fig. 31). This indicates that 4 curved
setae may not be the full complement for this level ; however, this one case may
not represent the typical condition. Also, in a number of larvae of 8 days, 8 hours
TABLE II
Distribution of setal types by segments at different stages
Setigerous segment
Larval stage
1
2
3
4
5
6
7
3 parapodia
3C
3S
3S
4 parapodia
3C
3S
3S
3S
5 parapodia
4C
3S
3S
3S
2B, 1L
6 parapodia
4C
3S
3S
3S
2B, 1L
2B, 1L
7 parapodia
4C
3S
3S
3S
2B, 1L
2B, 1L
2B, 1L
356 M. JEAN ALLEN
and older, a tiny slender rod was noted in both of the first setigerous sacs (Figs.
30 and 31). Its presence was not observed consistently throughout this age group.
As suggested by the tables, the setae once formed were retained throughout the
period of observation. This is in contrast to Wilson's analysis of the succession
of larval bristles in Nereis pclagica (1932) in which he found that as successive
setae formed, the ones more anterior began falling out.
DISCUSSION
Certain aspects of the development of the egg and of the early larvae of Diopatra
cuprca seem to be peculiar to this species, and in other instances to this genus or
to the closely related genus, Onuphis. The curious process by which the eggs are
formed in the ovary has been described by Andrews (1891b) and recently has been
briefly reviewed by Costello ct al. (1957). Lieber (1931) has described this proc-
ess for D. aniboinensis. Andrews (1891b) suggests that the algal-like strings of
"nurse" cells attached to the developing egg may have a supportive function while
the eggs are floating free in the coelom, rather than a nutritive one. However,
Treadwell (1921, page 81) states that in the eggs of Diopatra cuprca at Woods
Hole he was able to demonstrate a "definite communication pore between the ovum
and the first cell of the chain, indicating that they are true 'nurse' cells." Lieber
(1931 ) in a detailed study of oogenesis in Diopatra described and figured a cyto-
plasmic connection between the developing egg of D. aniboinensis and its attached
"nurse" cell and concluded that the cells were, in fact, nutritive in function and,
therefore, properly termed nurse cells. The communication pore noted by Tread-
well (1921) may conceivably represent the area where an amoeboid process of the
egg could contact the cytoplasm of the "nurse" cell.
Lieber ( 1931 ) has described a micropyle in the egg membrane of D. aniboincnsis.
The defect observed near the vegetal pole in some eggs of D. cuprca in the present
investigation may be a micropyle, although Andrews (1891b) makes no mention
of it in either D. cuprca or D. uiagna. These defects may instead represent the
remains of the communication pore noted by Treadwell (1921) in the developing
oocyte.
It has been noted that the ripe eggs of Diopatra cuprca appear to be perforated.
The canalicular nature of the membrane has been demonstrated in stained eggs
of Diopatra by Lieber (1931). A porous membrane is not restricted to the eggs
of Diopatra but has been noted in other polychaete eggs, for example, those of
Arcnicola cristata (Wilson, 1882).
Retention of the egg membrane as a larval cuticle (noted in D. cuprea) ap-
parently is not uncommon among polychaetes. Wilson (1882, page 295) states,
: 'The persistence in some cases of the chorion as the larval cuticle is a remarkable
occurrence entirely confined, so far as known, to the Chaetopods and Gephyrea,
and by no means universal among them." Examples of species which retain the
original egg membrane are Clyuienella torquata and Arcnicola cristata (Wilson,
1882), Nereis direr sic olor (Dales, 1950), and Thary.r inarioni (Dales, 1951).
The four anterior vacuolated plates of cells which have formed by the time
ciliation has been attained are peculiar to this form insofar as the writer knows,
and appear to originate from the four groups of prototroch cells.
DEVELOPMENT OF DIOPATRA CUPREA 357
The significance of the curious arrangement of yolk spheres into peripherally
located yolk plates has not been determined, for the main mass of yolk remains
in the central endodermal position (mid-gut region) of the trochophore. One pos-
sibility is that these peripheral plates may serve as a more efficiently placed food
supply for the rather precocious development of the setae and associated muscle
strands which differentiate from the mesoderm just medial to them.
As has been noted in the introduction there seems to be considerable confusion
in the literature concerning the identification of larvae and earlier stages ascribed
to Diopatra cuprea. It is well known that larval types are difficult to identify.
Two important characteristics used for distinguishing between larvae are the jaws
and setal types. The conspicuous asymmetry of the maxillary plates in Diopatra
cuprea has been noted (Figs. 29 and 30). Monro (1924), in his description of the
post-larval stage of D. cuprea, also pictures the unpaired, toothed plate associated
with the otherwise symmetrical maxillae. This asymmetrical jaw type is charac-
teristic of adult onuphids and eunicids. The functional significance of unpaired
maxillary plates in otherwise symmetrical jaws, which appear to work in scissors-
like fashion, is obscure. Comparing the diagram of the upper jaw pictured in
Monro (1924, Fig. 6, page 197) with the writer's photomicrograph of the jaws
of an 11 -day larva (Fig. 30), one may conclude that they are closely similar and
in all probability could have come from larvae of the same species when one con-
siders the difference in age. Monro (1924) includes a brief discussion of the
possible evolution of jaws within the eunicids and closely related groups.
Setae develop precociously in Diopatra cnprca. at least as compared with some
of the nereids, such as Nereis pelagica (Wilson, 1932) and Nereis divcrsicolor
(Dales, 1950). The importance of setal types in distinguishing between larvae is
indicated by the work of Wilson (1932), Krishnan (1936), Dales (1950), et al.
A comparison of the setae pictured here with the description and diagrams in
Monro's post-larval stage (1924) suggests that the larvae described by Monro
belong to a closely related species, if not to D. cuprea. Development of the first
setigerous segment (Monro, 1924, Figure 2, and text, page 195) is in agreement with
the findings described in the present study, but Monro indicates that from the second
through the fifth set all setae are of the short-tipped winged capillary type. The
view pictured is not clear (Fig. 3, page 195), and this setal type may or may not
fit the type shown in the present investigation (Figs. 32, 33, and 34). In con-
trast to Monro's larvae, the fifth set of setae observed in the present study has a
new setal complement which includes a bidentate acicular type which is retained
in segments 6 and 7 (Fig. 34). Beginning on the sixth segment of Monro's
larvae a setigerous type (Fig. 4, page 196) appears which probably could be de-
veloped from the bidentate acicular type described here ( Fig. 34 ) by the develop-
ment of a hook. However, to be comparable to the larvae described by the writer,
this hooked type should begin on the fifth parapodium instead of the sixth. Thus,
the two species may not be identical.
Wilson (1882) describes and figures some early stages in the development of
a polychaete which he identifies as Diopatra cuprea. These larvae, however, were
obtained from gelatinous egg masses, and Andrews (1891a, 1891b) states that these
early stages and larvae described by Wilson do not belong to Diopatra cuprea
but to Diopatra uiagna. Monro (1924) notes that Andrews does not give the
358 M. JEAN ALLEN
basis for his statement and Monro, therefore, questions its validity. Treadwell
(1921) has shown that the polychaetes described in the literature as D. magna
in reality belong to another genus which he has designated as Onuphis. Both
Diopatra and Onuphis are now accepted as distinct genera although they are
closely related ones (Dr. Marian H. Pettibone, personal communication; also see
Hartman, 1945, page 24, and Hartman, 1951, page 51, for keys separating these two
genera). Treadwell (1921) further points out the possibility that the larvae de-
scribed by Wilson are really those of Onuphis magna and seems inclined to agree
with Andrew's interpretation. A comparison of the ciliated larva pictured by
Treadwell from the gelatinous egg masses of Onuphis magna (1921, Plate 7, Fig-
ure 5) with that figured by Wilson (1882, Plate XXIII, Fig. 10) shows more
similarity between these two larvae than between Wilson's larvae and those of D.
cuprca described in the present study.
Comparing Wilson's larvae with the larvae pictured here, raised from the fer-
tilized eggs of D. cuprca, certain differences are noted. No stages in the present
study were observed that were as pear-shaped as Wilson's Figures 89 and 90
(Plate XXI), nor was any stage observed so markedly spotted with pigment as
the larva in Wilson's Figure 89. Further, the rudimentary apical tuft shown is
in marked contrast to the prominent apical tuft in the larvae here described. A
comparison of larvae with five sets of setae shows that there are differences be-
tween those of Wilson (1882, Plate XXIII, Fig. 10, and description on page 289)
and those pictured and described by the writer. In Diopatra cuprca, in the present
study, no dorsal cirri were observed, five occipital tentacles are present in normal
larvae at this setal stage, and the mid-dorsal tentacle is almost the same size as
the dorso-lateral (contrast Wilson's Fig. 10, Plate XXIII). Also a clearly defined
pharynx and well developed jaws are visible at this stage (Figs. 14 and 15 of the
present paper ; however, Wilson and Treadwell may have intentionally omitted
internal structures from their drawings ) . Further, the enlarged tip of the one
setal type shown in Wilson's larva (Plate XXI, Fig. 91) is different from any here
described for D. cuprca (Figs. 31 and 34), although it is possible that this type
might develop in a later stage.
Distribution of the two species in question provides further evidence concerning
the possibility of erroneous identification of their larvae. Both Diopatra cuprca
and Onuphis magna are found intertidally in the Beaufort, North Carolina, area
(Hartman, 1945 ) and in the Gulf of Mexico (Hartman, 1951 ) ; there is, therefore,
a chance of confusing the egg cases of the two genera in these areas. Thus far,
however, D. cuprca is the only onuphid found intertidally in the Woods Hole area
(Dr. Marian H. Pettibone, personal communication), so to date there is no pos-
sibility of confusion between these two onuphids (D. cuprca and 0. magna) in
the intertidal zone at Woods Hole. The writer is led to the conclusion, there-
fore, that the stages pictured by Wilson do not belong to Diopatra cuprca and
probably belong to Onupliis magna (D. magna of Andrews) as Andrews has stated.
If Andrews is correct and the evidence presented here indicates that he is
then the gelatinous egg masses found by Wilson belong to Onuphis magna.
Insofar as the writer knows, gelatinous egg masses of D. cuprca have never been
found in the Woods Hole area where this species is common. She herself has
never observed them and Mr. Milton B. Gray, who has collected D. cuprea for
DEVELOPMENT OF DIOPATRA CUPREA 359
a number of summers in the Woods Hole area (both for investigators and for
Course work), has never seen them (personal communication). Circumstantial
evidence presented by Monro (1924) indicates that the eggs of D. cuprea are laid
inside the tube (where the larvae develop) rather than in gelatinous egg capsules
lying free on the sand. However, the possibility remains that Monro is not deal-
ing with /). cuprea but with a closely related species. The one time normal spawn
jelly was observed in the present study, it dissolved readily in sea water. This
property of the jelly and the facts that cilia develop early and that the larva forms
a prominent apical tuft suggest that D. cuprea may have a free-swimming stage.
The writer, with the above observations in mind, would like to suggest that
the egg masses with developing larvae which have been noted along the Gulf of
Mexico (Hartman, 1951) as well as at Beaufort, North Carolina (Andrews, 1891b;
Hartman, 1945; Wilson, 1882), belong to Onuphis inagna and not to Diopatra
cuprea. Both species have been described as occurring together in these areas
although their distribution along the Gulf of Mexico is somewhat different (Hart-
man, 1951 ).
With the confusion of these larval types apparent in the literature, the brief
study of the setal types of D . cuprea included here may serve as at least one criterion
for distinguishing between the species of onuphids in the future. The usefulness
of setal types is apparent if one compares the table given by Krishnan ( 1936, page
521) for D. I'ariabills (Southern) with the tables included here for D. citprea.
In summary, one is led to the conclusion that the early stages and larvae de-
scribed by the several investigators cited probably do not belong to the species,
Diopatra cuprea, but to a closely related genus or species, in two instances probably
to Onuphis niagna which is the Diopatra inac/na of Andrews.
Further, this would seem to indicate that the descriptions of the writer for
Diopatra cuprea are the only ones which can be correctly attributed to this species,
with the possible exception of Monro's post-larval description which may belong
to D. cuprea. The possibility remains, however, that some investigation not here
cited has escaped the writer's attention.
The problem of activation of the egg of D. cuprea will have to be solved before
this egg can be used to any extent either for experimental purposes or for class
use. Some histochemical tests have been run on these stages (Allen, 1957) and
it is hoped that in working further with the eggs of D. cuprea some of the problems
noted will be solved. Further details of development may then be worked out to
serve as a basis for experimental and histochemical studies.
SUMMARY
1. Larvae of Diopatra cuprea (Bosc) have been raised, following artificial fer-
tilization, to a stage with seven sets of setae. Observations on living stages and
also on fixed and stained preparations have been described and photographed.
2. Cell lineage studies have not been made, but observations indicate that the
early cleavages are typical of those for spiral cleavage and that the ciliated stage
(age, three hours ) has a typical annelid cross and apical rosette. It, therefore,
seems justifiable to conclude that the development of Diopatra cuprea follows the
typical spiral pattern and mosaic development characteristic of other polychaetous
annelids.
360 M. JEAN ALLEN
3. Peculiarities of the development of this polychaete, and possibly of closely
related species, are the following : the peculiar algal-like nurse cells attached to
the developing oocyte (also characteristic of Onuphis eggs) when floating free in
the coelom, the amazing rapidity of development to the free-swimming stage (three
hours ) , the four plates of cells which appear to develop from cells of the prototroch
and their peculiar posterior extensions into at least four plates of yolk spheres,
and the asymmetry of the maxillary plates.
4. Very little can be found in the literature on the embryology of the genus,
Diopatra, and at least two authors have pointed out the possibility of error as to
species in the identification of the developmental stages. Evidence is presented
here which indicates that the early embryological and larval stages described by
other investigators have been erroneously assigned to Diopatra cuprea.
5. If the above is correct and it would appear that Diopatra cuprea is the only
onuphid found intertidally in the Woods Hole area one may conclude that the
investigation presented by the writer is probably the only study recorded in the
literature on the early developmental stages of Diopatra cuprea (Bosc). This is
exclusive of Monro's description of the later (post-larval) stage which, if not be-
longing to D. cuprea, is undoubtedly closely related to this species.
LITERATURE CITED
ALLEN, M. J., 1951. Observations on living developmental stages of the polychaete, Diopatra
cuprea (Bosc). Anat. Rec., Ill: 550.
ALLEN, M. J., 1953. Development of the polychaete, Diopatra cuprea (Bosc). Anat. Rec.,
117: 572-573.
ALLEN, M. J., 1957. Histochemical studies on developmental stages of polychaetous annelids.
Anat. Rec.. 128: 515-516.
ANDREWS, E. A., 1891a. Report upon the Annelida Polychaeta of Beaufort, North Carolina.
Proc. U. S. Nat. Mus., 14 : 277-302.
ANDREWS, E. A., 1891b. Reproductive organs of Diopatra. /. Morph., 5: 113-124.
BORRADAILE, L. A., AND F. A. POTTS, 1935. The Invertebrata. Second edition. The Macmillan
Co., New York.
BUMPUS, H. C, 1898. The breeding of animals at Woods Holl during the months of June,
July and August. Science, 8: 850-858.
COSTELLO, D. P., M. E. DAVIDSON, A. EGGERS, M. H. Fox AND C. HENLEY, 1957. Methods
for Obtaining and Handling Marine Eggs and Embryos. Marine Biological Labora-
tory, Woods Hole.
DALES, R. P., 1950. The reproduction and larval development of Nereis diversicolor O. F.
Muller. /. Mar. Biol. Assoc., 29 : 321-360.
DALES, R. P., 1951. Notes on the reproduction and early development of the cirratulid Thary.v
marioni (St Joseph). /. Mar. Biol. Assoc., 30: 113-117.
y HARTMAN, O., 1945. The marine annelids of North Carolina. Duke Univ. Mar. Station, Bull.
no. 2.
HARTMAN, O., 1951. The littoral marine annelids of the Gulf of Mexico. Publ. Inst. Mar.
Sci., Univ. of Texas, 2 : 7-124.
JUST, E. E., 1922. On rearing sexually mature Plat\nereis mcgalops from eggs. Amer. Nat.,
56 : 471-478.
KRISHNAN, G., 1936. The development of Diopatra variabilis (Southern). Zeitschr. wiss.
Zoo/. Leipzig, 147: 513-525.
LIBBER, A., 1931. Zur Oogenese einiger Diopatra-arten. Zeitschr. itnss. Zoo/. Leipzig, 138:
580-649.
DEVELOPMENT OF DIOPATRA CUPREA 361
MEAD, A. D., 1897. The early development of marine annelids. /. Morpli.. 13: 227-326.
MONRO, C. C. A., 1924. On the post-larval stage in Dia/mtni citprca. Bosc, a Polychaetous
Annelid of the family Eunicidae. Aim. Mag. Nat. Hist., scr. 9, 14: 193-199.
"SUMNER, F. B., R. C. OSBORN AND L. J. COLE, 1911. A biological survey of the waters of
Woods Hole and vicinity. Part 2. Bull. U. S. Bur. Fisheries. 31 : 545-860.
TREADWELL, A. L., 1921. Leodicidae of the West Indian region. Carnegie Inst. Wash., Pub.,
no. 293.
WILSON, D. P., 1932. The development of Nereis pclagica Linnaeus. /. Mar. Biol. Assoc 18 :
203-217.
WILSON, E. B., 1882. Observations on the early developmental stages of some polychaetous
Annelides. Stud. Biol. Lab., Johns Hopkins Univ., 2: 271-299.
A CONTRIBUTION TO THE BIOLOGY OF A DEEP SEA ECHINOID,
ALLOCENTROTUS FRAGILIS (JACKSON) 1
R. A. BOOLOOTIAN, 2 A. C. GIESE, J. S. TUCKER AND A. FARMANFARMAIAN
Hopkins Marine Station of Stanford University, California
In February, 1957, a hydrographic team 3 from the Hopkins Marine Station
accidentally discovered a bed of Allocentrotus fragilis (Swann, 1953) at a depth
of 68 to 98 fathoms in Monterey Bay, California. This discovery was made during
a routine hydrographic run. At the time a mid-water plankton haul with a stand-
ard one-meter net was in progress. The Hopkins Marine Station research vessel,
the "Tage," had apparently drifted with the onshore current. When the net was
surfaced, to their surprise and delight, the team found approximately two dozen
specimens of the deep sea urchin, Allocentrotus. This was the first time that the
animal had been obtained alive and intact in large numbers. At this spot the
fathometer indicated 80 fathoms and a radio "fix" recorded the position of the boat
to be 3637'54" N and 12201'12" W. All subsequent hauls were started from
this station.
Since a project on the biology of the shore sea urchins, Strongyloccntrotus
pur pit rat its and S. francisannts, was in progress at the Hopkins Marine Station, the
chance finding of a bed of the deep sea urchins was of immediate comparative inter-
est. Consequently, whenever possible, studies were made on the biology of Al-
locentrotus for comparison with Strongylocentrotus.
The oceanographic vessel, "Tage," was used for all work reported here. For
dredging a four-meter beam trawl was employed. The average dredging time was
twenty minutes. The entire sample, consisting of a variety of organisms, was
brought into the laboratory in live condition in a tub of sea water. The animals
were sorted and placed in separate tanks of running sea water. The species were
identified and at times the number of individuals counted.
The gonad index of the sea urchins, indicating the reproductive condition of
the urchins, was determined as in previous studies, as were also the biochemical
constituents of body fluid and tissues (Lasker and Giese, 1954; Bennett and Giese,
1955).
Habitat of Allocentrotus
Some of the physical features of the habitat of Allocentrotus should be con-
sidered in order to gain an understanding of the conditions under which this species
1 This research was supported by USPH Grant 4578C to A. C. Giese. We are indebted to
Dr. L. R. Blinks, Director of the Hopkins Marine Station, for making available the facilities
of the laboratory, to Dr. R. L. Bolin for facilitating the use of the "Tage," to Dr. D. P. Abbott
for sustained interest in the study, and to Mr. Joseph Balesteri, skipper of the "Tage," for his
cooperation.
- Now at the Department of Zoology, University of California at Los Angeles.
3 Under the direction of Professor R. L. Bolin of the Hopkins Marine Station and including
Mr. Thomas Fast and Mr. Robert Aughtry operating with the financial assistance of Grant
N60NR-26127 and Grant NSF-G-1780.
362
A DEEP SEA ECHINOID 363
lives in this area in Monterey Bay. By systematic grid dredging, the area of the
sea urchin bed was estimated to be about one square mile. The depth of the area
in which the urchins were taken varies between 55 to 90 fathoms, the shallow part
of the bed lying on the continental shelf, the deeper part bordering the Monterey
Canyon.
Dredges at various depths indicate that the larger animals tend to inhabit the
deeper regions near the Canyon, whereas the smaller animals are more frequently
found in shallower areas. These results are summarized in Table I.
The area nearest the Canyon is relatively flat and is composed of gravel and
sand overlying gray silt (Galliher, 1932a, 1932b). From time to time, however,
large boulders mainly of granite and shale, the largest of which weighed approxi-
mately 15 kilograms, were brought up in the dredge. In the shale young urchins
were frequently observed in their burrows, as illustrated in Figure IE. As the
shoreline is approached the configuration of the bottom is somewhat changed, con-
sisting mainly of granitic rock and coarse sand.
TABLE I
Sizes of Allocentrotus taken at various depths
Bathymetrical range Range in size of test diameter*
in fathoms in mm.
55-65 11.2- 21.3
60-65 11.2- 18.0
68 13.3- 29.4
65-90 55.0-103.3
* The measurement was made across the widest part of the test (the ambitus).
Olga Hartman (1955) has published a photograph of Allocentrotus taken at.
350 to 400 fathoms in the San Pedro Basin 1 1 miles northeast of Avalon, Catalina
Island, California. It was found in a sandy mud which appears to be relatively
flat except for small mounds.
As this species has been taken from 48 to 417 fathoms (Clark, 1912), the data
considered in this paper represent only a limited aspect of the habitat of Allocentro-
tus. It is possible that for the larger range over which it occurs, physical condi-
tions other than those described above may obtain.
Animals associated zvith Allocentrotus
Since the organisms found in the same habitat as Allocentrotus may play a role
in the ecology of the species, all of the organisms which came up in the beam trawl
were identified when possible and counts of their numbers were made to ascertain
their relative abundance. These organisms are listed in Table II. It is observed
that protozoans, coelenterates, annelids, nematodes. mollusks, arthropods, echino-
derms and fishes are found in the association. The interrelationships between
the various forms have not been studied.
Because of the random nature of the sampling it is difficult to say much about
the relative abundance of the various species in the natural habitat. However, the
crab, Mursia, is usually obtained, sometimes in large numbers as is the holothuroid,
Stlchopus and an unknown tectibranch. The starfishes Mcdiaster, Pycnopodia,
364
BOOLOOTIAN, GIESE, TUCKER AND FARMANFARMAIAN
FIGURE 1. A, An adult Allocentrotus fragilis 67 mm. in diameter. B, A test of Alloccn-
t rot us fragilis 73 mm. in diameter. C, A photograph of the aboral half of the shell of Allo-
centrotus showing the gonads. D, The Aristotle's lantern and the peripharyngeal coelom of
Allocentrotus. E, A specimen of Allocentrotus fragilis (15 mm. in diameter) imbedded in its
shale burrow.
Henricia, Pterastcr and Astro[>cctcn are also rather likely to be among the speci-
mens brought up in the trawl. From the numerous species and their relative
abundance it seems likely that the habitat of Allocentrotus is one with relative
abundance of food.
Olga Hartman (1955) found AUoccntrotns in deep waters (350-400 fathoms)
in association with a variety of animals (legend to plate 2A) : "A two-foot square
A DEEP SEA ECHINOID
365
sample from the bottom yielded glass sponge, many foraminiferans, 20 or more
species of annelids, many ophiuroids, and a large percentage of new or little known
animals." In her photograph of the benthos a crinoid and a sea star are seen among
the numerous Allocentrotus which appear to be spaced about a meter from one
another.
It is of interest to note that a rhabdocoel parasite similar to Syndesmus jrancls-
canus commonly found in the shore urchin (Lehman, 1946) was observed in the gut
of several specimens of Allocentrotus, and the specimens are of the same size as
those found in Strongylocentrotus. One, two or three at most, were found in the
gut and the incidence of infection was low.
Protozoans
Foraminiferans
Coelenterates
Psammogorgus
Metridium senile
TABLE II
Animals taken in association with Allocentrotus fragilis
Echinoderms
Stylasterias sp.
Astropecten californicus
Luidia foliolata
Annelids
Three different species of polychaetes
Nematodes
A variety of specimens
Mollusca
Rosea pacifica (octopus)
Numerous unidentified small gastropods
Arthropods
Crustaceans
Munidopsis sp.
Spirontocaris sp.
Mursia quadichaudii
Paguristes sp.
Echinoderms
Asteroids
Mediaster aequalis
Pycnopodia helianthoides
Pteraster tessalatus
Henricia aspera
Orthasterias koehleri
Ophiuroids
Gorgonocephalus eucnemis
Two other species of brittle stars
Holothuroids
Stichopus californicus
Vertebrates
Fishes representing the following families :
Liparidae
Agonidae
Zoarcidae
Ophidiidae
Cottidae
Batrachoididae
Scorpaenidae
Bothidae
Pleuronectidae
Petromyzontidae
Entophenus tridentatus
Rajidae
Raja sp.
Chimaeridae
Hydrolagus colliei
Nutrition of Allocentrotus
Although the Allocentrotus bed occurs in the euphotic zone (down to 200 meters
according to Sverdrup ct al., 1942), no conspicuous algae have ever come up in our
numerous dredgings. The large algae serve as the main food of the shore urchins
of the genus Strongylocentrotus (Lasker and Giese, 1954; Bennett and Giese,
1955). The sediments collected along with Allocentrotus in the dredge hauls con-
sist of a variety of decomposing organic materials in which strands of algae, diatoms,
366 BOOLOOTIAN, GIESE, TUCKER AND FARMANFARMAIAN
sponge spicules, nematodes, foraminiferan and other shells, as well as other protozo-
ans are found among numerous bacteria. Sometimes live nematodes and protozo-
ans were observed in the mud.
The gut usually contains numerous olive-green pellets measuring 1.2 to 2.8
mm. in diameter, relatively compact but soft in texture. When these pellets are
crushed and examined microscopically they are found to contain many small glassy
rings (desmids?), foraminiferans, sponge spicules, a variety of diatoms, sand
particles and unidentifiable organic particles. Acidification with HC1 indicates that
most of the skeletal particles are silicious since they do not dissolve. Treatment
with concentrated HNCX oxidized all the fluffy organic material leaving the silicious
diatom skeletons, sponge spicules and glassy rings. In the collection of July 25,
1958 the intestines of all the animals sampled were more completely filled with
pellets than in the other collections. The pellets were, in addition, a more vivid
green than in all the other cases. Extracts indicated the presence of a brown pig-
ment, fucoxanthin, plus a large amount of chlorophyll. The feeding was correlated
with a rich plankton bloom in the surface waters nearby. In the collection made on
August 14, 1958, some reddish pellets consisting entirely of organic debris and
bacteria were found among the green ones. The constituents of the gut pellets are
shown in Figure 2.
Specimens of AUocentrotus which survive the hazards of the trip to the surface
and arrive at the laboratory in good condition remain alive for many days. When
the animals are kept out of water for even a brief time they lose body fluid and air
is trapped inside the test, after which they float and die. Normal animals move
about the aquaria like Strongylocentrotus purpitratus, though less actively, and they
adhere less firmly so that they are more readily knocked off by even a small push.
They right themselves much more slowly than the purple sea urchin. Attempts
were made to feed AUocentrotus with boiled potatos, Phyllospadlx (eel grass) and
the algae, Uh'a, Iridaea, and Gigartina, as well as with animal matter such as
crushed mussel (Mytilus) and crushed deep sea crab (Mwrsia) after several days of
fasting. The animals nibbled at some of the algae and at Mytilus and Mursia,
dropping the material after a while, then going down to the bottom of the aquaria
to nibble again. It would appear, therefore, that AUocentrotus is more selective
than ,S\ pitrpuratits, which eats almost any organic material when hungry and
shows sustained intake for hours. However, it must be remembered that the speci-
mens are being tested at sea level and at about 15-16 C. whereas they come from
a deep sea environment where they are subjected to about 15 atmospheres of pres-
sure and temperatures of about 9 C. It is difficult to say what their behavior
might be in their natural environment.
It has been shown that the gonads of a purple sea urchin are probably the main
storage organs of the animal, the gonads in a gravid animal increasing to a size
which all but obliterates the body cavity left unoccupied by the gut and its contents.
The relative mass of the gonads in gravid AUocentrotus is much less than that of a
gravid Strongylocentrotus. At its peak the gonad of AUocentrotus is still a delicate
structure, both in size and in color (pale creamy- white in the male and yellowish
in the female). The gut of an AUocentrotus is generally well filled with pellets,
but it does not appear to be as full as the gut of the two species of Strongylocentrotus
studied. It appears, then, that food is generally less available in deeper waters
A DEEP SEA ECHINOID
367
FIGURE 2. Food pellets of AUoccnirotus as seen under low and high powers. A, Food
pellets as removed from the intestine (X 6). B, Crushed food pellets showing desmids (X 60).
C, Diatoms and sponge spicules in crushed food pellets (X60). D and E, Foraminiferans in
crushed food pellets (X 60).
368 BOOLOOTIAN, GIESE, TUCKER AND FARMANFARMAIAN
than on the shore, except after an unusually rich bloom of plankton as in the col-
lection of July 25, 1958.
Like the gonad of the two species of Strongylocentrotus tested, the gonad of
Allocentrotus contains a little stored glycogen (0.36 to 0.83 per cent or an average
of 0.57 per cent of the dry weight), considerable protein (about 30 per cent of the
dry weight), and a large store of lipid (an average of about 28 per cent of the dry
weight). The chemical constitution of the gonad of Allocentrotus is much like
that of the gonads of other species of sea urchins although it is smaller in proportion
to body size. The perivisceral fluid, which is possibly one of the channels for distri-
bution of the food from the gut, contains nutrients in solution much like the same
fluid in the other species of sea urchins tested. Total nitrogen amounted to 3.78 to
4.98 milligrams per cent, non-protein nitrogen to 1.28 to 1.34 milligrams per cent,
and a small amount of lipid is present. A variety of cells is present in the peri-
visceral fluid, resembling those of the other species of sea urchin (Boolootian and
Giese, 1958) and a clot forms much as in the other species of sea urchins tested
(unpublished data).
Healthy specimens of Allocentrotus kept in aquaria at about 15 C. in the
laboratory defecate very slowly. This may be an indication of a rather slow rate
of digestion but it may be the result of the abnormal conditions in the laboratory.
When animals with the gut loaded with food were brought in on July 25, 1958,
they defecated copiously. Defecation may therefore depend upon how full the gut
is at the time of collection.
All specimens collected sooner or later fall prey to a peculiar disorder. Small
spots of dark red color begin to appear on the surface of the test. These spots
then spread, covering the animal with large blotches of color. The tube feet
degenerate and the spines fall off after which the animal dies. Microscopic ex-
amination of the spots indicates that they are composed mainly of dead eleocytes,
the pigmented cells of the perivisceral fluid.
Reproduction
The first collection of Allocentrotus in Februarv of 1957 contained individuals
j
in full reproductive condition, the gonads of many males and females containing
mature gametes in large numbers. The eggs were readily fertilized and normal
development to the pluteus followed. Development was best at temperatures
between 9 14 C., cleavage being inhibited by higher temperatures. 4 The same
was true for the second collection in March of 1957. However, the gonads of the
animals collected in April no longer contained ripe gametes. Thereafter storms
and other difficulties prevented collecting the urchins until September of 1957.
The gonads of animals sampled in September, October, November and December
of 1957 and in January of 1958 were well developed and of relatively large size
until they spawned between January and the end of February, 1958, when the
next collection was made. The gonads during the second breeding season were
never as well developed as those of the first season, nor was as good a development
of the embryonic stages observed. 4
4 The results on development of Allocentrotus are being published by Dr. A. R. Moore in a
separate report. We are indebted to Dr. Moore for permitting us to quote here and in footnote
6 from his unpublished data.
A DEEP SEA ECHINOID
369
The reproductive state of an animal can be ascertained by measuring the ratio
of the volume of the gonad to the wet weight of the animal (Lasker and Giese,
1954). This ratio times 100 has been called the gonad index. The average gonad
indices determined in this manner are plotted in Figure 3. The course of the
FIGURE 3
A.
o
o
X
UJ
Q
O
<
z
o
I I I I
I
1 I
J I
I I I I
I i
JFMAMJJASONDJFMAMJJASOND
UJ
LL)
O
Q.
50
40
30
20
10
B.
"no data
J L
JFMAMJJ ASONDJFMAMJJASOND
UJ
OL
$
a:
LJ
a.
5
UJ
10
C.
J I
I I I I I I I I I I I I
J I
I
J I
J FMAMJJ ASONDJFMAMJ JASOND
FIGURE 3. A, Gonad index of Allocentrotus at different times from February, 1957 to
July, 1958. B, Variations in phytoplankton during the years 1954 and 1955 as determined by
Barham (1957). C, Variations of thermal monthly averages between 100-200 m. as reported
by Skogsberg and Phelps (1946) for the years 1936 and 1937. Same locality as that used in
the present study.
370 BOOLOOTIAN, GIESE, TUCKER AND FARMANFARMAIAN
curve (dashed line) from April to September, 1957 is not known but since in
1958 the gonads of animals obtained in July were just beginning to enlarge, a period
of reproductive quiescence may have occurred from April to the end of June, 1957
as happened in 1958. 3
All of the urchins used in determining the gonad index were mature, varying
in wet weight from 45.5 grams to 264.0 grams and in test diameter G from 55.0
to 96 mm. Even a population of mature animals of similar size shows considerable
variability in gonadal development at a given time. During the period when the
gonads of some individuals are well developed and large, the gonads of other
individuals are shrunken or undeveloped. The variability of gonad size is con-
siderably smaller when the gonads are immature or spent.
The great variability in the gonad index during the breeding season may
indicate : 1 ) that some individuals do not have access to adequate food to ripen
or to maintain their gonads, 2) that some individuals have just spawned while
others are ready to do so, or 3 ) that some individuals may be immature when
others are gravid. A histological study of the gravid and non-gravid gonads
might make it possible to decide between these alternatives.
DISCUSSION
It is interesting to compare the biology of Allocentrotus jrai/ilis to that of the
intertidal sea urchin, Strongylocentrotus purpuratns and to that of the subtidal
urchin, S. franciscanus. Whereas the inshore urchins generally graze on algae,
Allocentrotus appears to graze on whatever organic material occurs in the substrate,
but chiefly on organic detritus, bacteria, and microscopic animals and plants of the
organic "rain." 5". pnrpnratns is, on the other hand, omnivorous. When trapped
in a burrow with an opening smaller than the test diameter it feeds largely on the
detritus brought by sea water. In a sense, then, Allocentrotus represents an exten-
sion of this special feeding habit of S. purpuratus.
Allocentrotus lives in a community of invertebrates and fishes perhaps fewer
in species and in numbers than the urchins of the intertidal and subtidal zone,
although no decisive comparison can be made between the two communities because
of the paucity of data for the deep sea community. It is also singularly interesting
that a rhabdocoel containing hemoglobin should be present in the gut of the deep
sea urchin as in the gut of shore forms.
The data gathered in 1957-58 suggest that Allocentrotus has an annual breeding
season although the span of the cycle cannot be defined precisely at the present
time. During the fall and winter months from September, 1957 to January, 1958
the gonad index remained high. In both 1957 and 1958 the gonad index fell pre-
cipitously between February and March. It is of interest to correlate 1) growth
of gonads, and 2) spawning with physical conditions in Monterey Bay. Among
the possible variables are 1) light, 2) temperature, 3) salinity and minerals and
4) planktonic bloom which may be correlated with up welling.
5 Only one Allocentrotus was obtained on August 14, 1957 but this male had a gonad index
of 6.72 per cent, suggesting that the gonads were probably increasing in volume. Because of
the general variability of size of gonads in any sample, the measurement is only indicative.
6 The largest test diameter observed in specimens from Monterey Bay is 103.3 mm.
according to Dr. A. R. Moore.
A DEEP SEA ECHINOID 371
Although day-length has been correlated with breeding cycles of some inverte-
brates and vertebrates (Borthwick ct al., 1956), it does not seem likely that it is
a controlling factor for Alloccntrotus because of the low intensity of light at the
depths in which this animal lives. However, some photoperiodic animals are
affected by very low light intensities and to them the span of illumination is of
greater importance than the intensity of the light. The possible action of light in
timing the reproductive cycle of Alloccntrotus is not excluded.
Cyclic variations in temperature of the habitat of Alloccntrotus have been ob-
served (Skogsberg, 1936; Skogsberg and Phelps, 1946). The data for the years
1936 and 1937 are given in Figure 3C at a depth between 100 and 200 meters. A
seasonal rhythm is seen with low and fairly constant temperatures in spring and early
summer. In May the temperature range at 150 meters was 8.2 to 8.5 C. in 1936,
and 7.9 to 8.4 C. in 1937. In July the temperature at 150 meters began to rise,
reaching a maximum by December at which time it ranged from 9.6 to 10.1 C. in
1936, and was 9.3 C. in 1937. The difference between highest and lowest tempera-
tures is greater during upwelling of cold waters than during the period of warmer
waters. The temperature variations may be correlated with three major water
movements : the Oceanic period lasting from September to October, the Davidson
current period lasting from November through February, and the Upwelling period
occurring from late February through August. The Oceanic period and the
Davidson Current generally coincide with the high thermal phase and the some-
what lower chlorinity, although chlorinity variation is never large (Skogsberg,
1936). The onset of upwelling in late February coincides with the spawning of
Alloccntrotus and may act as the trigger for initiation of the spawning. The sub-
sequent warmer phase coincides with the period of growth of the gonads. As is
to be expected, surface temperatures were found to be more variable than deep
water temperatures according to Skogsberg and Phelps (1946) and the more recent
CCOFI report of 1958.
The upwelling in Monterey Bay is followed by a phytoplankton bloom (Bar-
ham, 1956), as seen in Figure 3B. It is possible that the phytoplankton is used by
the planktonic larvae of Alloccntrotus and by the metamorphosed young urchins
themselves when they reach the sea bottom. In this way the timing of events
in the breeding cycle may ultimately depend upon the food supply, the larvae ap-
pearing at the most favorable time for their growth, namely, when phytoplankton
is most abundant. All of these attempts to explain the breeding cycle of Allo-
centrotus must be considered as tentative hypotheses for which substantiating data
are still needed.
SUMMARY
1. Following a chance collection of a deep sea urchin, Alloccntrotus jragilis,
from a depth of 80 fathoms, it subsequently became possible to collect the urchins
on numerous occasions from the same area.
2. The area of the bed was determined by grid dredging and the nature of the
habitat determined to be relatively flat, gravel and sand underlaid with gray silt
containing organic detritus and microscopic organisms.
3. The deep sea urchin appears to graze on the bottom since the organisms and
organic debris of the bottom sediment appear in little pellets in its gut.
372 BOOLOOTIAN, GIESE, TUCKER AND FARMANFARMAIAN
4. Many types of invertebrates are associated with Allocentrotus f including
various other echinoderms. A variety of fishes is found as well.
5. Individuals with mature gametes were obtained in February and March of
1957 and during the period of September. 1957 to January, 1958. Spawn-out ap-
peared to occur between February and March during both years.
6. Attempts to correlate the life cycle of Allocentrotus with various environ-
mental factors led to the suggestion that upwelling may trigger spawning. The
planktonic larvae then presumably develop during the most effective time when
the planktonic blooms occur.
LITERATURE CITED
BARHAM, E. G., 1956. The ecology of sonic scattering layers in the Monterey Bay Area, Cali-
fornia. Ph.D. Thesis, Stanford.
BENNETT, J., AND A. C. GIESE, 1955. The annual reproductive and nutritional cycles in two
western sea urchins. Biol. Bull., 109: 226-237.
BOOLOOTIAN, R. A., AND A. C. GIESE, 1958. Coelomic corpuscles of echinoderms. Biol. Bull.,
115: 53-63.
BORTHVVICK, H. A., S. B. HENDRICKS AND M. W. PARKER, 1956. Photoperiodism. In: Radia-
tion Biology. A. Hollaender, ed., McGraw-Hill Book Co., N. Y. ///: Visible and
Near- Visible Light, 479-517.
CALIFORNIA COOPERATIVE OCEANIC FISHERIES INVESTIGATIONS (CCOFI) REPORT 1956-58,
Progress Report. State of California, Department of Fish and Game. Marine Re-
search Committee 7-56.
CLARK, H. L., 1912. Hawaiian and other Pacific Echini. Mem. Mus. Comp. Zoo/., 34 : 209-
' 383.
GALLIHER, E. W., 1932a. Sediments of Monterey Bay, California. Mining in California, 28:
42-79.
GALLIHER, E. W., 1932b. Sediments of Monterey Bay, California. Ph.D. Thesis, Stanford.
135 pp.
HARTMAN, O., 1955. Quantitative survey of the benthos of San Pedro Basin, Southern Cali-
fornia. Part I, Preliminary Results. University of Southern California Publications.
Alan Hancock Pacific Expeditions, 19: 1-185 (see especially Plate 2A, legend).
LASKER, R., AND A. C. GIESE, 1954. Nutrition of the sea urchin, Strongylocentrotus purpuratus.
Biol. Bull.. 106: 328-340.
LEHMAN, H. E., 1946. A histological study of Syndisyrinx franciscanus, gen. et sp. nov., an
endoparasitic rhabdocoel of the sea urchin, Strongylocentrotus frauciscanus. Biol.
Bull., 91: 295-311.
SKOGSBERG, T., 1936. Hydrography of Monterey Bay, California. Thermal conditions, 1929-
1933. Trans. Aincr. Philos. Soc., 29: 1-152.
SKOGSBERG, T., AND A. PHELPS, 1946. Hydrography of Monterey Bay, California. Thermal
conditions, Part II (1934-1937). Proc. Amer. Philos. Soc., 90: 350-386.
SVERDRUP, H. U., M. JOHNSON AND R. FLEMING, 1942. The Oceans. Prentice-Hall Inc., N. Y.
SWAN, E. F., 1953. The Strongylocentrotidae (Echinoidea) of the Northeast Pacific. Evolu-
tion, 7 : 269-273.
THE LARVAL DEVELOPMENT OF CALLINECTES SAPIDUS
RATHBUN REARED IN THE LABORATORY 1
JOHN D. COSTLOW, JR. AND C. G. BOOKHOUT
Duke University Marine Laboratory, Beaufort, North Carolina, and Department of
Zoology, Duke Unii'ersity, Durliain. N. C.
The crabs which comprise the family Portunidae include several commercially
important species and studies on their life history have been in progress for the
last 100 years. Of the British species only Port-units f>uber (L.) has been success-
fully reared in the laboratory through all larval stages to the first crab (Lebour,
1928). Larvae of Carcinus inacnas Penn. have been described by a variety of
workers but the complete development is not known from laboratory rearing. Of
the American species Callinectcs sapidus Rathbun is the most important com-
mercial crab in the Western Atlantic and Gulf of Mexico. Churchill (1942)
described the larval development of C. sapidus by reconstructing the sequence of
stages from planktonic material. Hopkins (1943, 1944), rearing the larvae through
the third zoeal stage, found that not all of the stages fit the description given by
Churchill (1942) and was of the opinion that the larvae described by Churchill
(1942) represented several different species. The complete larval development of
C. sapidus, from hatching to the first crab stage and beyond, was first reported from
laboratory rearing by Costlow, Rees and Bookhout (1959). While a brief account
is given of the number of stages, the duration of the intermolt periods, and the
time required for complete development, the larval stages are not described nor
is detailed information given on the various environmental factors under which
complete development occurred.
The present study has had two major objectives : one, to provide a detailed
description of all the larval stages of Callinectcs sapidus Rathbun reared in the
laboratory ; and two, to determine the effects of salinity and temperature on larval
development.
METHODS
Ovigerous Callinectcs sapidus females were obtained from the Beaufort Inlet
through the cooperation of Mr. David Beveridge, captain of the commercial
trawler "Beveridge." Additional females were obtained from crab pots placed in
waters of lower salinity. The crabs were placed in glass battery jars containing
running, filtered sea water of a salinity of 23-26 p.p.t. The battery jars were
tilted so that the slight overflow passed through a series of glass trays. When the
eggs hatched the larvae were carried into the glass trays by the overflow, removed
by large-bore pipettes as they collected on the light side, and segregated into
cultures of 50-75 zoeae per finger bowl. These were further subdivided into
1 These studies were aided by a contract between the National Science Foundation and
Duke University, G 4400. The authors wish to express their appreciation to Mrs. W. A.
Chipman and Mrs. C. King for their assistance throughout the study.
373
374
J. D. COSTLOW, JR. AND C. G. BOOKHOUT
plastic compartmented boxes with one zoea per compartment. Larvae which
hatched from these crabs (Series a, c, and d) were maintained at 25 C., 26.7 p.p.t.
with a photoperiod of approximately 12 hours light and 12 hours darkness. The
larvae which would have been designated "b" did not hatch.
To assure acclimation of the larvae to different salinities before hatching, other
ovigerous crabs were placed in battery jars which did not incline but were
partially filled with water of approximately the same salinity as the inlet water
during the summer months (32 p.p.t.). Four salinities were obtained from the
32 p.p.t. sea water by the gradual addition of appropriate volumes of distilled
water. The four salinities used were: 15 p.p.t., 20.1 p.p.t., 26.7 p.p.t. and 31.1 p.p.t.
The water used for the adult crabs was aerated but not changed. The crabs were
not fed and any fecal material which did appear was removed.
Some larvae which hatched at 20.1 p.p.t. were gradually changed to water of
10 p.p.t. Additional zoeae were hatched and maintained through most of the
larval period at 32 p.p.t.
TABLE I
Original number of Callinectes sapidus larvae maintained in 15 combinations of salinity and
temperature. Because the larvae reared at 25 C., 26.7 p.p.t. were hatched from three
different females at different times they are designated as a, c, and d. S~
per cent survival to first crab stage; * maintained
on shaker, 120 'mi H.
\p-p.t.
c.\
10.5
15.6
20.1
S
26.7
s
31.1
S
32.0
S
20
108
108
108
108
108
25
100
100
100
1.0
a) 18*
5.5
80
108*
108*
108*
c) 150*
2.7
150*
1.3
lOOOf
< 1
d) 100
8.0
30
108
108
108
108
t Diluted to 28 p.p.t. on day 41.
When hatching occurred in the jars without any overflow the zoeae were removed
with a large-bore pipette to finger bowls. The salinity of the water in the finger
bowls was identical to the water in which hatching had occurred. Both plastic
compartmented boxes and Syracuse watch glasses were used as rearing containers
for larvae within each salinity. Ten zoeae were maintained in each Syracuse
watch glass and 6 zoeae in each plastic compartment. Zoeae in each of the salinities
were maintained at three different temperatures : 20 C., 25 C., and 30 C. Zoeae
of all series were fed Arbacia eggs and Artcmia nauplii which were added each day
when the larvae were changed to freshly filtered sea water and clean receptacles.
Some plastic boxes were maintained on an Eberbach shaker (120/min.) at 25 C.
but the majority of the containers were stationary (Table I). The megalops and
crab stages were fed Artemia nauplii plus beef liver. The compartments containing
the zoeae were observed daily for exuvia and, at this time, the number of molts
and the mortality were recorded.
Drawings of the zoeal stages and megalops stage were made from the exuvia
of known molts and from larvae preserved at a known stage of development. All
LARVAL DEVELOPMENT OF CALLINECTES 375
figures were made to scale on graph paper with the aid of a Whipple disc inserted
in the ocular of a compound microscope. The detailed drawings of the appendages
of each stage are also drawn to scale, different from that used for the whole larva,
from appendages dissected out with glass needles.
RESULTS
Larval stages
First zoea: The characteristics of the first stage zoeae agree closely with those
given by Hopkins (1943). A small seta, described as between the dorsal and lat-
eral spines of the cephalothorax (Hopkins, 1943) was not found. The abdomen
has five segments plus a telson. As shown in Figure 1, A, B, the eyes are not
stalked. The conical antennule (Fig. 1, C) bears a total of 5 terminal processes,
the three aesthetes being longer and flatter than the two small setae. The proto-
podite of the antenna (Fig. 1, D) is elongated, bears two rows of minute spines on
the distal half, and the small exopodite terminates in two unequal setae. The
mandibles are small, with a broad cutting surface (Fig. 1, E). The endopodite
of the maxillule (Fig. 1, F) bears four terminal spines, equal in length, and two
slightly subterminal spines. The basal and coxal endites of the protopodite have
5 and 6 spines, respectively, and show slight bifurcation. The unsegmented endop-
odite of the maxilla (Fig. 1, G) also bears four terminal spines and two sub-
terminal spines. The basal endite of the protopodite bears four spines on each
bifurcation and three spines project from each lobe of the coxal endite. The
scaphognathite has three setae on the outer margin of the distal portion plus two
apical setae at the proximal tip. The first maxilliped (Fig. 1, H) has 4 natatory
setae (cut short in the figures) on the exopodite and a spine arrangement of 2, 2,
0, 2, 5 on the 5 segments of the endopodite. The second maxilliped also has 4
swimming hairs and a 1, 1, 4 spine arrangement on the three segments of the
endopodite (Fig. 1, I).
The second segment of the abdomen bears a short lateral knob and the third
segment has a short hook on each side. Segments 3 to 5 also have prominent
lateral spines which project caudally, overlapping the adjacent segment. A pair
of small setae project dorsally from all abdominal segments other than the first.
Each furcus of the telson bears a small dorsal spine and a larger lateral spine
(Fig. 1, A, B). The inner margin of each furcus has three spines.
The pattern of the chromatophores was consistent for all zoeal stages. The
location of those evident in Bourns-fixed larvae were : between the eyes ; posterior
to the eye and dorso-lateral to anterior part of gut ; dorsal to gut in posterior region
of cephalothorax ; below base of carapace spine ; mandible ; distal region of basop-
odite of first maxilliped ; middle of first abdominal segment, dorsal to gut ; margin
of third through last abdominal segments.
Second zoea: Eyes stalked. Number of aesthetes of antennule identical to first
stage. Endopodite of maxillule bears 4 terminal and 2 subterminal spines (Fig.
2, F) ; basal endite bears 7 spines and coxal endite has 7 spines; a small spine is
now present on outer margin of protopodite. Basal endite of maxilla (Fig. 2, G)
has 8 spines and coxal endite 6 spines. Five spines are present on distal margin
of scaphognathite and two project from apical tip. On third segment of endopodite
of first maxilliped, one spine is added (2, 2, 1, 2, 5) (Fig. 2, H). The exopodite
376
J. D. COSTLOW, JR. AND C. G. BOOKHOUT
FIGURE 1. Side (A) and ventral view (B) of first zoeal stage of Callinectes sapidits with
appendages. C, antennule ; D, antenna ; E, mandible ; F, maxillule ; G, maxilla ; H, first maxil-
liped; I, second maxilliped. Whole zoea, X 65 ; appendages, X 290.
LARVAL DEVELOPMENT OF CALLINECTES
377
FIGURE 2. Side (A) and ventral view (B) of second zoea of Callincctes sapidus with
appendages. C, antennule ; D, antenna ; E, mandible ; F, maxillule ; G, maxilla ; H, first maxil-
liped ; I, second maxilliped. Whole larvae, X 65 ; appendages, X 290.
378
J. D. COSTLOW, JR. AND C. G. BOOKHOUT
FIGURE 3. Side (A) and ventral view (B) of third zoea of Callincctes sapidus with
appendages. C, antennule ; D, antenna; E, mandible; F, maxillule ; G, maxilla; H, first maxil-
liped; I, second maxilliped. Whole larvae, X 43 ; appendages, X 170.
LARVAL DEVELOPMENT OF CALLINECTES
379
FIGURE 4. Side (A) and ventral view (B) of fourth zoea of Callincctes sapidus with
appendages. C, antennule ; D, antenna ; E, mandible ; F, maxillule ; G, maxilla ; H, first maxil-
liped; I, second maxilliped. Whole larva, X 43 ; appendages, X 170.
380 J. D. COSTLOW, JR. AND C. G. BOOKHOUT
bears 6 plumose swimming setae. Endopodite of second maxilliped has one addi-
tional subterminal spine (Fig. 2, I). This setation, 1, 1, 5, remains constant
through the remaining larval stages. Exopodite of second maxilliped hears 6
plumose swimming setae. Inner margin of each furcus of telson now bears one
additional spine without setules (Fig. 2, B).
Third zoea: Setation of antennule and antenna unchanged from previous stage.
The mandible (Fig. 3, E) has several small teeth in addition to the broad cutting
surface. Basal endite of maxillule bears 8 spines and 7 spines project from coxal
endite (Fig. 3, F). Basal and coxal endites of maxilla (Fig. 3, G) have 9 and 7
spines, respectively. Scaphognathite has 8 hairs on distal margin and 4 hairs
at apical tip. A second, subterminal spine added to the fifth segment of the endop-
odite of the first maxilliped gives a spine arrangement (2, 2, 1, 2, 6) which re-
mains constant in the remaining larval stages (Fig. 3, H). The exopodites of
both maxillipeds terminate in 8 swimming setae (Fig. 3, H, I). A sixth segment
has been added to the abdomen. It bears the small dorsal setae but does not have
lateral spines (Fig. 3, B).
Fourtli zoea: A slight swelling in the basal region of the antenna indicates the
beginning of the endopodite bud (Fig. 4, D). A small, unsegmented palp appears
with the mandible (Fig. 4, E). The basal endite of the maxillule bears 10 ter-
minal spines and one smaller subterminal spine (Fig. 4, F). Six spines project
from the terminal portion of the coxal endite and two more appear at the margin.
The basal endite of the maxilla bears 10 spines and 7 project terminally from the
coxal endite (Fig. 4. G). The exopodites of both the first and the second maxilli-
peds bear 9 swimming setae of unequal length (Fig. 4. H, I). The lateral edges
of the cephalothorax have three small setae (Fig. 4, A).
Fift/i zoea: The developing endopodite bud of the antenna (Fig. 5, D) is larger
than in the previous stage. The maxillule remains as in the previous stage but
setation of the maxilla is increased to 8 spines on the coxal endite (Fig. 5, F) and
the soft hairs on the Scaphognathite are increased to 20. The number of swim-
ming setae on the first maxilliped remains as in the previous stage (9) while the
second maxilliped now bears a total of 11 setae. Buds of the third maxilliped, chela,
and pereiopods are visible beneath the carapace. The number of setae pro-
jecting from the edge of the carapace has increased.
Si.vtli zoca: A fourth aesthete, subterminal to the original 3 aesthetes and 2
setae, is added to the antennule (Fig. 6, C). Hairs appear on the small, unseg-
mented palp of the mandible (Fig. 6, E). A plumose spine is added to the basal
segment of the endopodite of the maxillule (Fig. 6, F) and the coxal endite bears
a total of 9 spines. Spines on the basal endite of the maxilla (Fig. 6, G) have
increased to 13 and the marginal hairs of the Scaphognathite total approximately 25.
There are 11 swimming setae on the first maxilliped and 12 on the second maxilliped.
Pleopod buds appear for the first time on the abdominal segments 2 through 6
(Fig. 6, A, B). A small, non-plumose spine is added to the 8 spines within the
inner margin of the telson. The number of setae on the margin of the carapace is
also increased.
Seventh zoca: The terminal aesthetes of the antennule increase to 7 and 5
subterminal aesthetes have been added (Fig. 7, C). The basal portion of the an-
tennule is swollen and there is a slight indentation in the distal half. The devel-
LARVAL DEVELOPMENT OF CALLINECTES
381
FIGURE 5. Side (A) and ventral view (B) of fifth zoea of Calliticctcs sapid us with
appendages. C, antennule ; D, antenna; E, maxillule; F, maxilla; G, endopodite of first maxil-
liped; H, endopodite of second maxilliped. Whole larva, X 43 ; appendages, X 170.
382
J. D. COSTLOW, JR. AND C. G. BOOKHOUT
FIGURE 6. Side (A) and ventral view (B) of sixth zoea of Callincctcs sapidus with
appendages. C, antennule ; D, antenna ; E, mandible ; F, maxillule ; G, maxilla ; H, endopodite
of first maxilliped; I, endopodite of second maxilliped. Whole larva, X 43 ; appendages, X 170.
LARVAL DEVELOPMENT OF CALLINECTES
383
FIGURE 7. Side (A) and ventral view (B) of seventh zoea of Callinectes sapidus with
appendages. C, antennule ; D, antenna ; E, maxillule ; F, maxilla ; G, endopodite of first maxil-
liped ; H, endopodite of second maxilliped ; I, third maxilliped. Whole larva, X 43 ; appendages,
X170.
384
J. D. COSTLOW, JR. AND C. G. BOOKHOUT
FIGURE 8. Side (A) and ventral view (B) of eighth zoea of Callincctcs sapidus and
appendages. C, antennule ; D, antenna ; E, maxillule ; F, maxilla. Whole larva, X 32 ; ap-
pendages, X 135.
LARVAL DEVELOPMENT OF CALLINECTES 385
oping enclopodite bud of the antenna (Fig. 7, D) is approximately half the length
of the antenna. The basal endite of the maxillule (Fig. 7, E) bears 17 spines and
the coxal endite retains the 9 spines observed in the previous stage. The spines
of the basal endite of the maxilla number 14 and 10 spines are present on the coxal
endite (Fig. 7, F). On the scaphognathite approximately 29 soft, plumose hairs
fringe the outer margin. The swimming setae have increased to 14 on the first
maxilliped and to 13 on the second maxilliped (Fig. 7, A, B). The developing
thoracic appendages have increased in size and project below the margin of the
carapace.
Eighlh zoca: The aesthetes of the antennule are arranged in three tiers: 7 ter-
minal, 6 subterminal, and 5 in the most basal row (Fig. 8, C). Basal portion of
the antennule is more inflated and the endopodite is visible as a small knob. Endop-
odite of antenna (Fig. 8, D ) is now almost equal in length to protopodite and
shows evidence of segmentation. Basal endite of maxillule (Fig. 8, E) bears 21
spines and coxal endite has 15 spines. A second spine is added below the endop-
odite. Spines of the basal and coxal endites of the maxilla have increased to 15
and 10, respectively (Fig. 8, F). On the scaphognathite the plumose hairs have
increased to approximately 36. Swimming setae on the first maxilliped have de-
creased to 12 and 14 setae are found on the second maxilliped (Fig. 8, A, B ). On
the first maxilliped an epipodite, partially developed, bears short setae and soft,
non-plumose hairs (Fig. 9, A). Exopodite of the third maxilliped (Fig. 9, C)
bears two short terminal spines and the epipodite terminates in one small, non-
plumose spine. Chela and pereiopods are larger and project well beyond border of
the carapace. Pleopod buds (Fig. 8, A, B) bear short non-plumose hairs. Spines
on inner margin of telson total 10. Four small hairs project dorsally from posterior
margin of first abdominal segment.
Megalops: Rostrum pointed, longer than antennules but shorter than antennae ;
eyes stalked (Fig. 9, D, E). Appendages, eyes, and margins of carapace pro-
vided with small hairs.
Antennule (Fig. 10, A) now divided into peduncle of three segments and two
flagella. The unsegmented flagellum bears 6 non-plumose setae and the four seg-
ments of the other flagellum bear numerous aesthetes. The longer, terminal seg-
ment also bears two non-plumose setae. The antenna is composed of 1 1 segments,
some of which bear setae as shown in Figure 10, B. The mandible (Fig. 10, C)
has a palp of two segments with 11 bristles on distal segment. Endopodite of
maxillule (Fig. 10, D) has 4 spines on terminal segment and 6 spines on first
segment. The number of spines on the coxal and basal endites has increased to
17 and 25, respectively. Endopodite of maxilla (Fig. 10, E) reduced in size and
bearing only three spines. There is an increase in the number of spines on endites
of the protopodite and on the scaphognathite.
First maxilliped (Fig. 11, A) is considerably modified from swimming ap-
pendage of zoeal stages. Endopodite broader with 8 non-plumose setae on distal
border. Exopodite of two segments, with 6 terminal setae on second segment.
Epipodite well developed and fringed with long, non-plumose hairs. Second maxil-
liped (Fig. 11, B) has endopodite of 4 segments with stout spines on terminal seg-
ment. Exopodite is two-segmented with 6 terminal hairs. The epipodite is small.
Third maxilliped (Fig. 11, C) with large endopodite bearing numerous spines on
386
J. D. COSTLOW, JR. AND C. G. BOOKHOUT
FIGURE 9. Appendages of eighth zoea and side and dorsal view of megalops of Callinectes
sapidus. A, first maxilliped; B, second maxilliped; C, third maxilliped; D, side view of
megalops ; E, dorsal view of megalops ; F, ventral view of abdominal segments of megalops
(setae removed on alternate pleopods for clarity). Whole megalops, X32; appendages, X 135.
LARVAL DEVELOPMENT OF CALLINECTES
387
FIGURE 10. Appendages of megalops of Callinectes sapidus. A, antennule; B, antenna; C,
mandible ; D, maxillule ; E, maxilla. X 135.
388
J. D. COSTLOW, JR. AND C. G. BOOKHOUT
O.I
FIGURE 11. Appendages of megalops of Callinectes sapidns. A, first maxilliped ; B, second
maxilliped; C, third maxilliped; D, terminal segment of third maxilliped. X 135.
LARVAL DEVELOPMENT OF CALLINECTES
389
all segments ; exopodite unsegmented and bearing 6 terminal setae ; epipodite
fringed at distal portion by soft, non-plumose hairs. Spine on lateral surface of
basi-ischiopodite of cheliped (Fig. 9, D, E), and dactylopodite of fifth pereiopod
has 5 terminal spines. Cornua project from posterior edge of cephalothorax
TABLE II
Time of molting, expressed as days after hatching, for larvae of C. sapidus in salinity-temperature
combinations in which development was complete or partially complete
\p.p.t.
"ex
20.1
26.7
31.1
Molt I
Molt II
Molt III
Molt IV
Molt V
Molt VI
Molt VII
(to megalops)
Molt VIII
(to crab)
25
30
6-13
a) 7-9
c) 6-12
cl) 7-9
5-11
7-13
25
30
12-16
a) 10-12
c) 10-20
d) 10-12
11-16
11-19
25
30
17-27
a) 15
c) 17-26
d) 14-23
14-18
15-27
25
24-30
a) 19
c) 20-32
d) 18-26
20-29
25
28-34
a) 22
c) 24-39
d) 22-33
24-39
25
38
a) 27
c) 28-39
d) 26-38
29-43
25
43
a) 31
c) 35-49
d) 32-45
35-47
25
50
a) 37
c) 50-55
d) 39-53
45-55
(Fig. 9, E, F). Fifth abdominal segment retains lateral spines, projecting caudally
past the smaller sixth abdominal segment (Fig. 9, D, F). Endopodites developed
on all pleopods other than fifth pair. Exopodites of pleopods on segments 2
through 6 with 24, 23, 22, 21, and 12 long, non-plumose setae (Fig. 9, F). Four
small, curled spines are found on inner surface of endopodite of the pleopod of
390
J. D. COSTLOW, JR. AND C. G. BOOKHOUT
the second abdominal segment and three similar spines are present on endopodites
of remaining pleopods. Telson with 6 to 8 short spines on posterior border.
Larval development
Hatching was observed at all experimental salinities except 15 p.p.t. In water
of 20.1 p.p.t.-32 p.p.t. the zoeae hatched as first stage larvae and the so-called "pre-
zoea" was never observed. Complete development to the first crab stage occurred
in the four temperature-salinity combinations shown in Table I.
As shown in Table II, the time of molting of the three series of larvae main-
tained at 26.7 p.p.t., 25 C. (Series a, c and d) was similar. The first molt oc-
curred within the same period of time for larvae at 20.1, 26.7, and 31.1 p.p.t. At
these three salinities there was also little difference in the time of the later molts
(Table II ) and in the range of time for complete larval development (Table III).
The only difference in time required for total development was found in the
series of larvae hatched and reared at 32 p.p.t. After dilution to 28 p.p.t. on
day 41, at which time all the larvae had been either sixth or seventh stage zoea
TABLE III
Number of days observed for development of all zoeal stages (Z), duration of the megalops stage (M),
and time for total development to the first crab stage (T) for larvae of Callinectes
sapidus hatched and maintained at 25 C. in the salinities shown
20.1
26.7
31.1
32.0*
Z
M
T
Z
M
T
z
M
T
Z
M
T
43
7
50
a) 31
6
37
35-47
10-20
45-57
46
15
61
b) 35-49
7-9
44-56
d) 32-45
6-9
38-53
* Diluted to 28 p.p.t. on day 41.
for some time, some molted to the megalops stage and eventually metamorphosed
to the first crab on day 61.
The one series in which zoeae completed the first three molts at 30 C.,
26.7 p.p.t., shows no significant difference in the time of the molts in spite of the
additional 5 C. in temperature (Table II).
Mortality of C. sapid us larvae (Table IV) was highest during the first two zoeal
stages in all temperature-salinity combinations. In all salinities larvae never went
beyond the first zoeal stage when maintained at 20 C. At 10.5 and 15.6 p.p.t.
mortality was also highest during the first stage at all three temperatures. Larvae
maintained at one temperature-salinity combination, 25 C., 15.6 p.p.t., did molt
to the second stage but died within a few days (Table IV). Once the second molt
had been completed some of the remaining larvae usually lived to complete meta-
morphosis to the crab.
The number of zoeal stages of C. sapidus varied from 7 to 8. Most of the
larvae which molted to the megalops did so following the seventh zoeal stage but
one completed 8 zoeal stages and then metamorphosed to the megalops. The
majority of the eighth stage zoeae died without additional molts. The variation
LARVAL DEVELOPMENT OF CALLINECTES
391
TABLE IV
Mortality of larvae of Callinectes sapidns at different stages, expressed as per cent of
original number of zoeae, in those temperature-salinity combinations
which permitted at least partial development.
Vp.p.t.
C.\
15.6
20.1
26.7
31.1
Stage I
25
95
42
a) 72.2
c) 30.0
d) 11.0
53.3
30
95
58.3
60.1
Stage II
25
5
36
a) 16.7
c) 57.5
d) 42
22.8
30
5
37.0
37.0
Stage III
25
11
a) 5.5
c) 3.5
d) 10.0
12.0
30
2.7
2.8
Stage IV
25
8
a) 0.0
c) 2.0
d) 5.0
0.6
30
1.8
Stage V
25
1
a) 0.0
c) 0.0
d) 9.0
4.6
Stage VI
25
1
a) 0.0
c) 0.0
d) 9.0
0.6
Stage VII
25
0.0
a) 0.0
c) 0.0
d) 4.0
4.0
Megalops
25
0.0
a) 0.0
c) 4.3
d) 1.0
0.0
in number of stages occurred within one salinity-temperature combination (26.7
p.p.t., 25 C.) as well as in the other salinities. The megalops stage metamor-
phosed directly to the first crab stage.
DISCUSSION
Larval stages
The only existing description of all larval stages of Callinectes sap id us (Churchill,
1942) is based entirely on reconstruction from planktonic material. Hopkins
392 J. D. COSTLOW, JR. AND C. G. BOOKHOUT
(1943, 1944) was able to rear C. sapldns through the first three zoeal stages and
concluded that Churchill's (1942) description of the larvae included zoeae from
several species. Reconstruction of the stages in larval development is always
susceptible to this error in an area which includes more than one species. By
rearing zoeae, liberated in the laboratory from the egg mass of an identified female,
the species can definitely be known and confusion resulting from the mixing of
larvae from several species is avoided.
The larval development of many crabs has been reported to include a "pre-
zoeal" stage. The "pre-zoea" is described for C. sapidus by Robertson (1938) and
by Churchill (1942). In the present study the larvae, although varying con-
siderably in size, always hatched as first zoeae in salinities of 20.1, 26.7, 31.1 and
32 p.p.t. Lochhead, Lochhead and Newcombe (1942) observed that 90 per cent
of the eggs hatched as first zoeae under "favorable conditions" but that "pre-zoeae"
were obtained if conditions were "unfavorable." Sandoz and Rogers (1944) found
hatching to be associated with salinity : below 20 p.p.t. the per cent of larvae which
emerged as "pre-zoeae" increased.
The setation of the maxillipeds of C. sapidus larvae has been given by Churchill
(1942) and, for the first three stages reared in the laboratory, by Hopkins (1943,
1944). The results of the present study agree with previous findings for the first
two zoeal stages. Beginning with the third zoea, however, our description does
not agree with that given by previous workers. Churchill ( 1942) gives 6 and 7
setae for the first and second maxillipeds, respectively, Hopkins (1944) found 8
and 9 setae, and we observed 8 swimming setae on each maxilliped. Hopkins
(1944), describing a fourth stage zoea obtained from the plankton, gave the setation
of the first and second maxillipeds as 8 and 10 while we found it to be 9 and 9.
Robertson (1938) and Churchill (1942) put great emphasis on the cornua as
a distinguishing feature of the C. sapidus megalops. Aikawa (1937) described the
megalops of several species of Porhinus, obtained from the plankton, and included
the cornua in the figures for these species. Aikawa (1937) also mentioned the
hook on the basi-ischiopodite of the chela and the lateral spines on the fifth ab-
dominal segment of the megalops. Lebour (1928), describing the megalops of
Port unns pubcr reared from the egg in the laboratory and megalops of other species
of Portunus obtained from the plankton, did not figure or describe these three
characters for any species of Portunus.
The present description of setation of the maxillule and maxilla agrees with
Hopkins' (1943, 1944) findings for the first three stages. In many previous studies
on larvae of the Brachyura the zoeae have been staged very largely by differences
in the number of swimming hairs on the first and second maxillipeds. Aikawa
(1937) compares setation of the maxillule and maxilla for a great variety of
brachyuran larvae but includes only the first stage zoea. In each zoeal stage of
C. sapidus examined in the present study it was found that there was always a
progressive change in the setation of the maxilla. Setation of the maxillule was
also different, except for the fourth and fifth zoeae. Hence these appendages, and
others, may be important in staging larvae of different crabs. The significance of
these appendages as diagnostic characters, however, will have to await a com-
parative study of all stages in the larval development of other species of crabs.
LARVAL DEVELOPMENT OF CALLINECTES 393
Larval development
Although the effects of salinity and temperature on larval development of other
crabs have been studied (Coffin, 1958; Costlow and Bookhout, unpublished results),
Sandoz and Rogers (1944, 1948) give the only available data dealing specifically
with the blue crab, Callincctcs sapidus. In the present study on larvae of this
species the results agree closely with those reported for the first zoeal stage by
Sandoz and Rogers (1944).
If the salinity were reduced beyond 20.1 p.p.t. by dilution with distilled water,
the zoeae did not usually live beyond the first molt. Sandoz and Rogers (1944)
obtained some second zoeae at 20 p.p.t. and 25 p.p.t. (24-29 C.) but the few
which molted to the third stage did not live. In the present study the time of
molting (Table II) was quite variable, even within one salinity-temperature com-
bination. Sandoz and Rogers (1944) reported an average of from 67 days for
the first molt at 20 and 25 p.p.t., 24-29 C., although some larvae molted as late
as the eleventh day. In the present study the first molt was completed in from
5 to 13 days in several salinity-temperature combinations (Table II). The later
molts became more variable in time in all three salinities in which development was
complete.
In the present study isolated larvae did molt and successfully complete develop-
ment to the crab stage. Sandoz and Rogers (1944) did not observe any molting
among isolated larvae and all eventually died.
One series of larvae, hatched and maintained for 41 days at 32 p.p.t., was of
particular interest. The sixth and seventh stage zoeae were active but did not
molt to the megalops. On day 41 the larvae were divided into three groups. The
water containing one group of zoeae was reduced from 32 p.p.t. to 28 p.p.t. in
approximately 4 hours. All zoeae of this group died within 24 hours. Water
containing the second group of larvae was diluted to 28 p.p.t. over a period of
approximately 24 hours. Five days later one zoea molted to the megalops and
on day 61, metamorphosed to the crab. Larvae of the third group, retained at
32 p.p.t., died without any additional molting. While the number of larvae used
should not be relied upon for any definite conclusions, it may be pointed out that
the larvae hatched and reared at 31.1 p.p.t. completed metamorphosis to the crab
without dilution to a lower salinity. Thus it would appear that the threshold which
exists in the upper range of salinities is abrupt and well defined.
At 25 C. the duration of the megalops stage (6-9 days) was similar for larvae
maintained at 20.1 and 26.7 p.p.t. (Table III). In the higher salinity (31.1 p.p.t.)
10-20 days were required and in water diluted from 32 p.p.t. to 28 p.p.t., the
megalops persisted for 15 days before molting to the crab. Sandoz and Rogers
(1948) found little difference in the time required for the megalops to molt to the
crab in 20 p.p.t. and 31 p.p.t. The 2.6-2.9 days which they record, however, were
for stages obtained from the plankton and the exact age could not be known. If,
as suggested by Sandoz and Rogers (1948), the megalops were approximately 2-3
days old when first obtained, the total period of 5-6 days would correspond
closely with our results at 20.1 and 26.7 p.p.t.
Churchill (1942) estimated that zoeal development of C. sapidus in the Chesa-
peake Bay was completed in approximately one month. Zoeal development in
394 J. D. COSTLOW, JR. AND C. G. BOOKHOUT
the laboratory required a minimum of 31 days and a maximum of 49 days, at
various salinities. In the laboratory 7 zoeal stages and one megalops stage were
observed whereas Churchill (1942) described 5 zoeal stages and one megalops
from planktonic material.
The use of Artemia nauplii has proven successful in rearing a variety of decapod
larvae (Broad, 1957; Chamberlain, 1957; Knudsen, 1958; Coffin, 1958; Costlow
and Bookhout, unpublished results) and Cirripedia larvae have been reared from
hatching to settling and metamorphosis on Arbacia eggs (Costlow and Bookhout,
1957, 1958). The combination of Arbacia eggs and recently hatched Artemia
nauplii used in the present study provides a source of motile food of different
sizes. In our experience with other decapod larvae, also reared at different
salinity-temperature combinations, the zoeae were vigorous and fed actively.
C. sapidus larvae, even after completion of several molts, often appeared fragile
and less vigorous than larvae of other species. Algae have been used unsuccessfully
in attempts to rear the larvae of many decapods by previous workers. We have
found that while C. sapidus zoeae will ingest many of the unicellular algae and
live 10-13 days, the larvae never molt. Even though the gut is full of the cells,
and fecal pellets are numerous, further development does not occur. In the present
study algae were not used because zoeae which were provided algae have been
observed to feed less actively on Artemia nauplii. Dean (1958) has suggested that
what have been interpreted as differences in the nutritive quality of algae may
represent "resistance" to digestion.
The 7 zoeal stages described for C. sapidus may not represent the number of
stages present in development under natural conditions. A main criticism of
laboratory rearing has been that suboptimal conditions may produce "abnormal"
stages and give a picture of larval development which is not consistent with that
assumed to be found in the natural environment (Gurney, 1942). In the few
existing examples of successful rearing of Brachyura in the laboratory no reference
is made to "extra" or "abnormal" stages. Lebour (1930), dealing with larvae of
the Anomura, noted that 5 larval stages usually represent the normal development
of Galathca but that the fourth and fifth stages may be omitted. In the Macrura,
Templeman (1936) found a stage in the larvae of Homarus americanus, inter-
mediate in form between the recognized third and fourth stages, and attributed it to
unfavorable rearing conditions. More recently, Broad (1957) has shown that the
number of larval stages of Palacmonetes is directly associated with the availability
of food. Lebour (1928), discussing the primitive nature of the Brachyrhyncha
larvae, considers Portunus as the most primitive because of the many zoeal stages
(5) and the spine structure of the telson. The 7 zoeal stages described for C.
sapidus, a form closely allied to Portunus, may indicate a primitive adaptive quality
which has, in part, accounted for the success of this species all along the Atlantic
and Gulf coasts.
If larval development is complete, and the post-larval stage is reached, it appears
erroneous to refer to "abnormal" stages of development. Our present knowledge
of the factors involved in the physiology of larval development of the Brachyura
is too limited to predetermine the number of larval stages required for the develop-
ment of any crab.
LARVAL DEVELOPMENT OF CALLINECTES 395
SUMMARY AND CONCLUSIONS
The larvae of Callinectes sapidus Rathbun were reared in the laboratory from
hatching to the post-larval stages under conditions which combined 20 C, 25 C.,
30 C, and 6 salinities (10.5, 15.6, 20.1, 26.7, 31.1 and 32 p.p.t.). Of the 3,014
zoeae maintained in 15 different combinations of salinity and temperature 1-8 per
cent completed development at 25 C., in salinities of 20.1, 26.7, and 31.1 p.p.t.
The zoeal stages and megalops stage are described and figured. From this study
the following conclusions may be made :
1. Eggs hatched as first zoeae and the "pre-zoea" stage was not observed.
2. Seven zoeal stages and one megalops stage were observed in the complete
development to the first crab in the laboratory. An eighth zoeal stage was some-
times observed but usually did not complete metamorphosis to the megalops.
3. Setation of the maxillipeds and the maxillule showed a progressive increase
with each larval stage and may be useful in the staging of species obtained from
the plankton.
4. Development to the megalops required a minimum of 31 days and a maximum
of 49 days. The megalops persisted from 6-20 days in the salinities used.
5. There is no significant difference in time of zoeal development in water with
salinities of 20.1-31.1 p.p.t.
6. At a higher salinity (31.1 p.p.t.) a greater length of time is required for the
megalops to complete metamorphosis to the first crab than when reared in lower
salinities (20.1-26.7 p.p.t.).
7. Even though some zoeae completed development in salinities of 20.1-31.1
p.p.t. mortality was usually highest during the first two zoeal stages. Below
20.1 p.p.t. larvae rarely completed the first molt.
8. The large number of zoeal stages may not reflect development under natural
conditions. The 7 zoeal stages may, however, indicate a primitive adaptive
quality which has accounted for the success of Callinectes sapidus Rathbun along
the Western Atlantic and Gulf of Mexico coasts.
LITERATURE CITED
AIKAWA, H., 1937. Further notes on Brachyuran larva. Rec. Oceanogr. Wks. Japan, IX:
87-162.
BROAD, A. C., 1957. The relationship between diet and larval development of Palaemonetes.
Biol. Bull., 112: 162-170.
CHAMBERLAIN, N. A., 1957. Larval development of the mud crab Neopanope texana sayi
(Smith). Biol. Bull., 113: 338.
CHURCHILL, E. P., 1942. The zoeal stages of the blue crab, Callinectes sapidus Rathbun.
Chesapeake Biol. Lab., Publ. No. 49, pp. 1-26.
COFFIN, H. G., 1958. The laboratory culture of Pagurns samuelis (Stimpson) (Crustacea,
Decapoda). Walla Walla College Publ. No. 22, pp. 1-5.
COSTLOW, J. D., JR., AND C. G. BOOKHOUT, 1957. Larval development of Balanus eburneus in
the laboratory. Biol. Bull., 112: 313-324.
COSTLOW, J. D., JR., AND C. G. BOOKHOUT, 1958. Larval development of Balanus amphitrite
var. denticulata Broch reared in the laboratory. Biol. Bull., 114: 284295.
COSTLOW, J. D., JR., G. REES AND C. G. BOOKHOUT, 1959. A preliminary note on the complete
larval development of Callinectes sapidus Rathbun reared in the laboratory. Limn.
and Oceanography. In press.
396 J. D. COSTLOW, JR. AND C. G. BOOKHOUT
DEAN, D., 1958. New property of the crystalline style of Crassostrea virginica. Science, 128 :
837.
GURNEY, R., 1942. Larvae of Decapod Crustacea. Pp. 1-306, Ray Society, London.
HOPKINS, S. H., 1943. The external morphology of the first and second zoeal stages of the
blue crab, Callinectcs sapidus Rathbun. Trans. Amer. Micro. Soc., 62 : 85-90.
HOPKINS, S. H., 1944. The external morphology of the third and fourth zoeal stages of the
blue crab, Callinectes sapidtis Rathbun. Biol. Bull., 87: 145-152.
KNUDSEN, J. W., 1958. Life cycle studies of the Brachyura of Western North America, I.
General culture methods and the life cycle of Lophopanopcus Icucomanus leucomanus
(Lockington). Bull. So. Calif. Acad. Sci., 57: 51-59.
LEBOUR, M. V., 1928. The larval stages of the Plymouth Brachyura. Proc. Zool. Soc. London,
1928 : 473-560.
LEBOUR, M. V., 1930. The larvae of the Plymouth Galatheidae. I. Munida banffica, Galathea
strigosa and G. dispersa. J. Mar. Biol. Assoc., 17 : 175-186.
LOCHHEAD, M. S., J. H. LOCHHEAD AND C. L. NfiwcoMBE, 1942. Hatching of the blue crab,
Callinectcs sapidus Rathbun. Science, 95 : 382.
ROBERTSON, R. L., 1938. Observations on the growth stages in the common blue crab, Cal-
linectes sapidus Rathbun, with special reference to post-larval development. Thesis,
Univ. of Maryland, 46 pp.
SANDOZ, M., AND R. ROGERS, 1944. The effect of environmental factors on hatching, moulting,
and survival of zoea larvae of the blue crab, Callinectcs sapidus Rathbun. Ecology,
25 : 216-228.
SANDOZ, M., AND R. ROGERS, 1948. The effect of temperature and salinity on moulting and
survival of megalops and post-larval stages of the blue crab, Callinectes sapidus.
Va. Fish. Lab., unpubl. MS, 12 pp.
TEMPLEMAN, W., 1936. Fourth stage larvae of Homarus americanus intermediate in form
between normal third and fourth stages. /. Biol. Bd. Canada, 2: 349-354.
STUDIES OX THE FORM OF THE AMPHIBIAN RED BLOOD CELL
JOHN DAVISON
Department of Biology, Princeton University, Princeton, N. 7., 1 and Department of Biological
Sciences, Florida State University, Tallahassee, Florida -
To a student of cell form the erythrocyte is an ideal subject for investigation.
It is a free cell, not permanently involved in contact with other cells, and it has a
definite and relatively simple form. I recently published an account of a model
which was proposed as a partial explanation for the elliptical form of the amphibian
red cell (Davison, 1957). Since the model has served as a guide to the present
work, I will briefly describe its salient features as an introduction to these further
observations.
The blood cells of the newt Tritunis viridescens approximate thin elliptical
discs in form. Viewed as plane elliptical figures, triploid cells have approximately
1.5 times greater area than diploid blood cells, but are apparently no greater in
thickness, a relationship similar to that described by Fankhauser for 2n and 3n
skin epidermal cells (Fankhauser, 1952). Not only are the 3n cells larger, they
clearly have a different shape than 2n cells, being more eccentric regarded as
elliptical figures. Using the ratio of the major to minor axes (a/b) as an index
to cell form, 2n and 3n Tritunis red cells were found to have mean eccentricities
of 1.55 and 1.82, respectively.
It has long been recognized that liquid drops can, under the proper physical
conditions, simulate many protoplasmic structures (Thompson, 1942). Reasoning
that the blood cell exists in a system of cylinders, the blood vessels, I thought it
might prove interesting to examine the form characteristics of a fluid drop in con-
tact with a cylindrical surface. If one places a large (29 cm. in diameter) cylin-
drical glass vessel with the axis horizontal, and pours mercury on the inside of
the cylinder, the mercury will assume the form of a flat elliptical disc. Adding
more mercury to the pool increases both the area and the eccentricity of the drop
but does not appreciably increase its thickness. The model thus simulates the
form differences observed between 2n and 3n blood cells. In the model the mercury
is in contact with the cylindrical surface through the deforming force of gravity.
In the animal it is clear that the blood cells are applied to the wall of the capillary
but are not so oriented during their passage through larger vessels. No significant
differences were found in the diameter of 2n and 3n capillaries, an essential point,
since it is also clear from the model that the larger the cylinder the less eccentric
the fluid drop. The latter observations from the model suggest that changes in
capillary diameter should lead to alterations in red cell form, with an increase in
1 I would like to express my sincere appreciation to Dr. Gerhard Fankhauser of Princeton
University who so generously offered me the use of his laboratory and supported this work
through a grant from the Pfeiffer Foundation.
- Permanent address : Department of Biological Sciences, Florida State University, Talla-
hassee, Florida.
397
398 JOHN DAVISON
cell eccentricity following a decrease in capillary diameter and a decrease in cell
eccentricity following an increase in capillary diameter.
With this background in mind, the further objectives of the study may be
stated as follows :
( 1 ) To examine cell form when expressed as a continuous function of cell area,
especially with reference to the cross-sectional area of the capillary.
(2) To examine the effect of changes in capillary diameter on red cell form
under conditions of constant cell area.
(3) To quantitatively relate these variables.
ANIMALS AND METHODS
Since both diploid and triploid Spanish newts (Pleurodeles waltlii) were avail-
able, this animal was selected to examine cell eccentricity as a function of cell area.
Pleurodeles cells are less eccentric than those of Triturus, better permitting an
analysis of the manner in which the blood cell approaches the circular form. The
studies on adult Triturus followed the accidental discovery that cold-adapted
(8.5 C.) animals have much more eccentric blood cells than the same animals
maintained at room temperature (air conditioned 21 C.). Also one can con-
veniently measure capillary diameter in the tail fin of adult Triturus, especially the
males, while this is not possible in the heavily pigmented Pleur