THE
BIOLOGICAL BULLETIN
PUBLISHED BY
THE MARINE BIOLOGICAL LABORATORY
Editorial Board
HAROLD C. BOLD, University of Texas ARTHUR W. POLLISTER, Columbia University
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JOHN B. BUCK, National Institutes of Health
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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 C4 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 CHANG2 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 writh 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 wrould 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
0
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).
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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
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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
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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-
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FIGURE S. Influence of pressure on flash responses, at 25° C., to repetitive stimulation at
a frequency of one every three seconds. During two periods under pressure, stimulation was
discontinued as indicated in the figure. The complete series, A and B, is from a single speci-
men, with a rest period indicated by the break in the abscissa of B.
after 4000 psi). Out of the total series of experiments, a very few specimens
deteriorated and failed to recover at all (e.g., Fig. 6, A, after 5000 psi).
Fifth, excess flash intensities after decompression were not dependent on main-
taining repetitive stimulation during the period of sustained pressure (Fig. 5, A
and B).
PRESSURE AND MNEMIOPSIS FLASH
Sixth, qualitatively the same phenomena were observed when a high as well as
when a low frequency of stimulation and response were involved. A representative
example of a high frequency of stimulation, i.e., 3 per second, is shown in Figure
4, B, for comparison with the more commonly employed frequency of one every
three seconds. The higher frequency was inconvenient as a routine, both because
of the rapid fatigue always associated with it, and the difficulty of applying a desired
pressure in a fraction of a second between flashes.
7-
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FIGURE 6. Flash responses under various pressures, at 34° (A) and 30° C. (B), at a stimulation
frequency of one every three seconds. Two different specimens.
Finally, with reference to adaptation and facilitation under pressure, as well as
to excess flash intensities following release of pressure, a noteworthy observation
was made on a number of occasions, namely, that specimens which had lost their
excitability, through fatigue or other causes, could be rendered excitable again
merely by holding them under 3000 to 5000 psi for periods of one to five minutes.
Such treatments were not invariably successful, of course, inasmuch as deterioration
beyond the possibility of recovery sometimes occurred.
10
JOSEPH JIN CHANG AND FRANK H. JOHNSON
Urethane and pressure
At room temperature, 1.0 M urethane in sea water quickly abolished the
luminescent response. Lower concentrations of 0.5 down to 0.05 M caused in-
hibitions that varied in extent with the individual specimen, the amount of adherent
jelly, and duration of exposure to the drug. Although some specimens gave
luminescent flashes, at reduced intensity, in 0.5 M urethane, at least for a short
period of time, other specimens very rapidly lost their excitability in 0.25 M. In
0.2 to 0.15 M urethane the response of excised canals disappeared after a few
7-
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SECONDS
FIGURE 7. Flash responses, under various pressures, of a single specimen in sea water
containing 0.1 M urethane at 23.7° C. Stimulation frequencies were one every 3 seconds (A)
and 2 per second (B).
minutes and the cilia stopped beating, although in sea water without urethane the
luminescence and ciliary action would often persist for a couple of days. In 0.1 M
urethane the response was lost after about half an hour, but in 0.05 M it persisted
dimly for a longer time. Thus, urethane causes a progressively increasing inhibition
of the flash response, at a rate depending on the concentration added, being faster
the greater the concentration. In studying the influence of pressure, therefore, the
experiments wrere carried out within a short period of time after adding the drug.
The results showed that the generalizations described in the preceding section
with respect to the action of pressure in absence of urethane are qualitatively ap-
PRESSURE AND MNEMIOPSIS- FLASH 11
plicable in the presence of urethane. Representative data are illustrated in Figure
7, for 0.1 M urethane and two different frequencies of stimulation. The instability
of the system in the presence of urethane, especially in the higher concentrations or
at higher temperatures, made satisfactory experiments difficult to carry out, and the
quantitative significance of the results uncertain. The data indicate, however, that
no marked difference in volume change for the over-all process of the flash is
caused by urethane (see Fig. 3).
DISCUSSION
The Mnemiopsis flash is obviously limited by two types of processes, namely,
physiological excitation that leads to a luminescent response, and biochemical reac-
tions involved in light emission itself. While detailed information is not available
with respect to either of these processes, it is reasonable to believe that pressure can
alter the response by influencing the state of the activating system in the photogenic
organ just prior to, or at the time of stimulation, as it does in muscle (Brown,
1957). Moreover, it may be assumed with considerable assurance that the process
of light emission is limited by the activity of one or more essential enzymes. The
probability that Mnemiopsis luminescence is directly dependent on more than one
enzyme is suggested by the somewhat complicated effects of pressure described in
this paper, as well as by the inability to demonstrate a simple "luciferin-lucif erase
reaction," i.e., light emission on mixing a boiled and cooled aqueous extract of the
triturated photogenic organs with a cold-water extract of similar organs. Even
with a single limiting enzyme, pressure may affect the over-all process through
several mechanisms. Two mechanisms of potential importance are (1), by in-
fluencing an equilibrium between catalytically active and reversibly inactivated
states of the enzyme, the actual state depending upon temperature and the condi-
tions of the chemical environment such as presence of drugs, ions, or other agents
that act on the equilibrium, and (2), by influencing the catalytic process itself, i.e.,
the change from normal to activated states of the reactants. Where consecutive re-
actions are responsible for the measured results, the effects of increased pressure
are liable to become considerably more complicated, and transitory changes from
one steady-state to another can assume a variety of patterns, such as an initial
augmentation followed by an inhibition, or vice versa, with a converse pattern as-
sociated with the release of pressure.
In the present experiments the immediate effects of changes in pressure are at-
tributable to an immediate change in one or more specific reaction rate constants, or
possibly equilibrium constants. The phenomena of adaptation and facilitation under
pressure, as well as "overshoot" following decompression, are indicative of changes
in steady state concentrations of reactants, although effects on slowly changing
states of equilibria cannot be ruled out. Such equilibrium changes could pertain,
a priori, either to the process of physiological excitation or to enzymes involved in
light emission, but the fact that "overshoot" and excess luminescence following a
period under pressure are not contingent on continued stimulation during that
period argues against the process of physiological excitation as the major site of
action.
The pronounced instability of the Mnemiopsis system at high temperatures or in
12 JOSEPH JIN CHANG AND FRANK H. JOHNSON
the presence of urethane makes it unusually difficult to find evidence of any re-
versible, inactivation equilibrium change, analogous to those which limit other
processes that undergo less rapid destruction under the influence of these factors
(Johnson, Eyring and Polissar, 1954). In Mnemiopsis, if such equilibria exist,
they are obscured by the essentially irreversible processes, and the effect of pressure
on these equilibria becomes correspondingly difficult to detect.
Finally, some basic similarities should be pointed out with respect to the in-
fluence of pressure on the electrically stimulated flash in excised segments of the
Mneniiopsis organ, and on the much slower flash in excised notopodia of
Chaetopterus (Sie, Chang and Johnson, 1958). Thus, both exhibit initial reduc-
tions in flash maxima, followed by adaptation and facilitation under pressure, as
well as "overshoot" and excess luminescence on release of pressure. Parenthetically,
in view of the fact that the vertical distribution of certain ctenophores extends to
depths of some 2500 meters (Chun, 1903), where the pressure amounts to 250
atmospheres or about 3500 psi, it is interesting to surmise that the phenomenon of
adaptation of the flash response to increased pressure might enable a luminescent
ctenophore to descend gradually from the surface of the sea to such depths without
having the intensity of its flash reduced by the increase in pressure.
The similarities between the flash of Mnemiopsis and that of Chaetopterus, as
well as similarities in the effects of pressure on the two, are more impressive than
the differences, which are largely quantitative. The flash of the latter organism
is of the order of 100 times longer in duration, but in both organisms the decay after
peak intensity is exponential, and in both organisms the time-course becomes longer
with decrease in temperature. The Chaetopterus flash, as well as luminescence
intensity of the secreted slime, are somewhat less sensitive to increased pressure
than is the Mnemiopsis flash, while the effects of increased pressure are more sensi-
tive to temperature and to the presence of urethane.
The foregoing remarks are necessarily somewhat general. Further and more
specific interpretation of the observed phenomena must await more detailed knowl-
edge than is presently available concerning the total process of the flash response.
The effects of pressure on the luminescence of homogenates ("squeezates") of
Mnemiopsis have been studied and will be made the subject of a later communication.
SUMMARY
Using small segments of excised meridional canals of Mnemiopsis leidyi,
luminescent flashes induced by square wave electrical pulses of precise voltage and
duration have been accurately recorded with the aid of a photomultiplier-amplifier
and dual beam cathode ray oscillograph. Analyses of more than 8000 flashes, under
various conditions of temperature, hydrostatic pressure, urethane concentration,
and frequency of repetitive stimulation, have led to the following generalizations.
1. The time course of luminescence intensity in an individual flash at a given tem-
perature is not appreciably altered by increased pressures which greatly reduce the
flash maximum.
2. The latent period between time of stimulation and onset of response is like-
wise not significantly altered by pressure, within the sensitivity of the methods
employed.
. PRESSURE AND MNEMIOPSIS FLASH 13
3. In a series of consecutive flashes, at frequencies of one per 3 seconds to 4 per
second, the initial effect of increased pressure is always to reduce the maximum
intensity of the flash ; detectable reductions are caused by relatively slight pressures,
of less than 100 psi.
4. A series of pressure increases in increments of several hundred psi is less in-
hibitory on flash intensities than a sudden increase to the highest pressure involved.
Sudden increases of 1000 to 5000 psi temporarily abolish the flash, whereas with
gradual increases to these pressures, the flash may persist, though at reduced in-
tensity.
5. Under a sustained pressure, a process of adaptation frequently occurs,
whereby on continued repetitive stimulation the initially inhibited flash recovers and
then facilitates, sometimes to much higher intensities than prior to compression.
6. On sudden decompression, part way or all the way to atmospheric pressure,
the initial effect is always an increase in flash intensity over that occurring under
pressure, or sometimes over that occurring prior to compression. The only excep-
tion occurs when excitability has disappeared completely, as occasionally happens.
7. The recovery process after pressure assumes a variety of unpredictable pat-
terns; in some instances the first one to three flashes are excessively high ("over-
shoot"), followed by rapidly decreasing flash maxima, whereas in other instances
a gradual facilitation and decline take place.
8. Excess luminescence intensity in the recovery phase is independent of main-
taining repetitive stimulation during the preceding period under pressure.
9. Excitability that has been lost through fatigue or unknown causes can be
restored in some instances by subjecting the specimen to pressures of 3000 to 5000
psi for periods of 1 to 5 minutes.
10. Qualitatively the same results of pressure are observed at various tempera-
tures between 15 and 35° C. Any definite influence of temperature on the effects
of pressure is obscured by variations in the quantitative effect of a given pressure on
different specimens and on the same specimen in different physiological states.
11. Urethane, in concentrations between 0.05 and 0.5 M, causes a progressively
increasing reduction of flash maxima with duration of exposure of the drug, and
at rates that increase with drug concentration and temperature. Qualitatively the
same phenomena are observed with respect to the influence of pressure on the flash
jn the presence as in the absence of urethane.
12. The Mnemiopsis photogenic system is particularly sensitive to destructive
effects of urethane and of elevated temperatures, thereby obscuring the possible
existence of reversible thermal inactivation reactions and the possible influence of
pressure thereon.
LITERATURE CITED
BROWN, D. E. S., 1957. Temperature-pressure relation in muscular contraction. In: Influence
of Temperature on Biological Systems, F. H. Johnson (ed. ), pp. 83-110. Amer.
Physiol. Soc. Publishers, Washington, D. C.
CHANG, J. J., 1954. Analysis of the luminescent response of the ctenophore, Mnemiopsis Lcidyi,
to stimulation. /. Cell. Comp. Physiol., 44 : 365-394.
CHANG, J. J., 1956. On the similarity of response of muscle tissue and of lampyrid light organs.
/. Cell. Comp. Physiol., 47 : 489-492.
CHUN, C., 1903. Aus den Tiefen des Weltmeeres. Gustav Fischer, Jena. (Cf. p. 545.)
JOHNSON, F. H. (ed.), 1957. Influence of Temperature on Biological Systems. Amer. Physiol.
Soc., Publishers, Washington, D. C.
14 JOSEPH JIN CHANG AND FRANK H. JOHNSON
JOHNSON, F. H., H. EYRING AND M. J. POLISSAR, 1954. The Kinetic Basis of Molecular Biology.
John Wiley and Sons, New York.
MARSLAND, D. A., 1957. Temperature-pressure studies on the role of sol-gel reactions in cell
division. In: Influence of Temperature on Biological Systems, F. H. Johnson- (ed.).
pp. 111-126, Amer. Physiol. Soc., Publishers, Washington, D. C.
SIE, H.-C., J. J. CHANG AND F. H. JOHNSON, 1958. Pressure-temperature-inhibitor relations in
the luminescence of Chaetopterus variopedatus and its luminescent secretion. /. Cell.
Comp. Physio!., in press.
SPYROPOULOS, C. S., 1957a. Response of single nerve fibers at different hydrostatic pressures.
Amer. J. Physiol., 189 : 214-218.
SPYROPOULOS, C. S., 1957b. The effects of hydrostatic pressure upon the normal and narcotized
nerve fiber. /. Gen. Physiol., 40 : 849-857.
TASAKI, I., AND C. S. SPYROPOULOS, 1957. Influence of changes in temperature and pressure on
the nerve fiber. In: Influence of Temperature on Biological Systems, F. H. Johnson
(ed.), pp. 201-220, Amer. Physiol. Soc., Publishers, Washington, D. C.
OSMOTIC HATCHING IN THE EGGS OF SOME
FRESH-WATER COPEPODS
CHARLES C. DAVIS
Department of Biology, H7estern Rcsen'c University, Cleveland 6, Ohio
Hatching of the eggs of eucopepods apparently has been described only by
Marshall and Orr (1954, 1955) and Ziegelmayer (1927). The two papers differ
fundamentally in the interpretation that is given, for Ziegelmayer, working with 17
species of Cyclops, thought the outer egg membrane swelled over a period of 6 to 12
or more hours, developing a pressure between the inner and the outer membranes.
Subsequently, according to him, the outer membrane would burst, and the nauplius
emerge, closely surrounded by the inner membrane. On the other hand, Marshall
and Orr thought that the inner membrane swelled, and that the pressure developing
within it resulted in the rupture of the outer membrane. The inner membrane, con-
taining the unhatched nauplius, emerged through the opening. These authors
described a considerable space between the nauplius and the stretched inner mem-
brane. Observations were made by Marshall and Orr ( 1954 ) primarily on the
marine calanoid, Calanus finniarchicus, but they supplemented their study by the
examination of other marine copepods belonging to four sub-orders (Calanoida,
Cyclopoida, Harpacticoida, and Caligoida), and by the examination of two species
of fresh-water cyclopoids (Cyclops agilis and C. viridis).1
From the general appearance of the hatching process, both Ziegelmayer and
Marshall and Orr concluded that it was osmotically controlled. Although Ziegel-
mayer's paper was devoted largely to reports of experiments on the permeability
and changes of permeability of the membrane, he performed no experiments that
were aimed at proof of osmotic control. Marshall and Orr performed a rather
inconclusive experiment, in which they placed 15 Calanus finniarchicus eggs that
were nearly ready to hatch in sea water that had been diluted with a small quantity
of fresh water. Eleven of the eggs bulged and 7 hatched successfully, whereas in
the controls in undiluted sea-water only 3 out of 14 bulged and hatched.
In explanation of the onset of the hatching process, Ziegelmayer was convinced
that there was a change of the permeability of the membrane, caused by some in-
fluence (hormone?) from within (hence, from the enclosed nauplius). On the
other hand, Marshall and Orr (1954) suggested that it (p. 400) "might be that a
sudden increase of excretion by the embryo leads to an increased content of salts and
the imbibition of water."
MATERIALS AND METHODS
Ovigerous specimens of Diaptomus siciloides, D. ashlandi, D. oregonensis,
Cvclops bicuspidatus and Mesocyclops eda.r were taken from the plankton in
1 After the present paper was in press, it was discovered that P. Heegaard in 1947 (Con-
tribution to the phylogeny of the arthropods : Copepoda, in Spolia Zool. Mus. Hauniensis, 8 :
1-227) had given figures and brief descriptions clearly indicating that hatching in C aligns
curtits, C. rapa.r, and Lernacoccra branchialis occurs in a manner comparable to that described
by Marshall and Orr (1954).
15
16 CHARLES C. DAVIS
Hatchery Bay, Put-in-Bay, Ohio (western Lake Erie) in mid- June to mid- July,
1958 (air and water temperatures were 21° ± 1° C. during the period of collection
and observation — 23° C. for M. eda.r). Most of the observations, and all of the
experiments, were with Diaptomus ashlandi and D. siciloides. Specimens were
kept in U. S. Bureau of Plant Industry model watch-glasses until chosen for de-
tailed observations, at which time the egg sacs were removed from the mothers and
the eggs observed with a compound microscope magnifying lOOx and 443 X. No
coverslip was used, the objective being immersed directly into the water when
necessary.
Observations of the hatching procedure were supplemented by experiments de-
signed to test the validity of the osmotic theory of hatching. Sucrose solutions were
made up of the following concentrations: 1 M, 0.5 M, 0.4 M, 0.3 M, 0.2 M, 0.1 M,
0.05 M, 0.04 M, 0.03 M, 0.02 M, and 0.01 M. These solutions were used to ascer-
tain the approximate osmotic value of the fluid within the inner membrane, and to
test the permeability to water of the inner membrane and larval surface at certain
stages of the development of the nauplius in relation to the moment of hatching. To
avoid repetition, the detailed experimental procedures are more conveniently given
below under Results.
The work for this paper was undertaken at the F. T. Stone Laboratory, and was
supported by a research stipend furnished by the Ohio Division of Wildlife, through
the Ohio Natural Resources Institute, Charles A. Dambach, Director. Sincere
thanks are extended to Dr. Dambach, and to Dr. Loren Putnam, Director of the
Stone Laboratory, for their unstinted aid in providing space and equipment for the
accomplishment of the project.
RESULTS
1. Simple observations
No difference was observed in the hatching of the eggs of Diaptomus siciloides,
D. oregonensis, and D. ashlandi. Before hatching, the eggs averaged 109 p. in
diameter, and thus had a volume of 678,110 /A Individual eggs were separated
from each other by the material of the egg sac proper, as shown in Figure 1, which
depicts a portion of an egg mass of D. siciloides, with the eggs near the hatching
stage. In this preparation the eggs were spread apart with fine needles for ease in
viewing them, and one has been displaced from the egg sac. As shown, there are
spaces of small but variable magnitude between the egg sac material and the eggs
proper. Only a single membrane can be distinguished around the enclosed larva
but in reality there are two, as shown below. The contained embryo for the most
part fills the entire space within the membranes, although when viewed from the
dorsal aspect, the naupliar appendages are visible laterally, closely appressed against
the body. The red bi-crescentic naupliar eye is clearly visible anteriorly. The
nauplius was observed to twitch its legs from time to time as much as 24 hours
before hatching began.
The initiation of hatching was indicated by the appearance of a fluid-filled space
between the nauplius and the egg membranes. This was followed very quickly by
the bulging of the egg surface (Fig. 2). The outer membrane broke, probably due
to the internal pressure, and it could be seen that there was a second inner membrane
HATCHING IN COPEPOD EGGS
17
protruding through the opening (Fig. 3). In eggs which were isolated from the
egg mass, it was clear that the two halves of the outer membrane were pushed aside
by the emerging inner membrane. For some time a portion of the inner membrane
remained inside of one of the halves of the outer membrane (Fig. 4), but eventually
the entire inner membrane slipped out as a perfect sphere, and left the outer mem-
brane behind (Fig. 5). For all of this period the volume enclosed by the inner
membrane was increasing, so that when the stretched membrane slipped free of the
outer membrane it had an average diameter of 153 //, and a volume of 1,875,400 ju,3.
Thus the volume, compared to the original volume of the egg, increased in a ratio of
2.77:1. The nauplius was completely surrounded by a fluid-filled space.
During the extrusion of the inner membrane from the outer, the unhatched
nauplius typically remained completely motionless. In some instances it twitched,
but never more than it had for several hours previously. At first after extrusion,
200 JJ
FIGURE 1. A portion of an egg sac of Diaptounts siciloidcs with eggs near the hatching stage.
The sac was teased apart with fine needles, and one egg has been displaced from it. Note that
the individual eggs lie somewhat loosely within the secretion forming the egg sac. Also note
that the embryo is closely invested by the egg membranes, so that there is no space between the
membranes and the enclosed nauplius.
the three pairs of appendages were held as they were before the swelling of the
inner membrane. Although each larva that was observed at this stage was ex-
amined carefully, and larvae were seen from all possible angles, no trace of a third
membrane, close around the animal, could be detected. After a period of one or
two minutes, the appendages suddenly broke free from the sides of the body, and
became extended laterally in their usual free-living naupliar position. This was
observed many times. When the larva was viewed from a dorsal or ventral posi-
tion, it could be seen that the appendages broke free from the sides of the body in a
series of two or three short jerks. There was no evidence that this movement took
place through muscular contractions of the animal (though this was not precluded).
The appearance was that the appendages were, due to their structure and elasticity,
pulling in the direction of their normal naupliar position, and that suddenly some
tissue, membrane, or other material holding the appendages down tore loose from
18
CHARLES C. DAVIS
O.I
MM
FIGURES 2-5. Individual eggs of Diaptomus siciloidcs during hatching. Figure 2 : a fluid-
filled space has appeared between the egg membranes and the nauplius, and the membranes are
bulging on one side. The inner and outer egg membranes are not yet distinguishable. Figure 3 :
HATCHING IN COPEPOD EGGS
19
the strain. A few seconds after the appendages assumed their naupliar position,
the animal began to move them in the twitching manner characteristic of free-swim-
ming calanoid nauplii. Approximately a minute later the diaphanous membrane of
the sphere burst with great suddenness. The internal fluid, being under con-
siderable pressure, was forced almost explosively out through the breach, carrying
the nauplius with it. \Yhile the nauplius was within the sphere, the setae of the
appendages, and the setae at the posterior end of the animal, appeared as though
they could easily and readily perforate the delicate membrane, for they impinged
upon its surface as the animal continued its movements. However, this method of
escape apparently did not occur during normal hatching, for almost invariably the
larva escaped head first, whereas the setae touched the membrane at the opposite
O.I MM
FIGURE 6. The nauplius of Cyclops bicuspidatus within the sphere
formed by the inner egg membrane after extrusion.
side of the sphere. Upon close observation it could be seen that the end of each
of the setae was pliable and bent over when it touched the membrane. Penetration
by the sharp ends of the setae as suggested by superficial examination, could not
occur (see below in section 4 for further observations on the breaking of the
membrane).
The total time elapsing from the first indication of hatching to its completion
ordinarily varied from 7 to 8 minutes.
the outer egg membrane has burst, and the inner membrane is bulging out through the opening.
The outer and inner membranes are clearly seen. Figure 4 : the outer membrane covering the
anterior half of the emerging larva has slipped off, but the inner membrane remains within the
other half of the outer membrane. The naupliar appendages are closely appressed against the
sides of the body. Figure 5 : the inner membrane, expanded to its maximum diameter, has
slipped out of the outer membrane entirely, forming a perfect sphere. The naupliar appendages
have assumed their swimming position.
20 CHARLES C. DAVIS
Hatching was observed in single sets of eggs from Cyclops bicuspidatus and
Mesocydops edax. It occurred in much the same manner as in the 3 species of
Diaptomus, and took an average of 6^/2 minutes from start to fini'sh. As with
Diaptomus, the nauplius was passive during the swelling and protrusion of the inner
membrane from the outer membrane and from the egg mass, except for a few
twitches. The volume increase of the contents of the inner membrane was not quite
as extensive as in Diaptomus. Before hatching the eggs averaged 82 /j. in diameter.
The spheres averaged 112 p. Hence the average volume changed from 288,710 /u,3
to 735,655 ju,3, or a ratio of 1:2.55. The nauplius occupied a greater portion of the
contents of the sphere than was the case with Diaptomus (Fig. 6), and it did not
escape in the same explosive fashion. When the membrane surrounding the sphere
burst, there was a sudden release of pressure, and the sphere collapsed, opening, as
with Diaptomus, at the head end of the larva. In both of these cyclopoids, in all the
instances observed, however, the nauplius remained somewhat entangled in the
membrane, and escaped only after a short struggle.
2. Experiments indicating the osmotic nature of hatching
The general appearance of the hatching process in copepods strongly suggests
that it is osmotically regulated. However, this has not been proven incontrovertibly
by any experimental results heretofore reported.
In preliminary exploratory experiments, eggs of Diaptomus siciloides in the
beginning stages of hatching were immersed in a 1 M solution of sucrose, or in
double-distilled water. The permeability to water of the inner membrane and the
naupliar surface was clearly shown by the fact that in the sucrose solution the larvae
and the inner membranes shrank drastically from the outer membrane through the
osmotic loss of water to the hypertonic solution. Obviously the larvae were de-
stroyed (Fig. 7). In double-distilled water, on the other hand, hatching (D.
siciloides} was completely normal except that the average time consumed during
the hatching process was reduced to 6 minutes, compared to an average of 71/t>
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 2V2 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 (Af) 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 A4 is greater than Af, which in turn is greater than A0.
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 21/^> 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 wrere 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 1011 /A3). 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° /A3 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 few7
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
Af of this fluid greater than the A0 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 Af and As. 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. Af must be greater than A0, and greater than A, at the time the egg was
laid. No information exists at present bearing on the relation of As of the nauplius
to Af in these marine species just before hatching. A4 might either equal Afi, or it
HATCHING IN COPEPOD EGGS 27
might be less than Ar. 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 showrn 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 3C)
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
H2O + CO2
C.A.
HC03~
H+
t
H2C03
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 <?
9
cf
9
d"
9
1/25
9.2
7.5
10.0
13.6
1.4
1.2
44.9
31.5
5.9
10.8
65.1
67.0
2/21
7.6
7.2
20.5
22.0
3.8
3.0
34.2
30.2
5.7
6.6
69.7
71.3
7.1
7.0
3/31
4.4
3.0
12.9
19.4
3.2
2.1
30.7
27.7
3.0
10.2
74.7
77.8
9.5
8.1
4/20
3.5
1.8
19.5
16.1
3.1
3.0
31.1
24.1
14.0
4.1
78.1
76.2
7.4
4.8
5/30
5.9
5.7
14.5
15.4
3.0
2.3
27.0
27.9
10.6
10.3
71.1
58.5 :
2.6
6/17
3.8
4.6
19.8
19.8
2.4
2.0
22.7
23.2
5.2
7.1
70.1
61.3
4.7
2.4
7/31
5.6
10.0
18.5
19.0
2.3
1.8
26.3
26.4
5.7
5.2
68.2
55.0
1.9
2.2
8/30
6.7
6.7
16.3
13.2
2.4
2.6
21.5
18.4
1.0
1.0
63.2
74.3
3.7
2.7
9/27
12.4
15.5
15.5
18.7
2.4
1.8
34.0
26.3
1.6
1.9
68.7
64.9
5.5
3.5
10/31
11.9
12.8
22.4
21.2
2.8
1.5
35.2
35.4
3.4
3.9
70.0
73.5
3.2
2.7
11/28
14.0
14.4
10.5
15.9
2.0
2.0
33.4
35.1
7.8
6.9
69.0
67.0
5.3
4.4
12/18
17.5
16.6
15.9
20.1
3.7
2.3
36.2
39.8
3.2
3.6
66.1
66.3
7.1
5.8
Av.
16.4
24.5
2.7
2.1
31.5
29.0
4.8
6.0
69.5
67.7
4.6
3.8
* GI refers to gonad index obtained as denned in the text. NPN refers to non-protein
nitrogen.
appear in the data of this table. At times of the highest gonad index, the gonads
per unit weight tend to contain more lipid, protein, glycogen and ash and less water
(especially in the female) than at the time of low gonad index. A more significant
rendition of the data of Table I is given in Figure 1, because it shows the distribution
of each chemical in gonads of members of a population sample taken each month.
It will be observed that at all times of the year gonads of some individuals of a
population sample may have relatively large amounts of certain constituents, while
gonads of other individuals of the same population sample may have a relatively
small amount. Certain trends do appear but an average value which emphasizes
these trends gives a less true picture of the actual facts than the distribution plot.
Statistics calculated from the data are not a truthful representation of the data, be-
cause standard deviations and confidence limits are meant to apply to a population
52
20
16
Gonad l2
Index 8
%
0
Lipid
% dry
32
28
24
20
16
12
8
4
50
Protein 40
% dry wt. 30
20
10
22
20
18
Glycogen
% dry wt. I2
10
8
6
4
2
0
NPN 40
%drywt.3
2.0
1.0
GIESE, ET AL.
J25 F2I M2I A26 M30 JI9 J3I A29 527 031 N28 DI8
L
42 24
Lb
• MALES
— O— FEMALES
--•--BOTH OR
UNDEFINED
-T3-—
FIGURE 1. The distribution graphs illustrate the inhomogeneity of the population of sea
urchins throughout an annual cycle, not only in gonad index but in content of various organic
constituents in the gonads (last four graphs). Glycogen content of gonads shows greatest
variability, non-protein nitrogen (NPN), least. For explanation see text.
with a normal distribution, not to a skewed one such as is the population dealt with
here.
The over-all averages for the entire year disclose some interesting information
about the gonads (last line, Table I). The ovary is distinctly richer than the testis
in lipid and glycogen but the testis appears to be richer than the ovary in non-
protein nitrogen, protein nitrogen and possibly in salts (ash) and water, although
the few samples taken and their variability from month to month make any deduc-
tions on the latter two substances questionable.
ORGANIC PRODUCTIVITY OF SEA URCHINS
53
When a sample of animals is selected and the ones with a low gonad index are
compared with those with a high index, the contrasts in chemical constitution of
gonads during the course of the reproductive cycle are most clearly brought out as
seen in Table II. In addition to the chemicals discussed above, it is seen that the
RNA per unit weight of the ovary increases with its enlargement while the DNA
decreases ; in the testis the reverse is true, the RNA per unit weight decreases while
the DNA more than doubles.
These differences between ovary and testis are understandable in view of the
TABLE II
Chemical constituents in spent and gravid gonads of the purple sea urchin (in % dry wt.)
Sex and
condition
Gonad
index
NPN
Protein
RNA
DNA
TN
Reducing
sugar
cf spent
2.3
2.0
23.1
3.25
0.0014
5.7
0.007
—
2.0
27.4
3.46
0.0010
6.4
3.5
2.2
23.7
2.59
0.0018
6.0
Av.
2.9
2.1
24.7
3.10
0.0014
6.0
Gravid
17.8
1.3
35.0
5.0
0.0007
6.9
0.037
18.2
2.1
34.4
4.8
0.00065
7.6
18.9
1.0
39.4
3.8
0.00059
7.3
Av.
18.3
1.5
36.3
4.5
0.00065
7.3
<? spent
1.52
4.1
25.9
2.3
4.7
8.3
0.0036
2.84
2.1
36.0
2.4
4.1
7.9
2.43
2.2
36.0
2.1
4.4
8.0
Av.
2.26
2.8
32.6
2.3
4.4
8.1
Gravid
21.5
0.66
43.0
1.3
9.8
7.6
0.034
19.2
1.20
39.8
0.9
8.0
7.6
1.77
0.59
42.5
0.9
9.3
7.4
Av.
19.5
0.82
41.8
1.0
9.0
7.5
NPN
Protein
Lipid
Glycogen
TN
Water
9 spent
1.18
3.75
23.2
9.5
0.41
7.5
63.2
1.28
3.07
20.1
18.7
3.35
6.3
78.8
1.49
2.84
28.2
15.5
2.77
7.4
—
Av.
1.32
3.22
23.8
14.6
2.18
7.1
71.0
Gravid
21.8
1.83
33.5
18.1
7.98
7.2
17.0
1.92
26.8
19.9
2.35
6.2
64.9
17.7
2.41
42.1
21.3
4.54
9.1
70.0
Av.
18.8
2.05
34.1
19.8
4.96
7.5
67.4
d1 spent
1.43
3.03
38.2
17.4
2.63
9.2
—
3.0
3.14
33.4
14.2
6.41
8.5
76.2
1.42
3.34
32.4
20.5
1.48
8.5
74.9
Av.
1.95
3.17
34.7
17.4
3.51
8.7
75.5
Gravid
21.3
1.63
37.5
13.0
4.24
7.9
21.0
1.91
24.4
11.0
9.86
5.8
65.1
21.6
4.47
35.2
12.6
4.43
10.1
66.3
Av.
21.3
2.67
32.4
12.2
6.51
7.9
65.7
54
GIESE, ET AL.
gametes produced and their prominence in the gravid gonads. It will be remem-
bered that female sea urchins can usually be distinguished from male sea urchins
during all months of the year by the presence of eggs in the ovary, even though the
eggs may be small and immature. Only occasional specimens are indeterminate as to
sex, either just after spawn-out or because they have not yet matured (very small
ones, that is, less than 17 mm. in test diameter are always indeterminate for the latter
reason). Conversely, males can usually be detected by the presence of sperm in the
testis. Eggs contain considerable stores of food for the development of the embryo
while sperm contain only stores for the brief period of locomotion of the sperm
preceding fertilization. A priori, one expects eggs to be rich in lipids and glycogen,
whereas sperm are expected to contain some glycogen as food reserve for movement.
One also expects the eggs to contain more RNA than sperm but less DNA. As
can be seen from the data, these expectations are indeed realized. More suprising
is the fact that the spent or immature gonads also show contrasts in chemical con-
TABLE III
Increase in organic constituents of gonads of the purple sea urchin during
growth from shrunken to maximal size (in mg.; total ivt. in grams)
c?
9
Relative
Relative
increase
increase
Spent
Gravid
Spent
Gravid
Gonad index
1.42
21.6
15.2 X
1.18
21.8
18.5 X
Total wt. (arbitrary) in
grams
1.0
15.2 15.2 X
1.0
18.5
18.5 X
Total nitrogen, mg.
84
1170
14.0 X 65.5
1369
21.0 X
Non-protein nitrogen,
mg.
29.9
266 8.9 X
26.6
328
12.3 X
Protein, mg.
336.5
5639
16.7 X
243
6512
26.7 X
Lipid, mg.
174
1854
10.4 X
146
3663
25.1 X
Glycogen, mg.
35
989 28.2 X
21.8
917.6
42.1 X
Reducing sugar, mg.
0.036
5.16 143.0 X 0.07
6.84
98.0 X
RNA, mg.
23
152
6.6 X
31.0
832.5
26.8 X
DNA, mg.
44
1368
31.1 X
0.014
0.120
8.6 X
stitution, especially the large lipid content of immature or shrunken ovaries as com-
pared to immature or shrunken testes. Presumably the lipids are present in the
ovarian epithelium which gives rise to the eggs. Histochemical studies would be
interesting on ovarian and testicular materials at different times in the gonadal cycle.
It is not possible to ascertain productivity of organic materials in the gonads of
the sea urchin on a per unit weight basis, because all that is then observed is a
shift in emphasis on certain materials, which accompanies the onset of maturity,
i.e., a synthesis of some materials at a greater rate than that of others. Further-
more, the relative content of water in the ovary declines to some extent concomi-
tantly with a general increase in the total mass of other substances in the ovary.
Therefore, to ascertain organic productivity of the gonads it is necessary to take into
consideration the increase in mass of the gonads, as well as their change in chemical
constitution (per unit weight) during the growth from a spent to a fully gravid con-
ORGANIC PRODUCTIVITY OF SEA URCHINS 55
dition. Gonads increase in mass by a ratio which equals the gonad index of a gravid
animal divided by the gonad index of a spent animal. For a female this is 18.5-fold,
for a male it is 15.2-fold (using the data for maximal and minimal sizes of gonads
given in Table II). If the gonads of a spent animal weigh 1 gram, as they would
in fact for an average-sized animal of 90 grams total wet weight, then the ovaries
of a gravid female of this size would weigh 18.5 grams and the testes of a gravid
male of this size would weigh 15.2 grams. The content of each chemical constituent
in the spent and gravid gonads of animals of this size could then be calculated by
multiplying the weight of the gonad in grams by its per cent content of each of the
constituents given in Table II. Data so calculated are given in Table III. By
dividing the content of each constituent in the gonad of a gravid individual by the
content of that constituent in the gonad of a spent animal, the relative increase in
mass of the chemical constituent in question during the growth of the gonads from
the spent to the gravid state was calculated and the data are given in Table III.
For example, to obtain the content in NPN in a spent ovary its weight, 1000 mg., is
multiplied by the average fractional content 4 of NPN in spent ovaries, 2.66 per
cent or 0.0266, giving 26.6 mg. To calculate the NPN in a gravid ovary its weight,
18,500 mg., is multiplied by the average fractional content of NPN in gravid ovaries
-1.775 per cent or 0.01775. This gives a value of 328 mg. The increase in mass
of NPN from spent to gravid condition is then 328 divided by 26.6, which is 12.3
times.
The chemical constituents showing the most striking total increases during the
growth of the gonad observed in Table III are of course the ones which have also in-
creased on a per unit weight basis. It will be seen that the total amount of DNA in
the testis increases by about 31 X, the RNA in the ovary 27 X, the glycogen in the
testis 28 X , the glycogen in the ovary 42 X , the lipid in the ovary 25 X , the lipid in
the testis 10 X, the protein in the testis 17 X , the reducing sugar in the testis 143 X
and in the ovary 98 X .
DISCUSSION
It is interesting at this time to inquire about several matters concerning the
gonadal biochemical cycle in the purple sea urchin. To what extent is it possible
to explain the chemical diversity in gonads in a population of sea urchins selected at
random at any time during the year? How does the build-up of the nutrients in
the gonads occur ? What is the over-all productivity of the purple sea urchin ?
The variability of chemical constitution of the gonads of the sea urchin during
the year may be just another index of the failure to get synchronized spawning in
this species. At almost all times the population is rather inhomogeneous with re-
spect to the gonad cycle, some animals having fairly well-developed gonads while
others are poorly developed or spent. Only in March and April is the gonad index
rather low for most specimens and only in December is it consistently high. Bi-
ochemical inhomogeneity of different individuals may therefore reflect population
inhomogeneity in gonadal development. Even when animals of like gonad index
are compared, however, one finds biochemical differences. Perhaps an individual
just spending or one just building up to the same intermediate gonad index, may be
4 The average of the values for the two groups of spent animals in Table II, namely, 2.1
and 3.2 per cent, giving 2.65 per cent or 0.0265.
56 GIESE, ET AL.
quite different histologically and histochemically. Information on this as a possible
explanation of chemical inhomogeneity is lacking at the present time.5
Another factor which may play a role in the variability in chemical constitution
of the sea urchin gonad is availability of nutrients at different times during the year,
or at any one time, a difference in availability of nutrients to each individual in the
population. The relative immobility of the urchins which have bored their way
into the soft rocks makes them dependent upon what grows in their immediate
vicinity or what the waves may bring to them by chance. The gonad is the main
storage organ of the sea urchin, a little organic material also being stored in the
gut (Hilts and Giese, 1949). When an urchin is starved the gonad shrinks and
its gonad size may decline even without spawning. However, the intestines of
almost all urchins from the field are filled with algae ; therefore food seems to be
generally available. The purple sea urchin's willingness to eat almost any food,
animal or plant, when starved, makes it seem unlikely that it lacks in quantity of
food in nature. However, the food may have unequal nutritive quality at different
times. No evidence was collected upon this point, but young growing algae are
known to contain much protein while old ones are made up, to a considerable ex-
tent, of polysaccharides which are probably a much less available source of food
(Wort, 1955). The availability of nutrients may therefore vary even though
the bulk of food taken in may be the same.
The build-up of nutrients in the gonads must be a relatively slow process, yet
the increase in organic matter during a gonadal cycle is rather striking, indicating
effective digestion, mobilization, and conversion of food. Digestion appears to be a
rather slow process in the sea urchin, since algae may be defecated for several
weeks from a single gutfull in an animal deprived of further sources of food. While
the enzymes of the sea urchin readily handle proteins and starch, they attack few
of the polysaccharides of the algae (Lasker and Giese, 1954; Huang and Giese,
1958). However, bacteria may play a role in digestion since they readily hydrolyze
the algal polysaccharides in the gut of the urchin. Where the nutrients go when
they leave the intestine is not clear. The perivisceral fluid contains some protein,
reducing sugar, lipid and very little non-protein nitrogen. Most of the protein
forms striking fibrous clots. When these are filtered out the remaining fluid
appears to be protein-free (TCA negative) .6 It is possible that the continual dribble
of sugar, amino acids, and possibly lipids, from the intestine into the body fluid, is
adequate for the build-up of the reserves in the gonads. However, it is desirable
that someone explore other pathways of nutrient transport, particularly by wander-
ing amebocytes and by the haemal system which extensively vascularizes both the
gut and the gonads (Hyman, 1955).
To assess the over-all productivity of the sea urchin it is necessary to consider
not only the gonad cycle and the increase in organic material which occurs there, but
also other possible constituents which accumulate organic materials. The only
5 That the small size of the sample of the population is not the cause of the variability of
the gonads is shown by a study with larger sample sizes by Josef Miller of Monterey Penin-
sula College. He compared the gonad index of samples of 10, 20, 40 and 80 sea urchins. The
gonad index for a given population of sea urchins at a given season was almost the same,
within a few per cent, regardless of the sample size.
6 However, two protein peaks are disclosed in paper electrophoresis studies of fluid filtered
after clotting (Favour and Giese, unpublished).
ORGANIC PRODUCTIVITY OF SEA URCHINS 57
organ of considerable size in the sea urchin other than the gonad is the intestine,
but some tissue is also present in the water vascular system, the muscles of the spines
and pedicellariae, the dermal branchiae, the epidermis, the mesenteries, and the
coelomic lining. In an urchin of about 90 grams, all of these structures are esti-
mated to weigh about 7 grams.
If, for purposes of argument this figure is tentatively accepted, then the total
increase in organic material with one gonadal cycle is approximately three-fold.
Unfortunately we do not know how many gonadal cycles a single sea urchin can
undergo in one season. The fact that a population of sea urchins collected at almost
any time of the year, with the exception of the time of the highest gonad index and
the period just after the maximal spawn, shows individuals with widely different
indices (see Figure 1 and the figures in Bennett and Giese, 1955), suggests that
a single individual may spawn several times during the year. If this is true, several
times the above figure may be a more nearly correct estimate of production of
organic material. Since the sea urchin also grows in diameter and bulk, the true
figure must be larger on this account as well. We do not at present have sufficient
data to make a determination of the growth rate and the rate of incorporation of
nutrients into body material.
SUMMARY
1. Monthly determinations were made of the amount of lipid, glycogen, non-
protein nitrogen, protein, water, and ash present per unit weight in gonads of the
purple sea urchin, Strongylocentrotus purpuratus. Tests for reducing sugar, DNA
and RNA were made for gonads at the height of the reproductive season and after
spawning-out.
2. A change in relative proportions of the chemical constituents was observed
with maturation of the gonads. In the ovary protein, lipid, glycogen, reducing
sugar and RNA increase proportionally more than the over-all increase in bulk of
the gonad, while DNA and possibly water, increase proportionally less. In the
testis, glycogen, reducing sugar, DNA and possibly protein, increase proportionally
more than the over-all increase in bulk, while RNA, lipid, and possibly water, in-
crease less than the increase in total bulk.
3. A considerable increase in the total amount of all the organic constituents
tested here occurs during the growth of gonads. Thus, a gravid ovary is about
18.5 times the bulk of a spent one and a gravid testis is about 15.2 times the bulk
of a spent one.
4. The sources of nutrients and the possible transport are discussed with refer-
ence to the literature.
LITERATURE CITED
BENNETT, J., AND A. C. GIESE, 1955. The annual reproductive and nutritional cycles in two
western sea urchins. Biol. Bull., 109 : 226-237.
CERIOTTI, G., 1952. A microchemical determination of desoxyribonucleic acid. /. Biol. Chcm.,
198: 297-303.
CONWAY, E. J., 1947. Microdifrusion and Volumetric Error. C. Lockwood, London. 2nd
ed. ; pp. 13-132.
FISKE, C. H., AND Y. SUBBAROW, 1925. The colorimetric determination of Phosphorus. /. Biol.
Chem., 66 : 375-400.
GIESE, ET AL.
GOOD, C. A., H. KRAMER AND M. SOMOGYI, 1933. The determination of glycogen. /. Biol.
Chcm., 100: 485-491.
HERSHEY, A. D., J. DIXON AND M. CHASE, 1953. Nucleic acid economy in bacteria infected
with bacteriophage T2. I. Purine and pyrimidine composition. /. Gen. Physiol., 36 :
777-789.
HILTS, S. V., AND A. C. GIESE, 1949. Sugar in the body fluid and tissues of a sea urchin.
Anat. Rec., 105 : 140.
HUANG, H., AND A. C. GIESE, 1958. Tests for digestion of algal polysaccharides by some
marine herbivores. Science, 127 : 475.
HYMAN, L. H., 1955. The Invertebrates IV : Echinodermata. McGraw-Hill Book Co., Inc.,
N. Y., pp. 558-569.
IVERSON, R. M., AND A. C. GIESE, 1957. Synthesis of nucleic acid in ultraviolet-treated Es-
cherichia coli. Biochim. Biophys. Ada, 25: 62-68.
LASKER, R., AND A. C. GIESE, 1954. Nutrition of the sea urchin, Strongvlocentrotus purpuratus.
Biol. Bull, 106: 328-340.
LESLIE, I., 1955. The nucleic acid content of tissues and cells. In : The Nucleic Acids, Vol. II,
Academic Press, New York ; 576 pp.
MARKHAM, R., 1942. A steam distillation apparatus suitable for micro-Kjeldahl analysis.
Biochem, J., 36: 790-791.
MEYER, K. H., 1943. The chemistry of glycogen. Advances in Ensymol., 3 : 109-135.
OGUR, M., AND G. ROSEN, 1950. The nucleic acids of plant tissues. I. The extraction and
estimation of desoxypentose nucleic acid and pentose nucleic acid. Arch. Biochem., 25 :
262-276.
SCHMIDT, G., L. HECHT AND S. J. THANNHAUSER, 1948. The behavior of the nucleic acids
during the early development of the sea urchin egg (Arbacia). /. Gen. Physiol., 31:
203-207.
SCHMIDT, G., AND S. J. THANNHAUSER, 1945. A method for the determination of desoxy-
ribonucleic acid, ribonucleic acid, and phosphoproteins in animal tissues. /. Biol.
Chem., 161 : 83-89.
SCHNEIDER, W. C., 1945. Phosphorous compounds in animal tissues. I. Extraction and
estimation of desoxypentose nucleic acid and of pentose nucleic acid. /. Biol. Chem.,
161 : 293-303.
SEIFTER, S., S. DAYTON, B. Novic AND E. MUNTWYLER, 1950. The estimation of glycogen
with the anthrone reagent. Arch. Biochem., 25 : 191-200.
SOMOGYI, M., 1945. Determination of blood sugar. J. Biol. Chem., 160 : 69-73.
SOMOGYI, M., 1952. Notes on sugar determination. /. Biol. Chem., 195 : 19-23.
WORT, D. J., 1955. The seasonal variation in chemical composition of Macrocystis integrifolia
and Macrocvstis leutkcana in British Columbia coastal waters. Canad J. Bot., 33 :
323-340.
THE PHYSIOLOGY OF SKELETON FORMATION IN CORALS.
I. A METHOD FOR MEASURING THE RATE OF CALCIUM DEP-
OSITION BY CORALS UNDER DIFFERENT CONDITIONS
THOMAS F. GOREAU1
Department of Physiology, University College of the Wcsi Indies, and
The New York Zoological Society
The purpose of this study is to examine the rate of growth of reef-building corals
by measuring the calcium deposition in the skeleton with the aid of a new method
using radioactive calcium-45 as tracer. With this procedure it was possible to
determine calcification rates in the different parts of coral colonies, and to estimate
quantitatively the effect of light and darkness, zooxanthellae and carbonic anhydrase
inhibitors on skeletogenesis.
Numerous attempts have been made in the past to estimate the growth rates of
reef-building corals, mostly by letting weighed and measured coral colonies grow
in their natural habitat for periods of months to years (Agassiz, 1890; Abe, 1940;
Boschma, 1936; Edmondson, 1929; Kawaguti, 1941; Ma, 1937; Mayor, 1924;
Motoda, 1940; Stephenson and Stephenson, 1933; Tamura and Hada, 1932; Vaug-
han, 1919). Recently, Kawaguti and Sakumoto (1948) tried, by a chemical
method, to determine the rate of calcium uptake of corals in light and darkness.
Using calcium-45 as tracer, we have developed a rapid and precise method for
measuring the rate of incorporation of calcium into the coral skeleton under con-
trolled laboratory conditions (Goreau, 1957). The preliminary experiments, de-
scribed here, were carried out on the following coral species : Manicina areolata
(Linne), Cladocora arbuscula (Lesueur), Porites divaricata (Lesueur), Acropora
prolijcra (Lamarck), Madracis decactis (Lyman) and Oculina diffnsa (Lamarck)
from Jamaica, B.W.I.; Acropora conferta (Quelch) from Eniwetok Atoll; and
Montipora vcrrucosa (Lamarck), Porites compressa (Quelch), Pocillopora dami-
cornis (Linne) and Porolithon sp., a coralline alga, from Hawaii.
All the madreporarian corals used in these experiments are shallow-water forms
which contain zooxanthellae. Among these, Oculina diffnsa is the only species
which has not been collected from reefs, but it is common in Kingston Harbour
where it grows on rocks on a muddy bottom (Goreau, 1958). The Hawaiian
Porolithon listed above is a calcareous alga of the family Corallinaceae, representa-
tives of which are important reef builders in the Central Pacific (Emery, Tracey
andLadd, 1954).
PROCEDURE
Freshly collected coral colonies in good condition were put into glass vessels
containing filtered sea water and fitted with tight covers. Aeration, circulation and
pH were maintained by bubbling a slow stream of air through the water. The
1 Mailing address : Department of Physiology, University College of the West Indies, Mona
St. Andrew, Jamaica, B.W.I.
59
60 THOMAS F. GOREAU
temperature was kept to within 1° C. during the experiments (about 25° C. in
Jamaica and Hawaii, 28.5° C. in Eniwetok) by keeping the vessels partly immersed
in a water bath. After allowing the coral to acclimatize for twenty-four hours,
neutralized Ca45Cl2 was added to give about 20,000 c.p.m./ml. of sea water. The
amount of calcium thus added was less than five per cent of the total dissolved Ca++
already present. The initial activity was determined by counting 60-//.1 aliquots
taken from each vessel after one hour, to allow for complete mixing of the isotope.
In addition to the living corals, pieces of clean dead corallum from the same
species were included in each vessel to act as controls for measuring the inorganic
isotopic exchange rate of the coral skeleton during the experiments.
Samples of coral and water were repeatedly taken, starting with three hours
from the beginning of the experiment, by the following method : a coral colony,
together with its control, was removed from the vessel and small pieces were cut
off with scissors or cutting pliers. From five to fifteen replicate samples of about
one hundred milligrams each were taken at a time. Samples were collected only
from homologous parts of the colonies. This was particularly important in branch-
ing corals such as Acropora and Ponies where there were shown to be strong dif-
ferences in the rate of calcium uptake between the apical and lateral branch polyps.
The coral pieces were placed on filter paper to remove excess radioactive sea
water, then washed in five two-minute changes of slightly alkaline distilled water.
After this, each sample was dissolved in a separate tube containing two milliliters
dilute HC1, and heated to boiling. The coral suspension was homogenized to
disperse the organic matter. The contents of each tube were made up to five mil-
liliters with distilled water, and a 500-/xl aliquot was taken for Kjeldahl nitrogen
determination.
The calcium in each tube was precipitated as the oxalate by the method of Vogel
(1943), and filtered out on pre-weighed Whatman No. 42 filter paper planchets,
using a cone to spread the precipitate in circles of uniform diameter. The dried
and weighed samples were counted with an end window G-M tube, and the observed
activity corrected for self-absorption.
In the early stages of these investigations, the question arose of choosing a
suitable parameter on the basis of which the calcium uptake could be expressed. For
example, Mayor (1924) measured coral respiration in terms of tissue weight after
the corallum had been dissolved with nitric acid; Odum and Odum (1955) deter-
mined biomass by loss on ignition at 600° C. ; and Kawaguti and Sakumoto ( 1948)
measured calcification rates per gram coral. None of these methods was con-
sidered satisfactory. The writer had previously used organic nitrogen as a measure
of total cellular matter in corals (Goreau, 1956). The relationship of organic
nitrogen to tissue weight was determined for the polyps of Mitssa angulosa, a coral
from which fairly large skeleton-free pieces of tissue could be readily obtained. In
this species nitrogen constituted 2 per cent of the wet weight and 11.2 per cent of
the dry weight. All results, save those of the exchange controls which lacked tissue,
were expressed in terms of calcium deposited per milligram of nitrogen, on the
assumption that the nitrogen is a measure of the total coral (plus zooxanthellae)
protein present. Nitrogen was determined by the micro-Kjeldahl method of Ma
and Zuazaga (1942).
The amount of calcium taken up by the coral was calculated from the specific
activity of the sea water in the vessels. This was determined by counting 60-jul
SKELETON FORMATION IN CORALS
61
water aliquots spread to a constant diameter in lens paper circles mounted on micro-
scope coverslips and dried under a lamp. The observed count was corrected for
self-absorption and the specific activity of the water calculated from its calcium
content.
THE CALCIUM EXCHANGE IN THE SKELETON CONTROLS
Equilibrium exchanges of calcium between the skeleton and sea water were
determined on samples of dead coral devoid of tissue, and run at the same time as
the living experimental colonies. Isotopic equilibrium appeared to be established
2000
2
5
-i
o
e>
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
6.3±1.58 (9)
9.8±0.54 (10)
7.8±1.70 (11)
12.4±6.50 (12)
80 _i_ 2 if. M r\\*
6.1±0.20* (10)
5.0±1.00 (8)
7.4±2.10 (7)
7.2±5.00* (11)
0.7
0.01
<0.7
<O.OS
Acropora conferta
Monti pora verrucosa
Pocillopora damicornis
. Zit-J./O \iv)
11.9±5.60 (9)
10.3±3.90 (11)
1 n_i_n AQ I \ i\
9.7±3.4 (10)
6.8±2.1 (10)
0.3
<0.03
Madracis decactis
Oculina diffusa
Porolithon sp.
i.uiu.^y \L£)
1.6±0.38 (7)
8.8±0.58 (11)
0.8±0.15* (9)
3.3±0.55 (13)
0.01
<0.001
* Measurements made on individual polyps.
As seen by the standard deviations of the results, there were usually large varia-
tions in the calcium uptake rates of individual samples even if these were taken from
adjacent morphologically comparable regions of the same colony. This was never
true of the exchange controls. The scatter was not attributable to injury, as all
damaged corals were discarded, and the error in counting, weighing and nitrogen
determinations was kept below three per cent. In regard to this, our tentative
interpretation is that the calcification rates of individual polyps fluctuate, and that
some are in a resting stage while others are more or less vigorously growing.
THE EFFECT OF THE REMOVAL OF ZOOXANTHELLAE ON THE CALCIFICATION
RATE OF SOME REEF CORALS
All tropical reef-building corals contain zooxanthellae. Their presence as in-
tracellular symbionts in the tissue of the coelenterate host has resulted in a great
deal of controversy as to their possible role in the biological economy of the coral
reef and its component animals. Boschma (1924, 1925a, 1925b, 1925c, 1926, 1929)
SKELETON FORMATION IN CORALS
65
400
UJ
o
tr
h-
z
o
o
_l
<
o
o
300-
200-
100
LEGEND
•0— LIGHT
•t- DARK
10
20
30
TIME IN HOURS
FIGURE 3. The progressive incorporation of calcium into the skeleton of Acropora prolifera
in light and dark. The results are expressed as /j-g calcium taken up per milligram nitrogen.
The vertical lines drawn through the points represent the standard deviation of the means.
66
THOMAS F. GOREAU
concluded that corals could digest zooxanthellae in the lateral lobes of the mesenterial
filaments when no animal food was available. Yonge and Nicholls (1930, 1931a
and 1931b) demonstrated that, under the conditions of their experiments, corals
were unable to derive enough food from the zooxanthellae to prevent starvation if
deprived of their normal animal food supply. They also showed that zooxanthellae
could not be digested by corals due to the absence of carbohydrate-splitting digestive
enzymes and that these algae were extruded intact and in large numbers when the
coral was kept in darkness for long periods of time, or whenever the metabolic rate
of the coral was depressed, i.e., by starvation or high temperature. The question
of whether or not the reef-building corals are at least in part herbivorous, i.e., feed-
ing on their zooxanthellae, has recently been revived by Sargent and Austin (1954)
and Odum and Odum (1955) who concluded from their productivity studies that at
least some of the organic matter produced by zooxanthellae and boring algae may
be utilized by the coral host. Unfortunately, these authors were unable to verify
the existence of such an internal food cycle by experimental means. At the present
TABLE II
Calcium uptake by colonies of Oculina diffusa and Manicina areolata
in presence and absence of zooxanthellae
Number of samples in brackets
Species
Light
Dark
With zooxanthellae
Without zooxanthellae
With zooxanthellae
Without zooxanthellae
0. diffusa*
M. areolata**
1.63± 0.38 (7)
462.00±63.20 (11)
0.37=1=0.01 (6)
28.40±7.80 (9)
0.81 =fc 0.15 (9)
71.70±14.90 (8)
0.26±0.01 (5)
30.20±6.20 (10)
* Measurements made on individual polyps, in //g Ca mg. N : hr. l.
** Individual samples taken from different colonies, calcium uptake expressed in counts per
minute per milligram skeletal calcium at eighty hours.
time, it is still necessary to agree with the conclusions of Yonge and Nicholls (1930,
1931b) that reef corals are specialized carnivores, the exceptional proliferative
powers of which are probably due to an increased metabolic efficiency made possible
by the ability of the zooxanthellae to assimilate many of the metabolic waste pro-
ducts of the animal host.
The zooxanthellae per se are not necessary to individual coral polyps, nor do
they appear to be directly linked with the calcification process since they are absent
from deep sea and cold water corals, while they are present in many non-calcareous
tropical shallow water coelenterates.
We have determined the effect of the presence or absence of the zooxanthellae
on reef coral calcification in Manicina areolata and Oculina diffusa. Colonies of
these corals, which are normally yellowish or greenish brown in colour, were kept
in circulating sea water in darkened tanks for periods of about six weeks, to cause
gradual extrusion of the zooxanthellae. The experiments were run only when the
coenosarc of the corals became completely colourless and transparent, and when
small pieces failed to give the chlorophyll test on extraction with eighty per cent
SKELETON FORMATION IN CORALS
67
TABLE III
Calcification rates in different parts of branching coral colonies, in jug Ca mg. AT-1 hr.~l
Number of samples in brackets
Species
Apical polyps of primary branches
Lateral polyps
M. verrucosa
P. compressa
P. damicornis
A. conferta
11.8±3.90 (9)
7.8±1.70 (11)
6.8±2.65 (11)
8.2±3.76 (10)
1.38±0.50 (6)*
1.55=b0.20 (5)*
1.31±0.72 (6)*
1.87±0.92 (8)**
* Lateral polyps taken from base of branch.
** Apical polyps of secondary branches.
acetone. These decolorized corals were at all times fully expanded and appeared
to be normal, except for the lack of zooxanthellae.
The experiments were conducted in both light and darkness, as described in
the foregoing section. In the two species observed so far, loss of the zooxanthellae
caused the rate of calcium deposition to fall to very low levels as shown in Table II.
The results for Manicina areolata are expressed in terms of the specific activity
owing to the accidental loss of the nitrogen samples. The experiment on Ocidina
diffusa ran for eight days and the results are given in terms of the nitrogen content.
It is significant that removal of the zooxanthellae almost abolishes the response of
the calcification reaction to light which is seen in the normal controls containing
zooxanthellae.
Although the zooxanthellae seem to play an important role in determining
calcification rates in reef-building corals, certain, as yet unknown, physiological
factors operate to control the basic mineralization process in a manner which bears
no obvious relationship to the number of algae present in a given species. This is
illustrated by the fact that large apical polyps of some of the branching acroporid
corals contain few zooxanthellae but calcify several times faster per unit of tissue
nitrogen than the yellowish brown lateral polyps which are literally stuffed with
algae.
TABLE IV
Calcium-45 uptake of coral treated with 10~z M Diamox
in light and darkness, in ^g Ca mg. N~l hr.~l
Number of samples in brackets
Species
Light control
Light with Diamox
Dark control
Dark with Diamox
M. decactis
0.98±0.49 (12)
0.56±0.05 (10)
P. divaricata
9.80±0.54 (10)
4.80±0.55 (8)
5.00±1.00 (8)
3.3±0.20 (11)
C. arbuscula
6.30d=l.S8 (9)
3.40±0.95 (10)
6.10±0.20 (10)
3.6±0.55 (7)
0. diffusa
Zooxanthellae
1.63±0.38 (7)
0.30±0.02 (8)
0.81±0.15 (9)
Not measurable
(6)
No
0.37±0.01 (6)
Not measurable
0.26±0.01 (5)
Not measurable
zooxanthellae
(6)
(6)
68
THOMAS F. GOREAU
CALCIFICATION RATES IN DIFFERENT PARTS OF A CORAL COLONY
A glance at any living coral will show that there must be large variations in the
growth rates of different parts of the same colony, especially in branching species.
A field analysis of the differential growth pattern of reef corals was published by
Stephenson and Stephenson (1933). With our method, the growth rates in dif-
ferent parts of the same colony were quantitatively measured. Studies on four
species of branching corals, summarized in Table III, show that the calcification
rates of the apical parts of such corals are from four to eight times faster than growth
in the lateral and basal regions. Well developed calcification gradients are found in
corals which have a strongly oriented growth pattern. An example of this is seen
Calcification rates in different parts of a colony
of Acropora conferta
Direction of primary growth
A.
B.
C-
8-2 ± 3-76
l-9± 0-92
0-5 ± 0-10
Ca mg N"' hr"
10 cm
FIGURE 4. Calcification rates in three different parts of a colony of Acropora conferta.
Only the apical polyps were sampled, their relative positions being indicated by the circles on
the diagram. The calcium deposition rate is highest in the large pale apical polyps which are
oriented in the direction of primary growth, and marked by circle A. At positions B and C,
progressively further away from the growing edge of the colony, the calcification rate becomes
greatly reduced.
in the important Pacific reef -building coral Acropora conferta in which the primary
direction of growth is horizontally outward from a center, resulting in the formation
of large tabular colonies. The main growth occurs in the tips of numerous radially
outgrowing branches, the apical polyps of which are colored a pale pastel mauve.
The apical polyps of the secondary branches are still pale but smaller, whereas those
of the tertiary branches are almost indistinguishable from the yellowish brown
lateral polyps. The results of a typical experiment are summarized in Figure 4,
the location of the different branches being shown in the diagram.
SKELETON FORMATION IN CORALS 69
THE EFFECT OF A CARBONIC ANHYDRASE INHIBITOR ON CALCIUM
DEPOSITION IN CORALS
Wilbur and Jodrey (1955) demonstrated that shell formation in the oyster
Crassostrea virginica was greatly reduced in the presence of small concentrations of
certain heterocyclic sulfonamides which are powerful specific inhibitors of the enzyme
anhydrase. In a series of unpublished experiments we found this enzyme present
in all of the twenty-three coral species that were tested. Although carbonic an-
hydrase was also found in several species of sea anemones and zoanthidea, none of
which are calcareous, it was of some interest to determine whether the inhibition of
this enzyme had any effect on the calcification rates of corals. The inhibitor used
in these preliminary experiments was 2 acetyl-amino l,3,4,diathiazole-5-sulfonamide,
or Diamox. This compound was supplied through the kindness of the Lederle
Laboratories Division of the American Cyanamid Division. The experiments were
carried out by placing healthy coral colonies into a 10~3 M (approx. 1 : 20,000) solu-
tion of Diamox in sea water and adding calcium-45 twelve hours later. The experi-
ments were run in light and dark, each having a control without Diamox. All
corals used in these experiments could survive 1 : 20,000 Diamox for at least two
weeks, provided they were kept in the light. In darkness, survival time was re-
duced to about five or six days.
In Porites divaricata, a fast growing shallow-water coral that tolerates strong
light, treatment with 10 3 M Diamox in the light caused a fifty-one per cent fall
in the calcification rate. Exclusion of light caused the calcium uptake to fall a further
thirty-four per cent in the presence of Diamox, as shown in Table IV. It is in-
teresting to note that, in this species, the inhibitor had about the same effect as
exclusion of light, both causing a fall of about fifty per cent in the calcification rate.
This seems to indicate that, as far as their potentiating effect on the calcification
rate is concerned, the action of carbonic anhydrase and that of photosynthesizing
zooxanthellae are similar and probably synergistic.
In Cladocora arbuscula, a coral which grows best in a somewhat deeper and
shadier environment, exclusion of light appears to have relatively little effect on the
calcification rate, as shown in Table IV, and the per cent inhibition of the calcium
uptake produced by Diamox is about the same in light as in darkness. Thus, in
this coral, the zooxanthellae appear to play a much less important part in the
calcification process than carbonic anhydrase.
In Oculina diffusa, the relative effects of carbonic anhydrase and zooxanthellae
could be studied in more detail since it was possible to grow this coral without its
algae. In the presence of zooxanthellae, there was a fifty-nine per cent decrease
in calcium uptake on exclusion of light, whereas Diamox in the light caused ap-
proximately eighty per cent inhibition. In darkness, with zooxanthellae and in the
presence of Diamox, calcification could not be measured under the conditions of our
experiment. Similar results were found in light and darkness in zooxanthellae-less
colonies, where there appeared to be practically complete cessation of measurable
calcification in the presence of Diamox. These results indicate that in this species
carbonic anhydrase exerts a somewhat greater effect on the calcification rate than do
the zooxanthellae.
In all four species of reef corals so far tested, 10~3 M Diamox caused a forty per
cent to fifty per cent decrease of the calcification rate. This concentration of Diamox
70
THOMAS F. GOREAU
4-4-
from , sea water in coelenteron
CALICOBLASTIC
GASTRODERMIS
photosynthe
Ajn\ /-~\
zookqnthellaey
transport
M FABOLIC
CALICOBLASTIC J. EPIDERMIS
2HCO
Adsorbed on mucopolysaccharide
in organic membrane
Ca(HC03)2
Ca CO, + H_ _..
o d. o
(ppt)
FIGURE 5. Diagram to show possible pathways of calcium and carbonate during calcificatioi
in a reef-building coral. A diagrammatic cross-section of the calicoblastic body wall at th
base of the polyp is shown but the parts are not drawn to scale. The coelenteron and tr
SKELETON FORMATION IN CORALS 71
was sufficient to cause complete inhibition of carbonic anhydrase activity in coral
homogenates as measured by the method of Meldrum and Roughton (1933). It
is obvious that neither zooxanthellae nor carbonic anhydrase in themselves are es-
sential to the calcification process, since this still goes on in the absence of one or
both, though at a greatly reduced rate.
DISCUSSION
The experiments described in this paper show that calcium deposition by mad-
reporarian corals and other calcareous reef-builders can be determined under a vari-
ety of controlled conditions. The methods used here constitute a first step in the de-
velopment of an accurate procedure for the rapid measurement of calcification rates
applicable to further experimental studies of the physiology of skeletogenesis in
corals.
The question arises as to whether coral growth rates determined under laboratory
conditions can be compared to those found on the open reef under natural conditions.
Since the experiments described above were not designed to test this, we are now
conducting field studies, using a modified technic which will be described in a sub-
sequent paper. Preliminary results show that calcification rates of coral are some-
what higher on the open reef than reported here, and that our standard light source
was too bright for optimal coral growth. We have evidence that this latter factor
accounts for the small and sometimes insignificant dark-light growth differences
observed in some coral species as shown in Table I ; i.e., high light intensities could
partially inhibit coral growth. The quantitative relationship of light intensity and
other factors with coral growth is now under investigation.
A working hypothesis has been developed to help to interpret some of our re-
sults and to delineate the role played by the zooxanthellae and carbonic anhydrase
in skeletogenesis of the reef-building madreporarian corals. To be satisfactory,
such a hypothesis must account for : 1 ) the species-specific morphology of the
skeleton; 2) its formation external to the body proper; 3) its chemical composition
which is over ninety-nine per cent CaCO3 and less than one per cent MgCO3 (Vin-
ogradov, 1953) ; and 4) the crystalline nature of the mineral matter which is nearly
pure aragonite, according to Meigen (1903) and Chave (1954).
The calcification process is considered as a reaction in which Ca++ and CO.," are
brought to the calcification centers by separate pathways. The weight of histological
evidence now indicates that the mineralization process occurs outside the calico-
blastic epidermis (Matthai. 1918; Hayashi, 1937; Goreau, 1956) which secretes
an organic matrix that may act as a template on which the final stages of skeleto-
genesis take place. It is of interest that this organic matrix contains an acid
mucopolysaccharide-like substance (Goreau, 1956). This gives rise to the pos-
sibility that Ca++, taken up from sea water and transported across the body wall to
the external surface of the calicoblast, is adsorbed by ion exchange on an acidic
space lattice provided by the mucopolysaccharide in the organic matrix. Here
flagellated gastrodermis containing a zooxanthella are shown at the top of the figure, the
calicoblastic epidermis is in the middle and the organic membrane with crystals of calcareous
matter are at the bottom. The boring algae, the effects of which are problematical, have
been omitted for simplicity. The direction of growth is upward, i.e., calcium deposition is in
a downward direction.
72 THOMAS F. GOREAU
the Ca++ combines with HCO3~ by the following reaction :
(1) Ca++ + 2HCO3- ^- =-* Ca(HCO3)2.
The unstable product of this reaction then breaks down :
(2) Ca(HCO3)2 -f- -» CaCO3(ppt) + H2CO3,
with the formation of calcium carbonate and carbonic acid. As long as calcium is not
a limiting factor, the rate of formation of calcium carbonate will depend on the rate
with which the carbonic acid is removed from the site of calcification. This can be ac-
complished through the fixation of CO2 by photosynthesizing zooxanthellae and/or
the action of carbonic anhydrase. The proposed scheme is summarized in Figure 5.
It is expected, therefore, that if the zooxanthellae are prevented from photosynthesiz-
ing by keeping the coral in darkness, or if the algae are completely removed, the
velocity of calcification will decrease, due to slowing down of reaction (2) . Since car-
bonic anhydrase has an action which is, in this respect, physiologically equivalent to
that of the zooxanthellae, the inhibition of the enzyme will also result in a slowing
down of the calcification rate. The greatest decrease occurs when the corals are
kept in darkness in the presence of a carbonic anhydrase inhibitor. The fact that
calcification still goes on under these conditions simply shows that neither the enzyme
nor the algae determine the basic calcification reaction, but that they can exert a
strong influence on its over-all rate. This is in agreement with the work of Wilbur
and Jodrey (1955) who showed that carbonic anhydrase does not affect shell calci-
fication in the oyster unless the rate is limited by one of the following reactions :
(3) CO2 + H20 «= =* H2CO3:
or
(4) CO, + OH- , > 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 H2CO3.
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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 Na24. This ion was obtained with
Na,CO3 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 Na24 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), SO4 by the method of Nalefski and Takano (1950)
and NH4 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 Na24 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 Na24 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 NH4 ; for urine components except Ca,
NH4 and Cl ; for gill fluid components except NH4, 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
NH4
Total
mEq.+
Cl
SO4
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
0
0
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 Na2* 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 SO4 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
<:'!
<M
>.os
<.02
= .01
>.50
>.05
<:o!
X51
<: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, NH4 and SO4.
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 Na24. The
same quantity of the isotope was added to equal volumes of 100% and 175% sea
water. Since the Na23 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 Na24/
Na23 ratio in 100% sea water by the Na21/Na23 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 Na24 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 Na24 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
Na24 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 Na2* 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 wrater more readily. This finding, coupled with the low
Na24 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 Na23/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 Na24 for Na23 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; SO4, 191; while in 175% sea water
they are : Na, 65 ; Mg, 40 ; K, 88 ; Ca, 70 ; Cl. 61 ; SO4, 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; SO4, 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 SO4 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 NH4 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 NH4 (4 and 8 in 100% and
175% sea water). If NH4 were excreted by simple filtration, marked reabsorption
of all other ions would be required to give such high NH4 values ; hence it is prob-
able that NH4 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 SO4 in (175% sea
water) could result from marked reabsorption of water and other ions (except
NH4) ; they could indicate secretion of Mg and, at least in 175% sea water, also of
S04.
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 wrhen 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 SO4 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 NH4 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 NH4 and Mg (possibly
also SO4).
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 NH4 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 Na24 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 SO4 is outward in 100% sea
water but in 175% sea water the SO4 in gill fluid is higher than in either serum or
medium ; hence there might be some SO4 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 NH4 is absent from sea
water and is higher in stomach fluid than in serum, gastric secretion of NH4 is
probable ; however the concentration of NH4 in stomach fluid is less than in urine,
where more secretion is indicated, and greater than in gill fluid where NH4 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 Na24 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 SO4) 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, NH4, Cl and SO4 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, NH4 and SO4 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, NH4 and Cl are
lower and only SO4 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 SO4. 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, NH4 and SO4. In crabs from 175% sea water corresponding serum
electrolyte significance is found for Mg, Ca, NH4, Cl and SO4.
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 NH4 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 Na24, 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. Na24 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 Na24 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
0
9
Corpus rardiarum
4-6
10
0
1*
0
(10%)
Brain-cardiaca-allata complex
3
15
3
0
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
0
(77.3%)
Brain-cardiaca -allata
3
22
1
21
0
complex
(95.4%)
Brain
3
23
4
19
0
(82.6%)
Larval donors:
Corpus allatum (5th instar)
6
24
0
5*
19
(20.8%)
(79.2%)
Corpus allatum (4th instar)
6
27
6
0
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 ZnSO4, or Na2WO4. When cellobiose was used as the substrate,
the reaction was stopped by Na2WO4 and H2SO4, 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
<r
2
>0.2
Q
l
o ^
< o
o
0
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
0
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 AgNO3
or HgClL, 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
0
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
d0.8
•5-
3
2.
t— \ r\ ^
I
— U.O
> LiJ
_ C/}
1- 0
0 0
< 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 wras 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 -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
0
I I I
0
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 wras 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 wjas 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 writh 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 twro 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 %0. 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 RETINA1
L. H. KLEINHOLZ
The Biological Laboratories, Kccd College, Portland, Ore</on; and Marine Biological
Laboratory, Woods Hole, Mass.
Three sets of pigments are generally found in the crustacean retina, where they
may undergo photomechanical movements under control of nenrosecretory hormones
(Kleinholz, 1936; \Yelsh, 1939; Brown ct a!., 1952, 1953). The di'stal retinal
pigment and the so-called proximal pigment of the retinnlar cells are dark pigments,
presumed to be melanins or ommochromes, although few studies have been made of
their chemical nature. The retinal reflecting pigment has been called guanine, but
this identification has been based more on the analogy with the tapetal pigment
occurring in the eyes of some vertebrates than on chemical study (Welsh, 1932;
Kleinholz, 1936). I attempted, a score of years ago, to examine the chemical
nature of this reflecting pigment of the crustacean retina, but, beyond gathering
some information on solubility properties, efforts toward more specific characteriza-
tion proved abortive because of limited amounts of available material.
Development within the past decade of techniques for isolation and examination
of small amounts of biological material prompted a renewed attempt to identify
this reflecting material. This initial study, part of which has been reported in
preliminary form (Kleinholz, 1955), was done on the lobster, Hoinan/s amcricanns,
and the chelicerate, Liinuliis f>ol\'[>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'.;^ ' ' •' Vs ,
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 NH4OH ; 0.069r Li,CO, ; 0.2 M
borate buffer at pH 9.2; or a solution of 50f/r ethanol containing 2% NH4OH.
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 Rf 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 Rf 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 Rf 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
RF -30
.20
.10
.50
0
0
n o
o
o
o
.40
0
.30
0
.20
N-PROPANOL
NH4OH
U XP XA H
.10
0
0
0
0
N-BUTANOL
ACETIC
H20
U XP XA H
.80
.60
.40
.20
Q
0
0
0
0
.60
0
ETHYL ACETATE
PYRIDINE
H20
U XP XA H
.50
.40
.30
0
0
0 0
0
0 0
0
ISOAMYL
NA2HP04
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 0 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 Rf 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 Rf 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 Rf 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 Rf 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
1This 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 b5) 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 2y2 gm.), Samia cynthia (iy2 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
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FIGURE 1 (left). The CO-insensitivity of pupal respiration as a function of basal O2 con-
sumption. CO/Os ratio = 19:1. The oxygen tension was 5% in both CO and N2 mixtures.
The gas exchange of 18 diapausing Cynthia pupae was measured first in the N2 mixture and
then in the CO mixture. The per cent of CO-insensitive respiration is plotted against the
respiration in the N2 mixture.
FIGURE 2 (right). The CO-sensitivity of pupal respiration at reduced O2 tensions. The
average O2 consumption of four brainless Promethea pupae whose average O» consumption in
air was 33 mm.3/gm. live wt./hr. is recorded at each successive O2 tension in the O2-N? mix-
tures. Similarly the average O2 consumption of five other pupae, whose average O2 consumption
in air was 27 mm.3/gm- live wt./hr., is recorded at each O2 tension in the O2-CO-N2 mix-
tures. The rate of respiration in each gas mixture is expressed as per cent of basal rate in
air. The CO/Oa ratio was kept constant at 19:1 by adding appropriate amounts of N2 to
the O2-CO mixture. The right-hand vertical axis records the per cent of CO-insensitive
respiration. Oxygen consumption decreased at tensions below 2% and this so complicated
measurements of CO inhibition that values for O2 tensions below 2% could not be calculated.
The average weight of the pupae was 2 grams.
for the present, suffice it to note that in the presence of carbon monoxide the gas
uptake of pupae which have low respiratory rates in air is markedly increased and
that this fact complicates studies of carbon monoxide inhibition on these animals.
C. The CO-sensitivity of pupal respiration at reduced oxygen tensions
In the following experiment the CO-sensitivity of the respiration of a group of
Promethea pupae was measured at oxygen tensions ranging from 5 to 2 per cent
of an atmosphere. The results summarized in the lower curve in Figure 2 disclose
CARBON MONOXIDE AND RESPIRATION 141
that at low oxygen tensions pupal respiration becomes sensitive to carbon monoxide.
As the figure reveals, pupal respiration was not depressed by low oxygen alone down
to 2 per cent. In sharp contrast to this insensitivity of respiration to low oxygen
tensions in the nitrogen-oxygen mixtures, the respiration in 19:1 CO/O2 remained
constant only between 5 and 4 per cent oxygen and then progressively decreased
as the oxygen tension decreased. In other words, at low oxygen tensions pupal
respiration is inhibited by carbon monoxide. These observations suggest that cyto-
chrome oxidase is functioning at all times in the diapausing pupa but at higher
oxygen tensions its CO-sensitivity is in some manner masked. A similar experi-
ment performed on Cynthia pupae yield substantially the same results.
The cause of the slight stimulation of respiration shown in the figure in nitrogen-
oxygen mixtures containing 2 per cent oxygen or more is unknown. In 1 per cent
oxygen respiration fell to about 85 per cent of the basal rate. It is significant that
these measurements were conducted on Promethea pupae less than one half the
size of Cecropia pupae used by Schneiderman and Williams (1954a) in an experi-
ment appraising the effect of oxygen tension on pupal respiration. They reported
that the respiration of Cecropia pupae decreased when oxygen tension fell below
5 per cent. These contrasting results are explained by the fact that in the larger
Cecropia pupae the diffusion distances are greater than in Promethea pupae. There-
fore, the actual tension of oxygen within the pupal tissues is probably less for large
pupae than small ones. As a result, the respiration of large pupae is limited at
oxygen tensions which do not affect the respiration of small pupae.
D. The effects of sodium azide and antimycin A on pupal respiration
Three groups of five diapausing Cynthia pupae, whose average basal respiration
was 15.4 mm.3/gm. live wt./hr., were injected with sodium azide to internal con-
centrations of 10~5 M, 10"4 M, and 5 X 10~4 M. The average respiration of these
pupae on the day of injection was indistinguishable from the respiration of five con-
trol pupae injected with water. Since azide is an extremely soluble small molecule,
it is doubtful that impermeability is responsible for this insensitivity. Pupal res-
piration is thus relatively insensitive to azide as well as to carbon monoxide.
In a similar experiment, the effects of antimycin A on respiration were examined
in fifteen Cynthia pupae whose average basal respiration was 13.3 mm.3/gm. live
wt./hr. A control group of five received a 1 per cent ethyl alcohol solution, a second
group received antimycin A to an internal concentration of 10~7 M and the third
group received antimycin A to a final concentration of 10~6 M. Injecting the 1 per
cent ethyl alcohol had exactly the same effect as injecting distilled water and
promptly doubled the respiration. Compared with the ethyl alchol control, 10~7
antimycin A inhibited respiration about 20 per cent and 10~6 M about 30 per cent.
Similar concentrations of antimycin A commonly cause much higher inhibitions in
other organisms and the respiration of the diapausing pupa may be considered
relatively insensitive to this potent inhibitor of the cytochrome c reductase system.
2. DNP-stimulated respiration
A. The stimulatory effect of DNP
An important clue to the nature of the oxidative pathways of diapausing pupae
was uncovered in 1955 by Harvey and Shappirio (unpublished observations) who
142
CHARLES G. KURLAND AND HOWARD A. SCHNEIDERMAN
discovered that DNP stimulated the respiration of diapausing Cecropia pupae and
that this respiration was CO-sensitive. This result, which they generously shared
with us, agreed with the earlier observations of Bodine and Boell noted in the
Introduction, and suggested to us a number of experiments using DNP.
A series of Cynthia pupae were injected with DNP to internal concentrations
ranging from 5 X 10~4 M to 10~5 M. Figure 3 records the average initial stimula-
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FIGURE 3 (left). The effect of several concentrations of DNP on the respiration of dia-
pausing pupae. Five Cynthia pupae received injections of DNP to an internal concentration
of 5 X 10~* M, four received 10~4 M, four received 5 X 10"B M, four received 10~B M, and a
control group of four received distilled water. The average O2 consumption for the first three
hours after injection is recorded.
FIGURE 4 (right). The effect of DNP injection on the O2 consumption of diapausing pupae.
Four Cynthia pupae whose average O2 consumption was 13.0 mm.ygm. live wt./hr. were in-
jected with DNP to an internal concentration of 1 X 10"4 M. Respiration was measured for
three hours each day over a five-day period. The day of injection is denoted as day "0".
tion of respiration of several concentrations for the first three hours after injection,
while Figure 4 records the respiratory behavior of the 10~4 M group over a five-day
period. Dinitrophenol called forth an immediate and spectacular increase in oxygen
consumption which averaged 12 times the basal rate in the case of pupae receiving
5 X 10~4 M. As Figure 4 shows, in the group receiving 10~* M the initial accelera-
tion of respiration on the day of injection was followed by a decline on the following
CARBON MONOXIDE AND RESPIRATION
143
day. This was succeeded by a gradual increase of respiration over a three-day
period, to a peak on the fifth day after injection almost as great as the initial peak
respiration. The respiration returned to about normal approximately two weeks
later. The initial stimulation of respiration is doubtless due to the uncoupling effect
of DNP which causes an acceleration of the turnover rate of the components of the
respiratory chain (Cross et al., 1949; Chance and Williams, 1956). The secondary
effects which develop several days later, appear to be the result of (a) injury-stimu-
lated respiration provoked by injection through the cuticle (see Section 3 below) and
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FIGURE 5. The stimulation of O2 consumption by DNP as a function of basal respiratory
rate. (A) The percentage stimulation of O2 consumption of Cynthia pupae after injection
of DNP to internal concentrations of 5 X 10~* M and 10~* M is plotted as a function of basal
respiration. (B) The total DNP-stimulated respiration of the pupae in (A) is plotted as a
function of the basal respiration.
(b) the development of an "energy debt" metabolism (analogous to an "oxygen
debt repayment" (Kurland et al., 1958)) as the result of prolonged uncoupling of
phosphorylation by DNP. Comparable results were obtained with diapausing
pupae of Cecropia, Promethea and Polyphemus.
The time course of the respiratory changes recorded in Figure 4 is also typical
of pupae receiving 5 X 10~4 M DNP but the pattern differed somewhat in pupae
that received lower concentrations. Because the initial stimulation of respiration
was less, the fall in respiration recorded in Figure 4 was commonly absent. The
144 CHARLES G. KURLAND AND HOWARD A. SCHNEIDERMAN
5 X 10~4 M concentration is apparently close to the lethal level and occasional in-
dividuals died about a week after receiving that amount.
Further analysis of the effects of DNP disclosed that pupae with high initial
basal respirations were proportionately less stimulated by DNP than were pupae
with low basal metabolic rates. Figure 5 A shows that a Cynthia pupa with a basal
metabolic rate of 5 mm.3/gm. live wt./hr. experienced a 16-fold stimulation of
respiration after injection of DNP whereas a similar pupa with a basal respiration
of 30 mm.3/gm. live wt./hr. experienced only a 6-fold stimulation of respiration.
Thus, there is a steep decline in the per cent of DNP-stimulated respiration as basal
respiration increases. Figure 5B further reveals that DNP-stimulated respira-
tion approaches a limit as the basal respiration approaches 25 mm.3/gm. live wt./hr.
The significance of this limit will be considered in the Discussion.
B. The effect of carbon monoxide, azide and antimycin A on DNP-stimulated
respiration
Diapausing Cynthia pupae were injected with DNP and then exposed to carbon
monoxide. The results, summarized in Figure 6, reveal that about half the DNP-
stimulated respiration was inhibited by carbon monoxide. Further analysis of the
data from this experiment revealed that CO-sensitivity increased slightly as the
rate of oxygen consumption increased. Thus pupae with a DNP-stimulated res-
piration of 90 mm.s/gm. live wt./hr. had only 45 per cent of their respiration in-
hibited by carbon monoxide, whereas pupae with a DNP-stimulated respiration of
125 mm.3/gm. live wt./hr. had nearly 70 per cent of their respiration inhibited by
carbon monoxide.
The effect of azide on DNP-stimulated respiration of diapausing Cynthia pupae
is recorded in Figure 7. There was no significant initial inhibition of the respiration
when sodium azide alone was injected (see Section ID), but some of the DNP-
stimulated respiration was inhibited by this reagent. Indeed, as Figure 7B shows,
more than three-fourths of the DNP-stimulated respiration was inhibited by 5 X 10~4
Jl/ sodium azide. However, in group B only half of the pupae receiving injections
of DNP and none of the pupae receiving sodium azide survived for more than a
week, indicating these high concentrations of antimetabolites were ultimately toxic.
Comparable results were obtained with Cecropia pupae.
Experiments appraising the antimycin A-sensitivity of DNP-stimulated respira-
tion were conducted on a series of 15 Cynthia pupae which received 10~4 M DNP
and 10~6 M antimycin A. About 30 per cent of the DNP-stimulated respiration
was inhibited by this concentration of inhibitor. Thus, the respiration of DNP-
stimulated pupae is no more sensitive to antimycin A than the respiration of normal
pupae.
3. Injury-stimulated respiration
A. The C0-sensitivit\ of injury-stimulated respiration
As mentioned previously, integumentary injuries to pupae dramatically ac-
celerate respiration for one to three weeks ( Schneiderman and Williams, 1953a,
1953b). Moreover, this accelerated respiration is proportional to the extent of
injury and seems to be caused in part by diffusible substances released at the site
CARBON MONOXIDE AND RESPIRATION
145
of injury (Jankowitz, 1955; Schneiderman, 1957). Although the respiration in-
duced by a small incision into Cecropia pupae was not inhibited by carbon monoxide,
repair of extensive wounds was prevented by this gas (Schneiderman and Williams,
1953b, 1954b), suggesting that the cytochrome oxidase system was functioning in
injured pupae.
To investigate this possibility the following experiments were carried out. Four
Cynthia pupae were given a large injury by removing their pupal legs; two pupae
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CO
5X10 4M. DNP
XlCf 4M. DNP
FIGURE 6. The CO-sensitivity of DNP-stimulated respiration. Two groups of five dia-
pausing Cynthia pupae were injected with DNP to internal concentrations of 5 X 10~* M
and 10"4 M. All the pupae were exposed to 5% O2 and 95% N2 and then to 5% O2 and 95%
CO (CO/Os^ 19:1). The average respiration over a one-hour period is recorded.
were immediately placed in 7 per cent oxygen and nitrogen, and the other two
were placed in a corresponding atmosphere of oxygen and carbon monoxide. The
pupae were maintained in their respective gas mixtures for one week, and the
gas mixtures were renewed thrice daily.
As Figure 8 shows, injured pupae in the nitrogen mixture developed a charac-
teristic injury respiration; on the other hand, those in carbon monoxide mixtures
did not. Indeed, five days after injury both of the pupae maintained in carbon
monoxide had died. Thus carbon monoxide apparently caused death by preventing
146
CHARLES G. KURLAND AND HOWARD A. SCHNEIDERMAN
the development of injury respiration. Similar results were obtained with Prome-
thea pupae. These results are in general agreement with those of Schneiderman and
Williams (1954b), who reported that the repair of injury was CO-sensitive. How-
ever, their experiments failed to detect the CO-sensitivity of the respiration as-
sociated with repair of injury, presumably because they employed only small injuries.
Such CO-sensitivity was demonstrated by Harvey and Shappirio (Harvey, 1956)
who pointed out that after very large injuries respiration becomes sensitive to car-
bon monoxide. This is confirmed in the following experiment summarized in
UJ
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FIGURE 7. The azide-sensitivity of DNP-stimulated respiration. (A) Four diapausing
Cynthia pupae were injected with 10~4 M DNP, four with 2.5 M sodium azide, and four with
both reagents. The average Oa consumption over a three-hour period is recorded. (B) Five
pupae were injected with 5 X It)"4 M DNP, five with 5 X It)"4 M sodium azide and five with
both reagents. The average O2 consumption over a three-hour period is recorded. The
average initial oxygen consumption of the pupae in (A) and (B) was 16.8 mm.8/gm. live wt./hr.
Figure 9. Four brainless Cynthia pupae were injured by removing the pupal legs
and after three days, when they had developed a large injury respiration, the CO-
sensitivity of their respiration was determined. About two-thirds of the injury
respiration was inhibited by carbon monoxide. Similar results were obtained
with Cecropia pupae. It can also be seen in Figure 9 (as well as in Figure 6) that
the oxygen uptake of pupae respiring at a rapid rate was limited by the low oxygen
tension. This contrasts with the respiratory behavior of pupae with low metabolic
rates, where 5 per cent oxygen and 95 per cent nitrogen commonly stimulated
oxygen consumption (see Section 1C).
CARBON MONOXIDE AND RESPIRATION
147
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FIGURE 8 (left). The effect of injury and simultaneous exposure to CO on respiration.
Two injured Cynthia pupae were maintained continuously in 7% O» plus Ns and two were
maintained in 7% O« plus CO (CO/O« = 13:1). The average respiration of each pair over a
3-hour period is recorded as a function of time. The day of injury is denoted as day "0".
FIGURE 9 (right). The CO-sensitivity of injury respiration. The average respiration over
a 4-hour period of four brainless Cynthia pupae 3 days after injury in air, in 5% Oa and 95%
N«, and in 5% O2 and 957* CO (CO/O2 ratio = 19: 1).
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DAYS AFTER INJURY
FIGURE 10 (left). The effect of azide injection on the O= consumption of four groups of
five diapausing Cynthia pupae over a five-day period. The day of injection is denoted as
day "0".
FIGURE 11 (right). The effect of DNP on injury respiration. The average O2 consump-
tion over a 3-hour period of two injured diapausing Cynthia pupae prior to and after the in-
jection of water, and of two injured pupae prior to and after the injection of 5 X IO""4 M DNP.
148
CHARLES G. KURLAND AND HOWARD A. SCHNEIDERMAN
B. Injury-stimulated respiration in newly pupated Cecropia
Four Cecropia silkworms were injured within one day after pupation by re-
moving a rectangular window of pupal cuticle from their faces. No significant
stimulation of respiration was observed. Since injury respiration is characteristic
of diapausing pupae, and since the respiration of newly pupated Cecropia is much
greater than the respiration of pupae firmly in diapause, this result suggests that
the production of injury respiration is intimately associated with the extremely low
respiration of the diapausing insect (see Discussion).
C. The aside-sensitivity of injury-stimulated respiration
The effect of azide on injury respiration was examined by injecting a series of
diapausing Cynthia pupae with sodium azide at several concentrations. In this
experiment the injection itself served as the injury. The average daily respiration
of each group of pupae over a five-day period is plotted in Figure 10.
All pupae treated with 5 X 10~4 M sodium azide died within 10 days after injec-
TABLE 1 1
Effect of simultaneous injury and injection of DNP on the respiration
of six diapausing Cynthia pupae
Basal respiration,
mm.Vgrn./hr.
Treatment
Max. resp. as
% basal rate
Day of max.
25.0
Injury + H2O
258
2
12.0
Injury + H2O
521
2
31.0
Injury + H2O
234
3
26.5
Injury + DNP
449
6
40.5
Injurv + DNP
496
6
30.5
Injiirv + DNP
Died
—
tion, indicating either (a) that this concentration had a simple toxic effect, or
(b) that the development of injury-stimulated respiration was inhibited by azide
and this caused death, as was the case when injured pupae were exposed continuously
to mixtures of carbon monoxide and oxygen (see Section 3 A above). In lower
concentrations of azide, the inhibition of injury respiration was proportional to
concentration.
D. The effects of DNP on injury-stimulated respiration
The pupal legs were removed from a group of four Cynthia pupae. Three days
after wounding, when injury respiration had reached its maximum, two of the
pupae received injections of DNP to an internal concentration of 5 X 10~4 M, and
the remaining two received injections of water. The data summarized in Figure 11
show that 5 X 10~4 M DNP caused a significant acceleration of maximum injury
respiration ; however, the increase was proportionately much less than that en-
countered in DNP-treated diapausing pupae. Comparable results were obtained
with Cecropia and Promethea pupae.
In another experiment, six diapausing pupae were injured by removing their
CARBON MONOXIDE AND RESPIRATION 149
pupal legs ; half these pupae immediately received water injections, while the
remainder received injections of DNP to an internal concentration of 5 X 10"4 M.
The respiration of these pupae is summarized in Table II. The maximum respira-
tion of injured pupae treated with DNP was reached six days after the injury,
while those receiving injections of water displayed maximum respiration two or
three days after injury. Thus DNP delayed the development of injury respiration.
DISCUSSION
1. A new explanation for the insensitivity of pupal respiration to carbon monoxide
Studies noted in the Introduction have shown that the onset of pupal diapause
in giant silkworms is accompanied by a precipitous fall in the rate of oxygen con-
sumption, and that the low respiration of the diapausing pupa is virtually uninhibited
by carbon monoxide and cyanide. As judged by its insensitivity to these inhibitors,
nearly all of the respiration of the diapausing pupa appeared to proceed via path-
ways independent of cytochrome oxidase. Hence it was suggested that the respira-
tion of the diapausing pupa was mediated by a terminal oxidase other than cyto-
chrome oxidase, possibly a flavoprotein or an autoxidizable cytochrome of the b type
(Schneiderman and Williams. 1954a. 1954b). This suggestion was taken up by
various investigators (Cotty, 1956; Ito, 1955). The present experiments provide
an alternative explanation for the CO-insensitivity of pupal respiration ; namely,
that it is due to a great excess of cytochrome oxidase relative to trace amounts
of cytochrome c in most of the tissues of the diapausing pupa. This limitation of
cytochrome c leads to an unsaturation of cytochrome oxidase, and this in turn
leads to the insensitivity of pupal respiration to carbon monoxide and azide. Under
this view the principal factor underlying the lozv respiration of the diapausing
pupa is the limitation of cytochrome c in most of the pupal tissues, while the prin-
cipal factor underlying the CO- and aside-inscnsitivity of pupal respiration is the
excess of cytochrome c o.ridase in most of the pupal tissues. Thus, quantitative
changes in the relative amounts of respiratory enzymes after pupation are respon-
sible for both the low over-all respiration of diapause and for CO-insensitivity.
In other words, the basic differences between the respiratory enzyme systems of
diapausing and non-diapausing insects are quantitative, but they lead to qualitative
differences in the response of the insect to certain inhibitors. Contrary to earlier
opinions, cytochrome oxidase appears to be the principal terminal oxidase during
diapause as well as during all the other stages of the life history.
2. Preliminary theoretical considerations
It can be shown that an excess of cytochrome oxidase may lead to a virtual
CO-insensitivity of respiration that is actually mediated by cytochrome oxidase,
and in the final section of this discussion a brief theoretical analysis of this asser-
tion is presented. The argument offered is that when cytochrome oxidase is in
great excess and thus not saturated, a large fraction of the cytochrome oxidase
may be inhibited by carbon monoxide without affecting the rate of electron transfer
from cytochrome c. Stated in another way the greater the "saturation" of cyto-
chrome oxidase by cytochrome r. the greater the CO-sensitivity of respiration;
the less the "saturation" of cytochrome oxidase by cytochrome c, the less the CO-
150 CHARLES G. KURLAND AND HOWARD A. SCHNEIDERMAN
sensitivity. This conclusion seems intuitively acceptable and is proven in Section 9
(below). Recognizing this relation between CO-sensitivity and saturation of
cytochrome oxidase it is not difficult to interpret the several experimental results.
3. Carbon monoxide experiments
Evidence presented previously has shown that the specific target of carbon
monoxide in the insect at all stages is reduced cytochrome c oxidase (Schneiderman
and Williams, 1954a, 1954b). A principal factor determining the impact of carbon
monoxide on cytochrome oxidase is the CO/CX ratio: the higher this ratio, the
greater the proportion of reduced cytochrome oxidase molecules inhibited. The
CO/O2 ratios employed in the present experiments were usually 16:1 or 19:1
and ambient oxygen tensions were maintained at 6 or 5 per cent. Direct analysis
of the composition of the tracheal gas of normal diapausing pupae kept at these
oxygen tensions by a precise microgasometric method (Levy and Schneiderman,
1957, 1958) revealed that the actual oxygen tension within the tracheal system,
and hence within the insects' tissues, was about 1 per cent lower than ambient, that
is, about 5 or 4 per cent. Therefore in the present experiments the actual CO/O,
ratios within the pupal tissues approached 24:1. Since it has been shown that
a CO/Oo ratio of 16:1 causes a 50 per cent light-reversible inhibition of the cyto-
chrome oxidase activity of homogenates of the thoracic muscles of Cecropia moths
(Pappenheimer and Schneiderman, unpublished), we may conclude that the CO/0,,
ratios used in the present experiments were capable of inhibiting no less than 50
and probably as much as 75 per cent of the reduced cytochrome oxidase activity
of homogenates of the insect's tissues. However, as we have already noted in the
previous section, the inhibition in a homogenate where cytochrome oxidase is satu-
rated by added cytochrome c may be quite different from the inhibition observed
in the intact insect where the cytochrome oxidase may not be saturated by cyto-
chrome c. Let us now consider what our several experiments tell us about the
saturation of cytochrome oxidase in the diapausing pupa.
Perhaps the most crucial result is recorded in Figure 2. As the figure shows,
when the oxygen tension is reduced to 2 per cent in a mixture of oxygen and
nitrogen, the oxygen consumption of the pupa remains about the same as in air,
but the CO-sensitivity of the respiration is enhanced. The simplest interpretation
of this result is that cytochrome oxidase is present in excess over some rate-limiting
link in the respiratory chain, and only at low oxygen tensions does the cytochrome
oxidase-oxygen reaction become the limiting step in the respiratory chain, subject,
as a consequence, to inhibition by carbon monoxide.
The reasons for stimulatory effects of carbon monoxide on pupae with low
metabolic rates (cf. Fig. 1) are not yet clear. Similar stimulatory effects of carbon
monoxide have been reported by Bodine and Boell (1934a) for Melanoplus, by
Klein and Runnstrom (1940) for unfertilized eggs of the sea urchin, and by
others (cf. review by Needham, 1942, p. 496). Possibly it does not represent
stimulation of respiration but is simply gas uptake due to an actual oxidation of
CO by the tissues to CO,, (cf. review of Lilienthal, 1950). Perhaps it is some-
thing different altogether, such as an uncoupling action (Thimann et al., 1954). For
our present purposes suffice it to say that the phenomenon, although not yet ex-
plained, does not affect our interpretation of the basic action of carbon monoxide
CARBON MONOXIDE AND RESPIRATION
151
cytochrome oxidase and the argument that cytochrome oxidase is only partially
saturated in pupal tissues. Further evidence supporting this argument derives
from studies with DNP and azide which are considered in Sections 4 and 5 below.
Significant data revealing the degree of saturation of cytochrome oxidase in the
diapausing pupa are also to be found in the observation that CO-sensitivity of pupal
respiration increases with increasing basal respiration and is instantly enhanced by
DNP, and in the fact that the increased respiration that follows injury or the initia-
tion of adult development is inhibited by carbon monoxide. Moreover, we have
found that the increased respiration that follows a prolonged period of anoxia is also
sensitive to carbon monoxide. These results, which are summarized in Table III,
TABLE III
Summary oj tine effects of metabolic inhibitors on the respiration of diapausing pupae
in various physiological states and on developing adults
Effect
^ylnhibitor
Physio- >.
logical
condition \
CO
CO 'O2=about 20:1
DNP
Azide
Antimycin A
Diapause
respiration
Stimulation at low basal
rates
Slight or no inhibition
at modest basal rates.
Inhibition increases as
basal respiration in-
creases
Up to 50% inhibition at
low oxygen tensions
5 X 10-« M stimulates
respiration an average
of 12-fold and as much
as 16-fold
Stimulation less at high
basal rates
No immediate effect
at concentrations up
to 5 X 10-4 M
30% inhibition at
10-6 M
DNP-stimulatert
respiration
An average of 50% in-
hibition
—
30 to 70% inhibition
depending on concen-
tration of azide
30% inhibition at
10~6 \I
Injury-stimulated
respiration
No or slight inhibition
after small injury; up to
60 per cent inhibition
after large injury
Exposure immediately
after injury prevents de-
velopment of injury-
stimulated respiration
Stimulation by DNP in-
versely proportional to
size of injury-stimulated
respiration. After large
injuries, about 2-fold
stimulations by DNP.
Injection of DNP im-
mediately after injury
delays development of
injury respiration
Inhibition propor-
tional to concentra-
tion of azide
Developing adult
More than 50% inhibi-
tion (Schneiderman and
Williams. 1954a)
5 X 10-< M stimulates
respiration about 2-fold
—
—
lead to the conclusion that the fraction of respiration sensitive to carbon monoxide
is a function of the rate of oxygen consumption of the silkworm at all stages. This
implies that virtually any process which increases the rate of pupal respiration in-
creases the saturation of cytochrome oxidase and that, in the pupa, cytochrome
oxidase is in great excess and hence very unsaturated.
Recognizing the importance of low over-all respiratory rate as a factor in
CO-insensitivity, it is worthwhile considering certain diapausing insects whose res-
piration is not resistant to carbon monoxide or cyanide. Two species whose respira-
tion continues to be inhibited by carbon monoxide or cyanide during diapause are
prepupae of the larch sawfly. Pristophora (McDonald and Brown, 1952), and larvae
152 CHARLES G. KURLAND AND HOWARD A. SCHNEIDERMAN
of the horse hot fly, Gastrophilus (Levenbook, 1951). It is of considerable signifi-
cance that the respiration of these insects at 25° C. is many times greater than the
respiration of diapausing silkworm pupae. Thus the respiratory rate of Pristophora
is about 165 mm.3/gm. live wt./hr., while that of Gastrophilus is more than 100
mm.3/gm. live wt./hr.3 This compares with a respiratory rate for diapausing
silkworm pupae of 8 to 20 mm.3/gm. live wt./hr. Furthermore, in diapausing
silkworm pupae only the skeletal muscles, of which there are few, have a saturated
cytochrome c oxidase, and these account for only a small fraction of the insect's
total respiration. In diapausing species with high respiratory rates like Pristophora
and Gastrophilus it appears likely that ( 1 ) they have more muscular tissue and this
accounts for a larger fraction of their total respiration than do the muscles of
diapausing pupae, and (2) some of their non-muscular tissues may have a saturated
cytochrome oxidase. These factors could easily account for their sensitivity to
carbon monoxide.
4. The significance of DNP-stimulated respiration
The experiments with DNP demonstrate that in diapausing pupae cytochrome
oxidase is not fully saturated. As is well known, DNP increases the turnover of
the respiratory carriers, presumably because it is able to uncouple phosphorylation
from electron transfer, and so increases the demand for oxygen (Chance and Wil-
liams, 1956). The data in Section 2 reveal a striking 12- to 16-fold acceleration
of pupal oxygen consumption by 5 X 10~4 M DNP. This may be one of the largest
DNP stimulations ever recorded. It contrasts with the finding of Bodine and
Boell (1938) that the respiration of diapausing Melanoplus eggs was accelerated
a maximum of only 3.5 times by 3 X 10~5 M DNP, while further increase in concen-
tration produced a submaximal response. De Meio and Barren (1934) and
Maroney et al. (1957) have reported DNP stimulations in various invertebrate
tissues of only about two-fold. Aside from the magnitude of DNP-stimulated
respiration (which by itself suggests unsaturation of cytochrome oxidase), the CO-
sensitivity of DNP-stimulated respiration is of special interest. It indicates that
DNP accelerates the turnover of several carriers of the respiratory chain but has
a lesser effect on the turnover of cytochrome oxidase. This conclusion arises
from the fact that CO-sensitivity is a function of the saturation of cytochrome
oxidase. The CO-sensitivity of DNP-stimulated respiration tells us that DNP
increases the saturation of cytochrome oxidase. Thence it follows that DNP not
only accelerates over-all respiratory rate but alters the quantitative relationship
between cytochrome oxidase and the intermediate carriers in the respiratory chain.
One of these accelerated carriers is almost certainly cytochrome c, which may
be the most important rate-limiting carrier in the respiratory chain, a point we shall
consider further in Section 6 (below). Dinitrophenol appears to increase in some
way the effective turnover of this enzyme and increases thereby the saturation of
cytochrome oxidase. It is significant that both the absolute magnitude and the
CO-sensitivity of DNP-stimulated respiration were lower for pupae with low basal
respiration. Thus in the present experiments, although pupae with low basal
metabolic rates were proportionately more stimulated by DNP than pupae with
high basal metabolic rates, the latter developed a greater over-all respiration under
3 This last value was calculated from values obtained at 37° C. by assuming a Q™ of about 2.5.
CARBON MONOXIDE AND RESPIRATION 153
the influence of DNP. The data also reveal that CO-sensitivity reached a maximum
of about 70 per cent when DNP-stimulated respiration reached its maximum. We
interpret these findings to mean that pupae with low basal respiration have less
cytochrome c available to be turned over and, as a result, these pupae are not capable,
even under the influence of high concentrations of DNP, of completely saturating
their cytochrome c oxidase and thereby achieving maximum CO-sensitivity. These
DNP studies provide support for the argument that the low over-all respiration of
diapause is due to a low concentration of some respiratory component, probably
cytochrome c, whereas the CO-insensitivity is the result of the relatively high con-
centration of cytochrome c oxidase.
In our experience the respiration of developing adults is accelerated by DNP to
a much lesser extent than that of diapa vising pupae, usually about two-fold. This
fact suggests that in the developing adult, as contrasted with the diapausing pupa,
cytochrome oxidase is virtually saturated. Also, although development may be
delayed, developing adults survive concentrations of DNP which are toxic to
diapausing pupae, possibly because their higher metabolic rate enables them to
metabolize the DNP (cf. Cross et al. 1949).
5. The significance of aside-insensitive respiration
In these insects it seems safe to identify cytochrome oxidase as the main target
of azide CHorecker and Stannard, 1948; Stannard and Horecker, 1948). The
experiments summarized in Section ID disclosed that azide had no immediate
effect on diapause respiration at concentrations as high as 5 X 10~4 M. This re-
sult supports the conclusion drawn above, that cytochrome oxidase does not limit
pupal respiration. On the other hand, the sensitivity to azide of DNP-stimulated
respiration was quite striking. This is consistent with the argument that, under
the influence of DNP, cytochrome oxidase becomes more saturated.
6. The limiting link in the pupal respiratory chain
The present experiments provide only one clue to the identity of the limiting
link in the pupal respiratory chain. This is the fact that antimycin A — a potent
inhibitor at the concentrations we employed of the DPNH-cytochrome c reductase
system — had only a minor effect on normal pupal respiration and DNP-stimulated
respiration. This inhibitor is said to have as its specific target the Slater factor
which mediates the transfer of electrons from flavoprotein to cytochrome c (Potter
and Reif, 1952; Reif and Potter, 1953; Chance and Williams, 1956). The
insensitivity of pupal respiration to this reagent suggests that the limiting link in
the pupal respiratory chain lies between the Slater factor and cytochrome oxidase,
e.g., cytochrome c. Recent studies of Shappirio and Williams (1957a, 1957b)
indicate that the limiting link is very likely cytochrome c, for with very sensitive
spectroscopic techniques they were unable to detect this enzyme in most pupal
tissues although cytochrome oxidase was easily demonstrated. They also showed
that in homogenates of pupal tissues, cytochrome c is a rate-limiting link in the
oxidation of DPNH. Hence it seems safe to identify limiting concentrations of
cytochrome c as a principal cause of the unsaturation of cytochrome oxidase in
pupal tissues.
154 CHARLES G. KURLAND AND HOWARD A. SCHNEIDERMAN
7. Injury-stimulated respiration
The increased sensitivity to carbon monoxide and azide shown by pupae sup-
porting an injury respiration (Sections 3 A and 3C) indicate an increased satura-
tion of cytochrome oxidase after injury. The observation (Section 3D) that
5 X 10"* M DNP failed to accelerate injury respiration to the same degree as
diapause respiration supports the conclusion that cytochrome oxidase is virtually
saturated when injury respiration is at its maximum. What brings about this in-
creased saturation of cytochrome oxidase is not known with certainty but the pres-
ent experiments suggest that it is caused by a gradual synthesis of cytochrome r
which is provoked by injury. Recall that injured pupae treated with DNP were
delayed in developing maximum injury respiration when compared with injured
pupae receiving water injections. This suggests that integumentary injury in-
itiates some process which requires a supply of phosphate bond energy which was
uncoupled by DNP. The gradual development of maximum injury respiration
over a three-day period suggests further that this energy-demanding process in-
volves, in part, the synthesis of one or more of the respiratory chain components
and does not simply reflect increased turnover of pre-existing enzymes. We in-
terpret the increased CO-sensitivity of injury-stimulated respiration to indicate that
more cytochrome c is being synthesized than cytochrome oxidase. That augmented
protein synthesis does in fact follow injury has been demonstrated by Telfer and
Williams (1955), who showed that the incorporation of OMabelled glycine into the
pupal proteins was stimulated by injury to about the same extent as respiration.
It is not without interest that the synthesis of these respiratory components ap-
pears to be obligatory. Indeed, the data in Section 3A suggest that when synthesis
is prevented by prolonged exposure to carbon monoxide, the pupae fail to develop
an injury respiration and die. This obligatory synthesis of new respiratory com-
ponents may be imposed upon diapausing pupae because their capacity for wound
repair is restricted by their low metabolic rate. Apparently this repair process is
able to compete with the "maintenance" processes of the diapausing pupa, thereby
causing death when total energy production is reduced by carbon monoxide. In
this connection, it is noteworthy that newly molted pupae, whose respiratory rate
is considerably larger than that of pupae firmly in diapause, fail to show an injury
respiration. This reflects their capacity to underwrite the energy requirements of
injury without augmenting the respiratory chain. This capacity is also present in
developing Cecropia adults and we have also shown it in all stages of non-diapausing
species such as the bee-moth Galleria mellonella.
8. Conclusions
The several lines of evidence considered in the preceding sections persuade us
that earlier conceptions of the respiratory enzyme system of diapausing silkworms
need re-evaluation. The basic differences between the respiratory enzyme chains
of the diapausing pupa and the non-diapausing stages appear to be quantitative dif-
ferences and not qualitative differences as was suggested earlier (Williams, 1951 ;
Schneiderman and Williams, 1954a, 1954b). The CO-insensitivity of pupal respira-
tion does not stem from the activity of a CO-insensitive terminal oxidase, but re-
sults from a great excess of cytochrome oxidase relative to other components of the
CARBON MONOXIDE AND RESPIRATION 155
respiratory chain. None of our findings supports the renewed suggestions of
Wojtczak (1955) and Ito (1955) that tyrosinase functions as a terminal oxidase in
insects. Indeed, in view of the failure of potent inhibitors of tyrosinase like
phenylthiourea to inhibit respiration (Schneiderman and Williams, 1954a) and the
light-reversibility of the carbon monoxide inhibition of silkworm growth (Schneider-
man and Williams, 1954b) and respiration (Pappenheimer and Schneiderman, un-
published) this is not likely. The present data, coupled with the recent spectroscopic
findings of Shappirio and Williams (1957a, 1957b) and with the studies of Harvey
and Williams (1958a, 1958b) on the pupal heart, indicate that cytochrome oxidase
is the terminal oxidase during pupal diapause and cytochrome c is the limiting com-
ponent in the pupal respiratory chain.
In this perspective, the increased respiration following integumentary injury and
initiation of adult development reflects an increase in cytochrome c content which
occurs at a faster rate than any increase in cytochrome oxidase. Possibly the in-
crease in cytochrome c reflects its adaptive synthesis in response to changes in the
energy requirements of the tissues. These changes were induced on the one hand
by injury and on the other by the prothoracic gland hormone which initiated adult
development. Such an adaptive synthesis of cytochrome c has been suggested in the
case of regenerating rat liver by Drabkin (1955). However, while the data sup-
port the view that cytochrome c is the limiting link in the pupal respiratory chain,
they do not rule out the possibility that other factors, such as phosphate acceptors,
may exert short-term effects on pupal respiration.
In conclusion, it is worth recalling that many animals other than diapausing
pupae of the silkworm have a low respiration that is insensitive to carbon monoxide.
Moreover, in many of these, such as diapausing eggs of grasshoppers and silkworms
and unfertilized eggs of sea urchins, cytochrome oxidase is clearly present. The
usual explanation for CO-insensitivity has been that respiration proceeded along
tracks alternative to the cytochrome oxidase system (cf. Needham, 1942, p. 567).
It is noteworthy, however, that in interpreting some of the very first experiments
which showed this CO-insensitivity, Runnstrom (1930) suggested that cytochrome
oxidase was not saturated with its substrate and this was the reason for CO-
insensitivity in the sea urchin egg. In retrospect, it seems likely that this idea was
sound and that the CO-insensitivity of the respiration of many systems is probably
the result of an excess of cytochrome oxidase relative to some other component of the
respiratory chain.
9. Final theoretical considerations of carbon monoxide-insensitive respiration
The basic premise underlying the arguments offered in the earlier sections of
this discussion is that an excess of cytochrome oxidase can lead to a virtual carbon
monoxide-insensitivity of a cytochrome oxidase-mediated respiratory chain. This
is shown as followrs. It is well known that carbon monoxide combines only with
the reduced form of cytochrome oxidase (also called a3) :
(1) CO + a3++ ^ C0-a,++ ; K (C(>a:<++)
(C0)(a3++y
Equation (1) is the simple chemical equilibrium with a characteristic equilibrium
•constant that describes the interaction of reduced cytochrome oxidase (as++) with
156 CHARLES G. KURLAND AND HOWARD A. SCHNEIDERMAN
carbon monoxide. This equation tells us that at a given concentration of carbon
monoxide the amount of CO-a3++ complex formed is determined solely by the
steady-state concentration of reduced cytochrome oxidase.
Now the steady-state concentration of reduced (and oxidized) cytochrome
oxidase is determined by the rate of electron transfer to cytochrome oxidase, and
this, of course, is measured by the rate of oxygen consumption.
/^\ 4__i_ § /-\ +++ I /"k
\£) Q-3 I ^2 ~~ #3 ~T~ ^J% •
Equation (2) describes this steady-state between reduced and oxidized cytochrome
oxidase. It is important to note that equation (2) does not describe a simple
chemical equilibrium but a steady-state where the "apparent equilibrium constant"
depends on the rate of electron transfer through the respiratory chain. Thus, if
the rate of electron transfer to as+++ from the previous component in the chain is
very slow, most of the cytochrome oxidase will be in the oxidized state and the ratio
of a3++ to a3+++ will be small. Since the rate of electron transfer is measured by the
rate of oxygen consumption, the "apparent equilibrium constant" for equation (2)
will vary with the rate of oxygen consumption. This fact, incidentally, rules out the
use of the usual Warburg formulation to describe quantitatively the effects of carbon
monoxide on respiration, namely
N CO
o~ ^"oT
where "N" is the fraction of respiration not inhibited by carbon monoxide (War-
burg, 1927). For, this formulation assumes that all the oxidase is in the reduced
state, and hence that "the observed respiration is proportional to the amount of
enzyme not combined with carbon monoxide'' (Warburg, 1949. p. 78). Indeed,
Warburg points out that in view of this assumption it is remarkable that there are
cells for which his equation applies (p. 79).
When carbon monoxide is used as an inhibitor of cytochrome oxidase. the de-
gree of inhibition of respiration depends upon the new steady-state reached by the
system, in which both oxygen and carbon monoxide compete for reduced cytochrome
oxidase. In this steady-state, some of the cytochrome oxidase is in the oxidized
state, some is reduced and complexed with carbon monoxide, and the remainder is
reduced and transferring electrons to molecular oxygen, i.e., playing a role in res-
piration. The effect of carbon monoxide on respiration depends on the degree to
which carbon monoxide decreases the concentration of reduced cytochrome oxidase
that is transferring electrons to molecular oxygen. Since as++ must satisfy the equili-
brium conditions of equation (1) and the steady-state conditions of equation (2), it
becomes apparent that the amount of a3+++ plays a major role in determining how
much a3++ remains to function in respiration. We thus see that the effect of carbon
monoxide on respiration depends on the fraction of the total cytochrome oxidase in
the reduced state. In other words, the effect of carbon monoxide on respiration
depends upon the ratio of the actual rate of uninhibited respiration (as measured by
the concentration of reduced cytochrome oxidase) to the maximum potential rate
of respiration when virtually all the oxidase is kept in the reduced state (as measured
by the total concentration of cytochrome oxidase). This ratio,
++ _U /r ..+++*
a,++ + a-.
CARBON MONOXIDE AND RESPIRATION 157
the fraction of the total oxidase in the reduced state, is what we ordinarily refer to
as the "saturation" of cytochrome oxidase. When the saturation of cytochrome
oxidase is high, the carbon monoxide sensitivity is high, and when the saturation is
extremely low, the effect of carbon monoxide on respiration is insignificant. This
can easily be seen when we consider two extreme cases, bearing in mind equations
(l)and(2).
Let us examine a system in which the initial steady-state concentrations of aS+
and a,+++ are about equal (i.e., a high saturation). In such a system, with a 20:1
CO/O2 ratio an appreciable amount of CO-«3++ can form. When the new steady-
state is established in the presence of carbon monoxide, the ratio of the concentra-
tions of a?i++ to o,+++ is the same as before. However, the absolute concentration of
both these components has been reduced considerably since a large part of the
cytochrome oxidase is complexed with the carbon monoxide. As far as respiration
is concerned, the significant reduction in a,++ leads to a significant inhibition of
respiration by carbon monoxide.
By contrast, consider a system in which the initial steady-state concentration of
an+++ is much greater than the concentration of a,4+. The presence of a CO/O., ratio
of 20: 1 will lead to the formation of only a small concentration of CO-a3++ because of
the low concentration of a.,++. Indeed, when the difference between the concentra-
tions of rt3+++ and <7.,++ is very great (i.e., a very low saturation), the total pool of
cytochrome oxidase will not be significantly affected by carbon monoxide. As a
result, the steady-state concentration of a.^+ will not be significantly diminished by
the presence of carbon monoxide. Thus the CO-sensitivity of such a system is
small.
From the above analysis we learn that an excess of cytochrome oxidase relative
to other components of the respiratory chain will lead to CO-insensitivity of respira-
tion. The same conclusion was reached independently by Harvey and Williams
(1958b) using a different system and method of analysis.
One further theoretical consideration is crucial to the explanation offered above
for CO-insensitivity. If the inhibition of cytochrome oxidase by carbon monoxide
is a function of the total cytochrome oxidase present, then it must be possible for
the transfer of electrons from the carrier part of the respiratory chain to proceed
independently of a sterically specific arrangement of the chain components. In
their review. Chance and Williams (1956) have discussed this possibility. They
concluded that it was highly improbable that the chain components were fixed in
position, and they presented two alternatives. Either the chain components were
free to act by random collisions according to a modified law of mass action ; or, they
were fixed in such a manner that the prosthetic groups were free to rotate on an
axis and be brought into apposition with adjacent chain components. In either
case, electron transfer could proceed across chain components that were not im-
mediately adjacent to one another. Therefore, it seems possible for the carriers
of the respiratory chain of the dia pausing pupa to transfer electrons to a "pool" of
cytochrome c oxidase. This pool of cytochrome o.ridase can manage all of the
oxidations, even in the presence of inhibitors, as long as there is sufficient uninhibited
enzyme present to meet the needs of electron transfer. In short, it appears possible
for an excess of cytochrome oxidase in tissues to account for the CO- and azide-
insensitivity of respiration and of various physiological functions such as heart-beat.
158 CHARLES G. KURLAND AND HOWARD A. SCHNEIDERMAN
The arguments presented in the previous sections persuade us that this is the situa-
tion in most of the tissues of diapausing silkworm pupae.
The experiments reported in Section 1A were performed in collaboration with
Dr. Roger D. Smith. We gratefully acknowledge the helpful criticisms of Dr.
David P. Hackett, Dr. Conrad S. Yocum, Dr. Carroll M. Williams, and Dr. Howard
M. Lenhoff.
SUMMARY
1. To characterize the respiratory enzyme chain that functions during diapause,
the respiration of diapausing pupae of the Cecropia, Cynthia, Promethea and Poly-
phemus silkworms was measured in the presence of specific mixtures of oxygen,
nitrogen and carbon monoxide, after injection of various metabolic inhibitors and
after injury.
2. Pupal respiration is at best only slightly inhibited by carbon monoxide and
is often stimulated. Whatever CO-sensitivity there is occurs only in pupae with
high basal metabolic rates. Moreover, when respiration is accelerated by injecting
dinitrophenol (DNP), or by injury, this evokes an enhanced sensitivity to carbon
monoxide. Indeed, it appears that the fraction of respiration sensitive to carbon
monoxide is a function of the rate of oxygen consumption of the silkworm at all
stages.
3. Reducing external oxygen tension to 2% fails to inhibit oxygen consumption,
but increases markedly the CO-sensitivity of pupal respiration. Thus low oxygen
tensions seem to unmask CO-sensitivity.
4. Pupal respiration is insensitive to azide concentrations as high as 5 X 10~4 M.
However, the azide-sensitivity, like the CO-sensitivity, increases markedly when
pupal respiration is stimulated by DNP or injury.
5. Antimycin A at a concentration of 10~6 M inhibits less than one-third of
normal pupal respiration or DNP-stimulated respiration. Compared to other or-
ganisms diapausing pupae are resistant to this inhibitor of the cytochrome c re-
ductase system.
6. Dinitrophenol at a concentration of 5 X 10~4 M stimulates pupal respiration
an average of 12-fold and as much as 16-fold. These are among the largest DNP-
stimulations ever recorded. Although pupae with high basal metabolic rates are
less stimulated proportionately by DNP than are pupae with low basal metabolic
rates, they develop a greater over-all respiration under the influence of DNP.
7. Dinitrophenol-stimulated respiration is inhibited by carbon monoxide. The
higher the DNP-stimulated respiration, the greater the inhibition by carbon mon-
oxide. From this and other evidence it appears very likely that DNP accelerates
the turnover of one or several components of the respiratory chain while having a
lesser effect on cytochrome oxidase.
8. Dinitrophenol delays the appearance of injury-stimulated respiration, sug-
gesting that the development of this increased respiration requires phosphate bond
energy. Furthermore, exposure to carbon monoxide causes the death of injured
pupae indicating that injury respiration is obligatory and involves the synthesis of
new respiratory components.
9. Newly molted pupae not yet firmly in diapause do not respond to wounding
with an injury respiration and their respiration is sensitive to carbon monoxide.
These findings are correlated with their high respiratory rate.
CARBON MONOXIDE AND RESPIRATION 159
10. The modes of action of the several inhibitors within diapausing, injured,
and developing insects are considered in detail and a new explanation is proposed
to account for the CO-, azide-, and cyanide-insensitivity of pupal respiration.
11. It is concluded that the insensitivity of diapausing pupae to inhibitors of
cytochrome oxidase results from an excess of this enzyme over its functional require-
ments in the pupal respiratory chain. This concept is examined in detail and found
to be theoretically sound. Evidence is presented that the limiting link in the res-
piratory chain is cytochrome c. Thus, contrary to earlier conceptions, it appears
that cytochrome oxidase is the principal terminal oxidase during diapause as well
as during all the other stages of the life history, and that the CO-insensitivity of
pupal respiration stems from a great excess of cytochrome oxidase relative to
cytochrome c.
12. The increased CO- and azide-sensitivity of pupal respiration after injection
of DNP or injury results from an increase in the saturation of cytochrome oxidase
provoked on the one hand by an increase in the turnover rate of cytochrome c, and
on the other by the synthesis of cytochrome c.
13. It is suggested that the CO-insensitivity of the respiration of other organisms
may be the result of an excess of cytochrome oxidase relative to some other com-
ponents of the respiratory chain.
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PERIODICITY OF MITOSIS AND CELL DIVISION
IN THE EUGLENINEAE1
GORDON F. LEEDALE
Department of Botany, The Durham Colleges in the University of Durham, England
In the course of an investigation into the division cytology of flagellates of the
class Euglenineae, it became necessary to determine the time and rate of mitosis for
each of the forty species under examination. The present paper deals with the
periodicity of mitosis revealed in the twenty species studied in detail for this feature,
and relationship of the periodicity to the day-night cycle. An account of the
structure and division of the cell and nucleus will be published separately (see
Leedale, 1958a, 1958b).
MATERIAL AND METHODS
1. Species studied -
The three main sources of material have been my own wild collections, the Cam-
bridge Culture Collection of Algae and Protozoa, and the Sammlung von Algenkul-
turen, Gottingen. All species have been isolated by Professor E. G. Pringsheim
or myself, with the exception of Trachelomonas grandis which was isolated by Singh
(Singh, 1956) and sent to me by Professor H. C. Bold.
The names of species are corrected according to Pringsheim (1956) for the
genus Euglena, and to Huber-Pestalozzi (1955) for the remaining genera. Color-
less species are indicated by an asterisk.
* Astasia klebsii Lemmermann
Colacium mucronatum Bourrelly
Cryptoglena pigra Ehrenberg
* Distignia proteus Ehrenberg em. Pringsheim
Euglena acus Ehrenberg
Euglena deses Ehrenberg
Euglena gracilis Klebs (strain "T," green form)
* Euglena gracilis Klebs (strain "T," colorless form)
Euglena gracilis Klebs (strain "Z," green)
Euglena spirogyra Ehrenberg
Euglena viridis Ehrenberg
Eutreptia pertyi Pringsheim
Eutreptia viridis Perty
1 From a study carried out in the Botany Departments of Queen Mary College, London,
and The Durham Colleges; some of the results were included in a thesis approved for the
degree of Doctor of Philosophy in the University of London. My thanks are due to Dr.
M. B. E. Godward of Queen Mary College and Professor E. G. Pringsheim of the University
of Gottingen for their help and advice.
2 I would like to thank Professor E. G. Pringsheim, Mr. E. A. George of Cambridge and
Professor H. C. Bold for supplying me with material.
162
MITOTIC RHYTHMS IN THE EUGLENINEAE 163
* Hyalophacus ocellatus Pringsheim
Lepocinclis ovum var. buctschlii (Conrad) Huber-Pestalozzi
Lepocinclis steinii Lemmermann em. Conrad
* Menoidium cultellus Pringsheim
* Peranema trichophorum (Ehrenberg) Stein
Phacus pusillus Lemmermann
Phacus pyrum (Ehrenberg) Stein
Trachclomonas India Stein em. Deflandre
Trachelomonas grandis Singh
2. Cultivation
Cells were isolated from wild collections by the micropipette method (Prings-
heim, 1946a). All species except Peranema trichophorum were grown in soil-water
tubes (biphasic culture, Pringsheim, 1946a, 1946b) with a wheat grain, starch or
ammonium magnesium phosphate beneath the soil. Eutreptia spp. were grown in
tubes with 50% sea-water. Peranema trichophorum was grown in soil extract
containing 0.5% milk.
In addition to the biphasic cultures, green and colorless forms of Euglena gracilis
(strain "T") were cultivated in 0.2% Difco beef extract, or "SATBY" (0.1%
sodium acetate, 0.2% Difco tryptone, 0.1% Difco beef extract, 0.2% Difco yeast
extract, in distilled water).
Cultures were hung in a north-facing window or in temperature-controlled cab-
inets with either incandescent or fluorescent lighting on a time-switch. The cul-
tures were grown at a standard temperature of 20° C.
GENERAL FEATURES OF THE CULTURES
A biphasic culture of any species of the Euglenineae has a typical growth pattern.
Sub-culturing to a new tube with a heavy inoculum is followed by a lag-period of
two to three days during which time there are few or no divisions. This is followed
by a period of multiplication which is eventually slowed and halted by overcrowding
of the medium. There is an upper limit of number of cells per ml. of medium
(the "culture saturation point") at which cell multiplication falls to a low rate.
This effect is not caused by exhaustion of the medium ; if the cells of a "saturated"
culture are centrifuged off and the medium re-inoculated, the culture builds up as
quickly as before, and this can be repeated several times.
According to the size of the inoculum, the division rate, and the "culture satura-
tion point" of the species concerned, the increase in cell numbers may continue for
one to twelve months. It is the mitotic rhythms occurring during this period of
multiplication which are the subject of this paper.
MITOTIC PERIODICITY IN GREEN SPECIES
Fixations made at two-hourly intervals for several (not successive) 24-hour
periods showed that all green species of the Euglenineae had mitosis confined to
the dark period when growing in biphasic culture under natural light conditions.
The restriction of nuclear division to the dark period was examined in detail in
164
GORDON F. LEEDALE
4O-
o
"Seo
O
I/)
13
O
-^40
CJ
-4
A
B
o
cr>
6 (/>'
-4
-•2
8p.m.
Time
FIGURE 1.
fifteen green species. Fixations were made at half-hourly intervals from the onset
of darkness on one, two or three consecutive nights, the material for any one series
being taken from the same culture tube. Five hundred cells were counted in each
of two preparations from each fixation and the number of cells in mitosis and cell
division noted. The results of these counts were similar for all species and are
recorded graphically for six representative species in Figures 1 and 2.
Mitosis began one to two hours after the onset of darkness. In Euglena
spirogyra (Fig. 1, A), Euglena viridis (Fig. 1, B) and Eutreptia pertyi, mitosis
began at the same time on each of three successive nights. The mitotic maximum
MITOTIC RHYTHMS IN THE EUGLENINEAE
165
Time
FIGURES 1 and 2. The number of cells per thousand in mitosis at half-hourly intervals
during one, two or three consecutive nights, plotted as mitosis percentage against time. All
results are for biphasic cultures growing in the natural day-night cycle, the dark period be-
ginning at 8 PM. FIGURE 1. Green species : A, Euglena spirogyra; B, Euglena viridis. FIG-
URE 2. Green species : A, Phacus pusillus; B, Phacus pyrum; C, Trachelomonas bulla; D,
Trachelomonas grandis.
occurred from 2% to 4^ hours after the onset of darkness in all species. The
maxima for Euglena viridis (Fig. 1, B), Eutreptia pertyi and Phacus pusillus (Fig.
2, A) occurred at the same time on three successive nights; those for Euglena
spirogyra (Fig. 1, A) covered a two-hour period within three nights. The span
166 GORDON F. LEEDALE
of the nightly period during which mitosis occurred ranged from three to six hours
in the different species. The mean maximum percentage of cells undergoing mitosis
each night is recorded in Table I.
Recording the number of cells at each stage of mitosis in each fixation produced
a more detailed picture of the periodicity. The results for Euglena spirogyra for
one dark period (Fig. 3) illustrate the complete restriction of nuclear and cell
division to within a five-hour period, beginning approximately two hours after the
onset of darkness. Successive maxima of the mitotic stages occur, a wave of pro-
phases being followed by waves of metaphases, anaphases, telophases and cell
cleavage. This pattern was repeated in other cultures of the same species and by
other species, the relative size and span of the maxima varying according to the
duration of the stages of mitosis in the different species.
TABLE I
The mean maximum percentage of cells undergoing mitosis each night in green species
of the Euglenineae in biphasic culture at 20° C.
Species Mean maximum %
Colacium mucronatum 2.6
Cryptoglena pigra 1.8
Euglena acus 1.9
Euglena deses 2.2
Euglena gracilis "T" 4.2
Euglena gracilis "Z" 5.7
Buglena spirogyra 3.4
Euglena viridis 4.9
Sutreptia pertyi 3.5
Sutreptia viridis 2.3
Itepocinclis ovum var. buetschlii 6.8
Itepocinclis steinii 1.3
PJiacus pusillus 3.4
Rhacus pyrum 3.1
T*rachelomonas butla 1.8
Trachelomonas grandis 2.9
Further series of fixations over a period of one year showed that no matter at
what time of the clock the natural dark period began, mitosis began one to two hours
later, the percentage of cells dividing each night being of the same order for any one
species (at 20° C.). There was no variation in the mitotic rate in relation to
day-length.
Examination of the same culture over a period of several months showed the
multiplication period to be discontinuous. Weeks with divisions occurring every
night were interspersed with occasional days when no divisions occurred.
The introduction of an artificial dark period during the natural light period
affected mitotic periodicity in all the green species. If the artificial dark period
was begun three hours or less before the natural one, mitosis occurred, but in a
lower percentage of cells than usual. When the artificial dark period was introduced
six hours or more before the natural one was due to begin, divisions rarely occurred.
The shortest day-length after which mitosis would occur was approximately twelve
hours.
No mitosis or cell division could be induced in any green species in biphasic
MITOTIC RHYTHMS IN THE EUGLENINEAE
167
culture in any conditions or intensity of artificial lighting, either direct or diffused,
incandescent or fluorescent. Attempts to reverse the mitotic periodicity in a tem-
perature-controlled cabinet with lighting on a time-switch were unsuccessful, the
cells becoming quiescent with no divisions occurring. Similarly, no mitosis occurred
in either continuous light or continuous darkness. Returned to natural light con-
ditions after such treatment, the cells recovered their full division rate within a day
if the treatment had been short, but less quickly if the treatment was prolonged.
C
o
(fl
o
O 6-
4-
2-
10pm
12 M.
2a.m.
Ti me
FIGURE 3. Mitosis in Euglena spirogyra. The number of cells per thousand in prophase
(P), metaphase (M), anaphase (A), telophase (T) and cell cleavage (Cl) at half -hourly
intervals during one night, plotted as percentage of each mitotic stage against time. The
results are for a biphasic culture growing in the natural day-night cycle, the dark period be-
ginning at 8 PM.
168
GORDON F. LEEDALE
80-
6am 12N. 6pm 12M. 6am
darkness.
Time
FIGURE 4.
Once mitosis had begun, it proceeded to conclusion even if the dividing cell was
then subjected to light. However, if light was introduced less than an hour after
the onset of darkness, no mitosis occurred. If a dark period of more than one
hour followed a full-length day and artificial light was then introduced, some cells
underwent a complete mitotic division, though on first examination no cells could be
found in mitosis, not even in prophase.
Euglena gracilis was the only species in which the time and rate of mitosis in
biphasic culture could be compared with those in a rich liquid medium. The
MITOTIC RHYTHMS IN THE EUGLENINEAE
16')
(J
30-
20-
60f
40-
C
O
^20
o
-C
^20-
10-
A
B
C
iA i^.
43
2
41
-6
-4
-2
2
41
6a.m. 12N. 6p.m. 12M. 6am.
darkness
T
me
FIGURES 4 and 5. The number of cells per thousand in mitosis at half-hourly intervals
during one, two or three (not consecutive) 24-hour periods, plotted as mitosis percentage
against time. All results are for biphasic or milk cultures growing in the natural day-night
cycle. FIGURE 4. Colorless species : A, Astasia klebsii ; B, Distigma protcus. FIGURE 5.
Colorless species : A, Hyalophacus ocellatns; B, Menoidium cultellus ; C, Pcranema tricho-
phorum.
mitotic rhythm shown by the green form of strain "T" in biphasic culture was absent
in 0.2% beef extract or "SATBY" medium. During the period of rapid multiplica-
tion prior to crowding of the culture, a fixation at any time of day or night showed
from 5-6% (beef extract) or 8-10% ("SATBY") of the cells undergoing mitosis
170 GORDON F. LEEDALE
(at 20° C.)- At 30° C. the mitotic rate of Euglena gracilis "T" in "SATBY" was
25-30%. In biphasic culture, maximum division rates were obtained at 20° C. ;
raising or lowering the temperature by five degrees resulted in a fall in division rate.
MITOTIC PERIODICITY IN COLORLESS SPECIES
Fixations made at half -hourly intervals over 24-hour periods showed that a
constant rate of mitosis was not maintained in any colorless species of the Eugleni-
neae in biphasic culture, bursts of mitotic activity alternating with periods when
mitosis was almost completely absent.
The results for 24-hour series of half-hourly fixations are recorded for the five
species studied in Figures 4 and 5. In addition to these series where a division
maximum occurred at some time during the 24-hour period, numerous series con-
tained no divisions or a few divisions scattered throughout the period. Many
single fixations at different times of day or night contained cells in mitosis.
TABLE 1 1
The maximum percentage of cells recorded in mitosis at any one time in colorless species
of the Englenineae in biphasic or milk culture at 20° C.
Species Maximum %
Astasia klebsii 8.0
Distigma proteus 3.9
Hyalophacus ocellatus 1.9
Menoidium cultellus 4.7
Peranema trichophorum (in milk) 2.1
Mitotic maxima occurred at any time of the clock. In none of the five species
did the periods of major mitotic activity bear any relationship to the alternating
light and darkness of the natural day-night cycle. The recorded maxima for
Astasia klebsii (Fig. 4, A) occurred at 10 AM, 4:30 PM and 10 PM ; those for
Hyalophacus ocellatus (Fig. 5. A) at 9 AM, 3 PM and 9:30 PM. The time-spans
of the major periods of mitotic activity ranged from 3y2 to 8^2 hours.
The highest percentage of cells obtained dividing at any one time is recorded for
each species in Table II. The percentages of cells dividing at different times on
different dates were of the same order for some species (Fig. 5, A and B) but not
for Astasia klebsii (Fig. 4, A).
The irregularly spaced bursts of major mitotic activity in the colorless species
continued in alternating artificial light and darkness, in continuous light, and in
continuous darkness.
The colorless form of Euglena gracilis "T" growing in 0.2% beef extract or
•'SATBY" medium behaved as did the green form in these media, exhibiting no
periodicity of mitosis, regular or irregular. A continuous division rate of 6-7%
was maintained in "SATBY" at 20° C., the rate increasing to 30-35% at 30° C.
DISCUSSION
Mitotic rhythms have been recorded for higher plants by Lewis (1901), Kellicott
(1904), Karsten (1915), Laughlin (1919), Stalfelt (1919), Friesner (1920),
Tischler (1921), Abele (1925), Brown (1951) and Jensen and Kavaljian (1958).
MITOTIC RHYTHMS IN THE EUGLENINEAE 171
The rhythm, in most cases thought to be endogenous, has been related to the onset
of germination, the balance between cell elongation and division, or light periodicity.
Lewis (1901) and Karsten (1915) found that the times of the maxima altered when
light conditions were changed, but Friesner (1920) found the maxima were inde-
pendent of light changes. Stalfelt (1919) and Brown (1951) state that the mitotic
rhythm of higher plants is exogenously imposed by the day-night cycle, disappearing
when the plants are grown in continuous darkness. No evidence of mitotic rhythms
in higher plants was found by Winter (1929) or Gray and Scholes (1951).
Mitotic rhythms in animals have been recorded by Ortiz-Picon (1933), Carleton,
(1934), Cooper and Schiff (1938), Cooper and Franklin (1940), Blumenfeld
(1942, 1943), Bullough (1948) and Milletti (1950). The rhythm has been related
to the activity cycle, a higher division rate occurring when the animal is at rest
(Cooper and Schiff, 1938; Bullough, 1948; Milletti, 1950). Kalmus (1935) has
recorded an exogenous rhythm of cell division for Paramecium.
Twenty-four-hour rhythms of mitosis have been recorded for a number of algae.
Division occurring exclusively at night has been recorded for species of the genera
Cladophora and Stigeoclonium (Braun, 1851), Spirogyra (Braun, 1851; Famintzin,
1867; Sachs, 1874; Strasburger, 1880). Zygnema (Kurssanow, 1912), and Vauch-
cria, Hydrodictyonand Ulothri.v (Sachs, 1874), whilst Karsten (1918) found three
maxima in each 24-hour period for species of Closterium, Cosmarium and Mesotacn-
hmi. Wildeman (1891) found no mitotic rhythm in Spirogyra. The present
author has found mitosis almost entirely confined to the dark period in species of
Hydrodictyon, Ulothri.v, Mougcotia, Spirogwa, Zygnema, Closteriwn, Cosmarium
and Staurastrum in biphasic culture. The rhythm was exogenous in these species,
and mitosis could be produced at any time of the clock by adjusting the time of the
dark period in a culture cabinet. Some species of Spirogyra and Zygnema under-
went mitosis in continuous light.
A nocturnal periodicity of mitosis in euglenoid species has been mentioned by
Dangeard (1902) for species of Euglena, Phacus and Trachelomonas, Baker (1926)
for Euglena gracilis in a split pea infusion, Ratcliffe (1927) for Euglena spirogvra
in modified Doflein's medium, S. R. Hall (1931) for the parasitic Euglena leucops
Hall when in its host, a species of Stenostomum, Gojdics (1934) for Euglena deses
in 0.1^ beef extract, Johnson (1934) for Colacium vesiculosum Ehrbg. and Chu
(1946) for Euglena spp. in biphasic culture. Only sparse growth was possible in
several of the media recommended by these authors. Lackey (1929) has made
the only record of a division maximum at night in a colorless species (Entosi-
phon sulcatum (Duj.) Stein, grown in a cracked wheat medium) and suggests
it might be explained on phylogenetic grounds. This is to be doubted since
the nocturnal rhythm of the green species is not endogenous and no such rhythm is
present in the five colorless species investigated in the present study. Lackey re-
cords some divisions during the day and it is probable that his division maxima
occurred during the dark period by chance, without being related to it.
The mitotic maxima recorded for higher plants and animals are increases over
a continuous low division rate. As would be expected, in organisms composed of
many cells arranged in tissues, some of which are specifically concerned with cell
division, mitosis occurs throughout the 24-hour period in the division sites. Diurnal
rhythms, whether in areas directly affected by light or not (root-tip meristems in
172 GORDON F. LEEDALE
plants, bone marrow in animals), can be related to metabolic rhythms, maximum
mitosis occurring during the period of minimum activity.
In unicellular organisms the division of labor between cell growth and mitosis is
often in time rather than in space. A cell tends to divide during a period of minimum
activity of that particular cell. Thus green unicells and filamentous green algae
often have a rhythm of mitosis which is closely related to the rhythm of photosyn-
thetic activity in the day-night cycle.
Such a relationship is exhibited by the Euglenineae. Green species, when living
autotrophically, divide only in the dark, and an almost full period of natural day-
light is necessary before mitosis will occur in the ensuing dark period. A threshold
period of darkness is required for the induction of mitosis, but once induction has
occurred, the mitotic process will begin and proceed to completion, even though the
cell be subjected to light before its nucleus has begun the anterior migration which
is the first sign of approaching mitosis. This induction precedes prophase by a
period of up to one hour, since the threshold period for induction is approximately
one hour after the onset of darkness, whilst the first prophases in all species appear
one to two hours after darkness. The final inductions would then be occurring
approximately three hours later, since the last prophases appear four to five hours
after darkness (Fig. 3).
It has been shown in Euglena gracilis that the nocturnal periodicity of mitosis is
removed by the stimulus of a rich food supply, the heterotrophic ("chemotrophic")
mode of nutrition of this species in beef extract or "SATBY" being unrelated to the
day-night cycle.
Mitosis in colorless species of the Euglenineae in biphasic culture shows an
irregular periodicity which is not related to the natural day-night cycle. The
heterotrophic mode of nutrition of the colorless species is also independent of light.
The factor deciding which cells in a culture divide during any one period of
mitosis is probably cell age (reflecting cell size and cell maturity) in both green
and colorless species. If 5% of the cells of a biphasic culture of a green species
divide each night in turn, the span of a generation will be 20 days. In Euglena
gracilis in "SATBY" at 30° C., with a division rate of 25-35%, the generation span
cannot be more than 8-12 hours.
The nocturnal rhythm of mitosis is presumably present in green species in the
wild when the supply of nutrients is low. An influx of rich organic nutrients will
remove the periodicity and, when combined with optimum temperature and pH,
may result in the sudden euglenoid "blooms" which often occur in bog-pools, farm-
yards, ponds and lakes.
SUMMARY
1. The periodicity of mitosis and cell division has been investigated in 15 green
and 5 colorless species of the Euglenineae.
2. Green species in biphasic culture under natural light conditions have mitosis
confined to the dark period. Mitosis begins one to two hours after the onset of
darkness, each species having a predictable percentage of cells dividing each night.
There is a threshold period at the beginning of the dark period after which mitosis
cannot be inhibited by light. The mitotic rhythm is exogenous, being removed by
growth in artificial light or darkness (resulting in no mitosis), or in a rich organic
medium (resulting in continuous mitosis at a constant rate).
MITOTIC RHYTHMS IN THE EUGLENINEAE 173
3. Colorless species in biphasic culture under any light conditions have an ir-
regular mitotic periodicity, bursts of mitosis occurring at any time of the clock and
alternating with periods in which mitosis is almost absent.
4. It is suggested that the presence or absence of regular or irregular mitotic
periodicity is related to the different modes and rates of nutrition of green and
colorless species in various conditions of light and darkness, and in various media.
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THE HORMONAL CONTROL OF METABOLISM IN CRUSTACEANS.
IX. CARBOHYDRATE METABOLISM IN THE TRANSITION
FROM INTERMOULT TO PREMOULT
IN CARCINIDES MAENAS
BRADLEY T. SCHEER
Department of Biology, University of Oregon, Eugene, Oregon, and Laboratoire Arago,
Banyuls-sur-Mcr
Recent reviews of the metabolic events in the intermoult cycle of decapod crusta-
ceans, and of the hormonal control of these events, have emphasized the fragmentary
nature of our present knowledge (Knowles and Carlisle, 1956; Scheer, 1957).
Particular interest centers around the metabolism of carbohydrate, which is known
from the work of Renaud (1949) to undergo considerable changes in the course of
the intermoult cycle. The present report is based on a study of a laboratory
population of approximately 100 specimens of the crab Carcinides ( = Carcinus]
maenas, in which the content of total carbohydrate, total soluble polysaccharide,
blood carbohydrate, blood lipochromes, and total non-protein nitrogen was deter-
mined on the individuals in samples drawn at intervals from the population.
MATERIALS AND METHODS
The animals were taken from a lagoon north of Banyuls-sur-Mer and brought
into the laboratory on October 11 : they were maintained throughout the experiment
in large aquaria in running sea water, and fed regularly on mussels. Examination
of the animals showed them all to be in the hard-shelled condition (stages Co
through D., of Drach, 1939), but closer determination of intermoult cycle stage
was not made until the animals were killed for analysis. Most of the animals were
males, and only males were used for the studies reported, to avoid complications
arising out of sexual differences. On October 14, November 13, and December 1,
samples of 20 to 30 crabs, selected at random, were drawn from the group, and
the eyestalks were removed from every second animal. Mortality was very low.
It is probable that a few animals moulted during the period of the experiment, but
cannibalism prevented any certain determination of this.
Eight to ten days after the sampling, the animals were extracted for analysis.
The stage in the intermoult cycle was carefully determined, using the criteria of
Drach (1939). For this study, an exact determination of the division between the
end of the intermoult period (C4) and the beginning of the premoult period (D:)
was essential. Accordingly, microscopic examination of the external branchial
epipodite of the first maxilliped was made to determine the presence of newly formed
setae beneath the old integument of this appendage. The presence of even the most
rudimentary new setae was taken as an index of the beginning of stage Da. These
rudimentary setae can be detected only by careful microscopic examination under
good illumination by transmitted light.
175
176 BRADLEY T. SCHEER
A blood sample was taken by bleeding from a cut walking leg. The animal was
then quickly cut up into 50-75 ml. of 5% trichloracetic acid; Renaud (1949) had
already shown that the elaborate precautions to prevent glycolysis which are neces-
sary in mammals are not as important in crabs. The mixture of acid and tissue
was transferred to an electric blender (Cadillac Atomixer), and blended at 8000
rpm. for 3 minutes. The mixture was rapidly filtered with suction, and the residue
returned to the blender with a second portion of acid for a second extraction. The
blender and residue were washed with a third portion of acid. The combined
filtrates were then diluted in a volumetric flask, usually to 250 ml., and stored in
the refrigerator until analyzed, always within a few days.
Blood carbohydrate was determined on some samples by the anthrone method
of Roe (1955). One ml. of blood was collected by dripping from a cut walking
leg, into a calibrated tube. One ml. of 5% trichloracetic acid was added with mix-
ing, and the mixture was centrifuged. One ml. of the supernatant was then trans-
ferred to a second tube for colorimetric determination. Blood lipochromes were
determined on other samples. To 1 ml. of blood. 5 ml. of acetone were added.
The acetone solution was then extracted with 2 ml. of petroleum ether, the ether
layer was washed with water, dried with solid KOH, and diluted to 5 ml. with
petroleum ether. The concentration of lipochromes was then read in the spectro-
photometer at 450 m/x against a petroleum ether blank. The measurements are
given as optical densities, since the exact nature of the lipochromes involved is not
known.
Total carbohydrate was determined by the anthrone method (Roe, 1955). One
hundred microliters (/xl) of the extract were transferred to a tube with a micro-
pipette, and diluted to one ml. for colorimetric determination. Polysaccharide was
determined by the same method. To 1 ml. of extract, 5 ml. of 95% ethyl alcohol
were added, and the mixture allowed to stand overnight in the refrigerator. The
tubes were centrifuged, the precipitate carefully drained, and suspended in 10 ml.
of distilled water. A one-mi, sample of this suspension was used for colorimetric
analysis. The anthrone method has the advantage for this study that it determines
a variety of carbohydrates, and relatively few other naturally occurring compounds.
All results are expressed in terms of glucose equivalents.
Non-protein nitrogen (NPN) was determined on 10-ml. samples of the extract,
using the micro-Kjeldahl digestion method of Hiller et al. (1948) and distilling the
digested mixture into 0.1 N HC1 in an all-glass still. Ammonia nitrogen (NH3N)
was separated by distilling the undigested extract in the same still. The final de-
termination of ammonia in both cases was colorimetric, using the Nessler reagent.
RESULTS
In the first sample, examined 11 to 19 days after collection, 10 out of 16 animals
(63%} were in the C4 stage (late intermoult) of the intermoult cycle; the remainder
were in the Dt stage (early premoult). In the second sample, examined 43 to 45
iays after collection, the proportion of C4 animals was 52% (12 C4, 8 Dx, 3 D2).
In the third sample, the proportion was 32% (7 C4, 15 Dx). These values suggest
that the population from which the samples were drawn was undergoing a steady
progression towards the moult. The x2 test shows that the proportion of C4 animals
in the third sample is significantly less than in the first, at the 5% level of probability.
HORMONAL CONTROL OF METABOLISM
177
In the first sample, only one of the 10 C4 animals had blood clearly pigmented
with lipochromes, while 5 of the 16 Dj animals had blood so pigmented; no quanti-
tative determinations were made in this series. In the second sample, 5 of the 12 C4
animals and 6 of the 1 1 D1 animals had lipochromes clearly evident in the blood ;
quantitative measurements were made on these 1 1 animals, and are presented in
Table I. For the third sample, quantitative measurements were made on all the
animals, and are presented in Table I. From the results on the third sample, in
which traces of lipochrome are found in nearly all specimens, it appears that the
level for qualitative detection of lipochromes lies at about 0.05 on the density scale
used to express concentrations. On this basis, we would conclude that only one
of the 7 C4 animals of the third sample had substantial amounts of lipochrome in the
blood, while 7 of the 15 Dj animals had such amounts. If we apply the x" test to
TABLE I
Lipochromes in the blood of Carcinides maenas. Optical density at 450 m/j. of a petroleum
ether extract, volume 5 nil., from 1 ml. of blood. The values for sample 2 (see text)
represent only animals in which blood lipochromes were qualitatively evident
C4
Di
.Stage condition
Normal
Eyestalkless
Normal
Eyestalkless
Animal
Density
Animal
Density
Animal
Density
Animal
Density
Sample 2
22
0.072
24
0.064
31
0.150
35
0.070
36
0.065
20
0.050
34
0.077
23*
0.157
33
0.112
40
0.088
42*
0.076
Sample 3
45
0.063
44
0.016
47
0.000
48
0.000
40
0.010
46
0.020
55
0.035
50
0.055
51
0.015
54
0.010
57
0.030
52
0.020
53
0.012
50
0.026
56
0.050
61
0.105
58
0.090
63
0.015
60
0.010
65
0.066
62
0.210
* Stage D2.
these values, we find that the frequency of occurrence of easily observable amounts
of lipochrome in the blood is not significantly different from 1 in 10 animals for
the C4 stage in samples 1 and 3, but is significantly different, at the 10% level of
probability or better, for all the other groups. The 1:10 ratio observed in C,,
sample 1, is also significantly different from the 1:2 ratio observed in Dlt sample o.
The mobilization of lipochromes from the digestive gland to the integumentary
tissues is an important part of the preparation for the moult, and all of the D,
animals in this study showed deposits of pigment in the region of the membranous
layer of the integument; indeed, this characteristic appears to be a fairly reliable
means of detecting the beginning of the premoult period. The appearance of lipo-
chrome in substantial amounts in the blood may therefore be taken as an indication
of the beginning of preparations for moulting. It is clear from the results presented
that this mobilization begins before the first morphological signs of premoult (initia-
178
BRADLEY T. SCHEER
tion of new setae) appear. Moreover, we may conclude that the C4 animals in the
second sample were further advanced towards the premoult stage than were those
in the first or third samples. There is no conclusive evidence that eyestalk removal
has any effect on the mobilization of lipochromes.
The results of the carbohydrate determinations are presented in Table II. We
may first note the rather striking difference in carbohydrate content of normal
animals in stage Q between sample 2 and the other two samples. The mean values
TABLE II
Total carbohydrate and polysaccharide content (mg. glucose equivalent per gm. body weight]
of three samples from a population of Carcinides maenas,
and the effect of eyestalk removal
C4
D,
Stage
Normal
Eyestalkless
Normal
Eyestalkless
condition
No.
Carb.
Poly-
No.
Carb.
Poly-
No.
Carb.
Poly-
No.
Carb.
Poly-
sac.
sac.
sac.
sac.
Sample 1
7
2.14
1.31
6
5.64
4.04
11
3.63
3.00
13
3.34
2.06
11-19 days
8
0.59
0.24
9
9.50
7.48
19
2.93
1.85
18
6.48
5.42
12
2.38
1.70
10
8.59
7.67
20
6.10
5.24
21
6.19
5.05
15
2.18
1.12
14
13.5
11.0
16
3.03
1.96
17
4.52
4.01
Sample 2
22
8.88
8.48
24
9.45
9.20
31
4.83
4.83
35
15.7
13.9
43-45 days
25
12.7
8.57
27
9.53
9.53
32
16.4
16.4
41
5.45
5.25
26
1.13
1.13
29
7.40
7.32
34
8.84
8.52
43
14.4
12.8
28
15.7
14.8
30
12.8
12.3
40
14.4
13.7
23*
15.0
15.0
36
12.2
11.6
33
16.5
15.8
38*
14.8
14.2
42*
23.6
10.9
37
3.18
3.18
39
19.3
16.8
Sample 3
45
1.36
1.36
44
8.13
8.13
47
1.45
1.45
48
1.72
1.41
67-70 days
49
2.73
2.21
46
9.40
7.06
55
9.41
8.48
50
3.52
2.98
51
4.10
4.10
54
6.11
5.03
57
16.0
15.2
52
4.90
4.90
S3
2.28
1.88
59
12.8
11.1
56
12.7
11.0
61
24.4
21.6
58
11.4
11.4
63
15.7
11.9
60
2.49
1.95
65
10.7
9.53
62
10.6
8.33
64
18.9
16.3
* Stage D2.
for samples 1 and 3 are 2.06 and 2.62 mg. per gm. for total carbohydrate, while the
corresponding mean for sample 2 is 10.12 mg. per gm. The difference between the
means for sample 1 and 2 is significant at the 5% level on the basis of the t test.
This difference in means arises from the fact that all but one of the values from
sample 2 are greater than 8 mg. per gm., while none of the values from samples 1
and 3 is as great as 5 mg. per gm. Moreover, the single low value in sample 2 was
obtained from one of the animals (no. 26) which had no obvious lipochrome in the
blood. If our earlier conclusion, that a substantial fraction of the animals in stage C4
of the second sample were well on their way toward stage Dlf is correct, then we can
further conclude that one characteristic of this transition is a marked increase in
HORMONAL CONTROL OF METABOLISM
the carbohydrate content of the body. This conclusion is confirmed by the values,
for normal animals in stage Da, which are nearly all well above those for the C4
animals of groups 1 and 3. The difference between mean values for sample 3 for
C4 and D1 is significant at the 1% level on the basis of the t test. Renaud (1949)
had already observed a similar change in Cancer pagunts with a mean glycogen
content of 2.09 mg. per gm. for animals in C4. rising to 4.43 mg. per gm. by the end
of Dj. We may therefore conclude that the increase in carbohydrate content which
is characteristic of the transition from intermoult to premoult may occur during the
latter part of stage C4, before any morphological evidence of the transition is
apparent.
TABLE III
Non-protein nitrogen (NPN) and ammonia nitrogen (NH3N) in normal
and eyestalkless Carcinides maenas (nig. per gm. body weight)
C4
Di
Stage
condition
Normal
Eyestalkless
Normal
Eyestalkless
No.
NPN
NH3N
No.
NPN
NH3N
No.
NPN
NH3N
No.
NPN
NH3N
Sample 1
8
2.40
0.25
6
2.45
0.10
11
2.44
0.13
10
2.56
0.10
12
2.89
0.17
9
2.52
0.08
19
2.84
0.05
13
3.07
0.12
15
2.88
0.20
14
3.58
0.08
20
3.39
0.06
18
3.08
0.05
16
2.58
0.07
17
2.65
0.08
21
3.82
0.04
Sample 2
22
2.90
0.16
24
2.68
0.24
31
2.91
0.19
35
2.96
0.30
25
3.21
0.20
27
3.96
0.24
32
3.97
0.28
41
3.17
0.29
26
2.03
0.13
29
3.22
0.23
34
3.13
0.24
43
3.30
0.25
28
4.74
0.23
30
3.51
0.26
40
3.48
0.28
23*
3.17
0.32
36
2.82
0.25
33
4.07
0.27
42*
3.69
0.42
37
2.72
0.19
38*
3.03
0.26
39
2.64
0.27
* Stage D...
The second item to be noted from Table II is the fact that the carbohydrate
content of the eyestalkless animals in C4 is throughout at levels characteristic of D,
animals. Indeed, there was no eyestalkless C4 animal with a carbohydrate content
as low as 3 mg. per gm., and in all but two, the value was higher than 5 mg. per gm.
The differences in means for normal and eyestalkless animals in C4 were significant
at the 5% level for both samples 1 and 3, on the basis of the t test. We may there-
fore conclude that the operation of eyestalk removal causes an increase in carbohy-
drate content from the low values characteristic of C4 animals to the higher values
characteristic of the next stage in the cycle, D1. The same operation is clearly
without effect upon animals already in stage Dt if for some reason these animals have
low carbohydrate content, since there are several eyestalkless Dl animals with rela-
tively low carbohydrate values, and the distribution of values in normal and eye-
stalkless specimens in this stage is substantially the same. We may further infer
from our results, though conclusive evidence is lacking, that some endocrine factor
is secreted in the eyestalk during stage C4, and that secretion of this factor stops
towards the end of that stage. One effect of this factor would be the maintenance
180 BRADLEY T. SCHEER
of carbohydrate content at relatively low levels. Since Renaud (1949) has shown a
steady increase in glycogen content beginning in stage C,, we may suppose that the
secretion of the factor concerned decreases gradually rather than suddenly.
In general, the polysaccharide values follow the carbohydrate values rather
closely, and 80% or more of the carbohydrate is precipitated by alcohol. However,
in the C4 animals of the first sample, the polysaccharide averages only 62 % of the
total carbohydrate ; the eyestalkless individuals, and indeed all of the other groups,
had a higher ratio. Blood carbohydrate was measured for the animals of sample 1
only. The results are presented in Table III. Since it appears that the carbohy-
drate content of the blood does not reflect changes in the total carbohydrate of the
body, and is not influenced by any of the other factors considered here, we utilized
the blood samples from the second and third group for lipochrome studies.
The observation of Needham (1955) that increased nitrogen excretion follows
eyestalk amputation, led us to examine the nitrogen content of some of the extracts.
The results are presented in Table IV. There appears to be no systematic variation
in either NPN or NH3N, except that both sets of values, and especially the NH.,N
values, are generally lower in the animals of sample 1 than in those of sample 2. No
obvious explanation for this difference appears. In both samples, the extracts were
prepared 7 to 14 days after eyestalk removal, by which time Needham (1955) found
that nitrogen excretion had returned to normal levels. We conclude that no long-
lasting modification in nitrogen metabolism evident from NPN or NH3N content
of the animals is related to the variables considered here.
DISCUSSION
Perhaps the most important finding of this study is that metabolic changes
(mobilization of lipochromes, increased carbohydrate content) preparatory to the
moult precede in time the morphological changes (formation of new setae). This
may not be surprising, but it has not been emphasized before. We cannot on the
basis of the evidence available conclude that the metabolic changes are causally related
to the subsequent structural changes, but this is a reasonable inference. However,
the two metabolic changes observed do not seem to be directly related one to the
other. There is in general no complete correlation between increased blood lipo-
chrome and increased carbohydrate. Moreover, the increase in carbohydrate which
follows eyestalk removal is not in general associated with increased blood lipochrome.
The increase in carbohydrate content as the animal approaches a moult was al-
ready known from the study of Renaud (1949) on Cancer pagurus. Moreover,
Schwabe et al. (1952) had observed a marked increase in total glycogen, represented
by deposition in the digestive gland and epidermis, in the transition to the premoult
stage in spiny lobsters ; their data also suggest that eyestalk removal in stage C
increases the total glycogen of the body, while the same operation in stage D re-
sults in no change. However, they did not determine this quantity directly, and
neither of their methods, for determination of glycogen or for determining intermoult
cycle stage, was entirely satisfactory. The demonstration of an increased carbohy-
drate content following eyestalk removal therefore comes as a definite addition to the
long list of metabolic and other changes which are consequences of this operation
(Knowles and Carlisle, 1956; Scheer, 1957).
The absence of any changes in blood carbohydrate was something of a surprise.
HORMONAL CONTROL OF METABOLISM 181
Renaud (1949) found a steady increase in the reducing power of the blood from
C3 through D1 in Cancer payurus, but this increase was not evident when the blood
was treated with cadmium sulfate and sodium hydroxide, a procedure supposed to
eliminate non-glucose reducing substances. Recent studies in my laboratory by
McWhinnie (unpublished) on the blood of Hciniyrupsus mains have shown that
the blood carbohydrate, like the total carbohydrate in acid extracts of the body, in-
cludes several components, of which glucose is a relatively minor one. Using the
highly specific hexokinase glucose-6-phosphate dehydrogenase method, she found
glucose concentrations averaging below 2 mg. per 100 ml., with a maximum about
2.5 mg(/c in stage Q, and a slight decrease in stage C3, but no change as a result of
eyestalk extirpation. We had earlier found a decrease in the reducing substances of
spiny lobster blood (Schee^and Scheer, 1951) following eyestalk removal, but
others (Abramowitz ct al., 1944; Kleinholz and Little, 1949) found no such change
in crabs. It is clear that the problem of blood sugar regulation in crustaceans re-
quires further careful study with particular attention to specificity of methods.
The question now arises, what is the source of the increased carbohydrate in
late intermoult, and what alterations in metabolism are responsible for the increase.
Related to this is the question of the endocrine factors which we presume to be re-
sponsible for the increase. The evidence on which we can base hypotheses re-
mains fragmentary. Injection of eyestalk extracts increases blood reducing sub-
stances, and specifically the fermentable reducing substances (Abramowitz et al.,
1944; Kleinholz and Little, 1949; Scheer and Scheer, 1951). It would be unwise
at present to equate fermentable reducing substances with glucose, and we do not
know the metabolic relations among the various carbohydrates found in the blood,
nor indeed the identity of these substances. Scheer and Scheer (1951) showed that
injected glucose was removed from the blood more rapidly in eyestalkless than in
normal spiny lobsters, and that most of the carbon of this glucose could be recovered
in the water- and alcohol-soluble fraction of tissue extracts. From the work of Hu
(1958) we know that this fraction may contain, besides glucose, several oligosac-
charides of the maltose series. But the relation of these substances to synthesis of
polysaccharides or other aspects of carbohydrate metabolism remains obscure.
Present evidence, from plants and animals alike, indicates that glycoside linkages
in general are formed by adding one monosaccharide unit at a time to existing
nuclei by the agency of nucleotide coenzymes. Hu ( 1958) has shown that nucleo-
tides are present in crabs, and that carbon from administered glucose appears in
these compounds.
Whatever the intermediate steps, the increased carbohydrate content of late inter-
moult crabs may be derived ultimately either from protein or carbohydrate or both.
The evidence that, in fed crabs, there is no change in the non-protein nitrogen,
suggests that there is no fundamental alteration in the intensity of protein metabo-
lism. On the other hand, the evidence of Neiland and Scheer ( 1953) that, in fasting
crabs, protein is used in preference to carbohydrate, and in eyestalkless crabs, the
amount of protein used is greater, together with the evidence of Needham (1955)
that under conditions (trauma) in which protein breakdown is increased eyestalk
removal leads to a further increase, suggest that, in eyestalkless animals there may
be an increased conversion of protein to carbohydrate. The fact that, in such
animals, the utilization of glucose is also increased (Scheer and Scheer, 1951)
182 BRADLEY T. SCHEER
would lead one to place the site of the presumed endocrine effect in the process of
glycogenesis, rather than in that of gluconeogenesis. We may, therefore, postulate
that, during the C4 stage, there is a gradual transition from carbohydrate oxidation
to polysaccharide synthesis as a major pathway of carbohydrate metabolism. This
offers a possible explanation for the difference in glucose oxidation observed by Hu
(1958) and Scheer and Scheer (1951) using similar procedures with different
animals. The crabs used by Hu may have been in the early part of stage C4 or
even in C3, when carbohydrate oxidation is dominant ; the spiny lobsters used by
Scheer and Scheer (1951) may have entered into the phase in which carbohydrate
synthesis predominates. Neither author determined the intermoult stage with great
accuracy. Or, if we accept the view of Carlisle (Knowles and Carlisle, 1956)
that the intermoult cycle is qualitatively different in animals with prolonged inter-
moults (diecdysis, as in crabs in winter) from the intermoult in animals which
moult regularly throughout the year (anecdysis, as in Hawaiian spiny lobsters), we
might suppose that carbohydrate oxidation is primarily characteristic of diecdysis
and that in anecdysis polysaccharide synthesis predominates. These suggestions
can be tested by careful comparison of the fate of carbon from administered labeled
glucose in the various stages of moult cycles of both types.
The question of hormonal control is likewise a difficult one. Carlisle (Knowles
and Carlisle, 1956) has summarized evidence suggesting that two separate hormones
are concerned in the control of the intermoult period. Carlisle attributes diecdysis
to the action of the well-known moult-inhibiting hormone, and cites his own ob-
servations and some of ours (Scheer and Scheer, 1954) to the effect that this hor-
mone is not active in certain crustaceans, including British populations of Carcinides
maenas in the summer. We have no way of knowing whether the animals studied
in the present investigation undergo a cycle of the type characterized by anecdysis,
or one characterized by diecdysis. It would appear that, to make much further
progress with these problems, it will be essential to work with at least partly purified
hormone preparations, and to have full information about the type of cycle and
stage of the animals in the cycle. We earlier postulated ('Scheer and Scheer, 1951)
an eyestalk factor which restrains carbohydrate utilization for polysaccharide, and
specifically chitin, synthesis. This factor would be the same as the "diabetogenic"
factor of Abramowitz ct al. (1944), and we suggested that it might also be the
moult-inhibiting factor. The results reported here do not give us any reason to
alter this hypothesis, nor do they substantially strengthen it. However, at present
it seems best to consider that the effects of eyestalk removal noted by Neiland and
Scheer (1953) on protein catabolism, and the related effect noted by Needham
(1955), might result from the action of this same factor. For conclusive evidence
concerning this hypothesis, it is essential to have a hormone preparation of reason-
able purity which can be tested for its specific metabolic effects.
This work was done during tenure of a John Simon Guggenheim Memorial
Fellowship. The author wishes to thank the Fellowship Board for the grant which
made this work possible, and especially to express his appreciation of the generous
provision of materials and facilities for this work at Laboratoire Arago, and of the
many kindnesses and the helpful assistance of the director. Prof. G. Petit, and his
staff.
HORMONAL CONTROL OF METABOLISM 183
SUMMARY
1. A laboratory population of Carcinides maenas was sampled three times over
a period of 70 days, and the blood carbohydrate, blood lipochromes, total body
carbohydrate, total body polysaccharide, non-protein nitrogen, and ammonia nitrogen
were determined ; the effect of eyestalk removal on these quantities was also
examined.
2. During the course of the observations, there was a progression within the
population from late intermoult (C4) to early premoult (D) stages.
3. The change from intermoult to premoult was signalized by the appearance of
relatively large amounts of lipochromes in the blood and integument, and by an in-
crease in total body carbohydrate content. These biochemical changes preceded any
morphological signs of preparation for moult.
4. Eyestalk extirpation caused an increase in the body carbohydrate, but did not
alter the blood lipochromes. The increase in carbohydrate was observed only in
those animals which had not undergone the change spontaneously.
5. The other quantities measured showed no variation attributable either to the
stage in the intermoult cycle or to eyestalk removal.
6. The results are discussed with relation to the possible mechanisms of the
effects observed, and the hormonal factors concerned.
LITERATURE CITED
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DRACH, P., 1939. Mue et cycle d'intermue chez les crustaces decapodes. Ann. hist. Occanogr.
Paris, 19: 103-391.
HILLER, A., J. PLAZIN AND D. D. VAN SLYKE, 1948. A study of conditions for Kjeldahl de-
termination of nitrogen in proteins. J. Biol. Chcm., 176: 1401-1420.
Hu, A. S. L., 1958. Arch. Biochem. Biophys. (in press).
KLEINHOLZ, L. H., AND B. C. LITTLE, 1949. Studies in the regulation of blood sugar concen-
trations in crustaceans I. Normal values and hyperglycemia in Libinia emarginata.
Biol. Bull, 96: 218-227.
KNOWLES, F. G. W., AND D. B. CARLISLE, 1956. Endocrine control in the Crustacea. Biol.
Rev., 31 : 396-473.
NEEDHAM, A. E., 1955. Nitrogen-excretion in Carcinides maenas (Pennant) during the early
stages of regeneration. /. Embryol. Exp. Morphol., 3 : 189-212.
NEILAND, K. A., AND B. T. SCHEER, 1953. The influence of fasting and sinus gland removal
on body composition of Hcmigrapsus midus. Part V of the hormonal regulation of
metabolism in crustaceans. Physiol. Comp. Occol., 4 : 321-326.
RENAUD, L., 1949. Le cycle des reserves organiques chez les crustaces decapodes. Ann. Inst.
Oceanogr. Paris, 24: 259-357.
ROE, J. H., 1955. The determination of sugar in blood and spinal fluid with anthrone reagent.
/. Biol. Chem,, 212 : 335-343.
SCHEER, B. T., 1957. Recent Advances in Invertebrate Physiology, pp. 213-227. Univ. of
Oregon, Eugene.
SCHEER, B. T., AND M. A. R. SCHEER, 1951. Blood sugar in spiny lobsters. Part I of the
hormonal regulation of metabolism in crustaceans. Physiol. Comp. Oecol, 2 : 198-209.
SCHEER, B. T., AND M. A. R. SCHEER, 1954. The hormonal control of metabolism in crus-
taceans VII. Moulting and colour change in the prawn Lcandcr serratus. Pubbl. Stas.
Zool. Napoli, 25 : 397-418.
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THE LIFE-CYCLE OF THE DIGENETIC TREMATODE, PROCTOECES
MACULATUS (LOOSS. 1901) ODHNER, 1911 fSYN. P. SUBTENUIS
(LINTON, 1907) HANSON, 1950], AND DESCRIPTION OF
CERCARIA ADRANOCERCA N. SP.
HORACE W. STUNKARD1 AND JOSEPH R. UZMANX
U. S. Fish and ITildlifc Service
The genus Proctocccs was erected by Odhner (1911) to contain Distomum
maculatum Looss, 1901, from Labrus memla and Crenilabrus spp. at Triest. Odhner
had found the parasite in Blennius ocellaris at Naples. One adult specimen from
Chrysophrys bifasciata and two immature specimens from lulis lunaris taken in
the Red-Sea, were described as a new species, Proctoeces erythraeus. Dawes (1946)
listed P. erythraeus as a synonym of P. maculatus (Looss), but the species was
recognized by Manter (1947) on the basis of six specimens he had collected from
Calamus calamus and Calamus bajonado at the biological laboratory of the Carnegie
Institution at Dry Tortugas, Florida. Several additional species have been de-
scribed. Fujita (1925) reported a metacercaria from the Japanese oyster, Ostrea
gigas, as a new species, Proctoeces ostreae. The paper was translated by R. Ph.
Dollfus who noted (p. 57), "II est a souhaiter que des recherches chez les poissons
mangers de Lamellibranches, sur les cotes de la prefecture d' Hiroshima, permettent
de decouvrir des exemplaires completement adultes de Proctoeces ostreae Fuj.,
chez lesquels 1'extension des vitellogenes et les dimensions des oeufs puissent etre
observees avec precision ; il sera alors possible de savoir definitivement si P. ostreae
Fuj. doit ou non tomber en synonymic avec P. maculatns (Looss)." Yamaguti
(1934) described P. maculatns from Spams arics, Spams macrocephalus, Pagroso-
mus auratus, and Epinephelus akaara in Japan. Several specimens from Pagroso-
mus auratus, which differed from P. maculatus in larger size, larger eggs, and trilobed
ovary, he described as a new species, Proctoeces major. Yamaguti (1938) reported
P. maculatus from Semicossyphus reticiilatus and described a larva from the liver
of the pelecypod mollusk, Brachidontes senhausi, as an unidentified member of the
genus Proctoeces. Manter (1940) described Proctoeces magnorus from a single
specimen found in the intestine of Caulolatilus anomalus, taken at Cerros Island,
Mexico. Hanson (1950) identified two specimens collected from Calamus sp. at
Bermuda by the late F. D. Barker as Distomum subtenue Linton, 1907, a species
described originally from Calamus calamus in the same area. Comparison of these
specimens with those from Tortugas identified by Manter as P. erythraeus estab-
lished their identity, and P. erythraeus was suppressed as a synonym of Proctoeces
subtenue (Linton, 1907). Hanson corrected the statement of Manter (1947),
noting that it is the vitellaria, not the uterus, which never extends into the post-
testicular region. Yamaguti (1953) predicated that Xenopera Nicoll, 1915 is a
1 Mailing address : American Museum of Natural History, Central Park West at 79th
Street, New York 24, N. Y.
184
LIFE-CYCLE OF PROCTOECES 185
synonym of Proctoeces, and Xcnopcra insolitus from S pants australis was listed as
Proctoeces insolitus (Nicoll, 1915). Winter (1954) described Proctoeces mac-
rovitellus from the intestine of Cyinatogaster aggregatits, taken off the coast of
southern California. It is notable that the final hosts of these trematodes are
porgies and labroid fishes of temperate and warm seas ; hard-mouthed, bottom forms
that feed on mollusks.
Uzmann (1953) described Cercaria miljordensis, a microcercous trematode larva
from Mytilus edulis in both intertidal and subtidal areas of Long Island Sound and
along the cost of Connecticut. About seven per cent of the mussels were infected
in the years 1951 and 1952. Although the infection was heavy in the area around
Milford, Connecticut, Uzmann noted that the parasite had not been reported from
higher latitudes despite intensive study of M. edulis over a period of many years.
The sporocysts develop in the venous sinuses of the mussel, beginning in the late
fall and continuing during the winter, with the release of the cercariae in greatest
numbers in the late winter and spring. The infection largely destroys the gonad
of the host and development of the sporocysts precludes normal gametogenesis.
The intensity of the infection seriously impairs the vitality of the mollusk and may
be lethal under temporary or sustained periods of ecological conditions unfavorable
to the host. Uzmann described the behavior of the cercariae and reported un-
encysted progenetic larvae referable to the genus Proctoeces in mussels harboring C.
miljordensis infections. He stated (p. 449), "Morphological comparison of the
two forms is favorable, and if the apparent relationship truly exists, an abbreviated
life-cycle may be possible since the larval Proctoeces contain many eggs with well
developed, motile miracidia. Experimental studies are projected and it is hoped
that decisive information can be presented at a later date." Shortly thereafter,
Uzmann was transferred to the Seattle, Washington Laboratory of the U. S. Fish
and Wildlife Service.
Further significant information was provided by the work of Hopkins (1954)
who described infection of the hooked mussel, Brachidontcs recurvus (syn. Mytilus
recuri'its} taken in Barataria Bay, Louisiana by Cercaria brachidontis n. sp., a
species so similar morphologically to C. miljordensis that their relationship was im-
mediately apparent. Cercaria brachidontis develops in orange-pigmented sporocysts
which completely destroy the gonad of the mussel. Immature cercariae have small,
knob-like tails, similar to those of C. miljordensis, but they are not present on fully
developed larvae. Hopkins referred the species to the family Fellodistomatidae but
without generic designation.
After the text of this paper was written, the account by Freeman and Llewellyn
(1958) appeared, announcing the discovery of the adult stage of a digenetic trema-
tode in the renal organs of the lamellibranch mollusk, Scrobicularia plana taken
from the mud-flats of the Thames estuary, at Chalkwell in Essex and Whitstable in
Kent. The worms were identified as Proctoeces subtenuis (Linton, 1907) Hanson,
1950, a species which was known previously only as a parasite of the hind-gut of
marine fishes belonging to the families Labridae and Sparidae, which occur chiefly
in tropical and subtropical seas. The asexual generations were not discovered and
since the adult stages had not been recorded from fishes of the English coast, the
authors concluded that in British waters the life cycle had been abbreviated and
restricted to invertebrate hosts. Possible methods were considered by which the
parasite had been introduced. They reported (p. 446) that, "The eggs are enclosed
186
HORACE W. STUNKARD AND JOSEPH R. UZMANN
w
-J
O.
LIFE-CYCLE OF PROCTOECES 187
in a thin, light-brown capsule." This statement appears confusing, since the "cap-
sule" is obviously the egg-shell and an egg comprises the shell and its contents, ovum,
embryo, or miracidium. Although many eggs contained active miracidia, they
varied much in size (from 0.026-0.073 by 0.015-0.030 mm.). The use of his-
tochemical techniques disclosed the presence in the vitellaria of dihydroxy-phenols
and protein, which on oxidation combine to form the quinones of the egg-shell, but
the corresponding phenol-oxidase was not demonstrated. Deficiencies in the egg-
making apparatus may account for the small and abnormal eggs. Freeman and
Llewellyn gave a detailed account of the morphology of the parasite and noted the
extent of individual variation. They stated (p. 447), "Several hundred specimens
were examined, and it is apparent that many of the characters thought to indicate
specific differences probably represent intraspecific variations of the kind emphasized
by Stunkard (1957)." As a result of their investigation, P. erythraeiis Odhner,
1911 and P. inagnonts Manter, 1940 were suppressed as synonyms of P. subtcnuis.
Furthermore, they stated (p. 455) that, "The differences between P. insolitus
(Nicoll, 1915) and P. subteniiis, and between P. maculatiis (Looss, 1901) and P.
subtcnuis, require reexamination."
The findings of Freeman and Llewellyn amply confirm the postulate of Uzmann
(1953) and constitute an important contribution to knowledge of the biology of the
digenetic trematodes.
The studies begun by Uzmann at Milford were continued at Woods Hole,
Massachusetts by the appointment of Stunkard to investigate the parasites of clams
and their predators in New England. Infections by C. milfordcnsis were found in
M. cdnlis taken in the Woods Hole area, although the incidence of infection was
low, about 0.5 per cent. However, the findings of developmental stages, from
cercariae to adults, confirmed the prescience of Uzmann that C. inilfordcnsis is the
larval stage of a species of Proctoeces.
DESCRIPTIONS
Adults. (Figs. 3. 4)
The general morphology of the worms is portrayed in the figures. The cuticula
is unarmed ; the suckers large and powerful. The digestive tract shows no unusual
features. The excretory vesicle bifurcates at the level of the posterior testis ; both
the stem and crura are lined with a simple epithelium which is flattened when the
wall is distended. The flame-cell pattern of the adult worm was not studied.
The genital pore is lateral, situated usually between the acetabulum and the
pharynx. The testes are diagonally tandem, either adjacent or somewhat separated.
Sperm ducts arise at the anterior ends, pass forward and join to form a common duct
just before entering the cirrus sac. In the posterior end of the cirrus sac it forms a
coiled seminal vesicle, filled with spermatozoa, and then opens into a straight, thick-
walled muscular canal. This structure is lined with high, secretory cells, whose distal
PLATE I
Drawings of P. macitlatits from M. edulls; made from fixed and stained specimens and at
the same magnification.
FIGURE 1. Juvenile specimen; length 1.20 mm.
FIGURE 2. Specimen just reaching sexual maturity, 6 eggs in uterus ; length 2.00 mm.
FIGURE 3. Gravid specimen, eggs small and mostly misshapen ; length 2.65 mm.
FIGURE 4. Gravid specimen, eggs normal with developing miracidia, length 2.62 mm.
188
HORACE W. STUNKARD AND JOSEPH R. UZMANN
PLATE II
LIFE-CYCLE OF PROCTOECES 189
ends are filled with chromatic granules, and terminates in the cirrus which protrudes
into a long, hermaphroditic atrial duct. The area between the wall of the cirrus
sac and the thick-walled canal is filled with secretory cells, whose ducts pierce the
thick wall and discharge into the narrow lumen. The ovary is pretesticular, in the
anterior part of the posterior one-half of the body. The oviduct arises at its posterior
face and turns ventrad and mediad where it expands into a fertilization space, from
which Laurer's canal emerges and continues dorsad and anteriad to open at the
surface above the ovary. The oviduct then enters Mehlis' gland where it receives
the common vitelline duct and expands into the ootype, where the egg is formed.
The uterus passes posteriad to the end of the body where coiled loops on either
side are followed by a median trunk which passes forward below the cirrus sac
to open into the ventral side of the hermaphroditic duct, six to ten microns before the
genital pore. In many of the specimens the eggs are malformed, of varying sizes,
often about one-third as large as in more normal individuals.
Average measurements in millimeters of ten gravid, mounted specimens ; limits
in parentheses: length, 2.74 (2.4-3.2); width, 0.81 (0.6-0.92); acetabulum,
0.38 X 0.43 (0.35-0.46) ; oral sucker, 0.24 )< 0.30 (0.21-0.32) ; pharynx, 0.18 (0.16-
0.20) ; ovary, 0.19 (0.16-0.22) ; anterior testis, 0.18 (0.15-0.20) ; 'posterior testis,
0.19 (0.16-0.23) ; eggs, 0.055 X 0.026 (see text).
Juveniles. (Figs. 1,2)
Figure 2 shows a specimen just reaching maturity, which has 6 eggs in the
uterus. It is somewhat flattened as a result of pressure during fixation. Measure-
ments in millimeters are: length, 2.00; width, 0.80; acetabulum, 0.34x0.37;
oral sucker, 0.18x0.22; pharynx, 0.125x0.150; ovary, 0.15x0.14; anterior
testis, 0.17 X 0.15; posterior testis, same size.
Figure 1 shows a smaller and less mature specimen, also flattened during fixa-
tion. The acetabulum is almost exactly in the middle of the body ; the post-acetabular
region increases relatively in size with the development of the reproductive organs.
Measurements are: length, 1.2; width, 0.56; acetabulum, 0.21 X 0.275; oral sucker,
0.128 X 0.15 ; pharynx, 0.125 X 0.125 ; ovary, 0.057 ; anterior testis, 0.079 < 0.072 ;
posterior testis, 0.092 X 0.079.
Sporocysts and Cercariae. (Figs. 5, 6, 7, 8)
Descriptions of the sporocysts and cercariae were given by Uzmann (1953).
His observations have been confirmed and additional data are presented. There
are at least three generations in the mollusk. Figure 5 shows a sporocyst with two
PLATE II
FIGURE 5. P. inacit/atits, mother sporocyst with daughter sporocysts containing germinal
cells of the next generation ; length 0.34 mm.
FIGURE 6. P. macidatns, daughter sporocyst with developing cercariae; length 0.54 mm.
FIGURE 7. P. macitlatus, large daughter sporocyst with developing cercariae, Cercaria
milfordcnsis, length 1.18 mm.
FIGURE 8. P. maculatus, cercaria, from stained and mounted specimen, excretory system
added from sketches of living worms ; length 0.26 mm.
FIGURE 9. Cercaria adranoccrca n. sp., daughter sporocyst from G. gemma; length 0.48
mm. This drawing is at the same magnification as Fig. 7.
FIGURE 10. Cercaria adranocerca n. sp., stained and mounted specimen, excretory system
.added from sketches of living worms ; length 0.21 mm.
190 HORACE W. STUNKARD AND JOSEPH R. UZMANN
daughter sporocysts and in each of them there are heavily staining germinal cells of
the next generation. The cercariae are subcylindrical and taper toward anterior
and posterior ends. In addition to the three pairs of cephalic gland ducts reported
by Uzmann, two additional pairs are sometimes visible ; the cell bodies are lateral,
in the preacetabular area, but so far it has been impossible to demonstrate them with
certainty, either by the use of vital dyes or in permanent preparations. The flame-
cell pattern has been worked out and is shown in Figure 8. On either side a duct,
which contains patches of cilia, emerges from the excretory vesicle near its anterior
end ; it passes forward, loops backward and here receives the two collecting ducts.
The anterior one of these ducts receives the fluid from four flame-cells and capillaries
located in the anterior quadrant of the body; the posterior one from flame-cells
and capillaries in the posterior quadrant. The flame-cell formula is 2\(2 + 2) +•
(2 + 2)].
Cercaria adranocerca n. sp. (Figs. 9, 10)
In addition to the specimens from M. edulis, described above, dissection of some
three hundred Gemma gemma from the region of Boothbay Harbor, Maine, in Au-
gust and September 1957, disclosed two infections by sporocysts and microcercous
cercariae, similar to those from M. edulis and from Brachidontcs rccuruus. The
sporocysts were relatively few, oval to sausage-shaped ; the largest one measured
0.42 mm. long and 0.11 mm. wide after fixation while smaller ones, no larger than
a cercaria, contained a few germ-balls. The end that bears the birth-pore may be
extended as a long, tapering protrusion.
The cercariae were studied alive, unstained and after staining lightly with Nile
blue sulphate and with neutral red ; also after fixation as stained and cleared per-
manent mounts. Frequently, one adhered to the slide by the posterior end and
extended the body in all directions. The body is subcylindrical, typically more
rounded anteriorly than posteriorly. When extended, contraction of the circular
muscles may produce an annulate appearance. The body wall is relatively strong
for so small a larva. The cuticula bears rows of closely set, flattened spines. Ducts
of cephalic glands were sometimes visible in the region of the oral sucker, but their
number and the location of the cell bodies were not determined. Alive, cercariae
measured from 0.16 to 0.33 mm. in length and 0.044 to 0.09 mm. in width. The
tail is terminal, spherical, and measures 0.01 mm. in diameter; it is easily detached.
The oral opening is subterminal, the sucker is 0.032 to 0.043 mm. in diameter.
When the anterior end of the body is extended, the prepharynx is about one-half
the length of the pharynx which measures 0.014 to 0.018 mm. in diameter. The
ceca are relatively long, extending about midway between the acetabulum and the
posterior end of the body. The acetabulum is situated in the posterior portion of
the anterior half of the body and protrudes, although it is not stalked. It measures
0.025 to 0.036 mm. in diameter: the ratio in size between the oral and ventral
suckers is about 4:3; although the size increased greatly over the figures given
above when the cercaria was subjected to extreme pressure for the study of the
excretory system. The region between the excretory vesicle and the acetabulum
contains cells which stain deeply ; they are the rudiments of the reproductive organs.
The excretory pore is terminal, at the base of the tail ; the vesicle is oval and may
extend forward more than half the distance to the acetabulum. On either side, from
the anterior end of the vesicle, a collecting duct passes forward to the level of the
LIFE-CYCLE OF PROCTOECES 191
bifurcation of the digestive tract where it turns backward ; the recurrent portion
contains tufts of cilia and near the level of the acetabulum it receives anterior and
posterior collecting tubules. The arrangement is portrayed in the figure and the
flame-cell formula is 2 [(2 + 2) + (2 + 2)].
In flame-cell formula and vestigial tail, this species is similar to Cercaria mil-
ford ensis and Cercaria brachidontis. In form of the excretory vesicle and presence
of cuticular spines it more closely resembles C. brachidontis and although neither
can be included in the genus Proctoeces, it is probable that they are larvae of some
member of the family Fellodistomidae. The species, described as ne\v in this paper,
is designated Cercaria adranocerca (adrano, inactive, feeble).
Type and paratype specimens are deposited in the U. S. Nat. Museum. Helminth.
Collection, No. 56236.
DISCUSSION
The discover}'- of unencysted metacercariae and of developmental stages from
cercariae to gravid adults demonstrates that C. miljordens'is is the larval stage of
a species of Proctoeces. However, the progenetic worms are often not entirely
normal. In some of the specimens (Fig. 3). the eggs are misshapen and of varying
sizes, often not more than one-third as large as in other individuals. A similar
situation was reported by Freeman and Llewellyn (1958). It appears that the
female organs, especially the vitellaria, may be deficient or that the ova are not
fertilized, and such abnormal eggs do not contain miracidial larvae. For this reason,
the extent and development of the vitelline follicles may not provide sound data for
specific criteria.
Identification of these specimens presents disturbing problems. The descrip-
tion of P. maculatus by Looss ( 1901 ) is based on the largest of his specimens and
is illustrated by a good figure. The characterization of P. crythraeus was very
inadequate ; Odhner gave no figure or measurements and the species was distin-
guished from P. maculatus because in the single mature specimen the acetabulum
was one-third smaller, the eggs were smaller, and the vitellaria did not extend as
far posteriad, a condition which might be expected in a specimen just reaching
maturity. For this reason, Dawes (1946) suppressed P. er\thraeus as a synonym
of P. maculatHs. The six specimens taken by him from Calamus spp. at Tortugas
agreed with Odhner 's account and Manter (1947) recognized P. erythraeus as a
valid species, but there was no figure and as yet there is no complete description
of P. crythraeus. In the (1947) paper. Manter stated that his (1940) listing of
Proctoeces and Tergestia in the family Monorchidae was an error, since the family
name, Fellodistomatidae, was accidentally omitted.
In a report on parasites of Bermuda fishes, Linton (1907) published the descrip-
tion of a new species, Distomum subtenue, from Calamus calamus. Smaller, im-
mature specimens were found in other hosts, two in Iridio bivittatus, and one each
in Harpe rufa and Lachnolaimus ma.rimus. Although two small specimens are
reported on p. 106 from H '. rufa, the table on p. 87 shows that only one trematode
was found in this host. In the "Food notes" on the fishes, which accompanied his
account of their parasites, Linton stated that C. calamus feeds on mussels and crabs ;
the others on mollusks, crabs, sea urchins and annelids. All are bottom feeders
and Breder (1929), in describing these fishes, stated that the mouths of porgies
(C. calamus is the saucer-eye porgy) are (p. 180), "armed with strong jaw teeth,"'
192 HORACE W. STUNKARD AND JOSEPH R. UZMANN
and that the memhers of the Labridae are (p. 202), "usually provided with strong
canine teeth. . . . These fishes are provided with powerful pharyngeal teeth with
which they crush mollusks."
Comparison of Linton's description and figure of Distomum subtcnue with the
two specimens from Calamus sp. taken at Bermuda by Barker and the six specimens
taken from Calamus spp. at Tortugas by Manter, led Hanson to the conviction that
all were conspecific and accordingly she (1950) announced the specific identity of
Dist. subtcnue Linton, 1907 and P. erythracus Odhner, 1911. The species was
designated Proctocccs subtcnuc (Linton, 1907). Again, there was only a scanty
description and no figure. Manter (1954) identified five specimens from Latridop-
sis ciliaris, taken near Wellington, New Zealand, as Proctocccs subtenuc (Linton,
1907) Hanson, 1950. and listed the soecies from the Red Sea, Bermuda, Tortugas,
and New Zealand. If this determination is correct, the parasite is widely distributed
and infects different kinds of fish. The latter point is probably not significant since
the worms are progenetic and voting mature specimens could be taken from the
digestive tract of any fish which had recently ingested an infected host-mussel.
Dollfus fin Fujita. 1925) was undoubtedly correct in the prediction that mollusk-
eating fishes would be found to harbor the adult stage of Proctoeces ostreac, the
unencysted metacercaria discovered by Fujita. Since members of the genus
Proctoeces develop and may actually mature in bivalve mollusks, it seems certain
that fishes may acquire the infection by eating these mollusks, although another
method is of course not precluded.
Linton's (1907) description of P. subtcnuis is accompanied by a figure and al-
though done over fifty years ago it was, until the paper by Freeman and Llewellyn
(1958), the most complete account of the species available. The length and width
of the specimens and the sizes of the oral and acetabular suckers as given by Linton
are actually greater than the corresponding measurements given by Looss (1901)
for P. maculatus. Although P. sitbtemds may be specifically distinct, there is at
present no adequate basis for distinguishing between it and P. maculatus. The
progenetic specimens described in the present paper are almost certainly identical
with those described by Linton, and until they can be distinguished from P.
maculatus, should be assigned to that species.
Proctoeces is clearly a member of the family Fellodistomidae, the name of which
was confirmed in a letter by the late Charles W. Stiles and published in Stunkard
and Nigrelli (1930). Cable (1953) recognized four subfamilies: Fellodistominae
Nicoll, 1909; Gymnophallinae Odhner, 1905; Haplocladinae Odhner, 1911; and
Tandanicolinae Johnston, 1927. Dollfus (1947), however, had maintained that
Monascus Looss. 1907 has priority over Haplocladus Odhner, 1911 and that the
correct name of the subfamily is Monascinae. Finally Freeman and Llewellyn
(1958) pointed out that the excretory vesicle in members of the genus Proctocccs,
which has an epithelial lining, controverts the thesis of La Rue (1957) that in the
Anepitheliocystidia, in which the family Fellodistomidae is included, the definitive
bladder is not epithelial.
ABSTRACT-SUMMARY
Sexually mature worms from Mytilus cditlis, taken in Connecticut and Massa-
chusetts, are identified as Proctoeces maculatus (Looss, 1901). The specimens are
often sterile, which reflects the abnormal conditions of development in the molluscan
LIFE-CYCLE OF PROCTOECES 193
host. Similar worms were reported by Freeman and Llewellyn (1958) from
Scrobicularia plana taken in the Thames estuary, England, and identified as Proc-
toeccs subtenuis (Linton, 1907), but we regard P. subtenuis as identical with P.
inaculatits. Evidence is presented to show that Cercaria milfordcnsis Uzmann,
1953 is the larval stage of P. maculatns. The taxonomy of the species is discussed.
Cercaria adranoccrca n. sp. is described from Gemma gemma taken at Boothbay
Harbor, Maine. It is not congeneric with P. maculatns, but is referred tentatively
to the family Fellodistomidae.
LITERATURE CITED
BREDER, C. M., JR., 1929. Field Book of Marine Fishes of the Atlantic Coast from Labrador
to Texas. G. P. Putnam's Sons, New York and London.
CABLE, R. M., 1953. The life-cycle of Parvatrema borinquehae gen. et sp. nov. ( Trematoda :
Digenea) and the systematic position of the subfamily Gymnophallinae. /. Parasitol.,
39: 408-421.
DAWES, B., 1946. The Trematoda. Cambridge Univ. Press.
DOLLFUS, R. P., 1947. Sur Monascns filiformis (Rudolphi, 1819) A. Looss, 1907, trematode
de 1'intestin de Ccpola ntbcsccns (L.) en Mediterranee. Ann. Parasitol.. 22: 319-323.
FREEMAN, R. F. H., AND J. LLEWELLYN, 1958. An adult digenetic trematode from an inverte-
brate host: Proctocccs subtenuis (Linton) from the lamellibranch Scrobicularia plana
(da Costa). /. Marine Biol. Assoc., 37: 435-457.
FUJITA, T., 1925. Etudes sur les parasites de 1'huitre comestible du Japon Ostrca gigas Thun-
berg. Ann. Parasitol., 3: 37-59. Translation by R. Ph. Dollfus.
HANSON, MARY L., 1950. Some digenetic trematodes of marine fishes of Bermuda. Proc.
Helm. Soc. ll'ashington, 17: 74-89.
HOPKINS, S. H., 1954. Cercaria brachidontis n. sp. from the hooked mussel in Louisiana.
/. Parasitol., 40: 29-31.
LA RUE, G. R., 1957. The classification of digenetic Trematoda: a review and new system.
Exp. Parasitol., 6: 306-349.
LINTON, E., 1907. Notes on parasites of Bermuda fishes. Proc. U. S. Nat. Museum, 33 :
85-126.
Looss, A., 1901. Ueber einige Distomen der Labriden des Triester Hafens. Zentrbl. Bakt.,
29: 402-404.
MANTER, H. W., 1940. Digenetic trematodes of fishes from the Galapagos Islands and the
neighboring Pacific. Rep. Coll. A. Hancock Pacific E.rped., 1932-1938, 2(16) : 531-547.
MANTER, H. W., 1947. The digenetic trematodes of marine fishes of Tortugas, Florida.
Amcr. Midi. Naturalist, 38: 257-416.
MANTER, H. W., 1954. Some digenetic trematodes from fishes of New Zealand. Trans. Rov*
Soc. N. Z.. 82: 475-568.
NICOLL, W., 1915. The trematode parasites of North Queensland. Parasitol., 8: 22-41.
ODHNER, T., 1911. Zum naturlichen System der digenen Trematoden. III. Steringophoridae.
Zoo/. Ans., 38: 97-117.
STUNKARD, H. W., 1957. Intraspecific variation in parasitic flatworms. S\stem. Zool., 6 :
7-18.
STUNKARD, H. W., AND R. F. NIGRELLI, 1930. On Distomum vibe.v Linton, with special refer-
ence to its systematic position. Biol. Bull., 58 : 336-343.
UZMANN, J. R., 1953. Cercaria milfordcnsis nov. sp., a microcercous trematode larva from a
marine bivalve, M\tilus cdulis L., with special reference to its effect on the host. J.
Parasitol., 39: 445-451.
WINTER, H. A., 1954. Proctocccs inacroi'itcllus n. sp. de un pez embiotocido del Oceano
Pacifico del Norte (Tremat, Fellodistom.) . Ciencia, Mex., 14: 140-142.
YAMAGUTI, S., 1934. Studies on the helminth fauna of Japan. Part 2. Trematodes of fishes.
I. Jap. J. Zool., 5: 249-521.
YAMAGUTI, S., 1938. Studies on the helminth fauna of Japan. Part 21. Trematodes of fishes,
IV. Kyoto, Japan : pp. 1-139.
YAMAGUTI, S., 1953. Systema Helminthum. Part I. Digenetic trematodes of fishes. Tokyo,
Japan : pp. 1-405.
Vol. 116, No. 2 April, 1959
THE
BIOLOGICAL BULLETIN
PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY
TWO NEW GENERA OF DINOFLAGELLATES FROM CALIFORNIA1
ENRIQUE BALECH -
Scripps Institution of Oceanography. University of California, La Jolla, California
The coastal waters in the San Diego region support fairly abundant popula-
tions of phytoplankton. Armored dinoflagellates of this region were studied
extensively by Kofoid and his associates (1907-1933), but there are still numerous
undescribed or little known representatives especially among the smaller species.
In the present paper, two new genera and species are described. These were
originally isolated by Dr. Beatrice M. Sweeney in 1956-57 from coastal water at
La Jolla, Calif., and have since been maintained as laboratory cultures.
Acknowledgment is here made to Prof. Francis T. Haxo and Prof. Martin
\V. Johnson for their interest and for providing research facilities. The author
is indebted to Dr. Beatrice M. Sweeney whose cultures made this study possible,
to Mrs. Anne Dodson for valued technical aid and to Dr. K. A. Clendenning for
assistance in the preparation of the manuscript.
METHODS
The dinoflagellates were first examined alive. Fixed material was then studied
under an oil immersion objective and by phase contrast. To derive the general
plate formulae of the thecae, an individual cell was isolated under a cover-glass.
A drop of concentrated sodium hypochlorite solution was then passed slowly under
the cover-glass to destroy the protoplasm and to remove the cement which unites
the plates. This process was assisted by applying very gentle pressure to the
cover-glass, but great caution was necessary because of the fragility of the speci-
mens. With Scrippsiella, it proved helpful to store droplets of the cultures in a
wet chamber for a few hours. Under these conditions many of the cells shed
their thecae, to which the hypochlorite treatment was then applied. After testing
other methods, the following technique was adopted for the examination of
Fragilidiiim. Actively swimming individuals were killed by transferring them
into 5% formaldehyde with a micropipette. Individual specimens were next
isolated, and by applying gentle pressure to the cover-slip, the protoplasm was
1 Contribution from the Scripps Institution of Oceanography, New Series.
2 Permanent address : Casilla de Correo 64, Necochea, Argentina. This work was conducted
during the tenure of a John Simon Guggenheim Fellowship, 1957-58, at the Scripps Institute
of Oceanography, University of California, La Jolla.
195
Copyright © 1959, by the Marine Biological Laboratory
196
ENRIQUE BALECH
Scrippsiella sweencyi n. gen., n. sp.
a
FIGURE 1. Scrippsiella sivecneyi. a) A typical individual, ventral view, b) Ventral view
of the epitheca. c) Dorsal view of the hypotheca. d) Apical view of the epitheca. e) Antapical
view of the hypotheca. f) Sulcal region (S.a. : Anterior sulcal. — S.i. : left sulcal. — S.d. : right
sulcal. — S.p. : posterior sulcal). All figures about X 1500.
TWO NEW DINOFLAGELLATES
197
forced out of the theca through the cingular region. The hypochlorite treatment
was then applied to the empty Fragilidium theca, especially in studies of the sulcus
and cingulum.
Diagnosis. Small-sized, conical epitheca, rounded hypotheca, without horns.
Cingulum wide, cavazone, descendent, with displacement equal to two-thirds of its
width, without lists. The cingulum has six plates, five equal, preceded at the left
by a transitional one. Sulcus deep, of medium width, slightly curved to the right.
a
FIGURE 2. Scrippsiella siveeneyi, atypical plate patterns, a and b) Two epithecae,
apical view, c) Antapical view of a hypotheca. All figures about X 1500.
The sulcus has four plates, with the posterior plate largest. The pattern of the
major body plates is the same as that of an Orthoperidinium with three inter-
calaries. Cell length, 24—32.5 ^; transdiameter, 19-24 /t, chromoplasts numerous,
elliptical, generally brown-yellow. La Jolla, California.
Description. This organism resembles Peridinium trochoideum in its general
shape and size, and to some degree in its plate formula: 4', 3a, 7", 6c, 5" ', 2" ",
and 4s. Its epitheca is high and conical, most individuals deviating from a
198 ENRIQUE BALECH
rectilinear outline by a concavity near the apex, as shown in Figure la. The
hypotheca is almost hemispherical, and slightly shorter in length than the epitheca.
In the region of the girdle, there is a slight dorsiventral compression. In apical
view, the cells normally appear almost circular. The sulcus indents slightly into the
epitheca, is very deep, and of medium width. It does not reach the antapex when
in true frontal view.
The plate pattern of the major body plates is the same as that of an Ortho-
peridinium with three intercalaries. In the epitheca, the first apical plate (!')
is very narrow, with an asymmetrical rhombic shape and upwardly curved base.
Attached to its anterior end, there is an extremely narrow ventral apical plate.
The apex of the theca is horizontal, and is closed by a circular plate (apical pore
platelet) which indents the pentagonally shaped third apical plate (3'). Plates
2' and 4' are comparatively large, and generally 2' is a little wider than 4'. There
are three dorsal intercalaries. Plate 2a is usually pentagonal but is sometimes
hexagonal.
In the hypotheca, there are five post-cingulars and two antapicals. Plates 1"
and 5" ' are wide, and 3" ' is very asymmetrical ; its border with 2" ' is very long
in comparison with its border with 1" ". The two antapicals have a very restricted
connection with the end of the sulcus.
The cingulum has five plates of similar size, plus a transitional plate at the left
end which is somewhat different in shape and also a little higher than the other
cingular plates.
The sulcus of dinoflagellates is not easily examined, and has been neglected by
most protistologists for that reason. The sulcus of Scrippsiella sweeneyi is
exceptionally difficult to analyze, being about as difficult to study as that of
Hetcrocapsa triquetra. The anterior sulcal plate (S.a.) is narrow and a little
curved. It borders 7". Posterior to this plate are two smaller plates (S.i. and
S.d.). The shorter and broader of these two is the left plate (S.i.), which
extends very slightly beyond the distal end of the girdle. The right border of this
plate (S.i.) is thickened and ref ringent ; it is provided with poroids and at the
extreme anterior end there are two closely spaced pores. The right sulcal plate
(S.d.) narrows toward the posterior. The posterior plate (S.p.) is the largest,
forming the greatest part of the sulcus. Its right anterior border is strongly
oblique to the axis of the plate and articulates with S.i. The posterior right
border of S.p. is thickened.
The nucleus is round and located at the girdle level. Its diameter is about
one-third of the total cell length. The chromatin strands are less evident than in
most dinoflagellates. The chromoplasts are elliptical and numerous, sometimes
yellow-green but normally brown-yellow. Food is apparently stored as small
granules and also around the chromoplasts in bodies that resemble pyrenoids.
There is no pusule nor stigma.
The first external evidence of cell division is the formation of two discrete
longitudinal flagella with separate points of attachment. During division, the
cell escapes from the theca but retains a tough cellular membrane. The two
daughter cells remain attached to each other in an oblique plane. The posterior
cell is usually the smallest.
Locomotion is normally rapid, with a strongly rotatory motion. There is usually
TWO NEW DINOFLAGELLATES 199
one complete rotation of the organism during an advancement of one or two cell
lengths. Sometimes, when 5. sweeneyi cells reach the border of a drop, they
suddenly cast off their flagella. Generally they lose the transverse ribbon-like
flagellum first, which continues to beat in the detached state for a few seconds and
then vacuolizes. The longitudinal flagellum is about three times as long as the
cell ; it does not beat or vacuolize after detachment.
Occurrence. This organism was originally isolated on March 15, 1956, from
water collected off the S.I.O. pier at La Jolla, California, and has since been
observed frequently in locally collected water samples. It seems to be a year-round
inhabitant of the San Diego region, thriving especially in the summer months.
This species has also been observed in plankton net samples, its relative scarcity
in these being caused by its small size and poor retention on plankton silk.
Variations. The cingular and sulcal formula has been constant in laboratory
and field specimens : 6C and 4S. The cells varied in size, and in the laboratory
clonal culture used in the description, the cells also varied in shape and in plate
pattern. Deformed or aberrant forms of 5". szveeneyi were numerous in old
laboratory cultures, but these were not observed in plankton samples. The plate
formulae of the thecae from plankton samples were established in only a few cases,
so we do not yet know how much the plate formula of this organism varies in
nature. On the whole, the plate pattern has shown an amazing range of variation.
The normal plate formula is as stated above : 4', 3a, 7", 6c, 5" ', 2" ", and 4s.
It is generally assumed that the hypotheca is more conservative than the epitheca,
and this is true of the present organism. Deviations from the normal epithecal
plate configuration were observed in about 10 per cent of the specimens examined.
The range of variation encountered in the atypical specimens of 5\ szveeneyi was
rather exceptional for dinoflagellates, although similar variations occur in Pyro-
phacus Jwrologiciiiu. Plate formulae in these atypical specimens were:
(1) 4', 2a, 6";
(2) 4', 3a. 5";
(3) 4', 3a, 4";
A single specimen with 3', 3a and 5" showed an exceptional overgrowth of 1", which
reached the apical pore thus transforming 2' into la.
No alteration of hypothecal formula has been noticed in actively growing
cultures. In old cultures, I have observed hypothecal formulae of : 5" '. 2" ' and
one intercalary ; 4" ', 2" " ; and 3" ', 2" " and one intercalary. However, the plate
variation of the hypotheca has not exceeded two per cent in all examined specimens.
Discussion. The general characteristics of this organism place it in the
Peridiniaccae. If it were classified solely on the basis of its major body plates,
it would be included in the genus Peridinium. The cingular formula and sulcus
plates are characteristically different, however, from those of Peridinium. The
cingular and sulcal plates are conservative and important structural features con-
nected with the most dynamic parts of the cell. Undoubtedly this organism
belongs to a new genus. The species is also new. The only other known species
which bear general resemblances to the present organism are Peridinium subsalsum
Ost., and especially P. trochoideum (Stein) Lemm. Laboratory cultures of these
species \vere provided by Dr. Sweeney for comparative studies. Their assignment
200
ENRIQUE BALECH
to the genus Peridinium was clearly correct, but they bore only superficial re-
semblances to Scrippsiella sweeneyi. The genus is named after the institution
at which it was discovered, and the species is dedicated to Dr. Beatrice M. Sweeney
who made the original isolation and whose cultures made this study possible.
Fragilidium heterolobum n. gen. n. sp.
G
FIGURE 3. Fragilidium heterolobum. a) A typical individual, ventral view, b) Apical
view of the epitheca. c) Sulcal plates, d) Antapical view of the hypotheca. All figures
about X 1000.
Diagnosis. Medium-sized, roughly roundish pentagonal in ventral view.
Epitheca dome-shaped ; hypotheca asymmetrically bipedal, the left lobe being the
largest. Cingulum deeply impressed, subcentral, descending, displaced distally
about one girdle width, without lists. The cingulum has eleven sub-equal rec-
tangular plates plus a transitional plate at the right end. Sulcus narrow, only
slightly excavated, with six plates. Theca easily exuviated. Cell length 53-56 ^,
TWO NEW DINOFLAGELLATES 201
transdiameter 48-54 ^. Chromoplasts numerous, elliptical, brown. Genus char-
acterized by the high number of precingular, postcingular and cingular plates.
La Jolla, California.
Description
Plate pattern. The epithecal formula is 4', 9" and a "pore platelet." The
plate 1' is in general large and it has the most irregular form. It has connections
with seven plates: 1", 2", the pore platelet, 4', 8" and 9"; its border for 2" is
the smallest. Plate 1' is asymmetrically located, most of it being on the right
side of the epitheca; its width decreases gradually to the left. The other three
apicals are more regular. Apical 2' has six edges (for 1', 3', the pore platelet,
2", 3", 4")- The plate 3' touches 2', 4', 4", 5", 6" and the pore platelet. The
apical 4' touches 3', 1', 6", 7", 8" and the pore platelet.
The so-called pore platelet is relatively large, oval sigmoid, and placed obliquely,
to the median plane, i.e., the plane which passes through the sulcus, the joint of 1"
and 9", and the apex. This plate is variable but generally it has a convex left
side subdivided into two edges for 2' and 3', a concave side which touches 1', a
major pole for 4', and a minor one for the suture between 1' and 2'. The most
characteristic feature of this plate is a long and narrow reinforcement at the middle
of the plate, sigmoid, with a dorsal hook to the right ; it is variable sometimes
double. Along it there are sometimes a few very small pores.
The most characteristic precingulars are 1" and 9". The first, trapezoidal in
shape, is the smallest. The precingular 9", pentagonal, has two edges at the left :
the superior one for 1' and the posterior, reinforced, forms a part of the right
border of the sulcus. The precingulars 3", 5", and 7" are more or less quadrangular.
The hypothecal formula is 7" ', 2" " and Ip. The narrowest postcingular plates
are V" and 7"'. The latter is the smaller and is somewhat displaced posteriorly.
The antapicals are very asymmetrical, the left one being much longer. The
suture between 1" ' and 2" ' is irregular. Antapical 2"" contacts the sulcus more
than 1" ". which just barely touches it. The intercalary (p) is a large irregular
plate, bordered by the antapicals, 3" ', 4" ' and 5" '.
All plates of the epi- and hypotheca are smooth. Some spots of different
optical densities could be seen in a few specimens, especially in plate 1", with oil
immersion and phase contrast. There are sometimes pores located on the cingular
border of this plate.
The cingulum is formed by eleven subequal retangular plates, plus another
different plate at its right end. This C12 is curved, irregular, extending somewhat
into the sulcus, with a narrow left-posterior or sulcal end. For that reason this
plate could be named "transitional." The cingular plates lack sculptures.
The sulcus is narrow, has six plates, and is only slightly excavated. The
anterior sulcal plate has a very characteristic "boomerang" shape, with a posterior
concavity and a longer and narrower right arm. In its sinus there are two very tiny
platelets; the right one is the smaller. Behind the anterior plate and in contact
with 1" ' there is a long plate, with a little sinus at the middle of its right border,
where C12 ends. In connection with the latter, there is another small plate.
Finally, there is a posterior sulcal plate.
Protoplasm. The protoplasm is surrounded by a strong membrane and
contains more than one hundred elongate-elliptical chromatophores which are dark
202 ENRIQUE BALECH
yellowish-brown. Food is stored as numerous granules of variable shape, which
are generally small and located most abundantly in the peripheral layer.
The nucleus is large and compact, and is surrounded by a strong membrane. It
is elongated in the equatorial plane, is somewhat curved, and has very dense thin
threads of granular chromatin more or less perpendicular to the major axis of
the nucleus. At the concavity, I sometimes observed large masses that were not
distinctly granular.
The longitudinal flagellum extends beyond the antapex about two and a half
cell lengths ; it has a fast vibratory movement of short amplitude. The transverse
flagellum, very slightly flattened, is long and completely encircles the girdle. The
organism swims with a predominantly rotating motion.
Dimensions (in fixed and slightly distorted cells). Length 53-56 //,; trans-
diameter 48-54 ju,. In an individual with a length of 55.5 /A, the epitheca was
27.5 ju, and the hypotheca was 24 p in length.
Variations. I have observed some variation in form (cell length more or
less short in comparison with the transdiameter) and also in the plate pattern.
Sometimes 4" appears divided into two plates; thus the postcingular series some-
times has eight instead of seven plates. Occasionally there are eight instead of
nine precingulars, and in one individual, a very narrow 1' was observed fused with
the pore platelet.
The normal formula is: 4', 9". a pore platelet, 12c, 7'", 2"", Ip and 6s.
Discussion
The only difficulty encountered in the tabulation of this organism was the
rapidity with which it exuviated its plates. Most of the individuals were found in
a quiet state, short ellipsoidal in form, and without theca. The actively swimming
cells were of course difficult to measure and draw. Any attempt to stop them for
a moment led to cell deformation and ecdysis. This is accomplished in a very
peculiar way : all the plates separate from each other, but in general, they remain
surrounding the cell at a short distance, forming a regular assemblage. The plates
are very delicate.
The plate pattern of this species is fundamentally different from all of those
previously known (Balech, 1956; Biecheler, 1952; Dangeard, 1927; Graham, 1942;
Kofoid, 1907-33; Lindemann, 1928; Schiller, 1933, 1937). The differences are
in both the epi- and hypotheca. Since very little is known regarding the cingulum
and sulcus of most dinoflagellates, we cannot discuss the differences concerning these
regions. Nevertheless, it should be pointed out that the structure of the sulcus of
this species is different from that of all sulci already studied, and no other genus is
known with such a high number of girdle plates.
Other genera without epithecal intercalaries and with four apical plates are
Diplopsalis, Dolichodiniwn, Goniodinium, Glenodiniwn, Cladopyxis, and Ceratiuin.
The two latter are very different in form, bearing strong horns or arms, and with
many differences in plate pattern. Diplopsalis as defined by Lindemann (1928) is
actually an assemblage of several genera. But even on these terms, Diplopsalis
never has more than seven precingulars, five postcingulars and it lacks the posterior
intercalary. Glenodiniitm, as defined by Schiller (1933-37), is another very
heterogeneous assemblage with a very large variation of plate patterns. None of its
TWO NEW DINOFLAGELLATES 203
species has so many pre- and postcingular plates, and they also lack posterior
intercalaries. Dolichodinium seems to have only six girdle plates and has six
precingulars and six postcingulars instead of nine and seven. Goniodimmn is
perhaps the genus most closely related to Fragilidium, but it has only six precingu-
lars and six postcingulars ; instead of one posterior intercalary it has three inter-
calaries in the hypotheca. Until the discovery of Fragilidium, Goniodinium was
the genus with the highest known number (nine) of cingular plates (this number,
however, was not stated with certainty).
The high number of precingular, postcingular and cingular plates is sufficient to
characterize this new genus. The only other genera with seven postcingulars
are Glenodiniopsis and Heterodinimn. Pyrophacus is the only genus with a
higher number of these plates.
Fragilidium heterolobum was isolated from plankton at La Jolla (San Diego.
California) on March 20. 1957.
SUMMARY
1. Two new genera and species of dinoflagellates are described. Both were
originally isolated from plankton samples collected at La Jolla (San Diego,
California).
2. Scrippsiclla sweeneyi is a small species with the general tabulation of an
Orthoperidinium, but it differs in having six cingular plates. The structure of
the sulcus is also different. A great deal of variation in plate pattern was ex-
hibited by this organism.
3. Fragilidinm hetcrolobuin is a medium-sized species having a tabulation that
is quite different from all previously described dinoflagellates. It has a very high
number of cingular plates (twelve). The generic name refers to the characteristic
frequency and suddenness with which it sheds its plates.
LITERATURE CITED
BALECH, E., 1956. Etude des Dinoflagelles du sable de Roscoff. Revue Algologiquc Nile.,
Serie2(l-2) : 29-52.
BIECHELER, B., 1952. Recherches sur les Peridinicns. Supp. an Bull. Biologiquc dc France
et dc Belgiqnc. 36 : 1-149.
DANGEARD, P., 1927. Phytoplankton de la croisiere du Sylvana. Ann. Inst. Occanog., 4 (8) :
285-407.
GRAHAM, H. W., 1942. Studies in the Morphology, Taxonomy and Ecology of the Peridiniales.
Scientific results of Cruise VII of the Carnegie during 1928-29. Carnegie Inst. of
Washington Publication 542.
KOFOID, C. A., 1907. New species of dinoflasellates. Bull. Mus. Comp. Zool. Harvard, 50 :
163-207.
KOFOID, C. A., 1911. Dinoflagellata of the San Diego region. V. On Spiraulax, a new genus
of the Peridineae. Univ. Calif. Pub. Zool, 8 : 295-300.
KOFOID, C. A., AXD A. M. ADAMSON, 1933. The Dinoflagellata: the family Heterodiniidae
of the Peridinioidiae. Mem. 3fns. Comp. Zool. Harvard, 54 (1), 136 pp.
KOFOID, C. A., AXD J. R. MICHEXER, 1911. New genera and species of Dinoflagellates. Bull.
Mus. Comp. Zool. Harvard, 54: 265-302.
LINDEMANN, E., 1928. Pcridiuialcs. In: Engler and Prantl, Die Pflanzenfamilien. 2nd Ed.,
Vol. 2, 104 pp.
SHILLER, J., 1933, 1937. Dinoflagcllatae (Peridineae). In: L. Rabenhorst's Kryptogamenflora.
Vol. 10, Leipzig.
PHOTORECEPTION IN THE OPOSSUM SHRIMP, MYSIS
RELICTA LOVEN x
ALFRED M. BEETON 2
Department of Zoology, University of Michigan, Ann Arbor, Michigan
Little information is available on the physiology of photoreception in the
Mysidacea other than a few studies of phototaxis. Practically nothing is known
of their spectral sensitivity, dark-adaptation, or lower limits of vision. The two
observations that have been made on the response of the opossum shrimp, Mysis
relic ta Loven, have shown that they swim downward when subjected to light
(Dakin and Latarche, 1913) and are especially sensitive to a combination of high
temperature and bright light (Larkin, 1948). The present study of the spectral
sensitivity, dark-adaptation, and phototaxis of M. relicta was undertaken to add to
our information on the physiology of photoreception.
MATERIALS AND METHODS
Mysis relicta is ideal for a study of this nature, since its large size (average
length, 15.0 mm.) facilitates observation, and a laboratory population can be
easily maintained. The animals used in this study were collected in Lakes Huron
and Michigan.
All experiments were conducted in a cold-room under controlled light con-
ditions and at a constant temperature of 10° C. The dark-adaptation and special
sensitivity studies were carried out in an all-glass aquarium 12.0 by 8.0 by 3.0
inches, filled to 1 inch from the top. A 24-inch glass tube, with a 1-inch diameter,
was used in the experiments on phototaxis.
Under conditions of continuous darkness or light, the mysids normally rested
on the bottom of the tank, periodically making short excursions off the bottom.
If the experimental light was turned on when the mysids were swimming upwards,
they hesitated momentarily, turned, and swam rapidly to the bottom of the aquar-
ium. This momentary hesitation was found to be a reliable indicator for the
photic response.
The difficulty of observation in a dark room was met by using an infra-red
viewer and infra-red light source or a large Fresnel lens to focus low-intensity
red light (approximately 0.001 foot-candle) on the observer's eye. The red light
was produced by a neon glow lamp and a number 2404 Corning glass filter,
transmitting wave-lengths of 620 m//, and greater. It was permissible to use this
light for viewing, since preliminary studies had established that M. relicta was
almost completely insensitive to the red region of the visible spectrum in the 620-
1 Based on a thesis submitted in partial fulfillment of the requirements for the Ph.D. degree,
University of Michigan, 1958.
2 Present address of the author is : U. S. Department of Interior, Fish and Wildlife Service,
Ann Arbor, Michigan.
204
PHOTORECEPTION IN MYSIS RELICTA
205
450
400
450 500 550 600 650
Wave- length (mu)
700 750
FIGURE 1. Spectral distribution of a tungsten filament lamp (color temperature 2300° K) and
the spectral transmission of monochromatic filter combinations.
to 700-nijU. wave-band. Mysid behavior was the same when viewed by either of
the above methods.
A General Electric 7C7 tungsten filament lamp suspended 3 inches above the
water surface provided the light source for the spectral sensitivity studies. This
lamp has an average color temperature of approximately 2300° K and its lumen
output averages 45. Although the exact distribution curve of spectral energy was
not available for the lamp, a reasonably accurate curve was constructed by extrapola-
tion from data supplied by the Xela Park Laboratory of General Electric (Fig. 1).
400
450 500 550 600
Wave-length (
650 700 750
FIGURE 2.
Radiant energy output from a tungsten filament lamp (color temperature 2300° K)
through various monochromatic filter combinations.
206 ALFRED M. BEETON
Nine monochromatic-filter combinations were made with Corning glass color
filters and interference filters; Figure 1 gives the spectral transmission of these
filter combinations. The total energy output of a given filter combination was
calculated by multiplying the energy available (in 10-m/x-wide wave-bands) from
the lamp by the percentage transmission of the filter combination (Fig. 2). The
percentage transmission of the various filter combinations was obtained either
from data supplied by Corning (1948) or with a Beckman DU Spectrophotometer.
The range of intensities for a particular wave-band was obtained by the use of
evaporated-metal, neutral-density filters, having optical densities of 0.6, 0.9, and
1.0.
The reaction times of the mysids to the various intensities of radiant energy
were measured by a stop watch. Sufficient time was allowed between successive
tests to keep the mysids completely dark-adapted.
In the dark-adaptation experiments, the mysids were first subjected for 3
minutes to the intense light of a 1000- watt photoflood lamp. At the end of the
3-minute period all lights were turned off except the viewing light. Then the
mysids were subjected to flashes of light, approximately 0.1 second in duration,
of a given intensity. These flashes were spaced at 1- to 3-minute intervals. The
experimental light was a 6-watt, 9-foot-candle, 115-volt. tungsten-filament lamp
suspended 3 inches above the water surface. The intensity of the light was
altered by interposing various numbers of evaporated-metal, neutral-density filters,
each having an optical density of 1.0. The time in the dark, prior to first stimula-
tion, was measured by stop watch.
In the experiment on the phototactic response of M. relic ta, six individuals were
placed in a 24-inch glass tube, with a 1-inch diameter, lying horizontally to eliminate
any gravitational effects. The experimental light, a 7C7 lamp, was suspended
1 foot above the midpoint of the tube. After the mysids had been subjected for
measured intervals to total darkness or light, one-half of the tube was shaded and
the number of mysids in the unshaded half of the tube was recorded at 30-second
intervals for a 5-minute period. First the right and then the left half of the
tube was shaded to detect any bias in the distribution of the mysids. Control
runs, with neither half being shaded, were made at frequent intervals.
SPECTRAL SENSITIVITY
Earlier studies of spectral sensitivity
It has been well established that the first step in the response to light in any
animal is a photochemical reaction. Hecht's (1919, 1920, 1921) work on the
clam, Mya arenaria Verrill, contributed much toward establishing the photo-
chemical nature of photoreception. He demonstrated that the fundamental concept
of photochemistry, the Bunsen-Roscoe reciprocity law which holds that the photo-
chemical effect is equal to the product of time and intensity, could be applied to
the data of his studies.
Before a photochemical reaction can occur, light must be absorbed. The
absorption spectra of the visual pigments will therefore determine the effects on
the photoreceptor and in turn the response of the organism. The behavioral
response of an organism to different regions of the radiant-energy spectrum has
PHOTORECEPTION IN MYSIS RELICTA
207
given rise to the concept of "action spectra." For example, if a visual pigment
has its absorption peak at a wave-length of 500 111/1, the animal possessing this
visual pigment would be most sensitive to this wave-length. This concept has
led to a number of action-spectrum studies on invertebrates. Mast (1917) re-
TABLE I
Reaction time (seconds) of Mysis relicta to various wave-lengths of light at several intensities
( Unless noted otherwise the neutral-density filters transmitted 10 per cent of the light)
Wave-length
of peak
transmission
(HIM)
Number of
neutral-density
filters
Energy output of 7C7
lamp through filters
(microwatts/10 m/i/
lumens)
Average
reaction times
for 10 trials
(seconds)
Standard
deviation
395
0
2.20
0.77
0.18
395
*1
0.26
0.88
0.12
395
2
0.02
1.16
0.39
430
0
11.19
0.72
0.06
430
**1
2.80
0.84
0.06
430
*1
1.34
0.86
0.31
430
***2
0.34
0.92
0.16
430
****3
0.03
1.15
0.39
485
0
7.63
0.87
0.14
485
1
0.76
1.04
0.12
485
2
0.08
1.18
0.10
488
0
13.63
0.86
0.19
488
2
0.14
1.13
0.20
496
0
14.93
0.77
0.24
496
1
1.49
0.82
0.11
496
2
0.15
0.98
0.14
515
0
32.98
0.64
0.13
515
1
3.30
0.71
0.20
515
2
0.33
0.83
0.21
515
3
0.03
1.01
0.19
515
4
0.003
1.42
0.36
540
0
29.85
0.84
0.15
540
1
2.99
0.92
0.06
540
2
0.30
0.95
0.22
540
3
0.03
1.33
0.25
610
0
108.94
1.10
0.17
610
1
10.89
1.10
0.09
610
2
1.09
1.33
0.36
640
0
256.11
1.13
0.31
640
1
25.61
1.22
0.43
640
2
2.56
1.32
0.45
640
3
0.26
(No reaction)
* Neutral-density filter transmitting 12 per cent of the light.
** Neutral-density filter transmitting 25 per cent of the light.
*** Filters 1 and 2 combined.
* Neutral-density filter transmitting 10 per cent of the light combined with filters 1 and 2.
ported peak sensitivity to light at the following wave-lengths : 483 in/A in Euglena,
Arenicola, Trachelonwnas, and Lmnbricus ; 524 m/x in Pandorina and Eudorina;
503 nijit in Chlamydonionas, and blowfly larvae. Hecht (1921) established the
basis for future studies through his experiments on Mya arcnaria. This clam
208
ALFRED M. BEETON
had a maximum sensitivity at 490 m^u. Most insects apparently have two peaks
of maximum sensitivity, 365 m/*, and 492 niju, (Weiss, 1943).
Results obtained through the methods of these investigators are similar to
those secured by in vitro studies of the spectral absorption of squid rhodopsin
(Bliss, 1948). An electrical method has been employed to determine the spectral
sensitivity of the eyes of Limulus (Graham and Hartline, 1935), a grasshopper,
Melanoplus (Jahn, 1946), and the diving-beetle, Dytiscus (Jahn and Wulff, 1948).
These studies demonstrated, as did the behavior method of Weiss, that the
arthropod eye has a peak sensitivity in the green region of the spectrum.
100
10
en
x_
OJ
c I
o>
0
Eo.
o.oi -
0.001
0.6 0.8 1.0 1.2 1.4
Reaction time (seconds)
FIGURE 3. Reaction rate of Mysis rclicta to certain wave-lengths (m/ct) of light at various
intensities. (Each point represents the average reaction time for 10 trials.)
The only source of information on the sensitivity of mysids to various regions
of the spectrum is Hess' (1910) study of the phototactic response of a marine
mysid to certain regions of the visible spectrum. Sixty-four mysids were placed
in the dark and then subjected to a continuous spectrum of light. The mysids
swam toward the light and aggregated in certain regions of the spectrum : 40 in
the yellow-green, 19 in the blue and violet, and 5 in the red. When the position of
the spectrum was altered the mysids followed the yellow-green band.
PHOTORECEPTION IN MYSIS RELICTA
209
Spectral sensitivity in M. relicta
The dark-adapted mysids reacted to the different regions of the spectrum with
the typical photic response but the time of reaction varied with wave-length. The
mysids reacted most quickly at wave-lengths in the vicinity of 515 m/A and 395 m^t.
(Table I). The reaction times at wave-lengths of 610 m^ and 640 m/A were much
slower (Fig. 3) despite the fact that considerably more energy was available in
the red region of the spectrum than in the blue region (Fig. 2). If the mysid
eye were equally sensitive to all regions of the spectrum, the faster reaction time
0.7
0.8
en
T3
§0.9
O>
CD
E
c
o
o
<u
cr
.0
.2-
.3-
400 500 600
Wave-length (rim)
FIGURE 4. Action spectrum of the dark-adapted Mysis relicta eye.
would have been elicited by the higher energy of longer wave-lengths. The speed
of response decreased progressively as the wave-length increased from 395 niju.
to 488 m/j, (Fig. 3). Then the trend was reversed and the mysids responded
progressively faster as the wave-length increased. The reaction time at a wave-
length of 496 m/A was similar to that recorded at 430 m//, and the quickest response
was elicited by light with peak energy at 515 m/j.. The reaction to light with
wave-lengths longer than 515 m/z became progressively slower as the wave-length
increased.
The reaction times of the mysids to a wide range of intensities for a particular
210 ALFRED M. BEETON
wave-length were determined through the use of evaporated-metal, neutral-density
filters. Similar reaction times could be obtained for different wave-lengths by
altering the intensity. Approximately 1000 times more radiant energy was re-
quired in the red region than in the blue-green region of the spectrum before
equal reaction times could be obtained. The curves resulting from a plot of the
reaction time against the radiant energy available from the 7C7 lamp and the
filter combinations show that the reaction time varies inversely as the logarithm
of the light intensity (Fig. 3). It is apparent that the rate of response to dif-
ferent wave-lengths was caused by differences in the ability of the eye to absorb
energy at various wave-lengths.
The reaction times for a definite energy value of 3 as determined from the
curves in Figure 3 were plotted against the transmission peaks of the monochromatic
filter combinations to give an "equal-energy" curve. The resulting curve is the
"action spectrum" or spectral sensitivity curve for M. relicta (Fig. 4). The
mysids have a maximum sensitivity to light with a wave-length of approximately
515 m^u,; another peak of sensitivity occurs in the violet region of the spectrum at
or below 395 m^.. This suggests that the mysid eye contains at least two visual
pigments.
DARK-ADAPTATION
Earlier studies of dark-adaptation
All of the photoreceptors studied by various investigators have shown an in-
creased sensitivity to light after a period in darkness. This reaction has given rise
to the concept of dark-adaptation, a process that has been studied by several different
methods, including behavior. Studies on M . relicta have been made by a procedure
similar to a behavioral method employed by Hecht (1919) to study dark-adaptation
in Mya.
The behavior method for determining dark-adaptation has produced data that
are similar to those obtained by in vitro resynthesis of bleached rhodopsin (Chase
and Smith, 1939) and by measuring the electrical response of the eyes of Limn! us
(Hartline, 1930).
I could not find any published account of previous studies on dark-adaptation
in Mysidacea.
Dark-adaptation in M. relicta
The 3-minute exposure of the mysids to the photoflood lamp evidently caused a
bleaching of the visual pigment. The mysids did not respond to any light for at
least 30 seconds after the lamp was turned off. Response to the experimental
light after a period in darkness would indicate, therefore, that sufficient visual
pigment had been resynthesized for photoreception. The light intensity necessary
to elicit a response after a period in the dark was considered the "threshold
intensity."
The light intensity required to produce a reaction decreased with the increase
of time spent in the dark (Fig. 5). Several repetitions of this experiment gave
closely similar results. The increase in sensitivity was relatively fast during the
first 57 seconds, and then somewhat slower. The upper part of the curve probably
lies too far to the right, since the mysids did not start to swim up until at least
PHOTORECEPTION IN MYSIS RELICTA
211
30 seconds after the photoflood light was turned off ; consequently, it was im-
possible to determine their sensitivity during this period. Possibly the steeper
slope in the upper part of the curve can be attributed to a state of shock from
exposure to the intense light. The initial recovery of sensitivity of the mysid
eye, therefore, may progress at a rate other than indicated by the upper part of
the curve in Figure 5. Light intensity values of 9 X 10~2 foot-candle and below
10
1.0
CO
cz
o
o
£ I0:
en
d
O)
10'
-1 io4
i65h
10
r6
910
23456789
Time in the dark (minutes)
10
FIGURE 5. Dark-adaptation of Mysis rcllcta. Threshold intensity is plotted as ordinate
against time in the dark as abscissa. Inset is the plot of log of threshold intensity against
log of time in the dark.
and time in the dark gave a straight line when plotted on a logarithmic scale.
These curves closely resemble that for a bimolecular reaction.
Although a precise determination of the lowest limits of visual sensitivity could
not be made, a definite response to a light intensity of 10~6 foot-candle was estab-
lished. In all probability the mysids can detect even a much weaker light.
The question arises as to whether the stimulating flashes themselves have any
effect upon the dark-adaptation of the eye. Hartline (1930) believed that if
they are widely spaced in time, 3 to 5 minutes, they have little effect.
212 ALFRED M. BEETON
PHOTOTAXIS
Earlier studies on phototaxis
Photoreception is important for the normal orientation of mysids. Delage
(1887) established that the statocysts of mysids have a balancing function, but it
was subsequently demonstrated that if the statocysts are removed, the eyes make
possible normal orientation in the light (von Buddenbrock, 1914). The mysids
always kept their dorsal side oriented toward the light — a reaction termed the
"dorsal light reflex." Additional studies on three different species of mysids have
shown that orientation is governed by responses to a combination of gravitational
and light stimuli (Fraenkel, 1931; Foxon, 1940). Fraenkel suggested that light
stimulates the mysids to assume a position whereby a definite region of the statocyst
is stimulated by gravity.
The phototactic responses of mysids are varied and complex. Keeble and
Gamble (1904) reported that the phototactic sign changed from positive to nega-
tive when the mysids were moved from a white to a black background. Bauer
(1908) found that mysids remained at the bottom of an aquarium when light
came from above and were positively phototactic to a lateral light. Menke (1911)
concluded that Hemimysis lamornae was positively geotactic and negatively photo-
tactic. He also removed the statocyst and interpreted the mysids' subsequent
failure to leave the bottom as a pronounced negative phototaxis. Recent research
has shown that this behavior may be a "general position reflex" whereby mysids
utilize the tactile sense for orientation along with the "dorsal light reflex" (Foxon,
1940). Some differences in response can be related to age. Adults of Neomysis
vulgaris [N. integer Leach] are more strongly photonegative than are the young
(Lucas, 1936). Mysids have been shown to be telotactic; when two light rays are
crossed at right angles they react to one light source and ignore the other. Light-
adapted individuals also have shown a reversal in the phototactic sign by swimming
to and fro in the direction of the incident light (Fraenkel, 1931).
The observations of other workers that certain marine mysids possess a diurnal
rhythm in their chromatophore system (Keeble and Gamble, 1904) suggested the
possibility of a similar periodicity in the phototactic responses of M. relicta. Their
investigations of the chemical conditions of the mysid's tissues indicated the pos-
sibility of a metabolic periodicity. The liver and muscle tissues were alkaline in
the morning and became progressively acid during the day. Some of the experi-
mental work which Menke (1911) conducted led him to postulate that Hemimysis
lamornae was negatively phototactic during the day and positively phototactic at
night. The experiment described in the next section was devised to detect such a
periodicity in M. relicta. Although the results were not conclusive on that point,
other important findings warrant inclusion of these data in this paper.
Phototaxis in M. relicta
Whenever the control runs were conducted the distribution did not differ
significantly from random. The distribution did differ significantly (Chi Square
test) when one-half of the tube was shaded after the mysids had been in the
dark or light for a period of time. The mysids were photopositive unless they had
been subjected to total darkness for 10 hours; then they became photonegative
PHOTORECEPTION IN MYSIS RELICTA
213
(Table II). The photonegative condition persisted for only a short time as they
became light-adapted within 6 minutes. On August 15 at 11 :54 Eastern Standard
Time (EST) the mysids were definitely photonegative after being in the dark for
13 hours; after exposure to the experimental light for 6 minutes they tended to
be photopositive. On the same date at 21 :06 EST the mysids still responded
photopositively after approximately 4 hours in the dark. Similar results were
TABLE II
Phototactic response of M. relicta to a constant light intensity after periods in light and in total
darkness. {Observations were made of the distribution of 6 animals at 30-second intervals
for 5 minutes, except at 21:12 (5g minutes), 11 :55 (2\ minutes'}, 11 :58 (2\ minutes).
Numbers in parentheses indicate control run, i.e., entire tube unshaded. Asterisk
indicates significant Chi Square value at 5 per cent level. Experiments
were started at 20:54 EST on August 14, 1956, and completed
at 12:06 EST on August 18, 1957~]
Time
(EST)
Time in dark
Time in light
Number in portion of tube
Chi
Square
Hours
Minutes
Hours
Minutes
Shaded
Unshaded
20 :54
—
—
2
54
18
42
9.6*
11:54
13
5
—
—
51
9
29.4*
12:03
—
—
—
6
25
35
1.66
13:05
—
—
1
8
9
51
29.4*
13:11
—
—
1
14
17
43
11.27*
13:37
—
21
—
—
27
33
0.6
16:38
—
—
—
(30)
(30)
0.0
16:51
—
—
2
56
19
41
8.06*
21:06
4
10
—
— -
12
48
21.6*
21:12
—
—
—
6
28
38
1.51
21:17
—
—
—
—
(31)
(29)
0.066
10:19
12
57
—
—
36
24
2.4
10:25
—
—
6
43
17
11.26*
10:31
—
—
—
—
(28)
(32)
0.266
12:13
1
36
—
—
34
26
1.066
12:19
—
—
6
17
43
11.26*
12:24
—
—
—
(29)
(31)
0.066
22 :43
10
14
—
—
41
19
8.06*
22:48
—
—
—
5
35
25
1.66
21:28
3
38
—
—
26
34
1.06
21:34
—
—
—
6
21
39
5.4*
21:39
—
—
—
—
(34)
(26)
1.06
11:55
14
10
—
—
20
10
3.32
11:58
—
—
—
3
14
15
0.132
12:06
—
—
—
—
(33)
(27)
0.6
secured on August 16 at 10:19 EST and August 18 at 11:55 EST, the mysids
were photonegative after 13 and 14 hours in the dark. At 22 :43 EST on August 16
they were photonegative after 10 hours and 14 minutes in the dark. The fact that
the mysids could be photonegative either in the morning or at night indicates that
the phototactic response does not have a persistent diurnal rhythm. Existence of
the photonegative condition depends on the amount of time in the dark.
Experiments revealed also that if the light intensity was increased rapidly by
214 ALFRED M. BEETON
1 or 2 foot-candles, the mysids swam into the shaded area, although they previously
had been definitely photopositive. They adapted to this increased intensity within
2 minutes. Johnson (1938) obtained similar results with the copepod, Acartia
clausii Giesbrecht. These copepods were placed in a glass cylinder and subjected
to a step-wide change in intensity. The more rapid the change in intensity, the
greater was the response. The copepods were photopositive after a period in the
dark, but they swam away from the light when the intensity was increased.
DISCUSSION
The experiments indicate that My sis relicta has at least two visual pigments,
one with an absorption peak at 515 m/j. and another with a peak at or below 395 mju..
The maximum sensitivity at 515 m//, probably is important to the mysid for orienta-
tion in the environment, since light with a wave-band of 490 m/x to 540 m/u.
penetrates to considerable depth in Lake Huron (Beeton, 1958). The importance
of the sensitivity to violet light (395 HI/A) is not obvious, since light with a wave-
band of 300 niju, to 420 m/A does not penetrate into the deep-water habitat of the
mysids. Ultraviolet light has been found to cause negative phototaxis in Daphnia
pule.v (Moore, 1912), and D. tnagna (Baylor and Smith, 1957). In view of the
deleterious effect of ultraviolet light, it is not surprising to find that many arthropods
are sensitive to short wave-lengths and photonegative to ultraviolet light.
The mysids are able to "dark-adapt" at a relatively fast rate. This increase in
sensitivity is interpreted as a function of the amount of regenerated visual purple
available at each moment in the dark. The curve describing the progress of this
reaction resembles that for the equation for a bimolecular reaction. The literature,
however, reveals considerable disagreement as to whether the dark-adaptation data
are fitted best by the equation for a bimolecular or monomolecular reaction. The
reaction is probably bimolecular, but appears monomolecular when one of the
reactants is in excess. Some of the discrepancies in the results obtained by different
workers may possibly be explained, in part, by the data of Haig (1941). Haig's
data showed that the curve of recovery after adaptation to a low intensity is
decidedly different from that of recovery after adaptation to high intensity of
illumination. Hartline and McDonald (1947) presented a similar series of re-
covery curves for single visual elements of Limulus after exposure to light of
different intensities.
The change in the phototactic response of M. relicta is related to the prior
presence or absence of light. Time also is involved, since the mysids were photo-
positive unless they had been subjected to total darkness for 10 hours or more;
then they responded photonegatively.
Changes in the phototactic sign may be related to the activity of the organism.
The mysids were very active in the light and not as active in the dark ; possibly
the increased activity results in an acidic condition in the tissues. The acidity
would decrease with reduced activity. Daphnia, copepods, Gammarus (Loeb,
1918), and Hemimysis (Franz, 1911) can be made positively phototactic by
adding a weak acid to the water. The assumption of a metabolic change due to
differences in activity does not account for the rapid change from a photonegative
to a photopositive condition. The possibility remains that light may control a
chemical cycle in the organism. A compound, responsible for the photonegative
PHOTORECEPTION IN MYSIS RELICTA 215
•condition, may be broken down upon exposure to light. It may require a long
period of darkness for resynthesis of this compound.
Drs. David C. Chandler, Ralph Hile, and Stanford H. Smith reviewed the
manuscript. Dr. E. R. Baylor lent items of equipment and made invaluable sug-
gestions concerning the experiments.
LITERATURE CITED
BAUER, VICTOR, 1908. t)ber die reflektorische Regulierung der Schwimmbewegungen bei den
Mysiden mit besonderer Beriicksichtitung der doppelsinnigen Reizbarkeit der Augen.
Zeitschr. f. allgcm, Physiol., 8: 343-369.
BAYLOR, EDWARD R., AND FREDERICK E. SMITH, 1957. Diurnal migration of plankton crusta-
ceans. Rec. Adv. Invert. Physiol. ; University of Oregon Publication ; pp. 21-35.
BEETON, ALFRED M., 1958. Relationship of Secchi disc measurements to light penetration in
Lake Huron. Trans. Amer. Fish. Soc., 87: 179-185.
BLISS, A. F., 1948. The absorption spectra of visual purple in the squid and its bleaching
products. /. Biol. Chcm., 176: 563-569.
CHASE, AURIN M., AND EMIL L. SMITH, 1939. Regeneration of visual purple in solution. /.
Gen, Physiol, 23: 21-39.
CORNING GLASS WORKS, 1948. Glass color niters. Bulletin C-248. Corning, New York, 21 pp.
DAKIN, W. J., AXD M. LATARCHE, 1913. The plankton of Lough Neagh: a study of the seasonal
changes in the plankton by quantitative methods. Proc. Roy. Irish Acad., Scr. B,
30 : 20-96.
DELAGE, V., 1887. Sur une fonction nouvelle des otocystes commes organes d'orientation loco-
motrice. Arch. Zool. Expcr. Gen., Ser. 2, 5 : 1-26.
FOXON, G. E. H., 1940. The reaction of certain mysids to stimulation by light and gravity.
/. Mar. Biol, Assoc., 24 : 89-97.
FRAENKEL, G., 1931. Die Mechanik der Orientierung der Tiere in Raum. Biol. Rev., 6: 36-87.
FRANZ, V., 1911. Phototaktische lokomotions perioden bei Hemim\sis. Int. Rev. Hydrobiol.
(Biol. Suppl.}, 3: 1-23.
GRAHAM, C. H., AND H. K. HARTLINE, 1935. The response of single visual sense cells to lights
of different wave lengths. /. Gen. Physiol,, 18: 917-931.
HAIG, CHARLES J., 1941. The course of rod dark adaptation as influenced by the intensity and
duration of pre-adaptation to light. /. Gen. Physiol., 24: 735-751.
HARTLINE, H. KEEPER, 1930. The dark adaptation of the eye of Limuhts, as manifested by its
electric response to illumination. /. Gen. Physiol., 13 : 379-389.
HARTLINE, H. K., AND P. ROBB MCDONALD, 1947. Light and dark adaptation of single photo-
receptor elements in the eye of Limulus. J. Cell. Comp. Physiol., 30 : 225-254.
HECHT, SELIG, 1919. Sensory equilibrium and dark adaptation in Mya arenaria. J. Gen.
Physiol., 1: 545-558.
HECHT, SELIG, 1920. Intensity and the process of photoreception. /. Gen. Physiol., 2 : 337-347.
HECHT, SELIG, 1921. The relation between the wave length of light and its effect on the
photosensory process. /. Gen, Physiol., 3 : 375-390.
HESS, C., 1910. Neue Untersuchungen iiber den Lichtsinn bei wirbellosen Tieren. Pflueger's
Arch., 136: 282-367.
JAHN, T. L., 1946. The electroretinogram as a measure of wave-length sensitivity to light.
/. New York Ent. Soc., 54 : 1-8.
JAHN, T. L., AND V. J. WULFF, 1948. The spectral sensitivity of Dytiscus fascivcntris. J.
Ne^v York Ent, Soc., 56: 109-117.
JOHNSON, W. H., 1938. The effect of light on the vertical movements of Acartia clausii (Gies-
brecht). Biol. Bull., 75: 106-118.
KEEBLE, F. W., AND F. W. GAMBLE, 1904. The colour-physiology of higher crustaceans. Phil.
Trans. Roy. Soc. London, Scr. B, 196: 295-388.
LARKIN, P. A., 1948. Pontoporeia and Mysis in Athabaska, Great Bear and Great Slave Lakes.
Bull. Fish. Res. Bd. Can., No. 78, 33 pp.
216 ALFRED M. BEETON
LOEB, J., 1918. Forced Movements, Tropisms, and Animal Conduct. J. B. Lippincott, Phila-
delphia, 209 pp.
LUCAS, C. E., 1936. On certain inter-relations between phytoplankton and zooplankton under
experimental conditions. /. du Conseil, 11 : 343-362.
MAST, S. O., 1917. The relation between spectral color and stimulation in lower organisms.
/. Exp. ZooL, 22 : 471-528.
MENKE, H., 1911. Periodische Bewegungen und ihr Zusammenhang mit Licht mid Stoff-
wechsel. Pflneger's Arch., 140: 39-91.
MOORE, A. R., 1912. Concerning negative phototropism in Daphnia pulex. J. Exp. ZooL, 13 :
573-575.
VON BUDDENBROCK, W., 1914. tjber die Orientierung der Krebs in Raum. ZooL Jahrb., Abt.
Allg. ZooL, Physiol., 34 : 479-514.
WEISS, HARRY B., 1943. The group behavior of 14,000 insects to colors. Ent. News, 54:
152-156.
THE RESPIRATION OF UNFERTILIZED SEA URCHIN EGGS
IN THE PRESENCE OF ANTISERA AGAINST FERTILIZIN x
JOHN W. BROOKBANK
Department of Biology, University of Florida, Gainesville, Florida, and The Friday Harbor
Laboratories of the University of Washington, Friday Harbor, Washington -
It has been shown (Tyler and Brookbank, 1956a, 1956b) that antisera against
purified fertilizin, as well as antisera against homogenates of fertilized and un-
fertilized eggs deprived of the gelatinous coat material, increase the respiratory rate
of demembranated fertilized eggs, and cause an inhibition of cleavage. The
respiratory rate of blocked eggs increases to a maximum of 4 to 5 times the control
rate in 20 to 40 minutes, and subsequently decreases to the rate of the controls
during the next 40 minutes. Gradual cytolysis of the eggs occurs following 4-5
hours of exposure to undiluted antisera. The observed increase in the respiratory
rate in the presence of immune serum is apparently without parallel in the previous
literature. Haurowitz and Schwerin (1940) studied the respiration of pigeon
erythrocytes in the presence of immune rabbit serum and active and inactive com-
plement, yielding lysis and agglutination, respectively. No increase or decrease
in respiratory rate was noted in either case. Sevag and Miller (1948), studying
the respiration of E. typhosa (strain 0-901) in the presence of immune rabbit serum
and active or inactive complement, found that intact, sensitized cells consumed
oxygen at the same rate as the controls. However, in the presence of active
guinea pig complement, the cells lysed, with an accompanying transitory increase
(1.4-fold), which was followed by a decrease to one-fourth the control rate after
180 minutes. Harris (1948) measured the oxygen uptake of Salmonella cells
in the presence of agglutinating rabbit serum, and found no increase or decrease in
respiratory rate over a wide range of antiserum concentrations. Nowinski (1948)
investigated the possibility of an effect on respiratory rate by reticulo-endothelial-
immune-serum (REIS) acting on rat spleen slices, and by anti-chick brain serum
acting on chick brain homogenates (1949). No effect on respiration was observed
with REIS, and a slight inhibition of oxygen uptake of chick brain homogenates
was noted in the presence of anti-chick brain serum. MacDonald (1949) ob-
tained similar results with REIS using rat spleen slices in Thunberg experiments.
The purpose of the present experiments was to explore the effect of antisera
against fertilizin, which is, chemically, a rather well defined substance (Tyler, 1949,
1956), on the respiratory rate of unfertilized sea urchin eggs. Unfertilized eggs,
though normally respiring at a low rate, can be stimulated to respire at a much
1 This investigation was supported in part by a research grant (RG 4659) from the National
Institutes of Health of the United States Public Health Service. The author is also indebted
to Professor Albert Tyler for a critical reading of the original manuscript.
2 The author wishes to thank the Friday Harbor Laboratories of the University of Wash-
ington, Friday Harbor, Washington, for the use of space and equipment during the summer
•of 1957.
217
218 JOHN W. BROOKBANK
greater rate (4-5-fold increase) by parthenogenetic agents (Warburg, 1908;
Keltch and Clowes, 1947), by nitrophenols (Clowes and Krahl, 1934, 1936) and
other non-parthenogenetic substances, as well as by fertilization. In this connection,
it is noteworthy that Perlman (1954, 1957), and Perlman and Perlman (1957)
have reported that antisera against fertilizin, as well as antisera against extracts
of unfertilized eggs, are capable of activating the unfertilized eggs of Paracentrotus
lividus. The respiratory rate of eggs so treated therefore becomes of interest.
MATERIALS AND METHODS
Preparation of antigens and antisera
1) Lytecliinits variegatits (Cedar Key, Florida). Fertilizin antigens were pre-
pared from neutralized supernatant egg- water of acid- (pH 3.5) treated unfertilized
eggs. In the case of antigen number 4, the egg- water was dialyzed against distilled
water and injected without further purification. Antigen number 10 was prepared
by first precipitating the fertilizin of the egg- water (derived from a second spawn
of eggs) with 5/4 volumes of cold 95% ethanol (Tyler, 1949). The fertilizin
precipitate was then washed thoroughly with additional volumes of cold ethanol,
and vacuum-dried. The dried precipitate was dissolved in distilled water, and
used for injection. Both antigens had a final agglutination titer of approximately
1/1000 on homologous sperm.
A whole sperm antigen (number 11) was prepared from washed Lytecliinits
variegatus sperm (presumably free of seminal fluid) which were diluted to a
10% suspension by volume with sea water and frozen until used.
All above antigens were stored at -- 20° C. in one-mi, aliquots. After thawing
for use in injections and various tests, the material remaining in each individual
aliquot was discarded. This procedure avoided repeated freezing and thawing of
the antigens.
Antisera against the above antigens were prepared in rabbits according to
an immunization schedule described elsewhere (Tyler and Brookbank, 1956a).
Following a control bleeding, totals of 400 /xg N (no. 4), 200 /^g N (no. 10), and
6000 /^g N (no. 11) were injected into the animals over a period of three weeks.
The rabbits were bled by cardiac puncture 5 days following the final injection.
The antisera were recovered from the retracted clots, and dialyzed thoroughly
against sea water at 10° C., and stored at -- 20° C.
In addition, an antiserum against extract of washed, demembranated, fertilized
eggs of Lyt echinus pic t us was available, and was used in a number of experiments.
This antiserum had been previously shown to increase the respiration rate of
fertilized L. pictus eggs (Tyler and Brookbank, 1956b).3
2) Strongylocentrotus purpuratus (Friday Harbor, Washington). A single
fertilizin antigen was prepared by precipitation of the fertilizin from the egg-water
of acid- (pH 3.5) treated eggs by NaOH (in the ratio of 40 ml. 1 N NaOH per
liter of egg water; Tyler, 1949). The resulting precipitate was dissolved in sea
water, following neutralization of the alkali, and dialyzed against distilled water.
3 Due to an oversight on the part of the authors, Tyler and Brookbank (1956b) contains
an error on page 312, line 6. This line reads correctly if L. pictus is substituted for S.
purpuratus.
EFFECT OF ANTISERA ON RESPIRATION
219
TABLE I
The effect of antisera, normal sera, 0.1% sperm suspension, and hypertonic sea water
on the respiration of unfertilised Lytechinus variegatiis eggs
Experiment no.
Reagent employed
Aver, rate
(^l./min. /vessel)
Ratios of aver, rates
(increased/control)
1
No. 4 normal serum
0.28
No. 4 normal serum
0.28
Anti-no. 4
0.67
Anti-no. 4
0.67
2.4
Anti-L. pictus fertilized egg extract
0.73
9 fi
Anti-L. pictus fertilized egg extract
0.73
£tt\J
Buffered sea water
0.28
Buffered sea water
0.28
Buffered sea water
0.33
2
No. 10 normal serum
0.25
Anti-no. 11 serum
0.31
Anti-no. 10 serum
1.18]
4 1
Anti-no. 10 serum
1.13 J
T^ . 1
Buffered sea water
0.28'
3
No. 4 normal serum
0.33
No. 4 normal serum
0.28
Anti-no. 4
1.081
3c;
Anti-no. 4
1.03 J
.5
0.1% sperm suspension
1.07
3.5
Buffered sea water
0.33
Buffered sea water
0.28
Buffered sea water
0.22
4
No. 5 normal serum
0.30
No. 5 normal serum
0.22
Anti-L. pictus fertilized egg extract
0.921
^ 4.
Anti-L. pictus fertilized egg extract
0.84]
•J .^
Buffered sea water
0.27
5*
No. 11 normal serum
0.14
Anti-no. 10
0.95
6.7
Anti-5. pur pur at us fertilizin (three anti-
sera)
0.37
2.6
0.33
2.3
0.33
2.3
Buffered sea water
0.14
6
0.1% sperm suspension
0.83]
^ 9
0.1% sperm suspension
0.80]
\Jt£t
Hypertonic sea water
0.84]
7 3
Hypertonic sea water
0.84]
o.o
Buffered sea water
0.25
7
Anti-no. 10 undiluted
0.32
2.5
Anti-no. 10 1:1 dilution
0.26
2.0
Anti-no. 10 1:2 dilution
0.24
1.8
Anti-no. 10 1:4 dilution
0.20
1.5
No. 1 1 normal serum
0.16
Buffered sea water
0.10
Buffered sea water
0.12
* In experiment 5, one ml. of 20% egg suspension was used with vessels of 7-ml. capacity,
as opposed to 2.3 cc. of egg suspension with vessels of 15-ml. capacity in the other experiments.
Five-tenths ml. of antiserum or normal serum was used throughout all experiments. The condi-
tions in experiment 5 duplicate those which obtained during experiments with eggs of 5. purpuratus.
220 JOHN W. BROOKBANK
After dialysis, the fertilizin was precipitated with 5/4 volumes of cold ethanol,
washed, vacuum-dried, redissolved in distilled water, and used for injection into
each of three rabbits. This antigen had a sperm agglutination titer of approxi-
mately 1/500.
Following a control bleeding, the rabbits were injected according to a previously
described schedule (Tyler and Brookbank, 1956a) for a period of 21/4 weeks,
each receiving a total of 250 ju,g N. The animals were bled by cardiac puncture
4 days following the last injection.
Manometric methods
1) Lytechinus variegatus. The effect of the antisera on the respiration of
unfertilized eggs, deprived of all soluble fertilizin by acid (pH 3.5) treatment, was
followed, at 20° C., in a standard Warburg apparatus using vessels of approxi-
mately 15 ml. total capacity (k02 = 1.4-1.5 /tl./mm.). The eggs used in these
experiments were 90-100% fertilizable following the acid treatment. The set-
tled eggs were diluted to a 20% suspension, on the basis of settled volume, with
buffered sea water (0.01 M glycyl-glycine, pH 8.0). The suitability of glycyl-
glycine as a sea water buffer, and as a medium for the eggs, has been established
by Tyler and Horowitz (1937). The main chamber of the vessel contained 2.3 ml.
of egg suspension, the center well 0.2 ml. of 10% NaOH. and the side arm
0.5 ml. of the test solution (antiserum, normal serum, hypertonic sea water, or
sperm suspension). After the respiratory rate of the eggs in buffered sea water
had been established (usually after 40 minutes), the side arms were tipped and
the rate of respiration in the test solution was determined. At the end of each
experiment, the eggs were examined microscopically.
2) Strongylocentrotus purpuratus. During the two preliminary experiments
reported in Table II, the procedure followed was similar to that described above,
with the following exceptions: (1) The temperature employed was 18° C., the
maximum tolerated by these eggs. (2) One ml. of a 20% suspension, in buf-
fered sea water, was exposed to 0.5 ml. of serum, added from the side arm of
vessels of ca. 7-ml. total capacity (k02 = 0.7-0.8 jul./mm.). Thus, the ratio of
ml. of serum to the final volume in the vessel was increased, in these experiments,
from 0.18 to 0.33 ml. serum/ml. (3) In experiment 2, Table II, the egg sus-
pension was exposed to 1 mg% trypsin solution (crystalline, lyophyllized trypsin—
Worthington Biochemical Corp., Freehold, N. J.) for 10 minutes prior to sus-
pension in buffered sea water. This treatment prevented the elevation of the
fertilization membrane of test eggs inseminated in sea water following washing.
The eggs used in both experiments were 95-100% fertilizable after acid treatment.
Trypsin treatment reduced this figure to 30% in experiment 2, using the same
amount and concentration of sperm suspension for each test insemination.
RESULTS
As can be seen in Table I, all antisera against fertilizin increased the respiratory
rate of unfertilized Lytechinus eggs, including those antisera directed against S.
pupuratus fertilizin. The increase noted in heterologous antisera was somewhat
less than that observed using homologous antisera. Normal sera and antiserum
EFFECT OF ANTISERA ON RESPIRATION 221
against sperm effected no measureable increase in respiratory rate compared with
the rate observed in buffered sea water. The increased respiration in the presence
of antisera against fertilizin reached a maximum at 20-40 minutes following the
addition of the serum from the side arm. Following this, the rate declined toward
the control level, reaching this point in approximately 40 minutes. The rates
shown in Table I represent average rates over the entire period of exposure
(80 minutes) of the cells to the sera. Maximum rates ranged from 4 to 5 times
control rates. Serial dilution of the antiserum (in this case, the antiserum
directed against antigen 10 was employed) progressively lowers the value of the
maximum observed rate, as indicated in experiment 7, Table I. In experiment 5,
TABLE II
The effect of antisera against fertilizin on the respiratory rate of unfertilized
Strongylocentrotus purpuratus eggs
Aver, rate
Gtl./min./vessel)
Experiment No. 1
Antiserum from rabbit :
d. 0.15
e. 0.15 0.15 aver.
f. 0.15
Pre-injection control serum:
d. 0.14
e. 0.14 0.14 aver.
f. 0.14
Experiment no. 2 (trypsinized eggs)
Antiserum from rabbit:
d. 0.19
e. 0.21 0.20 aver.
f. 0.20
Pre-injection control serum :
d. 0.16
e. 0.17 0.1 6 aver.
f. 0.16
Aver, rate, buffered sea water (12 determinations) 0.17 (Range — 0.15-0.18)
dealing with the effects of heterologous antisera directed against the 5". purpuratus
fertilizin antigen, a higher proportion of antiserum relative to the amount of egg
suspension was employed in order to duplicate conditions existing during experi-
ments with 6". purpuratus eggs. The maximum rate observed with heterologous
antisera was about three times the control rate. The high (ca. 7 times the control
rate) rate observed with the homologous antiserum in this experiment presumably
reflects the higher concentration of antibody employed.
As controls, some aliquots of eggs were exposed to sperm suspensions (0.1%)
or to hypertonic medium (1 M final concentration with respect to NaCl) sea water,
added from the side arm of the vessel. The increased respiration observed as a
consequence of fertilization or treatment with hypertonic sea water approximates the
JOHN W. BROOKE ANK
increases obtained using homologous antisera against fertilizin (Table I). In-
creased respiration following exposure to hypertonic medium was first reported by
Warburg (1908), and confirmed by Keltch and Clowes (1947). The antiserum
against extract of fertilized Lytechinus pictus eggs, washed and demembranated
prior to homogenization, also proved effective in increasing the rate of respiration
of L. variegatus eggs (results included in Table I).
Two preliminary experiments using unfertilized S. purpuratus eggs yielded less
conclusive results. Slight increases in respiratory rate of questionable significance
occurred after the addition of homologous antiserum against fertilizin. The in-
creases in rate usually appeared within 30 minutes following addition of the sera,
and lasted for 15-20 minutes, after which time the rates returned to the control
level. Table II presents average rates during the time (60 minutes) the eggs
were exposed to the sera. In experiment 2, the eggs were trypsinized as described
above, on the assumption that a barrier exists at the egg surface which prevents
combination of antibodies with the necessary sites on the egg. This treatment did
not appreciably alter the results obtained during experiment 1. That the antisera
against S. purpuratus fertilizin contained antibodies capable of increasing respiratory
rate can be seen by the ability of these antisera to increase the respiration of
L. variegatus eggs (Table I). In addition, these antisera were capable of blocking
the first cleavage of demembranated fertilized 6". purpuratus eggs, indicating the
presence of antibodies against fertilizin (Tyler and Brookbank, 1956a, 1956b).
Furthermore, positive ring precipitin tests were obtained with the homologous
antigen. The problem of the failure of these antisera to cause an increase in the
rate of respiration of the unfertilized eggs of 6\ purpuratus comparable to that
observed using Lytechinus eggs remains unsolved at this writing. Since only two
experiments are available, it may well be that future work will resolve the ap-
parent difference between these two species.
Microscopic examination of Lytechinus eggs following these experiments re-
vealed no morphological evidence of activation, excepting, of course, the cases in
which the eggs had been inseminated. Samples of Lytechinus eggs treated with
immune serum or hypertonic sea water in the manometer vessels were transferred
to fresh sea water and observed periodically for 3 to 4 hours. No indications of
membrane elevation or cleavage were seen in these eggs, though control eggs
inseminated in the manometer vessels elevated fertilization membranes while in the
vessels, and developed normally following transfer to fresh sea water. In some
instances, Lytechinus eggs exposed to antisera against fertilizin were agglutinated
(experiments 2, 3) and some darkening of the cortical region was noted. In most
instances, the eggs tended to cytolyze in the antisera against fertilizin after 3-4
hours exposure.
DISCUSSION
The above results indicate that antisera against fertilizin are capable of tem-
porarily elevating the respiratory rate of unfertilized as well as fertilized (Tyler
and Brookbank, 1956b) Lytechinus eggs. The time course followed by the in-
increase to the maximum rate and the subsequent return to the control rate is
similar in both cases. Judging from the increased maximum rate in the presence
of a relatively greater amount of antiserum (Table I, experiment 5), it is probably
safe to assume that the rate obtained in a given experiment is a function of antibody
EFFECT OF ANTISERA ON RESPIRATION
concentration, provided the total volume and the number of eggs present remain
constant. This is also indicated by experiment 7 (Table I), in which serial dilu-
tions of antiserum against antigen number 10 were tested.
In comparing the results presented in this report with results obtained previously
by others, it is apparent that increased respiratory rate in the presence of specific
immune serum is seldom encountered, even over a rather wide range of biological
material. The report by Sevag and Miller (1948) represents the only instance
encountered by this author in which a temporary increase was observed. The in-
creased respiratory rate was found only upon lysis of the E. typhosa cells in the
presence of active complement, and was not observed when the cells were ag-
glutinated. Complement was not added to the sera employed in the present study,
nor were the sera heated to inactivate complement. Thus, the role of complement
in the system causing the increased respiration of Lytechinus eggs is not known,
though heating antisera against fertilizin to 56° C. for 30 minutes to inactivate C'l
and C'2 does not alter the cleavage blocking property of these antisera (Tyler and
Brookbank, 1956a). The unfertilized eggs do not cytolyze during the period of
measurement of respiratory rate, and remain intact for 3 to 4 hours following
removal from the manometer vessels. After this time, a gradual cytolysis be-
comes evident. The increased oxygen consumption does not appear, therefore,
to be associated with visible cytolytic changes in • the egg, since the maximum
respiratory rate in the presence of antisera against fertilizin is observed 20-40
minutes following the addition of the antisera from the side arm.
In considering the reports of Perlman (1954, 1957) and Perlman and Perlman
(1957) on the parthenogenetic properties of antisera against extracts of unfertilized
eggs and against fertilizin, one might be tempted to consider the increased rate of
respiration of unfertilized eggs in the presence of antisera against fertilizin, or
in hypertonic media, to be indicative of activation. This conclusion does not
seem warranted by the data presented in this report. No morphological evidence
of activation was encountered during the experiments, and one might more properly
consider the increased respiration of eggs so treated to be analogous to the increases
obtained in the presence of nitro-phenols (Clowes and Krahl, 1934, 1936; Krahl,
1950), methylene blue (Ballentine, 1940) and other substances which are not
considered parthenogenetic. With regard to the failure to observe activation of
eggs exposed to hypertonic sea water, it should be recalled that the time of exposure
of unfertilized eggs to the proper hyper- or hypotonic medium is critical (Harvey,
1940). Therefore, failure to observe morphological signs of activation under the
conditions prevailing in the manometer vessels is not surprising. In addition, a
wide range of hyper- or hypotonic media are capable of eliciting increased respira-
tion of the unfertilized eggs without effecting activation. The extent of the in-
creases obtained depends on the degree of hyper- or hypotonicity employed, in
much the same way as the extent of increases obtained with antisera against
fertilizin depends on the amount of antibody present (Table III).
In conclusion, it seems appropriate to consider the purity of the fertilizin
antigens used for injection. Special precaution was taken in the preparation of
the 6". purpuratns fertilizin antigen, and antigen number 10 (L. variegatus fertilizin),
to insure minimum contamination with material from the eggs. These antigens
were purified according to methods designed to yield electrophoretically homogene-
224 JOHN W. BROOKBANK
ous fertilizin, and were injected in exceedingly small amounts. The initial removal
of fertilizin from the eggs was carried out at pH 3.5. Eggs treated at this pH
for 2-3 minutes and returned to pH 8 develop normally, ruling against damage to the
eggs by this degree of acidity. Antigen number 4 (L. variegatus fertilizin) was ob-
tained in the same manner as number 10, except that the step involving precipita-
tion of the antigen with ethanol was omitted. Results obtained using antisera
against antigen number 10 paralleled those obtained using antisera against antigen
number 4 completely. Apparently antisera against purified fertilizin are capable of
temporarily raising the respiratory rate of the unfertilized egg. Results obtained
using antiserum against extract of fertilized eggs parallel those obtained using anti-
sera against fertilizin, since this antiserum also increased the rate of respiration of the
unfertilized eggs. Since antibodies most probably cause their effects through
combination with fertilizin at the egg surface, this last mentioned result seems to
indicate the presence of fertilizin haptens in the fertilized-egg antigen. The most
TABLE III
The effect of varying degrees of hyper- and hypotonicity on the respiration of unfertilized
Lytechinus variegatus eggs. Conditions the same as those prevailing for the
experiments in Table I. (Hypertonicity in terms of excess NaCl,
hypotoncity in terms of added distilled water)
Aver, rate
Tonicity /ul-/min. /vessel
Experiment 1 — hypertonic media
2.9 X sea water 0.97
1.85 X sea water 0.55
Sea water 0.20
Experiment 2 — hypotonic media
0.84 X sea water 0.50
0.75 X sea water 0.58
0.66 X sea water 0.67
Sea water 0.28
probable location of these fertilizin haptens is the hyaline layer (ectoplasmic layer)
of the fertilized egg, as proposed by Tyler and Brookbank (1956a).
SUMMARY
1. Homologous antisera against purified fertilizin, and against extract of fer-
tilized eggs (of Lytechinus pic tits} have been shown to temporarily increase the
respiratory rate of the unfertilized eggs of L. variegatus. Parallel experiments
employing antisera against fertilizin of Strongylocentrotus purpuratus and un-
fertilized 5". purpuratus eggs yielded essentially negative results. Further experi-
mentation is necessary before this apparent difference between the responses of the
eggs of these two species to antisera against fertilizin can be resolved.
2. Antisera against fertilizin of 6". purpuratus were effective in increasing the
respiratory rate of unfertilized L. variegatus eggs, indicating the presence of anti-
body capable of effecting increased respiration.
3. Normal sera and antiserum against sperm were without measurable effect
on the respiratory rate of L. variegatus eggs.
EFFECT OF ANTISERA ON RESPIRATION 225
4. None of the eggs treated with antisera against fertilizin showed morphological
evidence of activation.
LITERATURE CITED
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zation of marine eggs. /. Cell. Comp. Physiol., 15 : 217-232.
CLOWES, G. H. A., AND M. E. KRAHL, 1934. Action of dinitro compounds on sea urchin eggs.
Science, 80 : 384-385.
CLOWES, G. H. A., AND M. E. KRAHL, 1936. Studies on metabolism and cell division. I. On
the relation between molecular structure, chemical properties, and biological activities
of the nitrophenols. /. Gen. Physiol., 20: 145-171.
HARRIS, J. O., 1948. The respiration of Salmonella in the presence of agglutinating serum.
J. Bact.. 56 : 271-275.
HARVEY, E. B., 1940. A comparison of the development of nucleate and non-nucleate eggs of
Arbacia pitnctiilata. Biol Bull, 79: 166-187.
HAUROWITZ, F., AND P. SCHWERIN, 1940. Atmung aggutinierter und hamolysierter Erythro-
cyten. Enzyinologia, 9 : 95-96.
KELTCH, A. K., AND G. H. A. CLOWES, 1947. On the relation between oxygen consumption,
fertilization membrane formation, and cell division in artificially fertilized Arbacia eggs.
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KRAHL, M. E., 1950. Metabolic activities and cleavage of the egg of the sea urchin, Arbacia
pitnctiilata. A review, 1932-1949. Biol. Bull.. 98: 175-217.
MACDONALD, D., 1949. Influence of anti-organ sera upon metabolic processes : the effect of
anti-reticulo-endothelial-immune sera upon the dehydrogenase systems. Texas AY/>.
Biol. Med., 7: 332-335.
NOWIXSKI, W. W., 1948. Influence of anti-organ sera upon metabolic processes: Reticulo
endothelial-immune-serum (REIS) and the oxygen uptake of rat spleen. Texas Rep.
Biol. Mcd., 6 : 493.
NOWINSKI, W. W., 1949. Influence of anti-organ sera upon metabolic processes : Influence of
chick anti-brain serum upon the oxygen consumption of chick brain homogenates.
Texas Rep. Biol. Mcd.. 7 : 230-236.
PERLMAN, P., 1954. Study on the effect of antisera on unfertilized sea urchin eggs. Exp. Cell
Res., 6: 485-490.
PERLMAN, P., 1957. Analysis of the surface structure of the sea urchin egg by means of anti-
bodies. I. Comparative study of the effects of various antisera. Exp. Cell Res.,
13: 365-390.
PERLMAN, P., AND H. PERLMAN, 1957. Analysis of the surface structures of the >ca urchin
egg by means of antibodies. II. The J- and A-antigens. Exp. Cell Res., 13: 454-474.
SEVAG, M. C, AND R. E. MILLER, 1948. Studies on the effect of immune reactions on the
metabolism of bacteria. I. Methods and results with Ebcrtliella tvpliosa. J. Bact.,
55 : 381-392.
TYLER, A., 1949. Properties of fertilizin and related substances of eggs and sperm of marine
animals. Amcr. Nat.. 83 : 195-219.
TYLER, A., 1956. Physico-chemical properties of the fertilizins of the sea urchin Arbacia
pitnctiilata and the sand dollar, Ecliinarachnius panna. Exp. Cell Res., 10 : 377-386.
TYLER, A., AND J. W. BROOKBANK, 1956a. Antisera that block cell division in developing eggs
of sea-urchins. Proc. Nat. Acad. Sci., 42: 304-308.
TYLER, A., AND J. W. BROOKBANK, 1956b. Inhibition of division and development of sea-urchin
eggs by antisera against fertilizin. Proc. Nat. Acad. Sci., 42: 308-313.
TYLER, A., AND N. H. HOROWITZ, 1937. Glycyl-glycine as a sea water buffer. Science, 86 :
85-86.
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Zeitschr. f. pliysiol. Chcni., 57: 1-16.
A BICOLORED GYNANDROMORPH OF THE LOBSTER,
HOMARUS AMERICANUS
FENNER A. CHACE, JR., AND GEORGE M. MOORE
Division of Marine Invertebrates, U. S. National Museum, Smithsonian Institution,
Washington 25, D. C., and Department of Zoology, University of Neiv
Hampshire, Durham, Neiv Hampshire
Lobsters with sharply defined, bilateral color differentiation have been described
by several authors. Herrick (1896) mentioned the following variations of this
pattern in both the American and the European lobster : half normal color and
half light sky blue ; half normal and half pale red ; half greenish black and half
light orange ; half blue and half white ; and half light yellow and half bright red.
Schaanning (1929) gave a color figure of a European lobster that was light red
and dark blue. Templeman (1948) added records of two more bicolored American
lobsters, one normal and red, the other whitish red and purplish blue. Such color
variants have occasionally been referred to as gynandromorphs or hermaphrodites,
but there is no evidence that any of the previously recorded bicolored specimens
were also bisexual.
Only two cases of possibly complete hermaphroditism have been recorded here-
tofore for Homarus. Nicholls (1730) described and figured a specimen of the
European lobster, H. gaminarus, received from Newgate- Market, London, that
displayed all of the external and internal female characters on the right side and
all of the male structures on the left. Halkett (1919) collected a specimen of
H. americanus at Bay View, Pictou County, Nova Scotia, November 1917 "which
was absolutely male on the left side and absolutely female on the right side" ; this
specimen was sent to Queen's University, Kingston, Ontario, but apparently no
complete description of it has been published. Gordon (1957) described a speci-
men of H. gammarus from off Seahouses, Northumberland, that had all of the
characters of a perfect gynandromorph — female on the right side, male on the left
—except that there was no male opening on the left fifth pereiopod but, instead,
an imperforate indication of an opening on the coxa of the left third pereiopod ;
this specimen was not dissected, but Dr. Gordon suggests that "it probably has a
normal ovary on the right side and part ovary, part testis on the left side — or
a testis with ova in the anterior position." Herrmann (1890) discovered that
eggs are occasionally developed during spermatogenesis in the lobster testis but
he gave no indication that this was associated with any unusual external charac-
teristics. Finally, Ridewood (1909) recorded an ovigerous specimen of H. gam-
marus, presumably from off the Orkney Islands, that had genital openings on the
third right pereiopod and on the fourth and fifth left pereiopods, but dissection
disclosed only a normal paired ovary with apparently three oviducts, two of them
on the left side leading to the abnormally placed openings.
The specimen described below (U. S. Nat. Mus. Cat. No. 102241) seems to
be the first lobster to be recorded in which a color anomaly was associated with
226
LOBSTER GYNANDROMORPH 227
gynanclromorphism. The specimen was alive when presented to the Fish and
Wildlife Service at Woods Hole, Massachusetts, during the summer of 1954 by
a dealer operating between Boston and Cape Cod. Its place of origin is unfor-
tunately unknown ; it probably came from Massachusetts Bay but it could have
been shipped from New Hampshire, Maine, or even Canada. It died while being
photographed by John P. WTise, who offered it to the junior author for description.
After remaining in a freezer for about six months, it was transferred to formalin
for dissection ; the dissected portion is stored in ethyl alcohol, and the carapace,
abdominal tergites, tail fan, and chelipeds have been dried.
The specimen was about 10 inches (25 cm.) long from the tip of the rostrum
to the end of the telson. The carapace measures 86 mm. from the eye socket to
the hind margin. To the left of the midline of the animal, as well as on most of
the gastric and hepatic regions on the right side, the ground color was orange,
mottled and spotted with dark, greenish brown. The right side, posterior to the
mesogastric and hepatic regions, was similarly spotted and mottled but in shades
of blue over a lighter blue ground color, reminiscent of the colors of willow-pattern
china. The color pattern is indicated in Figure 1. The spotting and mottling
pattern seems to be continuous across the midline ; only the color is different.
The color transparency made from the living animal suggests that blue pigment
was largely lacking on the left side, and red, yellow, and possibly black pigments
were missing on the right side, although there is a greenish cast to the dark
mottling on the left and a pinkish tinge in some of the light blue areas on the right.
The left, crusher cheliped was colored like the left side of the body for the
most part, but the color photograph suggests that there were blue patches at the
outer, distal end of the merus, on the dorsal surface of the carpus, on the thick
portion of the hand, near the base of the fixed finger, and on the base of the
dactyl. The spines on this cheliped were bright red. The right, cutting cheliped
was blue, with a tinge of brown near the base of the fixed finger, and the spines
were almost pure white. The other pereiopods on the left side were orange with
greenish brown shadings similar to those on the adjacent portion of the carapace.
Those on the right side were very light, pinkish blue with darker blue shadings.
The left uropods were orange with greenish brown margins, and those on the
right, pale, pinkish blue with dark blue marginal bands. The fringe of setae on
the left uropods and on the telson to the left of the midline were reddish orange,
those on the right side, yellowish orange.
As in the three previously described gynandromorph lobsters, the specimen dis-
played female characters on the right side and male structures on the left. The
body was skewed to the right anteriorly and to the left posteriorly as shown in
Figure 1. This was almost certainly a result of the differential growth of male
and female lobsters. Templeman (1944) stated: "At all commercial sizes the
relative length of the carapace is less in the female than in the male. It remains
constant when the lobsters are small, but shows a definite and progressive increase
for those localities for which large lobsters were available for measurement. . . .
The ratio of the greatest width of the carapace to total length is in the smaller
relatively immature lobsters approximately the same for both males and females,
while in the larger animals it increases with size and more so in the male than in
the female." The skewing of the axis and the more swollen left (male) side
228
FENNER A. CHACE, JR., AND GEORGE M. MOORE
FIGURE 1. Dorsal surface of body of gynandromorph of Homarus aincricantis shuuing
asjTnmetry and color pattern. Drawn from color transparency of living animal.
FIGURE 2. Ventral surface showing opening of oviduct on coxa of third right pereiopod
and of vas deferens on fifth left pereiopod, asymmetrical thelycum, and characteristic female
and male first pleopods.
FIGURE 3. Abdominal somites viewed from the right (female) side. The left (male)
pleura are shown as if viewed from the inside.
LOBSTER GYNANDROMORPH 229
of the carapace are therefore readily understandable. The reverse skewing of
the abdomen probably also resulted from this differential growth, accentuated by
the proportionately larger abdominal pleura in the female, as shown in Figure 3.
The greater apparent length of the left (male) pleura of the fourth, fifth, and sixth
somites in this figure is misleading and is caused by the fact that the left (male)
pleura curve downward nearly vertically, whereas those on the right (female)
side extend obliquely outward, as shown in Figure 1. The distortion is less
striking than in Gordon's larger (11-inch) specimen of H. gaiiiinarns and in
Nicholls' specimen of the same species, the size of which was not given. This
might be expected from Templeman's (1944) findings that the differential growth
of males and females becomes progressively more marked with age.
The most noticeable disparity in our specimen is found on the ventral surface.
As shown in Figure 2. there is a female opening on the coxa of the right third
pereiopod and a male opening on the left fifth pereiopod, which is represented
only by the coxa and basis. The thelycum is distinctly asymmetrical, the right
(female) part being broad and nearly bare and the left (male) part narrower
and provided with long hairs. Neither element of this structure corresponds
exactly with its form in normal males and females, but the similarity to the con-
ditions in the appropriate sexes is more than superficial. Even the median plate
extending forward from the last thoracic sternite is modified as would be expected :
the right portion is longer and acute, like half of a typical female plate, and the
left portion is shorter and rounded as in the male.
The first right pleopod is typically female, flexible and long-haired, while the
left one is a rigid male intromittent organ. There is a well-developed appendix
masculina on the endopod of the left second pleopod, but none on the right member
of this pair. The second, third, fourth, and fifth pleopods on the left (male) side
are 37.5, 35.3, 33.5, and 29.0 mm. long, respectively, from the basal articulation
to the end of the endopod. Those on the right (female) side have corresponding
lengths of 37.2, 38.5, 38.0, and 33.3 mm. These figures agree remarkably well
with the proportionate lengths of these appendages in normal males and females,
as determined by Templeman (1944) : "In the male the swimmerets (including
protopodite and endopodite) on the second abdominal segment are the longest
and in the female those on the third and fourth are the longest and approximately
equal. The second swimmerets are not greatly different in length in males and
females. . . ." The sternal spines of the gynandromorph are about 2.5 mm. long
on the second, third, and fourth abdominal somites and about 2.0 mm. long on
the fifth somite. Data given by Templeman (1944) for New Brunswick speci-
mens indicate that the average length of the spine on the second somite in specimens
of comparable size is about 3.75 mm. in males and 0.5 mm. in females, and the
spine on the fifth somite is about 2.5 mm. long in males and 0.5 mm. in females.
The spines in the gynandromorph therefore seem to be intermediate in size, per-
haps more nearly approaching the male than the female condition.
Careful removal of the carapace and abdominal tergites of the specimen disclosed
a well-developed ovary filled with maturing eggs on the right side and a normal
testis on the left, as shown in Figure 4. An oviduct led from beneath the ovary
to the opening on the right third pereiopod and a typical vas deferens connected
the testis with the left fifth pereiopod. A few of the eggs in the ovary appeared
slightly discolored. Herrick (1911) stated that the presence of orange flecks
230
FENNER A. CHACE, JR., AND GEORGE M. MOORE
in the ovary, representing degenerating eggs that were not shed, is conclusive
evidence that a lobster has already spawned at least once, but the spots in our
specimen were not sufficiently distinct to permit an unequivocal determination that
spawning had occurred. As can be seen in Figure 4, a lobe of the ovary, probably
representing the connection between the two halves of the organ in a normal
female, was found just anterior to the heart. At this point the testis was inter-
rupted and the two parts of it were continuous with the intermediate portion of
the ovary. It appeared that the two portions of the testis were differentiated
parts of a single organ. There is no doubt that the testis was functional, for
sections showed active spermatogenesis. Normal, fully formed spermatozoa were
extracted from the vas deferens.
It is, of course, impossible to determine whether this specimen could have func-
tioned reproductively as either or both a male and a female lobster. Viable sperma-
tozoa and eggs were probably produced, but the unpaired intromittent organ and
the deformed thelycum might have prevented successful copulation with normal
males and females. The specimen must have been nearly or quite mature. As
testis
hepatopancreas
FIGURE 4. Dissection of lobster gynandromorph after removal of heart and stomach showing
well-developed ovary on right side and testis and vas deferens on left.
mentioned above, we were unable to determine whether or not it had spawned.
If it was caught off the Massachusetts coast, it was probably six or seven years
old according to Herrick (1911). Herrick (1896) also maintained that "very
few lobsters under 9 inches in length have external eggs, while only few have
attained the length of lO1/^ inches without having them." Templeman and Tibbo
(1945) concluded from the examination of New Brunswick specimens that the
length of males at sexual maturity is at least 5 cm. less than that of females. One
can hardly assume, however, that the size of a gynandromorph is directly com-
parable with that of a normal individual of either sex. Female lobsters probably
grow more slowly than males (Herrick, 1911), and one might therefore expect
the present specimen to be smaller than a normal male and larger than a normal
female of the same age, but the growth rates of abnormally bisexual crustaceans
may be complicated by hormonal or other factors.
We wish to thank John P. Wise for calling our attention to this unusual speci-
men. We also wish to acknowledge the assistance of members of the staff of the
LOBSTER GYNANDROMORPH 231
Division of Marine Invertebrates, U. S. National Museum, during the preparation
of the paper. Special thanks are due Charles E. Cutress of that staff for the
histological preparation and study of the reproductive organs of the specimen.
LITERATURE CITED
GORDON, I., 1957. A pseudo-hermaphrodite specimen of the lobster, Homants ganimarus
(Linnaeus). Ann. Mag. Nat. Hist., (12) 10: 524-528.
HALKETT, A., 1919. An hermaphrodite lobster. Canad. Field-Nat., 33 : 40.
HERRICK, F. H., 1896. The American lobster : A study of its habits and environment. Bull.
U. S. Fish Comm., 15 : 1-252.
HERRICK, F. H., 1911. Natural history of the American lobster. Bull. Bur. Fish., 29: 149-408.
HERRMANN, G., 1890. Notes sur la structure et le developpement des spermatozo'ides chez les
decapodes. Bull. sci. Fr. Belg., 22: 1-59.
NICHOLLS, F., 1730. An account of the hermaphrodite lobster presented to the Royal Society
on Thursday, May the 7th, by Mr. Fisher of Newgate-Market, examined and dissected,
pursuant to an order of the Society. Phil. Trans. Roy. Soc. London, 36: 290-294.
RIDEWOOD, W. G., 1909. A case of abnormal oviducts in Honiarus I'ttlgaris. Ann. Mag. Nat.
Hist., (8) 3 (13) : 1-7.
SCHAANNING, H. T. L., 1929. En eiendommelig varietet av hummer (Homants vulgaris).
Staranger Mus. Aarsh.. 1925-28, pt. 5 : 1-3.
TEMPLEMAN, W., 1944. Sexual dimorphism in the lobster (Homarus americanus). J. Fish.
Res. Bd. Canada, 6: 228-232.
TEMPLEMAN, W., 1948. Abnormalities in lobsters. Bull. Newfoundland Govt. Lab., No. 18:
3-8.
TEMPLEMAN, W., AND S. N. TIBBO, 1945. Lobster investigations in Newfoundland, 1938-1941.
Dept. Nat. Res. Newfoundland Res. Bull., No. 16: 1-98.
RE-EXAMINATION OF AN INHIBITOR OF REGENERATION IN
TUBULARIA
CHANDLER FULTON
The Rockefeller Institute, Nnt' }'ork, New York, and the Marine Biological Laboratory,
Woods Hole, Massachusetts
In the marine hydroid Tnlndaria the presence of hydranth structures has been
thought to prevent the development of new hydranths in nearby stem tissue. Two
preparations have been made from adult hydranths which inhibited the regeneration
of new hydranths on isolated stem segments. One of these (inhibitor ^vatcr> Rose
and Rose, 1941) was made by agitating adult hydranths in aerated sea water for
from 12 to 24 hours, while the other (hydranth extract, Tardent, 1955) was found
in the supernatants of homogenates of adult hydranths. These inhibitors of re-
generation were specific to hydranth tissue in that they were not obtained when
stems were treated in the same manner. They have been compared (Tweedell,
1958) and found to differ in a number of properties. The regeneration-inhibiting
substances in inhibitor water have been considered by a number of authors to
represent the substances normally responsible for physiological dominance in
Tnlndaria, and inhibitor water has been employed by Steinberg (1954) in an
experiment to indicate the mechanism of physiological dominance.
In the present investigation it was found that active inhibitor water could not
be prepared in the absence of bacterial growth, and as a consequence a re-examina-
tion of this inhibitor was undertaken.
MATERIALS AND METHODS
Freshly-collected Tubular ia crocca, provided by the Supply Department of
the Marine Biological Laboratory, was used in all experiments. Sea water was
filtered through paper shortly before use.
In preparing inhibitor water, an attempt was made to use methods comparable
to those used by previous workers (cf. Tweedell, 1958). Populations of adult
hydranths with 5 mm. of stem attached were isolated and washed thoroughly, and
then aerated in sea water for 24 hours at 17-22° C. After aeration, the hydranths
and debris were removed by filtration and the preparation was tested for its effect
on regeneration.
The bacterial population was estimated subjectively in early experiments by
turbidity and microscopical examination, and in later experiments was determined
using a Petroff-Hauser bacteria-counting slide. The bacterial population of
filtered sea water was found to be approximately 10r> per ml., which is too low for
accurate estimation with a counting slide. It was assumed that no bacterial
proliferation had occurred during the preparation of any given solution if the
bacterial population did not exceed this order of magnitude. It should be cautioned
that if mature male hydranths are used to prepare inhibitor water, turbidity may
in part result from the release of large numbers of sperm into the water.
232
TUBULARIA REGENERATION INHIBITOR 233
When it was desired to remove bacteria, the preparations were filtered through
an HA millipore filter (Millipore Filter Corp., Watertown, Mass.) or centrifuged
for 5 minutes at 30,000 g. Bacterial growth was prevented by the addition of
antibiotics. Penicillin and streptomycin were used at 100-125 ju,g./ml. ; sulfadiazine
was used at about 0.001 per cent (or saturation in sea water). At these con-
centrations, and in the cases of penicillin and streptomycin even at four-fold higher
concentrations, the antibiotics did not have any significant effect on the rate or
course of regeneration.
The solutions to be described were tested immediately after preparation for their
effect on the regeneration of freshly-cut, 7-mm. stem segments. Virtually all of the
stems in control groups regenerated, although as is usual with Tubularia there
was a considerable variation in the rate, even within a single group. A preparation
was considered to have inhibited regeneration if, during the time required for the
complete regeneration of the controls (emergence), all or a significant fraction
of the experimental group either disintegrated or healed but did not begin re-
generation. Stems in inhibitor water which regenerated were usually but not
always retarded.
RESULTS
Populations of 1.5-2 hydranths per ml. aerated in sea water regularly pro-
duced an inhibitor water which completely prevented regeneration. Occasional
batches of inhibitor water prepared at these or lower hydranth densities were in-
active, while populations of more than two hydranths per ml. usually gave a
preparation which caused disintegration of the stem tissue. However, considerable
variability was found in the activity of preparations made at the same hydranth
densities and under the same conditions (temperature, time, etc.), suggesting that
some factor other than those controlled was involved.
A number of observations suggested that the activity of inhibitor water was
due to bacterial growth. Preparations became quite turbid during the course of
aeration, and the condition of the hydranths deteriorated rapidly. The solution
developed a putrid odor. Hydranths killed by exposure to 30° C. for 15 minutes
rapidly disintegrated, but nevertheless produced active inhibitor water. When
active preparations were examined microscopically, a large, heterogeneous popula-
tion of bacteria was found. Removal of these bacteria often resulted in a reduction
but never in an elimination of the inhibitory activity of a preparation.
An estimate of the amount of bacterial growth which occurs in inhibitor water
preparations was obtained by preparing a series of 5 inhibitor waters at a density
of 1.5 hydranths per ml. and making counts with a bacteria counting slide at the
beginning and end of aeration. In this series, the bacterial density increased
during aeration from about 3 X 105 bacteria per ml. to about 108 bacteria per ml.
Bacteria were removed by centrifugation and each preparation tested for its effect
on the regeneration of 10 stems. The results are given in Table I. The increase
in bacterial number represents a minimum of 9 generations of bacterial growth.
It should be noted, however, that counts made at the beginning of aeration do not
include bacteria which are present in the hydranths and are released into the water
during aeration as the hydranths disintegrate.
In order to determine whether or not hydranths could produce active inhibitor
water in the absence of bacterial growth, hydranths were agitated in sea water
234
CHANDLER FULTON
containing antibiotics at concentrations sufficient to maintain bacteriostasis. The
results of three of the experiments with penicillin and streptomycin are given
in Table II. In the first two experiments shown (A and B), the amount of
bacterial growth was estimated subjectively. No detectable bacterial growth
occurred in any of the preparations in experiment A, and in spite of the fact
that the hydranth density was more than twice that necessary to produce complete
inhibition in the absence of antibiotics, the stems in both experimental groups all
regenerated at the same rate as the control stems. In experiment B, the hydranth
density was over four times that necessary to produce complete inhibition without
antibiotics. The preparation agitated at this hydranth density with antibiotics
(B4) produced some delay in the rate of regeneration. This delay may have
been due to the cytolysis of some of the hydranth tissue releasing the same sub-
stances present in hydranth extracts (see discussion).
These experiments indicate that in the presence of penicillin and streptomycin
in concentrations which suppress bacterial growth, inhibitor water cannot be col-
lected. However, it may be argued (Tweedell, 1958) that the antibiotics used
either prevent the hydranths from producing an inhibitor of regeneration or destroy
this inhibitor as it is produced. Three observations appear to exclude these
TABLE I
The number of bacteria present in five similar preparations of inhibitor water and the effect
of these preparations on regeneration. Observations were made at intervals for 4.3 days
Preparation
Control
1
2
3
4
5
Bacteria/ml. X 10"
0.0
2.3
3.3
3.7
5.5
7.4
Effect of preparation on stems
10/10 emerged within 2.4 days
10/10 did not begin regeneration
10/10 did not begin regeneration
10/10 disintegrated within 3.6 days
10/10 disintegrated within 1.4 days
10/10 disintegrated within 1.0 day
alternatives. ( 1 ) That penicillin and streptomycin do not destroy the inhibitors
was demonstrated by experiments in which bacteria were removed by centrifugation
and these antibiotics added to inhibitor water after preparation. In one such
experiment, there was no measurable reduction in the activity of the preparation
when antibiotics were added (Table II, C3) ; in another (not listed) there was
a slight reduction but not an elimination of the inhibition produced by the prepara-
tion (similar to that observed in other preparations when they were sterile filtered).
(2) Particularly important are three experiments with penicillin and streptomycin
and one with sulfadiazine in which bacterial growth occurred in the preparations
even though antibiotic was added at the beginning of aeration. Presumably bacteria
resistant to the antibiotics used developed in these preparations. In these cases,
the preparations inhibited regeneration in proportion to the amount of bacterial
growth which occurred in them (e.g., Table II, C5, 6), showing that, in spite of
the antibiotics, if bacterial growth occurred, an active regeneration inhibitor was
produced. (3) Three antibiotics — penicillin, streptomycin and sulfadiazine — dif-
fering greatly in chemical structure and presumed mode of action, were used alone
or in pairs to maintain bacteriostasis. Regardless of which antibiotic was used, if
bacterial growth was prevented the preparation failed to inhibit regeneration.
TUBULARIA REGENERATION INHIBITOR
235
To see if hydranths were a necessary component of the system, experiments
were done in which bacterial growth was allowed to occur in sea water in the
absence of hydranths. Dilute proteose-peptone solutions in sea water, aerated for
24 hours, and then sterilized by millipore filtration followed by the addition of
antibiotic, were potent inhibitors of regeneration, while control solutions in which
TABLE 1 1
Selected experiments which illustrate the activity of inhibitor water prepared with penicillin
and streptomycin. Bacterial density was either estimated (number represented by pluses
in the table) or counted directly using a bacteria counting slide (represented by number
per ml.). Abbreviations: pen., penicillin; strep., streptomycin
Experiment
Components added to sea water
Bacteria
per ml.
Stems
regenerated
vs. total
Mean time
of emergence
in days
Al
Pen. and strep.
—
10/10
2.5
2
4 hydranths/ml. + pen. and strep.
—
10/10
2.6
3
4 hydranths/ml. + pen. and strep.
—
10/10
2.5
Bl
None
—
10/10
2.3
2
8 hydranths/ml.
+ + +
0/10
—
3
Pen. and strep.
—
10/10
2.4
4
8 hydranths/ml. + pen. and strep.
—
10/10
3.1
Cl
None
ra. 105
10/10
2.4
2
2 hydranths/ml.
5 X 10s
0/10
—
3
2 hvdranths/ml., pen. and strep, added
after aeration*
0/10
—
4
Pen. and strep.
; 10*
10/10
2.3
5
2 hvdranths/ml. + pen.
2 X 10'
5/10
4.8
6
2 hydranths/ml. + strep.
1 X 108
2/10
2.3
7
0.1% proteose peptone + pen. and strep.
ca. 10r'
10/10
3.1
8
0.1% proteose peptone, pen. and strep,
added after aeration
3 X 108
0/10
—
* Penicillin and streptomycin were added to a portion of solution C2.
bacterial growth was prevented by the addition of antibiotic at the beginning of
aeration, at most, slightly retarded regeneration (e.g.. Table II, C7, 8; compare
Cl, 2).
To make certain that the inhibition produced as a result of bacterial growth
was not dependent on the presence of specific bacteria, preparations were made
236
CHANDLER FULTON
using Escherichia coli. Cultures were grown in a minimal medium (Davis and
Mingioli, 1950) from a small inoculum to 109 cells per ml. The bacteria were
removed by centrifugation, and the used medium diluted 1 : 5 in sea water containing
antibiotic. Such a preparation completely inhibited regeneration, while control
stems placed in a 1:5 dilution of sterile minimal medium with antibiotic regenerated
normally.
From these data it is clear that the activity of inhibitor water can be explained
on the basis of the bacterial growth which occurs in the medium, and that no other
inhibitors can be collected when bacteriostasis is maintained with antibiotics.
TABLE III
Summary of all experiments which indicate that inhibitor water is a by-product of bacterial growth.
Refer to the text for explanations of each experiment. Abbreviations: pen., penicillin;
strep., streptomycin; sulfa., sulfadiazine
Components added to sea water before aeration
Number of
experiments
Bacterial
growth
Inhibition of
regeneration
None
17
—
—
Hydranths
17
+
+
Hydranths*
6
+
+
Hydranths**
2
+
+
Heat-killed hydranths
4
+
+
Pen., strep., or sulfa.
14
—
—
Hydranths + pen., strep., or both pen. and
strep.
7
—
—
3
+
+
Hydranths + sulfa.
2
—
—
1
+
+
Stem lengths
2
—
—
Proteose peptone, pen. and strep.
3
—
—
Proteose peptone**
4
+
+
* Preparation sterile filtered after aeration.
* Preparation centrifuged after aeration, penicillin and streptomycin added to the super-
natant.
As an argument for the specific role of hydranth structures in producing in-
hibitor water it has been noted that a population of stems, aerated in sea water,
does not produce an inhibitor (Tweedell, 1958). After cutting, the ends of a
stem rapidly heal and secrete a thin layer of perisarc, so that very soon a cut stem
is entirely covered with chitin. Since no tissue is exposed, a preparation of stems
could not be expected to be a good medium for bacterial growth, and this might be
TUBULARIA REGENERATION INHIBITOR 237
the reason why no inhibitor was produced. Experiments were done in which
populations of clean stems were cut, washed, and aerated in sea water. Such
preparations did not support the growth of significant numbers of bacteria, and,
when tested on stems, permitted regeneration at the same rate as the controls.
A summary of the experiments which have been described, together with the
number of cases of each type, is presented in Table III. Cases in which verv
slight bacterial growth occurred in the preparations or in which the preparations
only produced a slight delay in regeneration (such as case B4, Table II) are
recorded as negative ( — ) in the table ; only definite cases of bacterial growth or
regeneration inhibition are recorded as positive ( + ). As the table indicates, the
inhibition of regeneration was always correlated with the growth of bacteria.
It is pertinent to mention certain experiments done with the regeneration in-
hibitor found in Tnbularia hydranth extracts. Such extracts were prepared by
homogenizing a population of adult hydranths and collecting the supernatant, as
described by Tardent (1955) and Tweedell (1958). It was found that the in-
hibition of regeneration produced by such extracts was not a result of bacterial
growth, in that when penicillin and streptomycin were added to the extracts to
maintain bacteriostasis the activity of the extracts was not affected in terms of
the proportion of stems inhibited by a given dilution of extract. It was found.
however, that in contrast to the original report of Tardent (1955), the inhibition
produced by Tnbularia tissue extracts was not specific to hydranth tissue. The
supernatant of homogenates from equivalent quantities of stem tissue also sup-
pressed the regeneration of stems. Tardent (personal communication) has ob-
tained the same result recently with Tnbularia larynx. Preliminary comparisons
on a wet weight basis indicate that hydranth tissue is about twice as active a
source of inhibitor as stem tissue. The lack of specificity of this inhibitor makes
it impossible, however, in the absence of further data, to adequately evaluate the
normal physiological role of the substances involved.
DISCUSSION
The results of the experiments with inhibitor water may be summarized as
follows. (1) Hydranths agitated in sea water produce bacterial growth and
inhibitors of regeneration. (2) If bacterial growth is suppressed with antibiotics, re-
generation inhibitors cannot be collected. (3) If antibotics are added at the begin-
ning of aeration but bacterial growth is not prevented, inhibitors can be collected.
(4) Bacterial growth in the absence of hydranths produces regeneration inhibitors.
These results, together with the appropriate controls, demonstrate that inhibitor
water as prepared in these experiments is a by-product of bacterial growth for which
the hydranths serve as inoculum and nutrient source. The results, however,
should not be taken to indicate that hydranths cannot produce any inhibitors of
regeneration, but rather that inhibitor water prepared as described by previous
workers contained no inhibitors which could not be accounted for as the products of
bacterial rather than hydranth metabolism.
If hydranths are agitated with antibiotics at densities several-fold higher than
those used to prepare inhibitor water (rf. Tweedell, 1958), occasionally such
preparations (e.g.. Table II, B4) retard regeneration even though bacteriostasis
has been maintained with antibiotics. It is interesting to note that in such cases
238 CHANDLER FULTON
bulbous outgrowths appear at one or both ends of many of the stems. These out-
growths are similar to those found in stems placed in Tubularia tissue extracts
(Tweedell, 1958; author's unpublished observations), suggesting that the cytolysis
of some of the hydranth tissue has released the substances found in hydranth extract
into the water.
Since this manuscript was originally submitted for publication, a paper by
Tweedell (1958) has appeared in which the results described in the present paper
are discussed. The results of this work were presented incompletely by Tweedell ;
the results as presented here answer the objections raised in his discussion. In
particular, the possibility that the antibiotics used had significant effects other than
that of maintaining bacteriostasis has been excluded by the results described above.
Tweedell notes that although bacteria were removed from some of his prepara-
tions by sterile filtration the preparations still inhibited regeneration. It is clear
from the present work that it is not the bacteria themselves, but rather the metab-
olites they release into the medium, which are primarily responsible for the
activity of inhibitor water. Removal of the bacteria from inhibitor water or
proteose-peptone solutions after aeration by filtration or centrifugation, or the
addition of penicillin and streptomycin to such preparations, in some cases reduced
the inhibitory activity of the preparation but in no case eliminated it.
SUMMARY
1. Rose and Rose (1941) found that adult Tubularia hydranths agitated in sea
water produced a solution, inhibitor water, which prevented regeneration. They
and subsequent workers have ascribed to this inhibitor a role in normal physiological
dominance. In the present investigation it has been found that considerable bacterial
growth occurs in the solution during the preparation of inhibitor water by the usual
methods, and that when antibiotics have been added to maintain bacteriostasis no
inhibitor can be collected. Experiments have excluded the possibilities that the
antibiotics used are preventing the production of the inhibitor or destroying it as it
is produced. It has been shown that metabolites produced by bacterial growth in
the absence of hydranths inhibit regeneration.
2. These data lead to the conclusion that inhibitor water represents the by-
products of bacterial growth for which the hydranths serve as source of inoculum
and as nutritive medium.
LITERATURE CITED
DAVIS, B. D., AXD E. S. MIXGIOLI, 1950. Mutants of Eschcrichia coli requiring methionine or
vitamin B12. /. Bact., 60: 17-28.
ROSE, S. M., AND F. C. ROSE, 1941. The role of a cut surface in Tubularia regeneration.
Physiol. Zool., 14 : 323-343.
STEINBERG, M., 1954. Studies on the mechanism of physiological dominance in Tubularia.
J. Exp. Zool., 127 : 1-26.
TARDENT, P., 1955. Zum Nachweis eines regenerationshemmenden Stoffes im Hydranth von
Tubularia. Rev. Suisse Zool., 62: 289-294.
TWEEDELL, K. S., 1958. Inhibitors of regeneration in Titbitlaria. Biol. Bull., 114: 255-269.
STUDIES ON THE STRUCTURE AND PHYSIOLOGY OF THE FLIGHT
MUSCLES OF BIRDS. 4. OBSERVATIONS ON THE FIBER
ARCHITECTURE OF THE PECTORALIS MAJOR
MUSCLE OF THE PIGEON
J. C. GEORGE AND R. M. NAIK
Laboratories of Comparative Anatomy and Animal Physiology, Department of Zoology,
M. S. University of Baroda, Baroda, India
Denny-Brown (1929), studying the red and white muscles of vertebrates, made
some observations on the "light" and "dark" muscle fibers in the breast muscle
of the pigeon. The later works on these two types of fibers have been reviewed
by George and Naik (1957). More recently, George and Naik (1958a, 1958b)
have shown that the red narrow fibers are rich in fat and mitochondria in sharp
contrast to the white, broad, glycogen-loaded fibers, which contain only a negligible
amount of fat and mitochondria. George and Scaria (1958a) histochemically
demonstrated higher lipase activity in the red narrow fibers. The Krebs' cycle
enzymes, too, seem to be localized in the narrow fibers (George and Scaria, 1958b).
These findings have stimulated considerable interest and called for a basic under-
standing of the nature and disposition of the fiber components of this muscle as
a whole. The present study, therefore, is an attempt to provide a comprehensive
picture of the pattern of fiber distribution and the nature of the metabolite load in
the different regions of the muscle.
MATERIALS AND METHODS
In order to obtain uniformly well developed pectoralis major muscle, only fully
grown wild pigeons, either shot or trapped from a single locality, were used through-
out for the present study.
Mapping the distribution of the two types of fibers in the muscle
Due to the bipectinate arrangement of the fasciculi, it was found convenient
to divide the muscle into twelve regions, each one extending to 10 mm. in length
along a hypothetical line, drawn midway between the origin of the muscle fasciculi
and the centrally placed tendon (as shown in Fig. 1 ) . From each of these regions
at the level of the aforesaid line, fresh frozen transverse sections were cut on a
freezing microtome. Subsequently the sections were treated in the following
manner. Transferring a fresh frozen section into distilled water or even saline
or isotonic sucrose solution resulted in uneven curling up of the section. Again,
the size of the muscle piece handled being large, some difficulties which were
encountered in the beginning in obtaining a good entire section, were completely
avoided by transferring the section directly into chilled 50% glycerol and mounting
it on a microslide in the glycerol solution. In the preparations thus made the
239
240
J. C. GEORGE AND R. M. NAIK
arrangement of the fibers in the section, however large, was faithfully maintained
with no distortions taking place. The glycerol-impregnated sections were thus
found to he ideal to manipulate. Moreover, the sections left in glycerol solution
and maintained at 0° C. can remain for more than a week without any perceptible
defect and thus could be utilized for future observations.
The desired region of the mounted section was projected on the screen of a
microphotographic camera at a magnification of 47 X and the photographic print-
ing paper exposed directly to the image. "Normal" bromide papers were found
FIGURE 1. Dorsal view of the pectoral is major muscle of the pigeon showing the hypo-
thetical lines 0-120 along which the distribution of broad fibers is recorded in Figure 2. The
squares A and B indicate the regions of the muscle used for studying the variation in metabolite
load and the structure at different depths of the muscle.
suitable. Using the sliding vernier on the stage of the microscope, continuous
photographic records of the distribution of the broad fibers were made (Fig. 5).
From such records by the method of random sampling, the mean value of the
number of broad fibers per square mm. was determined for every mm. depth of
the muscle. A survey of all the twelve regions was thus completed and a graph
plotted illustrating the continuous distribution of broad fibers per square mm. at
the distance of every 5 mm. along the line 0-120 (Fig. 1). The lines demarcating
the areas containing 30-50. 50-70, 70-90, 90-100, 100-120 and 120-140 and
120—150 broad fibers per square mm. were, drawn. The entire procedure was
ARCHITECTURE OF PIGEON PECTORALIS
241
_ 0 10 D-R 20 30 40 D-F 50 60
O DISTANCE ALONG HYPOTHETICAL LINE (mm)
110
120
D.F.
D.F.
FIGURE 2. Cross-sectional view of the pcctnralis major along the line 0-120 drawn in
Figure 1. The figures in the chart show the number of broad fibers per square mm. D.F.,
dorsal face of the muscle ; V.F., ventral face of the muscle.
NUMBER
OF B.F.
PER
mnV
200 400 600 800
NUMBER OF N.F. PER mm*
FIGURE 3. Relation between the number of broad fibers and the number of narrow fibers
per square mm. of transverse section of the muscle.
242
J. C. GEORGE AND R. M. NAIK
repeated on the f^ectoralis of three pigeons. The results obtained are summarized
in a graphical representation as shown in Figure 2. Since the individual varia-
tions in the pectoralis of different pigeons are considerable, the lines demarcating
different areas in the figure are not claimed to be absolute, but they do show the
% AGE OF
FAT
12
10
% AGE OF
GLYCOGEN3-6
3.2
RATIO OF 3-°
AREA 12
OCCUPIED
BY B.F. a9
TO THAT 0.6
OF N.F./rrm?
0.3
0 2 A 6 8
DEPTH OF MUSCLE (mm)
10
FIGURE 4. Variation in the percentage of glycogen and fat, in relation to the ratio of
the area occupied by the broad fibers to that of the narrow fibers per square mm., at different
depths of the muscle. The regions of the muscle marked A and B in Figure 1 were used.
generalized pattern of the distribution of the broad fibers in the pectoralis major
muscle of the pigeon.
For counting the broad as well as the narrow fibers in one and the same region,
the same procedure was adopted, except that the image of the section projected on
the screen was magnified to about a hundred times, and the sections from the
different typical regions of the muscle were used.
FIGURE 5. Negative prints of the transverse section taken from the region A (Fig. 1)
showing the continuous distribution of broad fibers (darker in color) at different depths of
the muscle. The numbers 1-10 on microphotographs indicate the depth in mm. from the
ventral to the dorsal face of the muscle.
243
244 J. C. GEORGE AND R. M. NAIK
Estimation of fat and glycoycn at different depths of the muscle
For the sake of convenience, the region of the muscle (marked A in Fig. 1)
on the posteriormost end of the keel was used throughout. In this region the
thickness of the muscle is only about 10 mm. and the variation in the distribution
of the broad fibers at the different depths of the muscle is gradual. From this
region A, a piece about 10 cubic mm. in size was cut out for the estimation of
glycogen and a somewhat bigger piece for the estimation of fat. From a region B
lateral to A, another piece was cut out and transferred to the freezing chamber
of the refrigerator and used later on for studying the distribution of the broad
fibers in this region by the method already described.
The muscle piece cut out from region A was mounted on the stage of a freezing
microtome so as to obtain horizontal sections. It was frozen hard, the outermost
epimysium was peeled off with a pointed forceps or sliced off by a superficial
stroke of the microtome knife, and 1-mm. thick slices of the muscle were serially
cut. Since all these horizontally cut sections were of uniform and known thick-
ness, each could be said to represent the nature of the muscle tissue at a known
depth. The thickness of the sections was not actually measured since the micro-
tome used was a brand new "Sartorius" model and all the possible precautions,
such as avoiding the fluctuations in the temperature, were taken so as to obtain
sections of uniform and accurate thickness. Each frozen section was immediately
transferred to a weighing bottle and dehydrated. The sections to be used for the
estimation of glycogen were dehydrated in a vacuum-desiccator at one atmosphere
pressure and maintained at 0° C., whereas for fat extraction, sections were de-
hydrated in an air-oven at 80° C., and finally in vacuum.
The dehydrated sections were weighed and their glycogen content was estimated
according to the method of Kemp ct al. (1954). For the quantity of the muscle
used for estimation (about 20-30 mg. per dry weight) it was found necessary to
dilute the glycogen extract in the deproteinizing solution to 10 ml. The color
developed was measured on the Beckman spectrophotometer (DU model) at 520 ^.
For the estimation of fat the dehydrated material was ground and, after weighing,
transferred to a fat-extraction thimble. The fat was extracted in the Soxhlet ap-
paratus with 1:1 ethanol-ether mixture (George and Jyoti, 1955). About 70-100
mg. of dehydrated muscle were used for each estimation.
The estimation of glycogen in the two types of fibers
Small pieces from the breast muscle of a decapitated pigeon were cut out and
dropped in previously chilled 80% methanol and left undisturbed at - - 10° C. for
24 hours. The fibers from the muscle thus preserved were teased out in methanol
under a binocular dissection microscope with watch-maker's forceps. The two
types of fibers were isolated and transferred to two separate containers containing
methanol and fitted with air-tight glass lids and stored in the refrigerator. Suffi-
cient numbers of fibers which would yield about 2-5 mg. in dry weight were iso-
lated and collected for each estimation. These fibers were then removed from
the methanol solution, dehydrated in vacuum and weighed on a microbalance.
Glycogen was estimated, as already mentioned, by the micromethod of Kemp et al,
(1954).
ARCHITECTURE OF PIGEON PECTORALIS
245
RESULTS
Figure 5 presents a typical picture of the distribution of broad fibers in the
muscle. In each fasciculus the broad fibers are mainly concentrated towards the
periphery. This pattern is maintained throughout the muscle. In regions of the
muscle where there are larger numbers of broad fibers or lesser numbers of narrow
fibers, the fasciculi have a smaller cross-sectional area with broad fibers closely
packed along their borders without any intervening narrow fibers. The number
of broad fibers per square mm. in the different regions of the muscle is shown in
Figure 2. The relation of the number of broad fibers to that of the narrow ones
per square mm. is shown in Figure 3. From both these, the number of broad
fibers, as well as the number of narrow fibers per square mm., in any region of
the muscle could be approximately determined.
The variation in the metabolite load and the number of broad fibers per square
mm. at different depths of the muscle are indicated in Table I, while in Figure 3
the same data are utilized to show the relation between the structure of the muscle
TABLK I
The number of broad fibers per square »/»/. and the percentage of fat and g/ycogen at
different depths of the breast muscle of the pigeon. (The portion of the muscle
marked A in Fig. 1 was used. The figures indicate the average values
of six sets of readings)
Depth of the
muscle in rnin.
(starting from the
ventral face)
Number of broad fibers
per square mm.
±S.D.
Percentage per dry weight of the muscle
±S.D.
Glycogen
Fat
0-2
90 ± 14
3.655 ± 0.275
10.289 ± 1.942
2-4
63 ± 8
.U75 ± 0.054
12.095 ± 1.056
4-6
48 ± 3
3.102 ± 0.127
14.632 ± 1.752
6-8
51 ± 4
3.409 ±0.184
13.250 ± 0.571
8-10
72 ± 9
3.588 ± 0.236
11.743 ± 0.572
and the metabolite load. The number of narrow fibers for the corresponding
number of broad fibers was calculated by using the formula of the regression line
in Figure 3 and the ratio of the area occupied by the broad fibers to that of the
narrow fibers in square mm. was determined by using the mean value of the
diameter of these fibers. The diameter of the broad fibers is 69.00 ± 14.00 /j.
( 1000) and that of the narrow fibers is 30.11 ± 6.56 p (2000). The figures given
in parentheses indicate the number of fibers measured from the fresh frozen sections
taken from the various regions of the muscle.
The values of the glycogen content of the broad and narrow fibers, calculated
on the dry weight of the muscle preserved in methanol, are, respectively, 10.240 ±
0.093// and 2.464 ± 0.311% (each value is the mean of three readings). Methanol
removes much of the fat (mainly from the narrow fibers) and some of the amino
acids.
DISCUSSION
It has been known that in many active muscles, the muscle fibers towards the
periphery become larger in diameter and lighter in color, compared to those in
246 J. C. GEORGE AND R. M. NAIK
the interior. In such muscles even in the individual fasciculus, the light fibers
are situated towards the periphery. In the pigeon breast muscle, the white broad
fibers and the red narrow fibers show a somewhat similar distribution pattern but
these fibers differ from the light and dark fibers of the other muscles in that they
are sharply differentiated into two distinct types without any intermediate forms.
The broad fibers are glycogen-loaded and poor in fat inclusions and mitochondria,
whereas the narrow fibers are fat-loaded and have a high mitochondria! content
and are poor in glycogen (George and Naik, 1958a, 1958b).
In a single muscle uneven distribution of metabolites has been long since realized.
To reduce such localized variation to the minimum, customarily a large piece of
muscle is utilized for the estimation of metabolites. Present work shows that in
a muscle like the pectoraHs major of pigeon having heterogeneous cellular elements,
variation in metabolites in the different regions of the same muscle and even in a
single fasciculus is quite large. Needless to say, what applies to glycogen and fat
might equally apply to other chemical constituents in which the two types of fibers
differ.
A general belief that the muscle fibers towards the periphery of the muscle
are more active than those in the interior and, due to higher activity, increase in
diameter, does not seem to hold good, at least in the case of the pcctoralis of
pigeon. Undoubtedly, the red fibers of pigeon breast muscle, due to their re-
markably well developed enzyme systems, play a major role in effecting the sus-
tained contractions of the muscle. In white fibers, on the other hand, the oxidative
processes are not developed or developed only to a negligible extent, in that the
dehydrogenase activity in these fibers, as shown by histochemical method, is neg-
ligible or nil (George and Scaria, 1958b). All the same, the white filters are not
inactive elements of the pigeon breast muscle. In the normal animal they show
no signs of atrophy. A glycerinated white fiber of pigeon breast muscle contracts
in the same manner as a glycerinated red fiber of the same muscle on the addition
of ATP. The study on the reactions of these two types of fibers to experimentally
induced disuse atrophy has yielded significant results. When the movement of
the humerus is restricted for three months by a plaster cast, the white fibers in
the deeper layer of the muscle show acute sign of atrophy whereas the red fibers
appear practically unaffected (George and Naik, unpublished data). These find-
ings suggest the possibility of some differences in the mechanical properties of the
two types of the fibers and in that case some physical factors may underlie the
distribution pattern of the two types of fibers in the muscle.
Denny-Brown (1954), has shown that a single nerve in the breast muscle of
pigeon can innervate both, the red as well as the white fibers. Since the activity
of these muscle fibers must be conditioned by the fundamentally different chemical
system in them, it is difficult to believe that the amount and the mode of activity
performed by these two types of fibers are the same. In what exact manner the
white fibers contribute to the activity of the muscle is far from clear and as a
prelude to such an understanding, an extensive study of these fibers is essential.
For such a study Figure 2 can be a useful guide. Moreover, the method used in
the present work to study the variation in the metabolite load in relation to the
variation in the fiber make-up of the muscle, can be used for studying the distribu-
tion of various constituents such as enzymes, amino acids and minerals in the
muscle.
ARCHITECTURE OF PIGEON PECTORALIS 247
\Ye are grateful to the members of the staff and the technicians of the Depart-
ments of Chemistry and Statistics, Faculty of Science, Baroda, for their unfailing
assistance in completing this work. One of us (R. M. N.) is indebted to the
Ministry of Education, Government of India, for the award of a Senior Research
Scholarship.
SUMMARY
1. The relative distribution pattern of the red and white muscle fibers in the
breast muscle of the pigeon is studied.
2. There exists a direct relation between the distribution of metabolites and
that of the two types of fibers in the different regions of the muscle.
3. Quantitative estimation of glycogen in the two types of filters confirms the
higher concentration of glycogen in the white fibers.
LITERATURE CITED
DEX \v-BRO\vx, D., 1929. The histological features of striped muscle in relation to its func-
tional activity. Proc. Roy. Soc. London. Ser. B, 104: 371-411.
DEXXV-BRUWX, D., 1954. As cited by Adams, R. D., ct al. in Diseases of Muscle. Paul B.
Hoeber, Inc., New York ; pp. 38 and 40.
GEORGE, J. C., AXD D. JVOTI, 1955. The lipid content and its reduction in the muscle and
hver of birds and bat during long and sustained activity. /. Aniin. Morph. Phvsiol.,
2: 38-45.
GEORGE, J. C., AXD R. M. NAIK, 1957. Studies on the structure and physiology of the flight
muscles of birds. 1. The variations in the structure of the pectoral is major muscle
of a few representative types and their significance in the respective modes of flight.
/. Anim. Morph. Pliysiol., 4: 23-32.
GEORGE, J. C., AXD R. M. NAIK, 1958a. The relative distribution and the chemical nature
of the fuel store of the two types of fibres in the pectoralis major muscle of the pigeon.
Nature, 181 : 709-710.
GEORGE, J. C., AXD R. M. NAIK, 1958b. Relative distribution of the mitochondria in the two
types of fibres in the pectoralis major muscle of the pigeon. Nature. 181 : 782-783.
GEORGE, J. C., AXD K. S. SCARIA, 1958a. Histochemical demonstration of lipase activity in the
pectoralis major muscle of the pigeon. Nature, 181 : 783.
GEORGE, J. C., AXD K. S. SCARIA. 1958b. A histochemical study of the dehydrogenase activity
in the pectoralis major muscle of the pigeon and certain other vertebrate skeletal
muscles. Quart. J. Micro. Sei. Cm press).
KEMP, A., J. M. ADRIEXXE AXD KITS VAX HEIJXIGEX, 1954. A colorimetric method for the
determination of glycogen in tissues. Biochcin. J.. 56: 646.
THE EFFECT OF OSMOTIC STRESS ON THE IONIC EXCHANGE
OF A SHORE CRAB
WARREN J. GROSS
Division of Life Sciences, University of California, Riverside, California
The decapod Crustacea have received considerable attention with regard to
their ability to regulate the inorganic ions of their blood (Krogh, 1939; Robertson.
1949, 1953, 1957; Prosser ct al, 1950). Prosser ct al. (1955) studied responses
of the shore crab Pachygrapsus crassipes to different concentrations of sea water.
The chief concern of their study was to determine the changes in the ionic con-
centrations of blood and urine which were effected by altering the concentration
of the external medium from normal. Determinations on the total losses and
gains of the respective ions between animal and medium were not made nor were the
effects of desiccation on ion concentrations in urine or blood determined. This in-
formation would be of special interest in the case of a semi-terrestrial crab such
as Pachygrapsus.
Gross (1958) demonstrated that when Pachygrapsus crassipes was placed
under osmotic stress, the principal exchanges of potassium were between the medium
and a source of potassium other than the blood, not mainly between blood and ex-
ternal medium. Also, evidence was produced that an extra-vascular pool partici-
pates in sodium exchanges between crab and medium. This paper will produce
further evidence that extra-vascular salt pools in Pachygrapsus contribute to ionic
exchanges with the medium, special attention being paid to calcium and magnesium.
The effects of desiccation on the ionic concentration of urine and blood in Pachy-
grapsus will be revealed and data confirming the findings of Prosser ct al. (1955)
will be produced.
MATERIAL AND METHODS
The subject of this investigation, Pachygrapsus crassipes Randall, was collected
at Laguna and Dana Point, California. All specimens were between molts, and
were mature, none being smaller than 20 gm.
Urine was sampled by inserting a micropipette into the excretory pores. Blood
was obtained by puncturing the cuticle of the leg joints with a micropipette.
Sodium and potassium were measured by means of a Beckman flame photometer.
Urine and blood were measured and diluted appropriately before being used
directly in the flame photometer (Gross, 1958). Samples as small as 0.05 ml.
thus could be analyzed to an accuracy of 2</f for sodium and 10% for potassium
at the minimum concentrations measured in this investigation. Thus, before and
after treatment samples of blood from the same crab could be analyzed for sodium
and potassium. Calcium and magnesium were determined by titration with ethyl-
enediamine tetra acetic acid (EDTA) a method described by Schwarzenbach ct al.
(1946) and Knight (1951). This method requires about 0.25 ml. of urine and
248
IONIC EXCHANGES IN A CRAB
249
about 0.50 nil. of blood. Because of the relatively large volume needed, repeated
blood samples on the same specimen were not taken. Urine samples were diluted
to 100 ml. and titrated directly. Media were titrated directly. Blood samples
were dialyzed against distilled water and the dialysate was titrated, a correction
being applied for the content of the dialysis bag. This process, which was not
necessary in the case of urine, gave a more distinct end-point than titrating the
diluted blood directly. Calcium and magnesium thus could be recovered within
an accuracy of 5% for the minimum concentrations measured.
TAHLK I
Effects of stress on ionic concentrations of blood and urine in Pachygrapsits
Treatment
50% sea \vater
1110% sea water
150% sea water
Desiccation
Mean
S.D.
No.
crabs
Mean
S.D.
Xo.
crabs
Mean
S.D.
Xo.
crabs
Mean
S.D.
X...
crabs
Sodium (mEq./l.)
Blood
397
24
37
*483
17.3
36
582
34
30
536
27.4
32
Urine
380
60
37
378
64.0
15
353
106
30
297
104
15
U/B ratio
0.96
0.14
37
0.78
0.14
15
0.63
0.16
30
0.56
0.17
15
Medium
232
464
696
Potassium
(mEq./l.)
Blood
7.36
1.4
37
*7.43
0.72
36
10.23
1.48
30
11.5
1.63
32
Urine
9.95
3.5
37
7.76
1.35
15
9.59
1.13
30
14.8
3.18
15
U/B ratio
1.45
0.50
37
0.82
0.19
15
0.94
0.33
30
1.34
0.50
15
Medium
4.9
9.8
14.7
Calcium (niEq./l.)
Blood
34.8
7.9
24
29.6
5.9
44
36.4
4.8
30
45.2
10.7
36
Urine
32.7
7.1
31
36.0
6.3
15
47.9
5.2
20
44.4
7.36
12
U/B ratio
0.98
0.13
23
1.17
0.20
15
1.33
0.18
29
1.07
0.33
12
Medium
10.0
20.0
30.0
Magnesium
(mEq./l.)
Blood
13.6
5.36
24
20.0
6.1
44
27.1
4.22
30
28.5
15.9
36
Urine
70.5
41.1
31
236
87
15
408
122
29
424
144
12
U/B ratio
5.62
4.52
23
13.6
5.3
15
15.4
4.44
29
23.6
10.9
12
Medium
52.0
104
156
* Gross (1958).
In order to measure the exchange of ions between animal and external medium,
crabs freshly removed from normal sea water were weighed, and blood was sampled
for sodium and potassium determinations. The crabs then were rinsed in water
of the salinity to which they were to IDC exposed, then immersed in a small volume
(50 ml.) of that same water for a period of about 24 hours. Adequate precautions
were taken against water loss by evaporation. Values concerning sodium and
potassium exchanges (Table II) have been reported previously (Gross, 1958)
250
WARREN J. GROSS
and include some data on animals immersed 24-48 hours in 100 ml. The crabs
could raise themselves out of the water and therefore were not completely immersed
at all times. After a period of about 24 hours, the animals were removed from
the media and their blood and urine sampled for the analysis of sodium, potassium,
calcium and magnesium. Likewise the media were analyzed for these ions.
Other crabs freshly removed from normal sea water were weighed, then desic-
cated for a period of about 48 hours for a loss of about 7% original weight. After
this treatment the blood and urine were analyzed for the above four cations.
TABLE II
Relative ion changes in blood and external medium caused by altering external medium from normal
Mean of ratios*
50%
sea water
S.D.
No.
crabs
150%
sea water
S.D.
No.
crabs
Vn
Blood change (rnEq./l.)
7 Zf.
0 8?
~>R
7 fi}
0 80
25
Medium change (mEq./l.)
I-
Blood change (mEq./l.)
0 S(S
0 4i
?0
1 00
0 70
24
Medium change (mEq./l.)
Pn
Blood change (mEq./l.)**
0 03
2 77
7 t
n 78
0 66
78
i^a
Medium change (mEq./l.)
~\l\rr
Blood change (mEq./l.)**
n QC
0 XO
71
0 fi-i
0 -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 showrs 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 betwreen 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 wras 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 0° such values
wrere 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 0 and +20°, oxygen consumption shows the usual logarithmic increase
FROZEN INTERTIDAL ANIMALS
261
\vith a Q10 of 2 to 3. Above this respiration decreases, probably due to thermal
injury. Below 0° the metabolic activity drops sharply with an apparent Q10 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
0
0
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 0 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 0° 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 0° 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
0 \
o \ o
\
RESPIRATION
vs.
SALINITY
LITTORINA
\ LITTOREA
0° \0
\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 0° 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 Q10 from the salinity of about 10. Above 0° the Q10 due
to the usual temperature effect on reaction rates is between 2 and 3. Combining
these one would expect a Qlf, 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 Q10 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 : 375—395.
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 Rf 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
0
MIGRATING PSEUOOPL ASMODIUM
(UNHYDROLYZED)
0
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
0
PRE-CULMINATION STAGE
(UNHYDROLYZEO I
0
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)
0 0
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
0
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. RIEGEL1
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 mm3.) 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 71/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%0; Ca, 0.01 \%0\ Mg, 0.020^-,; and Na. 0.078%0.
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 LD50 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 :
wt0 (= original body fluid weight) . . a ., . CA
X = - If the body fluid comprises 50 per
wt24 (= 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.
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BEADLE, L. C., AND J. B. CRAGG, 1940. Osmotic regulation in fresh-water animals. Nature,
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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.
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EDMONDS, E., 1935. The relations between the internal fluid of marine invertebrates and the
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Exp. Biol, 31 : 402-423.
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HUKUDA, K., 1932. Change of weight of marine animals in diluted media. /. Exp. Biol., 9 :
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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 : 468—491.
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.
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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
KNO3, 1 M 5.0 ml.
K2HPO4, 1 M 0.5 ml.
KH2PO4, 1 M 0.5 ml.
MgSO4, 1 M 2.0 ml.
Ca(NO3)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
H3BO3 1.43 g.
MnSO4-H2O 1.05g.
ZnCl2 0.05 g.
CuSO4-5H2O 0.04 g.
H2MoO4-H2O 0.01 g.
Distilled water to make 1000 ml.
"Iron solution" contained
Disoclium ethylenediaminetetra-acetate 0.5 g.
FeSO4-7H2O 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% CO2 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 106 cells mlr1) 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 I2 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 O2 (S.T.P.) per mg. total nitrogen per hour (Qo2 (N)).
Total N was estimated by sulfuric-acid digestion of an aliquot, with three succes-
sive additions of H2O2, 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.
0 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 (n2) 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 -- •• 1017/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 = 107 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 "±" sign 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 Qo2 (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 Qo2 (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 Qo2 (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 /*!. O2 (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 O10 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 writh 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
<r =72
CV =247.
P-i r-i n
LjJ
u
o
tr 60
LJ
CD
D
Z
40
I 5O 100 150 200 250 300 350 400 450 500
r r IN
20
J
1
MEAN=I6.I5
LARGEST SMALLEST
RANDOM I - CELL
MEAN =3^3
o- -- 1 5.4
CV =43.67.
1 n n
P-i
10
20 30
DIAMETERS IN
40
50
60
FIGURE 1. Histograms of mean diameters and products of major and minor radii. I-cells
were detected under 100 X and confirmed under 440 X as described in the Methods section.
As controls, myxamoebae were chosen at random for micrometric determinations.
306 MAURICE SUSSMAN AND HERBERT L. ENNIS
e) Micromanipulation of I-cells to test areas caused the induction of aggregates
within the test populations, whereas movement of R-cells at the same stage
of development did not.
The data to be presented provide subsidiary support for the contention that
the I-cells are the initiator cells and throw light upon their role in the aggregative
sequence.
METHODS
D. discoidenm, strain NC-4 wild type, was grown on SM agar medium in
association with Aerobacter aerogenes (Sussman and Noel, 1952). After 44
hours at 22° C., the myxamoebae had attained the stationary phase and were
harvested, washed by differential centrifugation, and dispensed on washed agar
plates (Sussman and Noel, 1952). Under these conditions the myxamoebae do
not increase in number and aggregate and fruit in normal fashion.
The procedure for I-cell identification has been previously given in detail
(Ennis and Sussman, 1958b). Initial recognition is accomplished at 100 X mag-
nification by size and flatness. Examination at 440 X reveals the great pseudopodial
activity, high rate of protoplasmic streaming, and granulation, and thereby con-
firms the diagnosis. A cautionary note is appended here. Occasionally, one
encounters moribund cells which typically attain enormous size before lysing.
However, these cells are perfectly round and hemispherical. They display no
motility and have lost their granules and vacuoles. After one has seen an I-cell,
there is no chance of confusing the two types and in any case, moribund cells are
extremely rare under the conditions of preparation and incubation described above.
RESULTS
A. Tune-lapse studies of aggregation
As mentioned, a high correlation was shown to exist between the positions of
I-cells and of subsequently formed aggregative centers. In these experiments,
washed myxamoebae were dispensed on washed agar at a population density of
200 cells/mm.2, optimal for center formation in this strain (Sussman and Noel,
1952). After 8 hours' incubation at 22° C., low power fields were chosen at
random and fixed in position on microscope stages. Those found to contain I-cells
were retained for further study. Camera lucida drawings or photomicrographs
were made at intervals until aggregation had begun and the centers were estab-
lished. In 50% of the fields, a center formed precisely at the position of the I-cell.
(Since in this stock, the ratio of centers formed to cells present is 1:2200, the
random chance of predicting that a center would form at a particular cell is 0.05%.)
In 30% of the fields a center formed near the I-cell. No centers formed in the
remaining 20%. In contrast, the incidence of centers in randomly chosen fields,
not examined for the presence of I-cells, was 25%. Thus the over-all chance of
a center appearing in a field containing an I-cell was three times greater than
random. The numerical data, given in detail elsewhere (Ennis and Sussman,
1958b), are here amplified by time-lapse series of camera lucida drawings and
photomicrographs.
INITIATOR CELL
307
Figure 2 illustrates the sequence of events when the center formed at the I-cell.
The first overt sign of impending aggregation was the appearance of large cell
clumps near the I-cell. Associations of more than two or three cells were never
encountered prior to this time and even these were purely transient. In the series
shown, the I-cell itself became part of a clump as its nearest neighbors began to
*(
too
o X
OIHN.
.0
0
t
„ *X •
O
••:••&
FIGURE 2. Time lapse camera lucida drawings of aggregation. The I-cell is the black
individual. Cell clumps, appearing first in C, were merely outlined. The two cross-hatches
mark the positions of dirt particles, used as points of reference. Respective times, in hours,
after deposition on washed agar : 10.8, 11.25, 11.7. 11.9. 12.4, 13.0, 14.4.
308
MAURICE SUSSMAN AND HERBERT L. ENNIS
_, , m
FIGURE 3. Time lapse photomicrographs of an aggregation. The arrows point to the
I-cell. In photograph No. 2, the I-cell was joined by a few neighboring R-cells to form a
tiny central clump. Photograph No. 3 was the last clearly discernible position of the I-cell.
INITIATOR CELL
309
0 0
o «
o '• • o
« O 0 »
O.IMH.
0° P
,0 •-^•:*7\v\-
c.
FIGURE 4. Time lapse camera lucida drawings of an aggregation. See legend of Figure 2 for
details. Respective times, in hours after deposition on washed agar : 12, 12.5, 13, 13.4, 14.
nestle against it. This occurred in all but a few of the aggregations studied. The
last clearly discerned position of the I-cell is in Figure 2-D. Its position was
barely visible in Figure 2-E as the I-cell enlarged and extended to the right. Mean-
while, the previously formed clumps enlarged and new ones appeared concen-
trically about and at progressively greater distances from the I-cell. At this time,
the loose cells and those in the clumps elongated and oriented radially. This
caused the clumps to attain the appearance of streams. The position of the ag-
gregative center then emerged clearly (Fig. 2-F) and is seen to have occupied the
last known position of the I-cell. Ultimately the streams moved into and joined
the center, producing the usual conical cell mass. Figure 3 is a series of
photomicrographs of another aggregation in which the center again formed at
the I-cell.
310 MAURICE SUSSMAN AND HERBERT L. ENNIS
A typical sequence in which a center was established near the I-cell is shown
in Figure 4. Again, the first sign of impending aggregation was the appearance
of cell clumps, although on this occasion, no clump formed around the I-cell. A
particularly large clump appeared at a distance of about 200 /A from the I-cell
(Fig. 4-C). The I-cell then moved into a small clump immediately above the
upper right reference mark. The cells elongated and oriented radially and a
center was established at a distance of about 100 /x from the last known position of
the I-cell. Thus the only real difference between the sequences shown in Figure 2
and Figure 4 is the appearance of the abnormally large clump, and this event always
preceded the establishment of a center near, rather than at, the I-cell. In 10 of 19
cases, the R-cells entered the aggregate but the I-cell remained outside. In the
other 9 cases the I-cell was also swept into the aggregate.
Figure 5 shows that a center need not form at a point along the previous path
of the I-cell. The four cases were chosen because the respective I-cells wandered
along relatively straight paths and could therefore clearly illustrate that such a
relation did not exist. Cases 3 and 4 are particularly pertinent in that the centers
did not form at the I-cells but did so at distances of 250 and 300 ju, respectively.
B. Aggregation after I-cell removal
The fact that I-cell removal can prevent subsequent aggregation was established
in previously reported experiments (Ennis and Sussman, 1958b). They also
showed that, to be effective, the removal must be accomplished at a very early
stage of the pre-aggregative period. Thus, I-cells were removed from drops con-
taining 500 myxamoebae within two minutes after they had been dispensed on
washed agar. The incidence of centers 16 hours later (at which time all aggrega-
tions were completed), was only 9% of the incidence in the control drops from
which I-cells had not been removed. If, however, the I-cells were permitted to
remain for about 5 minutes before removal, the incidence of centers rose to 40%
of the control value. Removal at 20 minutes increased the incidence to 67% of
the controls and removal at one hour was totally ineffective, i.e., equal percentages
of aggregates developed in the controls and in populations from which I-cells were
removed. Clearly, then, the presence of the I-cell in the immediate vicinity of the
R-cells, even for a few minutes, is sufficient to produce an inductive effect.
Experiments performed since then have indicated that the I-cells can exert this
inductive effect upon R-cells well outside of their immediate vicinity, albeit they
require more time to do so. Replicate samples of 6000 washed myxamoebae were
dispensed on washed agar at the optimal density of 200 cells/mm.2. The excess
fluid was absorbed by the agar and after one hour's incubation, an area, 1 mm.2,
was delineated at the center of each drop by scoring the agar surface lightly with
two pieces of razor blade, mounted parallel at a distance of 1 mm. The cells outside
of the square were brushed away, thereby leaving replicate samples of 200 myxa-
moebae at a density of 200 cells/mm.2.
Since the distribution of I-cells has been found to be 1 : 1940, one would expect
about 10% of the squares to have contained I-cells and accordingly to have ag-
gregated. As may be seen in Table I, precisely 10% of the squares so treated
did aggregate. Thus, it can be said that all of the aggregates observed must have
been contributed by those squares that contained I-cells and that no I-cells lying
<a
O.I MM.
FIGURE 5. Relation between aggregative centers and previous migratory pathways of
I-cells in four aggregations. In the top two, the centers coincided with the final positions
of the I-cells. In the bottom two, they did not. The respective times, in hours, at which the
first and last camera lucida drawings were made after deposition on washed agar : 10.8, 11.9;
9.8, 12.25; 9.8, 14.0; 10.0, 13.0.
311
312
MAURICE SUSSMAN AND HERBERT L. ENNIS
TABLE I
After the stated periods of pre-incubation, squares were scored on the agar surface and
outlying cells were brushed away. See text for details.
Pre-incubation period
in hours
No. of squares
No. with aggregates
%
1
120
12
10.0
4-5
120
20
16.7
6-8
120
59
49.0
10-12
120
109
91.0
outside the boundaries of said squares for the one-hour period and then brushed
away, appear to have exerted an inductive effect upon the R-cells lying within.
When, however, a period of 4-5 hours elapsed before the squares were delineated
and the outlying cells removed, the incidence of aggregates within the square rose
to 16.7%. After 6-8 hours the incidence was 49% and after 10-12 hours, 91%.
(At 12 hours, but not at 10, the cells were elongated and were beginning aggrega-
tion.) Thus, even at 4-5 hours, significantly more aggregates appeared than could
be accounted for simply by the presence of I-cells within the squares. The data
would therefore seem to force the conclusion that the I-cells lying outside the
squares must have exerted an inductive effect upon the R-cells within. It is
important to note that, since the total cell density was 200 cells/mm.2, the density of
the I-cells would have been about 1 per 10 mm.2. Thus the I-cells in exerting
their effect acted over truly fantastic distances.
The objection may be raised that not only the outlying I-cells were removed
but also the outlying R-cells. Why, then, could one ascribe the inductive effect to
the latter ? The answer lies in the fact that when replicate samples of from 250 to
2000 myxamoebae were dispensed at densities even greater than 200 cells/mm.2, a
Poisson distribution of aggregates was obtained in strict accordance with the dis-
tribution of I-cells. If R-cells had any inductive capacity of their own at these
densities, why then did not every sample aggregate regardless of whether an I -cell
was present or not?
In summary, the data indicate that the I-cell performs its mission at the early
stages of the pre-aggregative period. The immediate neighbors of the I-cell re-
quire its presence for only a few minutes and can then subsequently aggregate in
its absence. The more remote neighbors can also be affected if the I-cell is
alfowed to remain for a longer period of time. It is difficult to explain these
results save by the assumption of a diffusible "initiator substance."
C. The initiative capacity of R-cells
As mentioned previously, when I-cells were micromanipulated to test areas, they
could induce the test populations to aggregate, whereas R-cells at the same stage
of development could not (Ennis and Sussman, 1958b). In these experiments,
the I-cells and R-cells were micromanipulated to the test areas within 20 minutes
after they had been dispensed on washed agar. The question arose as to whether
or not R-cells which had been incubated for periods longer than 20 minutes prior
to micromanipulation might not display initiative capacity.
INITIATOR CELL
313
TABLE II
After the stated periods of £>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
0
0
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.
0
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 wrave 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 wTas 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
0
12
39
Cecropia
Cynthia
0
2
2
Cecropia
Polyphemus
0
2
1
Cynthia
Cynthia
0
0
5
Cynthia
Cecropia
0
0
20
Polyphemus
Polyphemus
0
2
0
Polyphemus
Cecropia
0
4
10
Totals
0
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 wfere 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
0
4
23
Cvnthia
Cvnthia
0
0
2
Cynthia
Cecropia
0
1
3
Polyphemus
Polyphemus
0
1
0
Polyphemus
Cecropia
0
0
1
Totals
0
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 wrere 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 Mg. 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
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338 CARROLL M. WILLIAMS
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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, ]]7oods 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 wrere fertilized arti-
ficially and almost \00c/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 3—4 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<jg. In Diopatra cuprca the unfertilized egg is oval. After
its growth period the average size of the egg is approximately 235-240 X 205-210
micra (Fig. 2). (Andrews, 1891b, gives the diameter of this egg as 400 micra ;
however, the above dimensions are based on repeated measurements by the writer. )
In living eggs the germinal vesicle is visible as a lighter region near the animal pole.
Surrounding it is an area of non-yolky cytoplasm in which are suspended bright
green pigment granules. External to these are yolk granules which increase in
number towards the vegetal pole. Their accumulation thus establishes a visible
animal-vegetal gradient and makes the egg very opacme. In reflected light under
•the dissecting microscope the eggs en masse in some batches are creamy yellow or
creamy white; in other batches, eggs have a greenish hue. With dark-field (under
low power ) the eggs are a rich yellowish cream color with brilliant green granules
obvious around the germinal vesicle. The differences in color apparent under the
dissecting microscope are due to the relative amounts of green pigment and yolk.
The egg has a clearly defined membrane, approximately 3 micra thick, which ap-
pears to be perforated by radial pores when viewed under the compound microscope.
A curious feature of the development of these eggs is the two strings of cells
attached to them during their growth period in the coelom. Andrews (1891b)
described these follicle cells and states (page 113) that "these objects were at first
mistaken for parasitic algae." These "nurse" cells are transparent (Fig. 1)
and bear a striking resemblance to blue-green algae. However, each algal-like cell
has a relatively large nucleus with a prominent nucleolus (Fig. 16). In the very
small oocytes bearing these "nurse" or follicle cells, the pigment is a brilliant
green as there is little or no yolk to mask it. These smaller eggs have a central
nucleus (Fig. 16). Subsequently, with the differential accumulation of yolk, the
nucleus becomes excentrically located, coming to lie near the animal pole (region
indicated in Figure 17).
Apparently the follicle cells are not lost until the end of the growth period in
oogenesis as a few full-size eggs have been observed with these algal-like strings
attached. Andrews (1891b) has observed that these cell strings are retained in
D. inagna until near the end of the growth period.
Fertilization. The egg is fertilizable at the germinal vesicle stage. The first
indication of fertilization is the lifting off of the egg membrane to form the fertiliza-
DEVELOPMENT OF DIOPATRA CUPREA 343
tion membrane. The perivitelline space is slight, being most obvious in the region
of the animal pole. The germinal vesicle becomes less and less distinct as the
perivitelline space forms. Usually the first polar body is given off within 20 min-
utes after insemination and the second within 30 minutes after insemination. A
small pigment-free area around the animal pole marks the position where the sec-
ond polar body will pinch off (Fig. 2). The polar bodies are small (Fig. 4), the
second polar body being somewhat larger than the first. Figure 17 shows a section
of an egg in metaphase I (the polar bodies are not visible).
Cleavage. Occasionally the two-cell stage may be observed 30 minutes after in-
semination but usually the first cleavage is not completed until 40 minutes or so after
insemination. The first cleavage furrow is meridional, cutting through the animal
pole first (Fig. 3) resulting in two blastomeres of unequal size, the AB being some-
what smaller than the CD blastomere (Figs. 4 and 18). There is some variation in
the size difference between the first two blastomeres. Blastomeres AB and CD
often divide about the same time as seen in living stages and sections. In some
cases the larger blastomere appears to divide first, as three-cell stages may be
observed (Fig. 19). It is possible, however, that these three-cell stages represent
abnormal development. The four-cell stage shows a cross furrow with the ar-
rangement of cells typical of spiral cleavage (Fig. 5). In living stages the nuclei
appear as lighter regions. The third cleavage results in an eight-cell stage with
four somewhat smaller micromeres being polar in position ( Fig. 6 ) . Cleavages
beyond these first few are amazingly rapid. A mid-cleavage stage is shown in
Figure 7. During late cleavage the blastomeres are held firmly together within
the original egg membrane, and a vacuolated peripheral area is appearing (Fig. 2SQ.
Earl\ ciliated stages (3 to 12 hours). These stages are approximately the^
size of the unfertilized egg. While there is no increase in mass, cells are continuing
to divide. Gastrulation in these very rapidly developing early stages may be
occurring by the time cilia have differentiated and probably takes place primarily
by epiboly.
Functional cilia penetrate the egg membrane within three hours after insemina-
tion. They appear to push through the pores noted above in the membrane of the
unfertilized egg. It is difficult to be certain of the ciliary distribution even when
using the phase contrast microscope, but cilia appear to cover the entire surface
except for two areas, a disc at the posterior end and the region around the future
apical tuft. The cilia thus appear to form a very broad band involving most of
the larva. At this stage peripheral vacuolated cells form four anterior plates which
surround, and appear distinct from, a central mound of denser cells ( Figs. 8, 9,
21, 23). A few small pigment spots may be observed in living larvae. Normal
larvae move in place for awhile but very shortly become surface swimmers. They
swim forward, at the same time spinning clockwise on the longitudinal axes when
viewed from the animal pole.
By slowing down swimmers with MS-222 and observing them with the phase
microscope (using dark-field which gives a strikingly beautiful picture), it is pos-
sible to get a "head-on" view of the former animal pole region. If the larvae spin
slowly enough, one can see what looks like a diagrammatic representation of both
the apical rosette (blastomeres ai.3-di.3) and annelid cross (compare Plate XI,
Figure 18, on Amphitrite in Mead's paper, 1897, page 311, or Figure 196-5, based
on Nereis, in Borradaile and Potts, page 283). Blastomeres a^-'^-d1- are less dis-
344
M. JEAN ALLEN
10
PLATE I
DEVELOPMENT OF DIOPATRA CUPREA 345
tinct but can be made out (blastomeres a1 ^-d1-2 and ai.3-cli.3 are apparently desig-
nated as a12-d12 and a13-d13 in Borradaile and Potts). The four anterior plates
of cells appear to arise from the four groups of prototroch cells and thus mark the
position of the prototroch proper beneath them. The apical rosette forms the tip
of the central mound of cells. Sometimes one or two globules (probably polar
bodies which have not yet disintegrated) are seen in the space between the central
mound and the membrane (Fig. 21).
The central mound in some 7-hour swimmers has grown almost to the animal
region of the membrane. The apical tuft, in some larvae at least, appears one to
two hours later. The cilia of the apical tuft have their origin from the central cells
at the tip of the mound (in the few cases measured, cilia were approximately 40
micra when first formed). Their origin is not surprising, for, as noted above, the
central cells make up the apical rosette which has been shown in other polychaetes
to become the apical organ of the trochophore.
Continuous with the four anterior vacuolated plates, but extending posteriorly,
are at least four yolk plates. Their formation leaves a space between them and the
medial endodermal yolk mass of the larva. Anterior vacuolated plates and pos-
terior yolk plates merge in the peripheral portion of the larval mass at about the
equatorial level. Yolk spheres similar to those in these curious thin plates can be
traced in serial sections from the posterior part of the larval mass peripherally
and anteriorly (where some are observed at the base of the vacuolated plates)
and then posteriorly just under the cuticle where they form thin plates. The
yolk plates thus appear to arise from the posterior part of the larval mass (original
vegetal hemisphere). The narrow spaces between each plate and the median mass
are continuous with each other posteriorly and are visible as slits in some 12-hour
larvae (and older ) when they rotate. All normal larvae of 12 hours have a
prominent apical tuft (one measured approximately 100 micra) which can be seen
under the dissecting microscope. Larvae swim rapidly about the antero-posterior
axis much as before with the apical tuft directed forward.
All figures are photomicrographs taken with a Makam camera. Figures 1 through 15 are
all of living stages, taken at X 100, without a cover glass except for Figure 15. Moving stages
were quieted with MS-222 (tricain). Figures 16 through 28 are all of sectioned material,
taken at X 352 except for 27 and 28 which were taken at X 220. Figures 29 through 34 are
all photomicrographs taken from "dry" mounts of larvae at X 430.
PLATE I
EXPLANATION OF FIGURES
FIGURE 1. Developing eggs before their growth period is completed, showing algal-like
strings of cells. FIGURE 2. Unfertilized egg showing lighter granular area at the animal pole
where the polar bodies will pinch off. FIGURE 3. Fertilized egg with cleavage furrow begin-
ning first at the animal pole. FIGURE 4. Two-cell stage, showing that the CD blastomere is
somewhat larger than the AB. Note the fertilization membrane and one of the polar bodies.
FIGURE 5. Four-cell stage viewed from animal pole, showing the cross furrow characteristic
of spiral cleavage. FIGURE 6. Eight-cell stage in two tiers, four slightly smaller micromeres
towards the animal pole. FIGURE 7. Early to mid-cleavage showing individual blastomeres.
FIGURE 8. Early swimming stage, approximately four hours old, showing two of the four
plates surrounding the central mound. FIGURE 9. "Head-on" view of stage similar to Figure 8,
showing four plates of cells (one at lower right clear) surrounding the mound. FIGURE 10.
Trochophore, approximately 28 hours old, with apical tuft and prototroch (haze at right
represents the beating cilia).
346
M. JEAN ALLEN
15
PLATE II
DEVELOPMENT OF DIOPATRA CUPREA 347
Trochophore stage, 24 hours old. Larvae of this stage are still approximately
the size of the unfertilized egg and are positively phototactic active swimmers,
rotating clockwise as in the preceding stages. Anteriorly, they have two red eye-
spots and a prominent apical tuft, 60-70 micra or more in length, consisting of sev-
eral long cilia surrounded by a ring of shorter cilia. The body is still covered with
cilia except for the small disc at the posterior end and a small area around the
apical tuft (Fig. 10). The cilia appear somewhat longer in the region of the
developing prototroch and telotroch. The denser central mass of cells represents
the differentiating yolk-laden mid-gut (Fig. 22). In living stages a slight inden-
tation observed on one side may represent the stomadeum. A few dark pigment
spots (green in dark-field ) in the region of the broad prototroch tend to mask
the pharynx in living larvae. In some larvae the slit-like spaces formed between
the posterior yolk plates and the underlying larval mass are still obvious ; in others,
growing cells have obliterated the slits so that the peripheral yolk plates are caught
between the mesodermal bands and the cuticle. The yolk plates are then visible
as a line of yolk spheres just beneath the cuticle (barely visible in Fig. 22). Thus,
posteriorly, the layers from inside out are the central yolk mass, mesodermal bands,
slits (in some instances), yolk plates, and larval membrane (Fig. 22).
Post-troclwplwrc stage, 36 lioiirs old. This stage is usually little longer, though
somewhat narrower, than the preceding and is characterized internally by the be-
ginnings of two to three sets of setae and the formation of glandular cells (prob-
ably mucous in nature; Figs. 24, 25, 26). The larvae are strongly positively
phototactic as evidenced by their swarming toward the light. They have prominent
red eyespots and a well developed apical tuft ( most of the cilia are approximately
85 micra, the longest measured being approximately 100 micra ) . The cytoplasm
at the level of the broad prototroch has a bubbly appearance due to refractile drop-
lets which tend to obscure the pharynx. Other external features are the narrow
telotroch, short cilia between the proto- and telotrochs, and possibly a posterior
tuft of cilia (a suggestion of this last was observed only twice, with the phase
microscope). Yellowish pigment may be observed scattered over the surface.
Visible under the cuticle posteriorly are the peripheral yolk plates. The gut from
an external view is similar to that in the preceding stage, forming a darker central
mass (yellow in reflected light). A posterior indentation may represent the proc-
todeum, as the hind-gut has not yet formed. The larvae appear to be flattened
slightly on the ventral surface. Serial sections reveal the pharynx, differentiating
setae, mucous cells, and four large posterior vacuoles which probably represent the
free ends of mucus-secreting cells (Figs. 24 and 26).
PLATE II
EXPLANATION OF FIGURES
FIGURE 11. Larva of 3% days with four sets of setae (the fourth set has retracted).
Visible are two eyespots at right, two posterior vacuolated cells at left, and darker mid-gut
region between the setae. FIGURE 12. Swimming larva, also 3% days old, with the fourth
set of setae just emerging. Note the beating prototroch at eye level and the telotroch at left.
FIGURE 13. Larva of approximately four days, showing four sets of setae, three dorsal tentacles
beginning to form, and black jaws visible through the body wall. FIGURE 14. Larva of six
days with five sets of setae and dorsal tentacles elongating. Note the eyespots, dark jaws, and
dark mid-gut region. FIGURE 15. Larva of seven days, with five sets of setae, photographed
with cover glass. Visible are two eyes, "knobby" tentacles, black jaws, light mid-gut region,
and two anal cirri.
348
M. JEAN ALLEN
f
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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 5l/2 to 71/. days. Larvae of 5l/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 8l/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 5l/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
l4l/2 clays, in two (from different batches ) by 171/2 days, and in one by IS1/-; 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 301/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 36°37'54" N and 122°01'12" 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
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O
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50
40
30
20
10
B.
"no data
J L
JFMAMJJ ASONDJFMAMJJASOND
UJ
OL
$
a:
LJ
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5
UJ
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I
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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 LABORATORY1
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 6—7 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: 284—295.
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 Pleurodeles adult. Capil-
lary visibility is good in the larvae of both species.
The animals were maintained either singly in small finger bowls or in groups
of 5 to 6 in large finger bowls, and fed with beef liver or live Tubijex. The cold-
adapted Triturus had been kept for several months in stainless steel trays in the
refrigerator and fed weekly on live Tubijex while at room temperature for a few
hours.
Experimental procedures were essentially identical for all animals as follows :
Blood was obtained by removing about 1 mm. of the tail tip with a pair of scissors
and permitting the tail to bleed directly into a drop of buffered saline on a glass
slide. The slide was examined immediately without coverslip and the outlines of
about 35 cells traced by means of the camera lucida. Placing a coverslip on the
preparation resulted in a certain amount of deformation so the practice was aban-
doned in favor of working quickly before any appreciable drying could take place.
Certain precautions that were taken should be mentioned. The slide should be
very clean to prevent deformation due to adhesion between the red cell and the
glass surface. All margins of the cell must come into focus at the same focal
setting, indicating that the cell is resting on one elliptical surface and not oriented
at an angle to the plane of observation. Following the tracing of a known linear
dimension from a stage micrometer it was possible to determine both the area and
the eccentricity of the red cell (area = y^-n-ab, and eccentricity == a/b, with a and b
the major and minor axes of the cell, respectively). A phosphate-buffered saline
(pH 7.4) was found to be a suitable medium for the cells, Q.7% NaCl being approxi-
mately isotonic for Plcnrodcles and adult Triturus while 0.6% NaCl was more
nearly isotonic for larval Triturus.
Capillary measurements were made by lightly anesthetizing the animal by short
term exposure to 0.1 % chloretone solution, rinsing in tap water and placing the
RED CELL FORM
399
animal on its side on a 5-inch square glass plate. Measurements of capillary
diameter were made on the tail fin margin by means of a calibrated ocular microm-
eter at about 430 X. Capillaries were identified as the smallest blood vessels con-
stituting a uniform size class when a given portion of the circulation was traced
from the arterial to the venous end, and through which the red cells pass in single
file. It is important that anesthesia be light as considerable capillary collapse can
occur in animals with partially arrested circulation. Measurements were restricted
to vessels through which blood was flowing in normal fashion. The measurements
on adult Triturus were carried out largely on males, not because of any sex differ-
ences but because of better visibility in the broader tail fin of the male. The animals
recovered from anesthesia in about one hour.
2.0 -•
B
1.5 --
1.0
300
U
600
90O
FIGURE 1. The relationship between cell area (^2) and cell eccentricity (a/b) in diploid
and triploid Pleurodclcs. The open circles represent the mean values for diploid and triploid
blood cells. Other points were obtained by breaking the total sample into classes of 50 fi2 and
plotting the mean values for area and eccentricity within each class. The arrow indicates the
mean value for the cross-sectional area of the capillary of the tail fin.
The relationship between red cell form and area was determined as follows with
mature larvae of Pleurodeles. Approximately 150 cells from diploid animals and
an equal number of cells from triploid animals were traced and the eccentricity and
area determined for each cell. The mean values for 2n and 3n blood cells were
determined from these samples. An additional 30 selected small cells and 30
selected large cells were measured in order to extend the analysis over the widest
possible range. The total sample was then arranged in order of increasing cell
area, and broken into size classes of 50 //,2. Within each size class the mean cell
area and eccentricity were calculated. Eccentricity (a/b} was then plotted versus
area for each size class together with the mean values for 2n and 3n blood (Fig. 1).
No significant differences were found between 2n and 3n capillary diameter and
400
JOHN DAVISON
the average capillary cross-sectional area for about 40 determinations is indicated
by the arrow in Figure 1.
Triturus came from two sources. The males used for the temperature studies
and the larvae represent stock originally from Farmville, Virginia. A small group
of female animals of uncertain origin were found to have less eccentric blood cells
than those of Farmville animals maintained at the same temperature. These females
were unusually large and probably represent a genetically distinct population. In
that regard it is interesting to note that the values reported for eccentricity in
Missouri animals are different from any of the findings in the present study
(Davison, 1957). Analyses were made of cell form, cell area, and capillary cross-
sectional area for each of the following groups of animals : Farmville males at
21° C, Farmville larvae at 21° C., females of uncertain origin at 21° C., Farmville
males adapted to 8.5° C., and the same males during the adaptation period following
transfer to 21° C. From 4 to 10 animals were measured from each group. Blood
cell findings represent the means of from 90 to 200 measurements and mean capillary
TABLE I*
Source
Temp.
0 C.
Cell
area ^
Capillary
area //2
a/b
K in the expression
(a/b) -1 =X(Aoeii/A«»p.)
Triturus
Farmville males
21
583
149
.69
0.177
Farmville males
8.5
576
113
.99
0.195
Farmville males
Farmville larvae
21 (6 days)
21
590
523
170
211
.64
.44
0.184
0.178
? females
21
579
242
.39
0.163
Pleurodeles
Mature larvae (2n)
Mature larvae (3n)
21
21
482
720
184
184
1.36
1.52
0.138
0.134
* Standard deviations for cell area were uniformly about 20% of the mean and about 10%
of the mean for capillary diameter and a/b.
size was calculated from 40 to 100 measurements. The same animals were used
for capillary and blood cell analyses. The time course experiment following transfer
from 8.5 to 21° C. was carried out on a group of 6 Triturus on which daily
measurements were made for a period of 6 days. The pertinent tabular data derived
from these studies appear in Table I.
RESULTS
Cell eccentricity as a function of cell area is plotted in Figure 1. For values
of a/b greater than about 1.3, a/b is essentially linear with respect to area and would
pass through the origin if extrapolated. For values of a/b less than 1.3, eccentricity
rapidly approaches 1 (circular form) as the area of the cell approaches the mean
cross-sectional area of the capillary (indicated by the arrow in Figure 1). It is
reasonable that if the red cell is no larger than the capillary it can pass through
without deformation, accounting for the circular form of the smallest blood cells.
Somewhat larger cells may be deformed as they pass through but not actually
applied to the wall of the capillary, an interpretation which may account for the
RED CELL FORM
401
curvilinear portion of Figure 1. Still larger cells slide through the capillary with
one elliptical surface applied to the capillary wall, with their form determined in
accordance with the linear portion of Figure 1 . The graphic information in Figure
1 may be given a somewhat more intuitive presentation as a series of forms in
Figure 2. The central circle represents the capillary area (184 p~) while the sur-
rounding blood cell forms were constructed from the data of Figure 1 at areas of
300, 500, 700 and 900 p.-. In this and all other reconstructed cell forms the blood
cells were assumed to be perfect ellipses in plane view and were first constructed
on coordinate paper employing the basic property of ellipses that .r- /a'2 + y~/b2 = 1
(a and b are the major and minor semi-axes of the ellipse).
Figure 3 illustrates the reconstructed form of larval and adult Farmville Tri-
turus cells and capillaries. The larval blood cell is slightly smaller and less eccentric
while the larval capillary is larger.
FIGURE 2. The information in Figure 1 was used to reconstruct the form of the blood cells
employing the property that x-/aT- + y-/bz = 1. The central circle is the capillary (184 fj.2).
The other figures represent cell forms at 300, 500, 700, and 900 /u2. The lower line represents
Figure 4 indicates the form differences observed in Triturus males maintained
at 8.5° C. (left) and 21° C. (center). The right hand figure illustrates red cell
form and capillary size in Triturus females of uncertain origin (21° C.). It is clear
from both Figures 3 and 4 that the larger the capillary the less eccentric the blood
cell, a result previously suggested from considerations of the model system. The
product of (a/b) -- 1 and capillary cross-sectional area (/4cap.) approximates con-
stancy for adult Triturus in which cell area (^Ceii) is essentially constant. That is :
The study of eccentricity versus cell area in Pleurodeles indicates for the linear
portion of the curve (a/b greater than 1.3) that :
(a/6) - 1 - k'Ac,u.
402
JOHN DAVISON
iou
FIGURE 3. The form of the blood cells and capillaries of adult (left) and larval (right)
Farmville Triturus reconstructed from the tabular data in Table I. The larval blood cell is
slightly smaller.
10 U
FIGURE 4. The mean forms of blood cells and capillaries in Triturus adults constructed
from the data in Table I. Farmville males at 8.5° C. (left), Farmville males at 21° C. (center),
and females of unknown origin at 21° C. (right). All cells have approximately the same area.
RED CELL FORM
403
These two expressions may be combined to give an equation relating all three
variables : a/b, Acen, and ACiip_ :
(a/b}-l=K(Acen/Acap.).
The extent to which this equation adequately describes the relationship between
these variables is clear from the uniformity of the constant K calculated from the
data in Table I. Triturus values vary from 0.163 to 0.195, while the Pleurodeles
values are somewhat smaller being 0.138 and 0.134 for diploids and triploids,
respectively.
B
FIGURE 5. The time course of the adaptation from 8.5° C. to 21° C. in Farmville male
Triturus. Closed circles are capillary cross-sectional area measurements and open circles red
cell area measurements (right ordinate). Triangles are eccentricities of the red cells (a/b)
(left ordinate). Notice the break in the right ordinate (^2)-
A point of considerable interest is the time at which red cell form is determined.
Two possibilities might be considered. The form of the cell might be determined
at the time it first enters the circulatory system with subsequent changes in the
capillary environment having no further effect on cell form. If this were true,
average cell form should change slowly following a change in capillary diameter,
with the total time period for the change equal to the life span of the erythrocyte.
On the other hand, if red cell form is plastic, one might expect a more rapid
response in red cell form following a change in capillary diameter, a result which
would support the concept that red cell form is constantly subject to the forces
acting on the cell during its passage through the capillaries. The latter view is
404 JOHN DAVISON
clearly favored by the time course data represented in Figure 5. Following trans-
fer of the animals from 8.5 to 21° C., the increase in capillary diameter is complete
within 3 days, while the change in red cell form is largely complete within the
same period of time. There is, however, a clear lag in the cell form response and
a somewhat greater time for the complete form transformation. It is interesting
to note that there is a small transient increase in cell area corresponding to the
time period when cell form is most rapidly changing. This increase may represent
a temporary change in the osmotic properties of the cell dependent on alterations
of the cell surface.
DISCUSSION
The quantitative and temporal relationship between red cell form and capillary
size clearly supports the concept that the form of the cell is determined, at least in
part, by the size of the capillary through which the cell is passing. Alterations in
capillary diameter lead to changes in cell form with kinetics supporting a view that
the red cell form is plastic and not fixed. Since the reasoning leading to these
analyses was influenced by considerations of the model system, a comparison be-
tween the model and the biological systems may be useful. I would like to point out
first, however, that model systems serve only to guide rationale, and certainly should
not be taken as literal representations of biological reality.
(1) There are gross differences between the relative sizes of the "capillary"
and the "cell." In the model the cylinder is many times larger than the mercury
drop. In the living system the cell is elliptical only if its area exceeds that of the
capillary cross-section, while in the model eccentricity steadily increases with drop
area since the drop is always in contact with the cylindrical surface.
(2) There is abundant evidence that the interior of the amphibian cell is fluid
like the mercury drop. Norris studied the manner in which displaced nuclei re-
turned to the center of the cell and concluded that except for the nucleus the cell
interior is liquid with the shape of the cell conferred by an outer envelope (Norris,
1939). Dawson presumed a liquid interior based on the observation of Brownian
movement in the cell interior (Dawson, 1928). Based on microsurgical findings,
Seifriz described the cell interior as essentially liquid with a plastic and elastic
cell envelope approximately 0.8 p. in thickness (Seifriz, 1926). The envelope thick-
ness agrees well with more recent estimates based on polarized light analyses of
human red cells (Mitchison, 1953).
(3) The model system is static while the living system is of course dynamic since
the cells are constantly moving through the circulatory system. It is of interest to
note that although the blood cells are ellipses with symmetrical ends as observed
at rest, they clearly do not have this form while passing through the capillaries. The
advancing end of the cell is more rounded than the trailing end so that if the
cell could be removed and flattened it would approximate a pear shape in plane
view. This configuration may also be imitated with the model by tipping the
cylinder and permitting the mercury drop to slowly flow along the surface of the
cylinder. The dynamic form of the blood cell may be interpreted as further evi-
dence for a fluid red cell interior.
In summary, then, it appears that red cell form is a consequence of physical
forces operating between the cell and the capillary wall. In answer to the classical
dilemma of whether form determines function or function determines form one may
RED CELL FORM 405
arrive at the inadequacy of either of these alternatives and conclude on the basis
of these studies : form determines form.
SUMMARY
1. A mercury drop in contact with a cylindrical surface takes the form of a
flat elliptical disc. Increasing the volume of the drop causes an increase in the
area and eccentricity of the drop but causes no appreciable increase in thickness.
With constant drop volume, the larger the cylinder the less eccentric the fluid drop.
2. Analyses of blood cell form and capillary diameter in Triturus and Pleurodclcs
disclosed the following relationships. The red cell is circular if its area does not
exceed that of the capillary. Eccentricity increases first in a curvilinear and then
in a linear fashion as the red cell increases beyond the cross-sectional area of the
capillary. Under conditions of essentially constant red cell area, eccentricity is
inversely related to the cross-sectional area of the capillary.
3. Based on the experimental findings the following equation may be derived
relating red cell area, capillary cross-sectional areas and eccentricity (a/b) :
4. Evidence for the physical nature of the red cell was discussed in relation
to the model system.
LITERATURE CITED
DAVISON, J., 1957. A fluid drop model of the elliptical red blood cell. Expcrientia, 13 : 472.
DAWSON, A. H., 1928. The segregation apparatus of the amphibian erythrocyte and its possible
relationship to the Golgi apparatus. Anat. Rcc., 39 : 137-152.
FANKHAUSER, G., 1952. Nucleo-cytoplasmic relations in amphibian development. Internal.
Rev. Cyt., 1 : 165-193.
MITCHISON, J. M., 1953. A polarized light analysis of the human red cell ghost. /. Exp. Biol.,
30: 397-432.
NORRIS, C. H., 1939. The tension at the surface and other physical properties of the nucleated
erythrocyte. /. Cell. Comp. Physiol., 14: 117-133.
SEIFRIZ, W., 1926. The physical properties of erythrocytes. Protoplasma, 1 : 345-375.
THOMPSON, D'ARCY, 1942. Growth and Form. The Macmillan Company, New York.
THE CONTROL OF REPRODUCTION IN DIPLOPTERA
PUNCTATA (BLATTARIA)1
FRANZ ENGELMANN
Department of Anatomy, Albert Einstein College of Medicine, Nciv York 61, N. Y.
In cockroaches, as in many insect species studied, a hormone released by
the corpora allata is necessary for the maturation of eggs (Scharrer, 1946; Engel-
mann, 1957). A variety of intrinsic and extrinsic factors, such as pregnancy,
egg resorption, or involution of the corpus luteum (Liischer and Engelmann, 1955;
Engelmann, 1957), food supply (Scharrer, 1946; Johansson, 1955, 1958; von
Harnack, 1958), and probably humidity and temperature are involved in the con-
trol of the function of the corpora allata. Furthermore, in some cockroach
species, mating accelerates the growth rate of the eggs and, therefore, increases the
total number of eggs produced during the lifetime of the animal (Griffiths and
Tauber, 1942; Roth and Willis, 1956; Wharton and Wharton, 1957).
In Diploptera punctata, a viviparous cockroach, the influence of mating, an
extrinsic factor, on egg maturation plays a more dominant role than in related
species. In virgins, the maturation of the first batch of eggs takes several weeks
or even months (Roth, personal communication), whereas after mating only 10 days
are required for egg maturation (Roth and Willis, 1955). Since a variety of
factors known to condition the activity of insect ovaries reach the gonads via brain
and corpora allata (Scharrer, 1958) it seems probable that the stimuli exerted
by the act of mating influence the ovary by the same route.
The present paper deals with the elucidation of the control mechanism govern-
ing female reproduction in Diploptera. Particular emphasis was given to the study
of the pathways by which the mating stimulus reaches the ovary.
MATERIAL AND METHODS
All experimental animals were taken from a stock colony and maintained in finger
bowls at room temperature of 22—26° C. They were fed dog chow and water.
Daily records were kept of the reproductive activity of the females. The presence
of a spermatophore in the bursa copulatrix of the female indicated mating, that of
an egg case in the brood sac ovulation.
Experiments such as castration, allatectomy, excision of the gonapophyses, and
severance of nerves were performed under CO2 anesthesia. The nervi corporis
cardiaci or the nervi corporis allati were severed either by frontal or occipital
approach.
For the histological investigation of the corpora allata and for the determination
of the completeness of the severance of nerves an organ complex consisting of
brain, corpora cardiaca, and corpora allata was fixed either in Bouin-Duboscq's or in
Zenker's fluid. The sections were stained in Gomori's chrome hematoxylin phloxin
1 Supported by U.S.P.H.S. Grant C-3413 administered by Dr. Berta Scharrer, and by a
travel grant from Deutsche Forschungsgemeinschaft.
406
REPRODUCTION IN DIPLOPTERA 407
or in aldehyde fuchsin as modified by Halmi. The volume of corpus allatum tissue
per one million nuclei was determined by a formula given in an earlier paper
(Engelmann, 1957). The data obtained, expressed as mean values for both corpora
allata of a given specimen, indicate the degree of activity of the corpora allata;
these values were correlated with the observed length of the oocytes.
RESULTS
1. The cyclic activity of ovary and corpus allatum
In ovoviviparous or viviparous cockroaches there is no space for additional
eggs within the brood sac of the female during the development of the embryos.
Egg maturation in the ovaries must, therefore, be temporarily suppressed. Since in
other species of cockroaches egg maturation depends on the activity of the corpora
allata, we first looked for a possible relationship between the structure of the corpora
allata and ovarian activity in Diploptcra during an interval encompassing the first
pre-oviposition, first pregnancy, and second pre-oviposition periods. The results
which are reported in the following paragraphs provided a basis for experimental
work.
In Diploptera mating takes place a few minutes after emergence (Roth and Willis,
1955). Within 9.43 ± 0.22 days thereafter (53 animals observed in our labora-
tory) the largest oocyte of each ovariole grows from about 0.55 mm. to a size
of approximately 1.5 mm. and then ovulates. Large amounts of yolk are deposited
in the oocytes during their period of growth.
No eggs ripen in the ovaries during pregnancy, which follows ovulation and
which lasts for 75.49 ± 1.47 days (45 animals ; see also Willis et al, 1958). There
is merely a slight growth of the oocytes, but no yolk whatsoever is deposited until
the second or third day before parturition. Then a more rapid growth of the
oocytes accompanied by yolk deposition begins, so that on the day of parturition
the largest eggs in the ovarioles measure about 0.87 mm. (Fig. 1). Within the next
three or four days the eggs grow to full size; 5.98 ± 0.17 days after parturition
these mature eggs ovulate (45 animals).
The activity of the corpora allata, as judged by the amount of cytoplasm, cor-
relates with the activity of the ovaries. The relative and absolute amount of
cytoplasm in these glands gradually increases during the first pre-oviposition period
until a maximum of activity is reached at the fourth day after mating (Fig. 1).
Values of less than 0.95 mm.3 of total tissue per one million nuclei indicate that this
gland is inactive; more than 1.45 mm.3 tissue per one million nuclei represents a
highly active gland. Between these figures there are varying degrees of activity.
The cells of the corpora allata maintain a high activity for about four days, during
which time most of the yolk is deposited in the oocytes. Shortly before ovulation,
a decrease in the activity of the corpora allata is observed, so that on the day of
ovulation the corpora allata contain only a small amount of cytoplasm.
During pregnancy, when the ovaries are quiescent, the corpora allata are in-
active, as shown by histological criteria (Fig. 1 ; see also Table II, a) ; the distribu-
tion of the nuclei in the corpora allata is similar to that on the day of emergence,
i.e., 0.84 mm.3 gland tissue per one million nuclei. Shortly before parturition an
activation of the corpora allata can be observed which gradually increases until
408
FRANZ ENGELMANN
the second or third day after parturition (Fig. 1). After reaching a peak of ac-
tivity, the cytoplasmic content of the glands decreases. The corpora allata are
nearly inactive again at the time of the second ovulation.
The second pre-oviposition period is three days shorter than the first. The
growth rate of the oocytes, however, is about the same in both periods (Fig. 1).
The shortening of the second period is merely due to the fact that yolk deposition
in the second batch of oocytes begins already during the late days of pregnancy.
The average peak of corpus allatum activity in the second reproductive period is
below that of the first, but this difference is statistically insignificant. The reason
for the variability of the data is, at least in part, that the onset of the second period
of activity is not as clearly defined as that of the first period. Therefore, the
1234
1st Egg maturation
8 9
Days
Pregnancy
1234
2nd Egg maturation
5 6
Days
FIGURE 1. Diagram indicating the morphological changes signalling cyclic activity in the
corpora allata of Diploptcra (upper curves), which are correlated with the growth of the
oocytes (lower curves) during the first and second pre-oviposition periods.
corpora allata of some females may have surpassed their highest activity when
those of other females, fixed after the same interval, are still maximally active.
2. The junction of the corpora allata in the adult female
The role of the corpora allata in the ovarian control of Diploptera was further
elucidated by the removal of these glands. Allatectomy was performed two to
four days after mating. In all nine animals thus operated the deposition of yolk
in the oocytes beyond the stage characteristic of the day of operation was pre-
vented. Twenty days after mating degeneration of most of the oocytes of the al-
REPRODUCTION IN DIPLOPTERA 409
latectomized animals had begun. It appears that by the fourth day after mating
not enough corpus allatum hormone has been released into the circulation to permit
the completion of egg maturation. Thus in Diploptera, in the same way as in
other species of cockroaches, egg maturation depends on the activity of the corpora
allata not only during the initial phase, but also during the time when most of the
yolk is being deposited in the oocytes.
The activity of the accessory glands of the female genital apparatus also de-
pends on the function of the corpora allata, since no secretory material is detectable
in the lumen of these glands in animals allatectomized shortly after mating. There
is yet another type of secretory cell that seems to be under the control of the corpora
allata as the following observation indicates. Around the sixth day after mating
the spermatophore is extruded from the bursa copulatrix (53 animals). At the
same time an aqueous fluid appears in the genital apparatus, which probably facili-
tates the extrusion of the spermatophore. In allatectomized females the spermato-
phore stays within the bursa, and the genital apparatus remains dry. In four nor-
mally mated females, in which for undetermined reasons the corpora allata did not be-
come active, as shown by the failure of eggs to mature, the spermatophores re-
mained in the bursa copulatrix for 18 days at which time the females were fixed
for further investigation.
Thus it appears that the hormone released by the corpora allata serves at least
three different functions in the adult female of Diploptera. It causes the deposi-
tion of yolk in the oocytes, it activates the accessory glands of the female genital
apparatus, and it stimulates an as yet unknown type of cell presumably present in
the bursa copulatrix, whose secretion facilitates the extrusion of the spermatophore.
The function of the accessory glands and of the secretory cells in the bursa seems
to be regulated directly by the corpora allata, since this mechanism is not disturbed
by ovariectomy.
3. The control of the activity of the corpora allata by the brain
In Diploptera, as in many other insect species studied, the brain innervates the
corpora allata via the nervi corporis allati, which are a component of the nervi
corporis cardiaci. Severance of these nerves was attempted in 44 virgins of Diplop-
tera, one to 10 days after emergence. Activation of the ovaries, i.e., deposition of
yolk in the oocytes, resulted in 39 of these animals, whereas in the five remaining
roaches no ovarian activity was found. A histological study of the brain-corpora
cardiaca-corpora allata complex of all 44 experimental animals was undertaken to
check for the completeness of the severance of the nerves. In 16 of these animals,
fixed nine to 33 days after operation, no severance or other disturbance of the in-
nervation of the corpora allata could be detected histologically. This group of 16
virgins includes the five cases mentioned above that matured no eggs ; the remaining
11 had matured their eggs. In all of these 16 virgins the corpora allata were in-
active at the day of fixation. Obviously, the nerve severance had failed in this
group. But how can egg maturation in 11 of these virgins be accounted for?
Perhaps in these cases the nerves were squeezed or stretched during the operation,
a situation which interfered with nerve conduction and thus led to temporary activa-
tion of the corpora allata. Apparently the brain must have resumed the inhibition
of the corpora allata a few days later.
410 FRANZ ENGELMANN
In 21 animals of the operated group an interruption of the nerves which in-
nervate the corpora allata could be detected histologically ; all of these animals had
active corpora allata, as determined by histological criteria. In all, egg maturation
took place as a result of the activation of the corpora allata within a period similar
to that normally observed after mating. From this it seems that mating does not
cause a higher activity of the corpora allata than that which results from severance
of the nerves that transmit inhibitory impulses to the corpora allata. One might
conclude, therefore, that mating prevents the flow of inhibitory nerve impulses. In
the remaining seven animals, which also matured their eggs, the success of the
operation could not be determined, because parts of the tissue were lost during the
histological procedure. It appears that severance of the nervi corporis allati in
virgins results in activation of the corpora allata, which indicates that in unmated
young females the brain restrains the function of the corpora allata by way of the
nervi corporis allati.
It was of further, interest to determine whether the corpora allata severed from
the restraining center in the brain remain active permanently or only for a period
sufficient to induce the maturation of one batch of eggs. In order to test this point,
15 virgins with severed nervi corporis allati were fixed 14 to 31 days after their
first ovulation had taken place. In eight of these animals only one egg maturation
had occurred ; the size of the next oocytes in line in the ovarioles was about 0.40
mm. at the time of fixation. This size is comparable to the measurements in preg-
nant females at a similar interval after ovulation. The corpora allata of these eight
animals were inactive as determined by histological criteria. The innervation of
the corpora allata in these animals seems to have been incomplete. A few intact
nerve fibers were observed in the sections. It may be that these had been squeezed,
and thus prevented from inhibiting the corpora allata for a short time, or these
nerve fibers may have regenerated and thus may have resumed inhibition of the
corpora allata. This might have been the case also in four additional virgins in
which egg maturation had begun a second time, but had not progressed beyond an
initial phase; here the largest oocytes measured on the average 0.62 mm. 20 to 30
days after the preceding ovulation, whereas in normal pregnant females, after a
similar interval, the oocytes measured only about 0.40 mm. In these operated
virgins the resumption of the inhibition of the corpora allata may have taken more
time and, therefore, a second growth period of the oocytes had been initiated. Only
in three specimens, in which the separation of the corpora allata from the brain
was complete at the time of fixation (interval over 40 days), the glands were active
(average of 1.32 mm.3 gland tissue per million nuclei). In these three animals,
the corpora allata had induced the growth of a second batch of eggs to full size
(1.50 mm.) within 24 to 30 days after the first ovulation.
Thus, severance of the corpora allata from the restraining center in the brain
results in a sustained activity of the glands. This activity persists at least long
enough for two successive batches of eggs to mature. In the three animals with
completely severed corpora allata the maturation of the second batch of eggs took
considerably longer than that of the first. This may have two reasons. In the
first place yolk deposition begins when the oocytes measure about 0.55 mm. which
means that the oocytes next in line after ovulation have first to grow from about
0.35 mm. to 0.55 mm. This initial growth of the oocytes results in a prolongation
REPRODUCTION IN DIPLOPTERA
411
of the pre-oviposition period which immediately follows ovulation. A second
factor may be a restraining influence coming from the corpus luteum which, during
its involution, seems to inhibit the growth of the next oocyte in line, as was reported
in Leucophaea (Engelmann, 1957).
4. The activation of the corpora allata by afferent stimuli
In mated females of Diploptera the growth of the oocytes accompanied by the
deposition of yolk began about one day after emergence, and was completed within
10 days thereafter (53 animals; see also Table I, a). Among 45 virgins, on the
other hand, the shortest interval in which egg maturation occurred was 37 days
(one animal) ; at 60 days after emergence eight additional virgins had ovulated.
TABLE I
Experiments during first pre-oviposition period
Operation
(days after
emergence)
Fixation
Number
of
animals
Corpus
allatum
(mm. 3/l
million
nuclei)
Ovary
(length of
the largest
oocytes
in mm.)
(Days after
operation)
(Days after
emergence)
a. Control : mated females
—
—
4-9
12
1.49±0.08
1.32±0.08
b. Severance of ventral nerve
cord. No mating
1-3
3-23
4-24
8
0.95 ±0.03
0.60 ±0.02
c. Severance of ventral nerve
cord followed by mating
1
6-20
7-21
8
0.85±0.02
0.61 ±0.02
d. Artificial mating stimuli
5-19
3-14
17-26
8
1.03 ±0.08
0.72±0.04
e. Excision of gonapophyses
1-5
8-11
9-14
12
1.33 ±0.14
1.11±0.10
f. Control: virgin females
—
—
5-31
8
0.84 ±0.02
0.59±0.01
Numbers following ± are standard errors.
Of the remaining 36 animals 13 virgins ovulated within a period of two to five
months, whereas 14 animals had not ovulated when they either died or were dis-
continued for other reasons. Nine virgins had not ovulated even after five months.
It is not definitely known whether or not all virgins eventually produce mature
eggs without mating. It is obvious, however, that mating is essential for a normal
growth rate of the oocytes maturing after emergence.
One may now ask, whether mating is equally essential to induce the normal
growth rate of eggs maturing after the first parturition. Therefore, 24 females
were isolated at parturition. The next ovulation occurred 5.83 ± 0.28 days there-
after. In eight additional females, in which mating was allowed to take place on the
first day after parturition, the pre-oviposition period took 5.75 ± 0.37 days. Thus,
mating does not accelerate the growth of the eggs maturing after parturition.
From this it appears that giving birth may serve as a stimulus replacing that of
412
FRANZ ENGELMANN
mating from the induction of egg maturation at a normal rate. Furthermore, there
is sufficient viable sperm stored in the spermathecae from the mating before the
first pregnancy, so that additional batches of eggs can be fertilized.
If parturition activates the corpora allata and consequently the ovaries, how is
it possible that yolk deposition in the second batch of oocytes begins a few days
before parturition (Fig. 1)? The following observation may give an answer to
this question. During the late days of pregnancy, when the abdomen of the female
is distended by the ootheca, the animal rhythmically contracts the abdomen. The
resulting movements of the egg case in the genital apparatus presumably initiate
the activity of the corpora allata occurring before parturition. The observation
that mating, as well as parturition, and presumably movements of the egg case in
the genital apparatus, activate the corpora allata suggests that sensory receptors
located in the genital apparatus receive those stimuli. It is likely that movements
of the young hatching within the vagina (Roth and Willis, 1955) enhance the
TABLE II
Experiments with pregnant females
Operation
(days after
ovulation)
Fixation
Number
of
animals
Corpus
allatum
(mm.3/l
million
nuclei)
Ovary
(length of
the largest
oocytes
in mm.)
(Days after
removal of
ootheca or
parturition)
(Days after
ovulation)
a. Control : pregnancy
—
—
4-62
11
0.83 ±0.02
0.40±0.01
b. Removal of ootheca
0-41
3-31
19-58
13
0.95±0.03
0.56±0.06
c. Removal of ootheca followed
by mating (within 4 to 6
days)
35-38
8-15
45-50
5
1.35±0.12
1.20 ±0.19
d. Control: parturition
—
4-5
—
5
1.10±0.08
1.14±0.06
Numbers following ± are standard errors.
activity of the corpora allata resulting from the rhythmical contraction of the ab-
domen prior to parturition. It is not known, however, whether the initial activa-
tion of the corpora allata by the movement of the egg case in itself would be suf-
ficient to bring about complete egg maturation. From the facts reported here and
in the preceding paragraphs it appears that in Diploptera afferent stimuli are solely
responsible for the induction of a normal growth rate of the oocytes in the ovaries.
This conclusion was further substantiated by an additional experiment. After
the removal of the egg cases from five pregnant females maturation of the next
batch of eggs took at least 41 days when no mating was permitted. On the other
hand, if mating was allowed after egg case removal, the oocytes matured within 9
days after mating (Table II, c). The time required for the maturation of the eggs
after removal of the egg case and subsequent mating, and that after mating following
emergence are practically the same. Again, an external factor accounts for the
induction of egg maturation at a normal rate.
REPRODUCTION IN DIPLOPTERA 413
If stimuli, received in the genital apparatus during mating and parturition, acti-
vate the corpora allata, a transmission of these stimuli to the brain and corpora
allata via the ventral nerve cord has to be postulated. In order to test this point the
ventral nerve cord was severed in 16 virgins 1-3 days after emergence. Eight of
these animals were allowed to mate afterwards, whereas another eight animals were
kept isolated as controls. None of the operated and mated females showed matura-
tion of the eggs. The corpora allata must have remained inactive and, conse-
quently, in these animals yolk deposition was completely prevented (Table I, c).
The same was the case in the eight females, which did not mate after severance of
the abdominal nerve cord (Table I, b). From these experiments one may con-
clude that stimuli received in the genital apparatus during the act of mating, and
presumably also during parturition, are transmitted via the ventral nerve cord to
the brain and corpora allata.
Further experimental proof for the conclusion that stimuli are received in the
genital apparatus seemed desirable. Therefore, a small artificial spermatophore,
made of glass, was pushed into the bursa copulatrix of virgins (Table I, d) . Most of
these artificial spermatophores remained in the bursa copulatrix for the duration of
the experiment, but in some cases they were extruded and had to be replaced. A
few days after successful implantation of the "spermatophores" the animals were
sacrificed in order to check the degree of maturation of the oocytes and to determine
histologically the state of the corpora allata. Among eight animals five had be-
gun to deposit yolk in the oocytes. This result is not as clear-cut as that after
mating (Table I, a), which might be due to the fact that a glass spermatophore is
too smooth to afford sufficient stimulation. Furthermore, normal mating may last
up to several hours during which time the movements of the male provide a more
efficient stimulation of the genital apparatus than that caused by deposition of a
smooth artificial glass spermatophore.
Apparently mating alone, without the deposition of a spermatophore, also induces
activity of the corpora allata (Roth, personal communication). On the other hand,
from the fact that yolk deposition in some of the experimental animals was in-
duced by artificial mating, it appears that mechanical stimulation during the act of
mating or during parturition tends to activate the corpora allata and in turn to
induce maturation of the oocytes.
As stated above, it was thought that sensory receptors on the gonapophyses of
Diploptera are involved in the perception of these mechanical stimuli. For this
reason in 12 virgins all three pairs of gonapophyses were excised in order to pre-
vent the perception of stimuli, when mating was allowed afterwards. However,
none of the virgins thus operated upon accepted a male within the duration of the
experiment. Nevertheless of the 12 operated females, 10 were found to have de-
posited yolk in the oocytes after a few days ; in some females the eggs had reached
nearly full size (Table I, e). Probably the afferent nerves were stimulated by the
excision of the gonapophyses and this resulted in activation of the corpora allata
and of the ovaries. The conclusion that sensory receptors on the gonapophyses
receive the stimuli during the act of mating or parturition seems, therefore, justi-
fied. This experiment does not exclude the possibility that other parts of the
genital apparatus supplied with sensory receptors may also be involved in the per-
ception of mating stimuli. As the following experiment shows, this proved actually
414 FRANZ ENGELMANN
to be the case. In three virgins with gonapophyses excised shortly after emergence
mating eventually took place 21, 28, and 52 days after operation. Nine to ten
days later, i.e., after an interval normally required for egg maturation in mated
females, ovulation took place. Thus, we conclude that sensory receptors on the
gonapophyses and on other parts of the genital apparatus receive the stimuli ex-
erted by mating and parturition.
5. The effect of mature eggs on the corpora allata
In other species of roaches mature eggs seem to exert an inhibitory effect on
the corpora allata (Engelmann, 1957). The question arises whether in Diploptera
mature eggs, either before ovulation or during embryonic development in the brood
sac, likewise inhibit the corpora allata in their function. For this reason, the egg
cases were removed from the brood sac during anesthesia in 13 females of Diplop-
tera, at different stages of pregnancy. These animals were kept isolated to pre-
vent mating and were fixed three to 31 days thereafter in order to check the degree
of activity of the corpora allata and of the ovaries. None of these 13 females ma-
tured their eggs in the ovaries within the tested period (Table II, b). Additional
five females, in which the oothecae were removed three to five days after ovulation,
were kept until the next ovulation occurred. This took place 42, 51, 54, 57, and
71 days after the removal of the oothecae. Thus, the period required for egg
maturation after removal of the egg case is roughly comparable to that observed
in virgins. In other words, in this experimental series in which appropriate af-
ferent stimulation was lacking, the corpora allata were retarded in their activity,
but not completely suppressed.
By contrast, egg maturation is inhibited throughout the period of pregnancy
which in Diploptera lasts 75 days. Therefore, the absence of mating or equivalent
stimuli alone would not account for this complete state of quiescence in the ovary
which in turn signals complete inactivity of the corpora allata. An additional in-
hibitory action seems to come from the ootheca and, in analogy with the known
situation in Leucophaea, it may be due to a humoral factor.
As to the mechanisms which, in the absence of appropriate stimuli such as
mating, keep the corpora allata of adult females of Diploptera partially suppressed,
nothing can be added to the already discussed inhibitory nervous link between brain
and corpora allata. It may well be that the factor eliciting this inhibition acts
independently of the inhibitor released by the egg case.
Further support for the concept that a restraining factor from the eggs is not
the sole or even primary afferent link governing the inhibition of the corpora allata
of Diploptera was obtained by castration experiments Eleven females were ovari-
ectomized two to three days after mating. Yolk had already been deposited in the
oocytes, which indicated that the corpora allata had been activated by mating.
Twelve to 44 days after this operation the females were fixed in order to check
histologically the activity of the corpora allata. The glands were completely in-
active as indicated by the calculation of an average of 0.86 mm.3 of gland tissue per
million of nuclei. From this it again appears that an as yet unknown mechanism
residing outside the ovary inhibits the corpora allata. These findings in Diplop-
tera are in contrast to observations in several other species of insects including
Leucophaea (von Harnack and Scharrer, 1956) where ovariectomy results in hy-
peractivity of the corpora allata.
REPRODUCTION IN DIPLOPTERA
415
Brain ^^
Corpora
Ovary
Egg case
Genffal apparatus
FIGURE 2. Diagram illustrating the control of reproduction in Diploptera. Stimuli exerted
by mating or parturition, received in the genital apparatus, are transmitted to the brain where
they inhibit the normal flow of restraining impulses from the brain to the corpora allata. The
corpora allata then release gonadotropic hormone which induces egg maturation. During
pregnancy, complete inhibition of the corpora allata is accomplished, presumably by a humoral
principle originating in the developing eggs. For further details see text.
DISCUSSION
The control of egg maturation in the viviparous cockroach, Diploptera, depends
on the following sequence of events (Fig. 2) : Stimuli exerted by mating, which
are received by sensory receptors on the gonapophyses or other parts of the genital
apparatus, ascend via the ventral nerve cord to the brain and act to inhibit the normal
flow of restraining impulses from the brain to the corpora allata. The corpora
416 FRANZ ENGELMANN
allata then release a hormone that induces the maturation of the eggs. Parturition
provides stimuli adequate for eliciting egg maturation in the second or subsequent
cycles, and no mating is then required. In virgins, the corpora allata seem to be
completely inhibited at first, but become slowly activated after some time and
eventually induce egg growth. During pregnancy another controlling factor origi-
nating in the ootheca makes the inhibition of the corpora allata complete.
As in Leucophaea (Scharrer, 1952; Engelmann and Liischer, 1956), and in
Oncopeltus (Johansson, 1958), the brain in Diploptera restricts the function of
the corpora allata by way of the nervi corporis allati. This can be concluded
from the fact that severance of these nerves in the species mentioned results in
persistent activity of the glands.
The fact that the brain inhibits the corpora allata in Leucophaea and Diploptera
at certain periods suggests that extrinsic and intrinsic "information" is integrated in
the brain and then passed on as messages to the corpora allata. In Leucophaea
the presence of mature eggs in the brood sac causes the brain to inhibit the corpora
allata by way of the nervi corporis allati during pregnancy. No eggs mature in
the ovary during this period. On the other hand, in Diploptera, the brain converts
stimuli received in the genital apparatus with the result that inhibitory impulses to
the corpora allata cease. The corpora allata then become active and induce egg
maturation. In both cases the brain seems to be the regulating center for the
function of the corpora allata, and there is evidence that also in other insect species
the brain functions as a controlling center for a variety of different endocrine func-
tions (Wigglesworth, 1934; Scharrer, 1958).
Extrinsic factors such as mating and parturition, are particularly important for
the proper control of the reproductive cycles in Diploptera. In other species of
cockroaches, at least one of these factors, namely mating, is involved in reproduc-
tion. In Periplaneta americana, for instance, mating accelerates growth of the
eggs and consequently more eggs are produced during the animal's lifetime if it is
permitted to mate (Griffiths and Tauber, 1942; Roth and Willis, 1956). In this
species, however, the influence of mating seems less pronounced than in Diploptera.
The same is true for Leucophaea maderae where mating also enhances the activity
of the corpora allata during the first pre-oviposition period. Egg maturation in this
species is accelerated by mating only by about one-third of the period required in
virgins (Engelmann, unpublished data) in contrast to the observation in Diploptera
where the acceleration of egg maturation upon mating amounts to at least five
times ; on the average even more.
In the second and subsequent reproductive cycles of Diploptera the stimuli
normally occurring in parturition are essential in replacing those of mating. In
Periplaneta and in Leucophaea, on the other hand, no comparable stimulation seems
to be required as is shown by the following observations. In females of Periplaneta,
isolated after their first mating, egg maturation in successive batches took only
slightly longer than that in females which were continuously kept with males
(Griffiths and Tauber, 1942). In this species parturition does not occur and,
therefore, mating would have to replace the effect of giving birth as described in
Diploptera. In contrast to the situation in Diploptera, mating in Leucophaea after
removal of the egg case did not shorten the time needed for egg maturation (Engel-
mann, unpublished data). At the present time no explanation can be given for the
REPRODUCTION IN DIPLOPTERA 417
fact that in Periplaneta and in Leucophaea mating influences egg maturation only
during the first pre-oviposition period and not thereafter. From this it seems that
Diploptera is unique among cockroaches with respect to the degree to which stimula-
tion of the corpora allata and consequently activation of the ovaries depends on
afferent nervous stimuli.
In females of Diploptera the normal mating act can be simulated by "artificial
mating," a fact which indicates that mechanical stimulation of parts of the genital
apparatus is the essential factor involved. It might be worth mentioning in this
connection that in some mammals such as the ferret, rabbit, weasel, cat, mating
likewise is essential for the completion of egg maturation and ovulation. In the
cat, ovulation can be brought about even by artificial stimulation of the cervix
uteri by means of a glass rod (Greulich, 1934; Porter et al., 1957). Here, upon
mechanical stimulation of an area in the genital apparatus, the brain activates the
hypophysis, which in turn induces ovulation by releasing gonadotropic hormones ;
this is analogous to the situation in Diploptera. One should point out that in the
cat maturation of the egg nucleus and the rupture of the follicle is induced by
mating, whereas in Diploptera it results in deposition of yolk in the oocytes. These
are two fundamentally different processes related to different reproductive mecha-
nisms, but both are induced by mating and both finally lead to ovulation.
In Diploptera, as in Leucophaea, we observe alternating activity and inactivity
of the corpora allata correlated with periods of egg maturation and of quiescence
of the ovaries. In this respect the reproductive patterns of two related species are
strikingly similar, but as was shown the regulation of these reproductive cycles is
achieved in different ways. In the female of Diploptera the corpora allata seem to
be maintained at a low level of activity. For every activation of these glands,
sufficiently high to induce egg maturation within a normal period of time, a stimula-
tion of the genital apparatus has to take place. On the other hand, in most virgins
of Leucophaea the corpora allata appear to be only slightly inhibited during the first
pre-oviposition period. During pregnancy, in Diploptera, a substance furnished by
the mature eggs in the brood sac inhibiting the corpora allata seems not to be the
primary restraining principle. In pregnant Leucophaea, however, such a sub-
stance originating in the eggs plays a dominant role in the control of the corpora
allata (Engelmann, 1957). Thus, in both related species we find humoral as well
as nervous afferent stimuli involved in the regulation of reproduction. They seem
to operate independently of each other. They are effective in both species, but in
Diploptera the nervous factor is the more important, whereas in Leucophaea the
reverse is the case.
The author wishes to express his graditude to Dr. B. Scharrer for her valuable
discussions during the preparation of the present paper. I thank Dr. L. M. Roth,
Natick, Massachusetts, for providing me with a colony of Diploptera punctata.
SUMMARY
1. In the viviparous roach, Diploptera, the factors controlling the maturation
of the eggs were studied during an interval encompassing the first pre-oviposition,
first pregnancy, and second pre-oviposition periods. During egg maturation the
corpora allata become active as judged by histological criteria (increase of cyto-
418 FRANZ ENGELMANN
plasmic content) ; they become and stay inactive during pregnancy. Ovarian in-
activity after allatectomy also demonstrates that active corpora allata are responsible
for the deposition of yolk in the oocytes. In addition, the corpora allata control
the activity of the accessory sex glands, and of an as yet unknown type of cell
presumably located in the bursa copulatrix, the secretion of which facilitates the
extrusion of the spermatophore.
2. In virgins, the brain inhibits the function of the corpora allata by way of
the nervi corporis allati, and thus prevents or delays the maturation of the eggs.
Severance of these nerves results in a sustained activity of the corpora allata with
the result that several successive batches of eggs mature.
3. In females which have not yet borne young, mating is essential for a normal
rate of egg maturation. The effect of mating can be replaced by parturition which
provides adequate stimuli to induce egg maturation. Thus, in an animal that has
just given birth, mating does not step up the growth rate of the eggs. The stimuli
exerted by mating or parturition are transmitted to the corpora allata by way of
the ventral nerve cord and the brain.
4. Artificial mating (mechanical stimulation with a small glass spermatophore)
also results in activation of the corpora allata and in subsequent maturation of
the eggs. Similarly, excision of the gonapophyses induces egg maturation, pre-
sumably because the afferent nerves are stimulated by this procedure. It seems
likely that sensory receptors on the gonapophyses and possibly on other parts of
the genital apparatus receive the stimuli exerted by mating and parturition.
5. The corpora allata of adult females of Diploptera are restrained to the extent
that egg maturation occurs only after considerable delay unless this inhibition is
eliminated by appropriate afferent stimuli. Complete inhibition of the corpora
allata, and thus of the ovaries, during pregnancy is achieved by the eggs developing
in the brood sac.
6. Ovariectomy does not result in sustained activity of the corpora allata as
is the case in some other insect species.
LITERATURE CITED
ENGELMANN, F., 1957. Die Steuerung der Ovarfunktion bei der ovoviviparen Schabe Lcuco-
phaea madcrae (Fabr.). /. Ins. Physiol., 1: 257-278.
ENGELMANN, F., AND M. LUSCHER, 1956. Die hemmende Wirkung des Gehirns auf die Corpora
allata bei Leucophaca madcrae (Orthoptera). Verh. dtsch. zool. Ges. Hamburg, 1956:
215-220.
GREULICH, W. W., 1934. Artificially induced ovulation in the cat (Felis domestica). Anat.
Rec., 58: 217-224.
GRIFFITHS, J. T., AND O. E. TAUBER, 1942. Fecundity, longevity and parthenogenesis of the
American roach, Pcriplaneta americana L. Physiol. Zool., 15 : 196-209.
VON HARNACK, M., 1958. Histophysiological studies on the corpus allatum of Lcucophaea
madcrae. II. The effect of starvation. Biol. Bull, 115: 521-529.
VON HARNACK, M., AND B. SCHARRER, 1956. A study of the corpora allata of gonadectomized
Leucophaea madcrae (Blattaria). Anat. Rec., 125: 558.
JOHANSSON, A. S., 1955. The relationship between corpora allata and reproductive organs
in starved female Lcucophaea madcrae (Blattaria). Biol. Bull., 108: 40-44.
JOHANSSON, A. S., 1958. Relation of nutrition to endocrine-reproductive functions in the milk-
weed bug Oncopeltus fasciatus (Dallas) (Heteroptera : Lygaeidae). Nytt Mag. Zool.,
7: 1-132.
LUSCHER, M., AND F. ENGELMANN, 1955. Ueber die Steuerung der Corpora allata-Funktion
bei der Schabe Leucophaca maderae. Rei>. Suisse Zool., 62 : 649-657.
REPRODUCTION IN DIPLOPTERA 419
PORTER, R. W., E. B. CAVANAUGH, B. V. CRITCHLOW AND CH. H. SAWYER, 1957. Localized
changes in electrical activity of the hypothalamus in estrous cats following vaginal
stimulation. Amer. J. Physiol., 189: 145-151.
ROTH, L. M., AND E. R. WILLIS, 1955. Intra-uterine nutrition of the "beetle-roach" Diploptera
dytiscoides (Serv.) during embryogenesis, with notes on its biology in the laboratory
(Blattaria: Diplopteridae). Psyche, 62: 55-68.
ROTH, L. M., AND E. R. WILLIS, 1956. Parthenogenesis in cockroaches. Ann. Ent. Soc. Amer.,
49: 195-204.
SCHARRER, B., 1946. The relationship between corpora allata and reproductive organs in adult
Lencophaca maderae (Orthoptera). Endocrinol., 38: 46-55.
SCHARRER, B., 1952. Neurosecretion. XI. The effects of nerve section on the intercerebralis-
cardiacum-allatum system of the insect Leucophaea maderae. Biol. Bull., 102 : 261-272.
SCHARRER, B., 1958. Neuro-endocrine mechanisms in insects. 2. Internat. Sympos. Neurosekre-
tion (Lund, 1957). Springer- Verlag, Berlin-Gottingen-Heidelberg, 79-84.
WHARTON, M. L., AND D. R. A. WHARTON, 1957. The production of sex attractant substance
and of oothecae by the normal and irradiated American cockroach, Periplaneta
amcricana L. /. Ins. Physiol., 1 : 229-239.
WIGGLESWORTH, V. B., 1934. The physiology of ecdysis in Rhodnius prolixus (Hemiptera).
II. Factors controlling moulting and 'metamorphosis.' Quart. J. Micr. Sci., 77: 191-
222.
WILLIS, E. R., G. R. RISER AND L. M. ROTH, 1958. Observations on the reproduction and
development in cockroaches. Ann. Ent. Soc. Amer., 51 : 53-69.
SOMITE GENESIS IN THE CHICK. II. ANALYSIS OF
NUTRIENTS FROM YOLK1
RONALD C. ERASER
Department of Zoology and Entomology, University of Tennessee, Knowillc , Tennessee
A previous communication has disclosed that there are a number of components
in the albumen of the avian egg which collectively are used by the embryo in the
development of somites (Fraser, 1957). These components were indicated to be
such small molecular forms as glucose, alanine and a heat-labile substance. More-
over, the protein moiety of the albumen may possibly be used to a limited extent
as well in this function.
Because the yolk provides the natural environment of the early chick blastoderm,
it seemed advisable to assay this portion of the egg for its nutritional value with
respect to the formation of somites. Studies by Needham (1931) and Romanoff
and Romanoff (1949) have indicated that the yolk of the avian egg is chemically
complex, containing such diverse molecular species as proteins, phospholipids,
coenzymes, amino acids, etc.
It has become apparent from many studies that the embryo does not use in-
discriminately all of the materials made available to it at any one period during
development. For example, the transitional nature of energy sources has been
pointed out by Needham (1950). Fraser (1956) has shown that the early blasto-
derm will starve on a fat diet, although there is much of this food substance present
in the unincubated egg.
With respect to the utilization of certain nutrients for specific morphogenetic
events, there exists but little information. Wilde (1955) has revealed the use of
various portions of the phenylalanine molecule by neural crest cells of the amphibian
in melanogenesis. Similarly, the development of the heart and of the brain of the
chick embryo have been shown to have different sugar requirements (Spratt, 1950).
The present paper represents an attempt to assay the yolk of the chicken egg for its
nutritional value, specifically in somite genesis. Other papers to follow will cover
other facets of this morphogenetic event.
MATERIALS AND METHODS
While a few of the eggs used in the present investigation were from Rhode
Island Red chickens, most were from White Leghorn hens. Nutritionally, the
two breeds appear equivalent, although the rate of development of the former is
perceptibly slower during the earliest phases of embryogenesis. The eggs were
stored at 18° C. until incubated, in all instances within a week after they had been
layed. Eggs were incubated at 38.0° C., while explants were cultured at 37.8° C.
1 This investigation was supported in part by research grant G-3486 from the National
Science Foundation.
420
CHICK SOMITE FORMATION 421
General methods for the culture of the early chick blastoderm have been outlined
previously (Spratt, 1947; Fraser, 1957). Definitive primitive streak (DPS)
blastoderms were removed from the eggs following approximately 21 hours of
incubation, and placed on semi-solid media. After camera lucida diagrams had
been made of them, the embryos were incubated for a period of 22 hours, after
which time diagrams were again made for the purpose of recording the extent of
development. The embryos were then fixed on the gel with Gerhard's fixative
and prepared for whole mounts. Delafield's hematoxylin was used as a stain.
Greater accuracy could be obtained in counting somites in stained preparations.
Approximately six hundred explants were cultured in the course of this investigation.
PREPARATION OF MEDIA
The reader is referred to a previous communication (Fraser, 1957) for an out-
line of the general procedure in preparing the media used in this study.
Yolk dialysate. One hundred and fifty ml. of yolk from unincubated fertile
eggs were dialyzed against 75 ml. of chick Ringer's solution in cylinders for two
days at 5° C. The dialysate was then collected and stored under refrigeration
until used.
Dialyzed yolk. One hundred ml. of whole yolk were dialyzed in the cold for
three days against large volumes of the Ringer's solution. Dialysis proceeded in
10-liter flasks, the saline contents of which were changed on three occasions. The
contents of the casings, representing the large molecule fraction of whole yolk, were
then collected for use.
Boiled dialysate. Fifty ml. of freshly prepared yolk dialysate were gently
refluxed for 10 minutes prior to use.
Ether partition of yolk dialysate. Fifty ml. of freshly prepared yolk dialysate
were shaken on three occasions with 15 ml. of redistilled ethyl ether in a separatory
funnel. The pooled ether phases were condensed at 45° C. under vacuum to near
dryness. Five ml. of chick Ringer's were added and the remaining ether was
distilled. Ether was removed from the aqueous phase in the same manner.
Acid hydrolysis of dialyzed yolk. The preparation of amino acids by the hy-
drolysis of dialyzed whole yolk used in the present study has been described by
Block et al. (1958). Twenty ml. of dialyzed yolk were boiled under reflux with
40 ml. of 8 N sulfuric acid for 20 hours. To this, hot saturated barium hydroxide
was added until a pH of 11 had been reached. After distillation in vacua had
removed the free ammonia, the excess barium was precipitated by an equivalent
amount of 1 N H2SO4. The BaSO4 was centrifuged off and the supernatant fluid
reduced in volume to dryness. The residue was then taken up in 10 ml. of chick
Ringer's solution for use.
CHROMATOGRAPHIC ANALYSIS OF YOLK DIALYSATE
Twenty ml. of the yolk dialysate, prepared as indicated above, were reduced in
volume to zero under mild heat from an infra-red lamp, and while agitated by a
jet of air. The excess salt was removed by solvent extraction as outlined by
Harris (1953). Drops of the concentrated aqueous phase, following ether extrac-
tion, were applied to Whatman No. 1 paper, measuring 18 by 22 inches. Two-
422
RONALD C. FRASER
dimensional chromatograms were prepared by descending chromatography with
water-saturated phenol as the first solvent and lutidine-collidine- water (3:1:1 by
volume) as the second. After drying, the papers were sprayed with a 0.1% nin-
hydrin solution in 95% ethanol. On drying in an oven, the spots that appeared
were compared with those on the amino acid map of Dent (1948). Similar chro-
matograms were made of the dialysate concentrate following oxidation with hydro-
gen peroxide, a procedure that is necessary for the identification of cysteine.
Figure 1 illustrates the identification of the free amino acids in yolk. In all
there are thirteen such compounds readily distinguishable by this assay method.
Spots numbered 1, 2 and 3 appeared only from a preparation following oxidation.
tyrosine
leucmes
cysteic
acid
valine
threonine
prolme
alonme
glutamic ospartic
acid acid
lysine ornithine
09
08 0.7
-^
0.6 O.5 0.4
DUPMHI
0.3 0.2 0-1
O Ld
Q
cvj
0 ^
O
FIGURE 1. Two-dimensional chromatogram showing the amino acids detected
free in yolk dialysate.
Grey colored spots indicated by numbers 2 and 3 do not coincide with any shown
on Dent's map, and hence are unidentified at this time. The presence of cysteic
acid, however, indicates the presence of free cysteine.
BARIUM AND ALCOHOL FRACTIONATION OF YOLK AND ALBUMIN DIALYSATES
To establish more precisely the requirements of the young chick blastoderm
from yolk and albumen in the formation of somites, a barium and alcohol frac-
tionation of these food sources was undertaken. The procedure has been indicated
in a general way by Harris (1953).
The pH of 15 ml. of yolk dialysate was brought to 8.2 by the careful addition
CHICK SOMITE FORMATION
423
of 0.1 M NaOH. To this were added 2 ml. of 1 M Bad,, the pH of which
had previously been adjusted to 8.2. The resulting mixture was placed in the
freezing compartment of a refrigerator for 30 minutes and then centrifuged. The
pellet was washed with five drops of BaCU (pH 8.2) and then brought into solution
with a small amount of doubly distilled water. To this was added a stoichiometric
amount of 1 M Xa2SO4 to precipitate all of the barium. The resulting supernatant
was retained as the "barium insoluble" fraction.
The supernatant from this procedure was further fractionated by ethanol. The
barium-soluble solution obtained was treated with sufficient molar sodium sulfate
to precipitate all of the barium. The supernatant was reduced in volume to 10 ml.
and chilled. To this were added 40 ml. of chilled absolute ethyl alcohol, thus
bringing the concentration of the alcohol to 80 per cent. After further chilling for
30 minutes the preparation was centrifuged, and the pellet (the "barium-soluble,
alcohol-insoluble" fraction) was washed with 5 drops of cold 80 per cent ethanol.
The supernatant from this fractionation is termed the "barium-soluble, alcohol-
soluble" fraction.
TABLE I
Assay of barium and alcohol fractions of yolk and albumen for nitrogen and phosphorus
Yolk
Albumen
Fraction
p
Gig./ml.)
Gig./ml.)
P/N
(ng./ml.)
Gtg./ml.)
P/N
Barium-insoluble
104
10
10.40
3.2
8
0.40
Barium-soluble, alcohol-insoluble
10.8
9
1.20
12.5
26
0.48
Barium-soluble, alcohol-soluble
8.8
664
0.01
8.6
98
0.09
Whole dialysate
129
742
0.17
25.7
128
0.20
The three fractions thus obtained were placed individually in Stender dishes
and the volume of each reduced to zero with mild heat and air agitation. Excess
salt was removed and the volume again reduced to zero. To each were added
4 ml. of chick Ringer's solution and the resulting fractions set aside for incorpora-
tion into media.
A similar fractionation was carried out on albumen dialysate.
ASSAY OF BARIUM AND ALCOHOL FRACTIONS FOR PHOSPHORUS
AND NITROGEN CONTENT
Umbreit ct al. (1949) have listed the phosphorylated intermediates known to
be present in the fractions derived from barium and alcohol treatment. In order
to determine the relative concentrations of the various esterified compounds in the
fractions used in the test media, an analysis was made on the fractions for nitrogen
and phosphorus.
The results of such an analysis are shown in Table I. Total phosphorus was
determined by the method given by Umbreit et al. (1949) after Fiske and Sub-
barow (1925). The nesslerization method of Koch and McMeekin (1924) for
nitrogen was employed. Phosphorus and nitrogen determinations were made, as
424
RONALD C. ERASER
indicated in the table, on the whole dialysate, the "barium-insoluble," "barium-
soluble, alcohol-insoluble," and "barium-soluble, alcohol-soluble" fractions. Prepa-
rations from both yolk and egg white were assayed.
CONTENTS OF THE MEDIA
The contents of the media used in the present investigation are given in
Table II, along with the amounts of each expressed in milliliters. Phenol red,
in concentration of 0.01 per cent, was used as an inside pH indicator. Moreover,
this dye also serves by coloring the medium, thus making it easier for the observer
TABLE II
Components of media used in the experiments and somite development
in ex planted chick embryos
Pf.ni
Aver. no.
Test material
Volume
of test
material
Ringer
Phenol
red
rcni-
cillin
strepto-
mycin
Phos-
phate
buffer
Bicar-
bonate
buffer
Total
volume
No.
embryos
pairs
somites
± stand.
error
None
.
33
2
2
2
1
40
_>.s
0
Whole yolk
20*
23
2
2
2
1
50
28
7.0 ±0.6
Yolk dialysate
20
13
2
2
2
1
40
56
7.4 ±0.3
Dialyzed yolk
20*
23
2
2
2
1
50
42
0
Dialyzed yolk + glucose
28**
15
2
2
2
1
50
22
0
Boiled yolk dialysate
20
13
2
2
2
1
40
45
2.0 ±0.5
Dialyzed yolk hydrolysate
2
1.3
0.2
0.2
0.2
0.1
4
30
0
Dialyzed yolk hydrolysate
+ glucose
2.8***
0.5
0.2
0.2
0.2
0.1
4
38
3.2 ±0.5
Ether-soluble fraction
2
1.3
0.2
0.2
0.2
0.1
4
30
0
Saline-soluble fraction
20
13
2
2
2
1
40
32
7.9 ± 0.3
Free amino acids + glucose
17f
16
2
2
2
1
40
41
2.5 ± 0.3
Barium-insoluble fraction of yolk
3.5ft
3.1
0.4
0.4
0.4
0.2
8
48
1.5 ±0.2
Barium-soluble, alcohol-insoluble
fraction of yolk
3.5
3.1
0.4
0.4
0.4
0.2
8
52
0.4 ± 0.2
Barium-soluble, alcohol-soluble
fraction of yolk
3.5
3.1
0.4
0.4
0.4
0.2
8
40
1.8 ±0.4
Barium-insoluble fraction of
albumen
3.5
3.1
0.4
0.4
0.4
0.2
8
38
0
Barium-soluble, alcohol-insoluble
fraction of albumen
3.5
3.1
0.4
0.4
0.4
0.2
8
45
2.2 ± 0.5
Barium -soluble, alcohol-soluble
fraction of albumen
3.5
3.1
0.4
0.4
0.4
0.2
8
36
1.0 ±0.3
* Twenty-five ml. of whole or dialyzed yolk were shaken with 50 ml. of Ringer's. Twenty ml. of the froth-free
liquid were decanted for use.
:* Eight ml. of 1% glucose plus 20 ml. of dialyzed albumen prepared as above.
** Four-fifths ml. of 1% glucose plus 2 ml. of dialyzed yolk acid hydrolysate.
t One ml. of a 100 mg. % solution of each of the 13 amino acids found free in yolk dialysate plus 4 ml. of a 2%
glucose preparation. Final concentrations: each amino acid, 2.5 mg. %; glucose, 200 mg. %.
ft The same amount (3.5 ml.) of the barium and alcohol fractions of both yolk and albumen was used.
to see the explanted embryos. The preparation of the penicillin-streptomycin
solution used to prevent bacterial growth, and the phosphate and bicarbonate
buffers has been described previously (Fraser, 1956). Throughout the period of
culture the pH of the media remained near 7.6. The concentration of the agar
used in preparing the semi-solid gels was 250 mg.%. The Ringer's solution was
prepared in accordance with the formula given by Spratt (1947), with NaCl
content reduced to 123 millimolar (Howard, 1953). In previous communications
(Fraser, 1956, 1957) the details of preparing the media have been given. When
the volume of the medium was small, 4 or 8 ml., it was poured into depression
slides supported on cotton rings within petri dishes. The media of larger volume
were placed in watch crystals held in similar fashion. The cotton rings supporting
CHICK SOMITE FORMATION
425
the containers of media were moistened with sulfadiazine (0.25 per cent) as a
further precaution against microbial contamination.
RESULTS AND DISCUSSION
The development of somites in DPS explants cultured on the media outlined
above for 22 hours is given in Table II.
One of the most striking observations is that the food materials used in this
morphogenetic process are present free in the dialyzable (small molecule) fraction.
FIGURE 2. Photograph of a DPS blastoderm cultured for 22 hours on dialyzed whole yolk.
Note absence of any appreciable development.
FIGURE 3. Photograph of an embryo of similar age cultured for 22 hours on a medium
containing yolk dialysate. Note the formation of somites.
The same was found to be true when the egg white was assayed (Fraser, 1957).
Somites developing in explants on the dialyzable fraction of whole yolk were
equal in number to those in explants cultured on the yolk medium itself. On the
other hand, no somites formed in blastoderms cultured on the large molecule
moiety, even when glucose was added to the medium. This observation would
indicate, in contrast to the conclusion of Taylor and Schechtman (1949), that the
embryo in culture cannot use native protein alone from either yolk or albumen
426 RONALD C. FRASER
for early morphogenesis. Figures 2 and 3 show these results. Blastoderms cul-
tured on dialyzed yolk appear the same as those cultured on dialyzed albumen or
on a saline-agar (non-nutrient) medium.
The chick embryo cultured in vitro can, however, use the protein moiety of
yolk following hydrolysis of the polymers, when supplemented by glucose as an
energy source. This observation would suggest that at this stage of development
the embryo lacks sufficient proteolytic enzymes for its early morphogenic require-
ments, and relies almost entirely ( if not completely ) on molecules of simpler form
found free in its environment.
There is, as yet, no complete characterization of the small molecule nutrients
used by the chick blastoderm for somite development. Ether fractionation reveals
that all materials used are in the aqueous phase. The inability of the very early
embryo to use fat as a substrate for development has been noted previously ( Eraser,
1956, 1957). It is equally true that some of the somite-forming capacity of yolk
dialysate is curtailed by heat. A similar heat-labile fraction has been found in
albumen dialysate (Eraser, 1957). There is further support for the presence of
a heat-labile component in the observation that somite development is somewhat
depressed in explants cultured on a medium containing all of the amino acids
found free in yolk.
As one would expect, the bulk of nitrogen, signifying the presence primarily
of amino acids, is found in the barium- and alcohol-soluble fraction of both yolk
and albumen. In view of the fact that there is some development of somites in
explants cultured on the amino acids detected in this portion of the avian egg, it
is not surprising that this fraction derived from barium and alcohol precipitation
should yield a similar result. This is borne out by observation.
There has been, however, some dispute as to whether the early chick blasto-
derm can utilize phosphorylated carbohydrate intermediates for its development.
Needham and Nowinski (1937) have come to the conclusion that such substances
cannot serve as nutrients for the early chick embryo. Novikoff, Potter and Le
Page (1948), on the other hand, have demonstrated the presence of phosphorylated
intermediates in embryonic chick homogenate. Eraser (1956) has reported the
presence of cytochrome oxidase in blastoderms as young as the beginning streak
stage. The great importance of glucose in the development of the early chick
embryo has been repeatedly demonstrated (Spratt, 1949; Eraser, 1954). More-
over, unpublished work from this laboratory has indicated that only a small portion
of carbohydrate metabolism in the young avian embryo is directed through the
phosphogluconate shunt. It is evident, therefore, that unless some other, much
less likely, mechanism for sugar oxidation exists, carbohydrate must be handled
in the conventional phosphorylated manner. If this assumption is true, we might
well expect that phosphorylated intermediates, when offered to the embryos, would
be metabolized. This contention is borne out by observation that somite develop-
ment proceeds to a limited degree in explants cultured on media containing these
carbohydrate phosphate esters.
Umbreit ct al. (1949) have listed the various phosphorylated compounds pre-
cipitated by barium and alcohol fractionation. According to them, most of these
intermediates in carbohydrate metabolism are brought down in the barium-soluble,
alcohol-insoluble fraction. Comparison of P/N ratios would indicate that such
CHICK SOMITE FORMATION 427
fractionation precipitates different compounds in albumen than in yolk. The wide
discrepancies in phosphate values point to this conclusion. Differences in somite
counts in embryos cultured on these fractions add further support. Finally, em-
bryos were cultured on media containing the barium-soluble, alcohol-insoluble
fractions of both yolk and albumen clialy sates which had been boiled. No somites
formed in blastoderms explanted on either of these media. There was no sup-
pressing effect, however, by heat on the barium-insoluble fraction of yolk. We
might conclude from this, then, that insofar as the development of somites is
concerned, the explanted chick embryo utilizes at least one different component
which is present in yolk but not in egg white.
Finally, w7e should attempt to ans\ver the provocative question why it is that
less than the maximal number of somites form in embryos cultured on certain
media. Actually the total number of somite pairs found in explants cultured
under optimal conditions on any yolk or albumen medium is somewrhat less than
that seen in embryos grown in ovo for a similar period of time. We can there-
fore conclude that the in vitro culture technique falls short in providing the embryo
with appropriate environmental conditions other than of a nutritive nature. But
insofar as somite development in explanted embryos alone is concerned, such dif-
ferences must have a nutritional basis, since physical environmental conditions are
presumably equivalent.
The experiments in this and the former paper in this series ( Fraser, 1957)
have shown that the formation of somites in the chick embryo cultured in vitro
is dependent on a number of chemical constituents in the food supply. We might
therefore profitably think that a submaximal number of somites in explants results
when one or more of the nutritional requirements for the maximal formation is
lacking. This deficiency may then act to limit the number of somites which could
develop within a specified period of time. It is only when all of the components
needed for this morphogenetic event are present that the maximum number of
somites will form.
SUMMARY
1. Definitive primitive streak chick embryos have been cultured in vitro on
media containing various fractions of whole yolk. Other embryos were grown on
agar gels containing fractions derived by barium and alcohol fractionation of yolk
and albumen dialy sates. Following 22 hours of incubation, the blastoderms were
mounted and examined for the development of somites.
2. The nutritional components of whole yolk are all present in the saline-
soluble, dialyzable moiety, although the embryo can use the acid hydrolysate of
dialyzed yolk, when accompanied by glucose, for this morphogenetic process.
3. The results, derived from an assay of barium and alcohol fractionation of
both yolk and egg white, indicate that the early chick embryo can use, to limited
extent, certain phosphate esters of carbohydrates in the formation of somites.
There appear to be different phosphorylated materials in yolk than in albumen used
by the embryo.
4. Insofar as whole yolk utilization is concerned for the formation of somites,
there is evidence that the chick embryo grown in vitro uses: (1) glucose, (2) the
amino acids found free in yolk, (3) a heat-labile, uncharacterized factor, and (4)
428 RONALD C. ERASER
certain phosphorylated carbohydrate intermediates. It may also use, but to a
very limited extent, the products of proteolysis of yolk proteins.
LITERATURE CITED
BLOCK, R. J., E. L. DURRUM AND G. ZWEIG, 1958. A Manual of Paper Chromatography and
Paper Electrophoresis. Second ed. Academic Press, New York.
DENT, C. E., 1948. A study of the behaviour of some sixty amino acids and other ninhydrin-
reacting substances on phenol-collidine filter paper chromatograms, with notes as to
the occurrence of some of them in biological fluids. Biochcin. J., 43 : 169-180.
FISKE, C. H., AND Y. SUBBAROW, 1925. The colorimetric determination of Phosphorus. /.
Biol. Chcm.. 66: 375-400.
ERASER, R. C., 1954. The utilization of some carbohydrates by in vitro cultured chick blasto-
derms in wound healing. Biol. Bull., 106: 39-47.
ERASER, R. C., 1956. The presence and significance of respiratory metabolism in streak-form-
ing chick blastoderms. Biol. Bull, 111: 77-91.
ERASER, R. C., 1957. Somite genesis in the chick. I. Partial characterization of stimulatory
factors in egg white. Growth, 21 : 29-43.
HARRIS, M., 1953. Partial purification of growth factors in the dialyzable fraction of chick
embryo extract. Growth. 17: 147-162.
HOWARD, E., 1953. Some effects of NaCl concentration on the development of early chick
blastoderms in culture. /. Cell. Comp. Physio!.. 41 : 237-259.
KOCH, F. C., AND T. L. MCMEEKIN, 1924. A new direct nesslerization micro-Kjeldahl method
and a modification of the Nessler-Eolin reagent for ammonia. /. Am. Chcm. Soc.,
46: 2066-2069.
NEEDHAM, J., 1931. Chemical Embryology. Cambridge Univ. Press. Cambridge.
NEEDHAM, J., 1950. Biochemistry and Morphogenesis. Cambridge Univ. Press. Cambridge.
NEEDHAM, J., AND W. NOWINSKI, 1937. Intermediary carbohydrate metabolism in embryonic
life. I. General aspects of anaerobic glucolysis. Biochcin. J., 31 : 1165-1184.
NOVIKOFF, A., V. POTTER AND G. LE PAGE, 1948. Phosphorylating glycolysis in the early
chick embryo. /. Biol. Chcm.. 173: 239-252.
ROMANOFF, A., AND A. ROMANOFF, 1949. The Avian Egg. J. Wiley, New York.
SPRATT, N. T., JR., 1947. A simple method for explanting and cultivating early chick embryos
in ritro. Science, 106: 452.
SPRATT, N. T., JR., 1949. Nutritional requirements of the early chick embryo. I. The utiliza-
tion of carbohydrate substrates. /. Ex p. Zool., 110: 273-298.
SPRATT, N. T., JR., 1950. Nutritional requirements of the early chick embryo. II. Differential
nutrient requirements for morphogenesis and differentiation of the heart and brain.
/. E.i-p. Zool, 114: 375-402.
TAYLOR, K. M., AND A. M. SCHECHTMAN, 1949. In ritro development of the early chick embryo
in the absence of small organic molecules. /. E.\'f>. Zool., Ill : 227-253.
UMBREIT, W. W., R. H. BURRIS AND J. H. STAUFFER, 1949. Manometric Techniques and Tissue
Metabolism. Burgess Pub. Co., Minneapolis.
WILDE, C. E., JR., 1955. The urodele neuroepithelium. II. The relationship between phenylal-
anine metabolism and the differentiation of neural crest cells. /. Morphol., 97 : 313-
344.
PRESENCE OF THE RED EFT WATER-DRIVE FACTOR
PROLACTIN IN THE PITUITARIES OF TELEOSTS
WILLIAA1 C. GRANT, JR.i AND GRACE E. PICKFORD 2
Department of Biology, Williams College, Williamstown, Massachusetts, and Biiif/liam
Oceanographic Laboratory, Yale University, New Haven, Connecticut
The lactogenic hormone (prolactin), on account of the complexity and diversity
of its functions, continues to excite speculation concerning its relation to changing
target organs during the course of vertebrate evolution. The role of this hormone
in the promotion of lactation in mammals, its effect on the secretory activity of
the crop gland in pigeons, and its association with certain aspects of parental care
in both mammals and birds are well known. In addition, prolactin is thought to
act as a hyperglycemic agent in some higher vertebrates and may be important in
regulating various events in the reproductive cycle, such as ovulation and pro-
gesterone secretion. Recently, Juhn and Harris (1958) have shown that the
lactogenic hormone may participate in the production of new plumage in birds
through stimulation of the feather papilla.
The problem of prolactin in the lower vertebrates has been reviewed by Atz
(in Pickford and Atz, 1957) with special reference to fishes. There is con-
siderable evidence that this hormone plays a physiological role in the poikilotherms,
although direct evidence for its presence is scanty. Blair (1946) found that
prolactin (of uncertain purity) stimulated the production of new melanophores in
toads, while Pickford and Kosto (1957) have shown that highly purified inter-
medin-free prolactin promotes melanin synthesis (but not new pigment cell forma-
tion) in the partially depigmented melanophores of hypophysectomized killifish,
Fundulns hetcroditus. The synergic action of prolactin on the melanocyte-
stimulating effect of intermedin (new pigment cell formation) was also demonstrated.
The acceleration of molting in prolactin-treated newts, Dicmyctylus ( = ;Triturus)
viridesccns, observed by Chadwick and Jackson (1948), may have resulted from
stimulation of the endogenous release of thyrotropin rather than to a specific action
of prolactin since the recipients were not hypophysectomized. The lactogenic
hormone stimulates the secretion of jelly by the oviducts of anurans (de Allende,
1939; Houssay, 1947 and later papers cited by Pickford and Atz, 1957; de Allende
and Orias, 1955). Positive results could be obtained even after castration or
hypophysectomy and the gonadotropins were ineffective. The gonadotropic effects
of prolactin on the European newts, Triton cristatus and T. alpestris, and the
stimulation of sperm release in male dogfish, Scyliorhinus caniculus, reported by
Tuchmann-Duplessis (1948, 1949) and Carlisle (1954), respectively, are inter-
esting but require further investigation.
1 Supported by a research grant (A1786 C-l) from the National Institutes of Health,
U. S. Public Health Service.
2 Supported by a grant from the National Science Foundation (NSF G4001).
-129
430 W. C. GRANT, JR. AND G. E. PICKFORD
A most curious and interesting reaction to prolactin was described by Chadwick
(1940) in tbe immature, terrestrial (red eft) stage of Dicmyctylus viridcscens.
The red efts migrated to water within a few days after injection of the hormone
and developed the olive pigmentation and strongly keeled tail of the aquatic adult.
Similar results were reported by Tuchmann-Duplessis (1949) in experiments with
immature terrestrial stages of Tritunts alpcstris. In a further study of this
problem, Grant and Grant (1956, 1958), using hypophysectomized efts to exclude
the reflex release of other pituitary factors, have confirmed that prolactin is directly
responsible for initiating the change in habitat preference. Hypophysectomized
efts receiving intraperitoneal injections varying from 8 to 0.04 mg. of highly
purified prolactin (C. H. Li) migrated to the water compartment of the vivarium
but failed to develop the pigment and morphological characteristics of the complete
"water-drive syndrome." The total response observed by Chadwick must therefore
involve other hormonal factors. In developing a more decisive test for the water
hormone, Grant (1958 and unpublished data) has estimated that a minimum dose
of 1.4 jug. of prolactin is necessary to elicit the response in 50% of the hypophysecto-
mized efts tested.
Although the indirect evidence cited above is strongly in favor of the hypothesis
that prolactin plays a natural role in the physiology of the lower vertebrates, direct
evidence for the presence of this hormone in the poikilotherm pituitary is limited
and requires confirmation. Leblond and Noble (1937), using the pigeon crop
test, obtained weakly positive results of an indecisive nature with pituitary implants
of turtle (Kinostcrnon odoratum), frog (Rana pipiens} and some species of teleos-
tean fishes (Amciuriis nebulosus and Lepomis gibbosus) although others were
negative. Foglia (1940) found that pituitaries of the toad, Bufo arcnanim,
stimulated the crop gland of the pigeon. Carlisle (in Medawar, 1953) reports
that dogfish pituitary contains a factor which promotes lactation in the mammary
gland, but the presence of oxytocin may be suspected. More recently Lehrman (in
Pickford and Atz, 1957) has demonstrated prolactin-like activity in pollack
pituitary brei (Pollachius virens) by means of the pigeon crop test. However,
negative results were obtained with a lyophilized powder derived from hake, pollack
and cod (Wilhelmi, Lot FSOx) (Lehrman, personal communication). The data
of Fonseca Ribiero and Tabarelli Neto (1943), with alcohol-preserved pituitaries
of the teleost Prochilodns harti, depend on the validity of the anuran oviduct test.
Chadwick (1941), using the red eft test, demonstrated the presence of a "water-drive
factor" in the pituitaries of the toad, Bufo americanus, the water snake, Natrix sp.
and the chicken. The pituitaries of two salamanders (Plcthodon mctcalfi and
Desmognathus juscus) and of two snakes (Diadophis sp. and Tlianmoplus sp.)
gave negative results.
The present investigation was undertaken to obtain further information on the
distribution of prolactin among the lower vertebrates. Pituitary glands of pollack,
Pollachius virens, carp, Cyprinus carpio, and killifish. Fittidit/tts hctcroclitns, were
tested for the presence of the water-drive factor by means of the red eft test.
MATERIALS AND METHODS
The pituitary material was collected and prepared by one of us (G. E. P.).
The wet weight of an average gland varies with the species, the size of the fish, the
WATER DRIVE FACTOR IN TELEOSTS
431
stage of the reproductive cycle, and possibly also the sex. In any given sample,
fishes of both sexes and different size ranges were taken together so that the
approximate number of glands injected per eft is less meaningful than the actual
weight of the material (Table I). In the case of Fundnlns it should be noted,
however, that unpublished data of B. Kosto have shown that the mean weight of the
pituitary expressed in terms of body weight is less in winter (sexual regression)
than in spring. The mean index, Pituitary Wt./Fish Wt. in mg. per cent was 0.36
in September and October, 0.51 in April. (Both sexes combined, no significant sex
differences were noted.)
Pollack (Lots 1, 2, 3, 4) : Glands were collected from fish in the pre-spawning
condition at Wilson's Beach, Campobello Island, on June 28, 1954. Fish were
brought in in the morning and the glands removed within two hours, wrapped in
TABLE I
Doses and calculated numbers of pituitaries involved for
each lot of teleost material tested
Lot numbers
Number of test
animals each lot
Total dose
in mg.
Approx. number of
donor pituitaries
per dose
Pollack : No. 1
4
10.0
0.8
2
20.0
1.8
No. 2, 4*
4
10.0
1.1
2
20.0
2.2
No. 3
4
10.0
1.2
2
20.0
2.4
Carp: No. 5, 6, 7*
4
20.0
0.8
2
40.0
1.6
Fundulus: No. 9
4
19.9
70.0
2
39.8
140.0
No. 10
4
19.5
28.0
2
39.0
56.0
No. 11
4
19.7
20.0
2
39.4
40.0
No. 12
2
19.8
24.6
* Each sample tested with same numbers of efts.
"Parafilm" in lots of 5 glands each, and frozen immediately. After four years
storage in a closed can in the deep freezer, it was found from their appearance that
many of the glands were partially desiccated. The average weight was approxi-
mately 50% of the expected wet weight (ca. 20 mg.). Therefore, in making up
dilutions for comparison with carp and Fundulus pituitaries (not semi-desiccated)
it was assumed that 100 mg. = 200 mg. wet weight. Pollack glands were weighed
in lots of ca. 100 mg. and homogenized in 2 ml. of 0.6% NaCl. The brei was kept
frozen until used. Carp (Lots 5, 6, 7) : The glands were taken from fish collected
in the Connecticut River on October 22, 1957. Although late in the season, some
of the males had flowing sperm and many of the females had well-developed ovaries.
After eight months storage in closed vials in the deep freezer the glands were
weighed in lots of 200 mg. each (9-12 glands) and homogenized, as in the case of
the pollack material, in 2 ml. of 0.6% NaCl.
432 W. C. GRANT, JR. AND G. E. PICKFORD
Fundulus (Lot 9) : A total of 1700 glands weighing 199.5 mg. was prepared
from freshly captured fish collected near New Haven, Connecticut, in October and
November, 1957, plus a few from aquarium-kept fish of the same batch killed in
December, 1957. All were in a state of complete sexual regression. The material
was frozen in closed vials to prevent desiccation. The brei was prepared seven
months later ; homogenization was difficult, probably on account of the tough neural
processes and diminished glandular material at this season of the year, but a smooth
homogenate was finally prepared and diluted to 2 ml. in 0.6% NaCl.
(Lot 10) : The greater part of the material (280 glands) was taken from pre-
spawning fish captured near New Haven, Connecticut, ca. May 1, 1955 and kept
up to ten days in the laboratory; 80 glands from freshly captured, early spawning
fish caught June 7-8, 1955, were added to bring the total to the required weight.
The material was stored for two years in closed vials in the deep freezer and the
brei was prepared in the usual manner from a total of 360 glands weighing 195.5 mg.
(Lot 11) : Two hundred glands weighing 197.5 mg. taken from freshly captured
fish at the beginning of the spawning season, June 7-8, 1955, were prepared as
described above.
(Lot 12) : Three hundred and twenty glands weighing 129.0 mg. taken from
freshly captured fish during the period of sexual regression in September and
October, 1955 and kept frozen for two and one-half years in closed vials were
homogenized in 1.3 ml. of 0.6% NaCl so that the concentration was equivalent to
that used in the preceding samples.
In addition to the above material, tests were conducted on a lyophilized prepara-
tion (Wilhelmi, Lot FSOx) derived from a mixture of hake, pollack and cod. The
bulk of the glands were taken from hake (Urophycis tennis) in post-spawning
condition. The material was collected at Wilson's Beach, Campobello Island, New
Brunswick, in the summer of 1952.
A number of efts collected near Petersham, Massachusetts in August and Sep-
tember, 1958 were hypophysectomized and kept for a period of two weeks before
treatment. The animals varied in weight from 0.63 to 1.48 gm. and all were
considered to be well removed from the naturally occurring water-drive phase of
their life cycle. Each animal received intraperitoneal injections on each of two
separate days of fish pituitary brei delivered at a standard volume of either 0.1 ml.
or 0.2 ml. per injection. After the first injection the efts were placed in containers
with a land and a water area and the time of their migration to water was noted.
Changes in weight and length during a period of four weeks after the first injection
were recorded in most instances. The data for the doses administered and the
calculated number of pituitaries involved are given in Table I.
RESULTS
The results of the above experiments are summarized in Table II.
(a) Tests with pollack: Most animals tested with pollack pituitary failed to
show the water-drive response. In all respects they appeared as non-treated hy-
pophysectomized efts, their skin dark and dry as layers of cornified epithelium built
up in the absence of normal molting factors presumably involving TSH. Two ani-
mals receiving brei from Lot 1 gave a partial response. Their skin sloughed off in
rough patches and the efts entered water at irregular intervals, remaining there for
WATER DRIVE FACTOR IN TELEOSTS
433
several hours at a time. One animal, however, treated with Lot 4 material, gave a
positive reaction by assuming the water habitat for a period of a week, during
which time all of the old cornified epithelium sloughed off revealing the smooth,
non-granular skin characteristic of the aquatic phase. Changes in weight and length
were erratic but some individuals (8) undoubtedly grew slightly.
(b) Tests with carp: Fourteen out of the 18 individuals treated with carp
pituitary gave a positive response on an average of about ten days following the
TABLE M
Results of water drive studies following treatment of efts with
various teleost pituitary preparations
Donor material
and lot numbers
No. test
animals*
Wt. change
per cent
4 weeks
Length
change
per cent
4 weeks
Results
Days to
water
average
Positive
Negative
Fundulus (post-
spawning)
Lot No. 9
6
5 (1 dead)
0
9.2
not recorded
Lot No. 12
2
2
0
8.0
Fundulus (chiefly
pre-spawning)
Lot No. 10
6
+ 7.41
+ 6.35
6
0
7.6
Fundulus (early
spawning)
Lot No. 11
6
not recorded
0
5
Pollack (pre-
spawning)
Lot No. 1
6
loss and gain
2 (partial)
4
Lot No. 2
6
loss and gain
0
6
Lot No. 3
6
loss and gain
0
6
Lot No. 4
6
loss and gain
1
5
8.0
Carp (late
spawning)
Lot No. 5
6
+8.41
+ 4.46
4
2f
12.0
Lot No. 6
6
5 (1 dead)
7.2
Lot No. 7
6
+8.70
+3.20
5 (1 dead)
12.6
F80x Wilhelmi—
lyophilized hake,
pollack, cod
6
not recorded
0
6
Controls
16
-5.77
none
0
16
*See Table I.
f See text.
initial injection and remained in water for periods of from two to three weeks. The
four unresponsive individuals may be discounted. Two receiving Lot 5 brei failed
to give even a partial response, but one of these was poorly injected so that some of
the preparation was lost, while the second individual was suffering from an acute
fungus infection. An additional two animals died shortly after the second injection,
before any response could have been expected. The efts showed a mean in-
crease in weight and length. All animals molted normally.
434 W. C. GRANT, JR. AND G. E. PICKFORD
(c) Tests with Fimduhis: Injection of pituitary brei from fish in sexual regres-
sion (Lots 9 and 12) yield positive results in all animals tested, with the exception
of one which died shortly after the first injection. The animals went to water
between eight and nine days after their first treatment and molted normally. It is
interesting to note that Lot 10, largely prepared from laboratory-kept animals in
the early pre-spawning condition, also gave positive results, while tests with Lot 11
brei from spawning animals were entirely negative. Efts receiving Lot 10 increased
significantly in weight and length ; the other preparations were not studied in
respect to the growth response. Animals receiving Lot 11 molted regularly, in-
dicating that the preparation contained at least some endocrine activity.
Injections of the lyophilized preparation FSOx gave negative results in all
animals tested.
Controls remained in the terrestrial phase, did not molt, lost weight and showed
no change in length.
CONCLUSIONS
These tests present convincing evidence that a prolactin-like substance is a
naturally occurring factor in the pituitary glands of teleost fishes. Carp brei
(Lots 5, 6, 7) gave a positive response as did material prepared from Fundulus
(Lots 9, 10, 12) in the pre- and post-spawning conditions. The nature of this
response indicates that this material, administered in doses of 10 or 20 mg., con-
tained lactogenic hormone well above the estimated threshold of 1 .4 ^g. for initiation
of the water-drive response. That there was no apparent difference in the nature
and time of response between animals receiving 10 and 20 mg. of brei agrees with
Grant (unpublished data) who has shown that at or above the 3 /Jig. level total
response can be expected in all efts treated, provided they are in a healthy condition.
One of the animals receiving pollack preparations showed positive water-drive,
while two others gave a partial reaction. Although the response was very weak in
this case, the presence of prolactin in small amounts is indicated. This is in agree-
ment with the work of Lehrman (in Pickford and Atz, 1957) who obtained positive
results with pollack brei on the pigeon crop test. Both the present series of tests
and those of Lehrman's gave negative results with the lyophilized powder FSOx.
The fact that pituitaries taken from animals near the end of the spawning season
and from those in the post-spawning condition, gave the strongest response is in-
teresting. Lot 11 brei from early spawning killifish gave negative results while
pollack brei from pre-spawning animals produced a very weak response. In the
latter case the possibility of deterioration of the sample, which was partially de-
hydrated after four years' storage in the deep freezer, cannot be excluded, but the
glands taken from Fundulus in June, 1958 are believed to have been in perfect
condition. Further experiments are needed to exclude a possible unexplained
deterioration of the sample. These tests may indicate a seasonal depletion in pro-
lactin associated with the reproductive cycle, but the results are not decisive. Nega-
tive results with the lyophilized powder, chiefly from post-spawning hake, conflict
with this hypothesis.
SUMMARY
It has been demonstrated, by means of the red eft water-drive test, that a
prolactin-like hormone is present in the hypophysis of teleostean fishes. Pituitary
WATER DRIVE FACTOR IN TELEOSTS 435
extracts from late spawning carp (Cyprinus car pi o} and pre- or post-spawning
killifish (Fundulus heteroclitus} gave a positive response in all instances. Pollack
pituitary brei (Pollachius virens] from pre-spawning fishes gave only a weak re-
sponse, and wholly negative results were obtained with an extract of Fundulus
pituitary glands from fish taken at the beginning of the spawning season. While
these data suggest a possible period of depletion during the early spawning phase of
the sexual cycle, the findings require confirmation. The experiments also demon-
strated that the pituitary of the three species investigated contains a growth-
promoting factor and a molting hormone, presumably somatotropin and thyrotropin,
respectively.
LITERATURE CITED
DE ALLENDE, I. L. C., 1939. Accion de la prolactine sobre el oviducto de los batracios. Rev.
Soc. Argent. Biol, 15: 190-193.
DE ALLENDE, I. L. C., AND O. ORIAS, 1955. Hypophysis and ovulation in the toad Bufo arenarum
(Hensel). Ada. Physiol. Latino Amer., 5: 57-84.
BLAIR, A. P., 1946. The effects of various hormones on primary and secondary sex characters
of juvenile Bufo jotvlcri. J. Exp. Zool., 103 : 365-400.
CARLISLE, D. B., 1954. The effect of mammalian lactogenic hormone on lower chordates. /.
Mar. Biol. Assoc., 33: 65-68.
CHADWICK, C. S., 1940. Identity of prolactin with water drive factor in Triturus viridcsccns.
Proc. Soc. Exp. Biol. Mcd., 45 : 335-337.
CHADWICK, C. S., 1941. Further observations on the water drive in Tritnrus viridcsccns. II.
Induction of the water drive with the lactogenic hormone. /. Exp. Zool., 86: 175-187.
CHADWICK, C. S., AND H. R. JACKSON, 1948. Acceleration of skin growth and molting in the
red eft of Triturus viridcsccns by means of prolactin injections. Anat. Rcc., 101 :
718.
FOGLIA, V. G., 1940. Accion de la hipofisis del sapo Bufa arenarum Hensel sobre el el buche
de la paloma. Rev. Soc. Argent. Biol., 16: 559-562.
FONSECA RIBEIRO, O., AND J. F. TABARELLi ME™, 1943. A<jao da hipofise de peixe sobre o
oviduto do sapo, Bufo marinus (L.). Rev. Fac. Mcd. Vet. S. Paulo, 2: 99-102.
GRANT, W. C., 1958. Water drive response in efts of Diemyctylus viridcsccns to varying dose
levels of prolactin. Anat. Rcc., 131 : 561.
GRANT, W. C., AND J. A. GRANT, 1956. The induction of water drive in the land stage of
Triturus viridescens following hypophysectomy. Anat. Rcc., 125: 604.
GRANT, W. C., JR., AND J. A. GRANT, 1958. Water drive studies on hypophysectomized efts
of Diemyctylus viridcsccns. I. The role of the lactogenic hormone. Biol. Bull., 114:
1-9.
HOUSSAY, B. A., 1947. Ovulacion y postura del sapo Bufo arenarum Hensel. V. Transporte
de los ovulos por el oviducto y el utero. Rev. Soc. Argent. Biol., 23 : 275-287.
JUHN, M., AND P. C. HARRIS, 1958. Molt of capon feathers with prolactin. Proc. Soc. Exp.
Biol. Med., 98 : 669-672.
LEBLOND, C. P., AND G. K. NOBLE, 1937. Prolactin-like reaction produced by hypophysis of
various vertebrates. Proc. Soc. Exp. Biol. Mcd., 36: 517-518.
MEDAWAR, P. B., 1953. Some immunological and endocrinological problems raised by the evo-
lution of viviparity in vertebrates. Symp. Soc. Exp. Biol., 7 : 320-338.
PICKFORD, G. E., AND J. W. ATZ, 1957. The Physiology of the Pituitary Gland of Fishes.
N. Y. Zool. Soc. New York.
PICKFORD, G. E., AND BERNARD KOSTO, 1957. Hormonal injection of melanogenesis in hypo-
physectomized killifish (Fundulus heteroclitus). Endocrinology, 61: 177-196.
TUCHMANN-DUPLESSIS, H., 1948. Developpement des caracteres sexuels du Triton traite par
des hormones hypophysaires gonadtropes et lactogenes. C. R. Soc. Biol., 142 : 629-630.
TUCHMANN-DUPLESSIS, H., 1949. Action de 1'hormone gonadtrope et lactogene sur le com-
portment et les caracteres sexuels secondaires du triton normal et castre. Arch. Anat.
Micr. Morph. Exp., 38: 302-317.
DEVELOPMENTAL CHARACTERISTICS OF LOW TEMPERATURE
CHICK BLASTODERMS. I. INFLUENCE OF THE HYPOBLAST
ON DEVELOPMENT IN VITRO1
JOHN R. HARRISON 2
Department of Zoology, Miami University, Oxford, Ohio
This is the third paper dealing with investigations of the developmental charac-
teristics of chick blastoderms incubated in ovo for extended periods at 25° C. Pre-
vious papers (Harrison and Klein, 1954 ; Harrison, 1957) reported on the formation
and dispersal of the primitive streak during low temperature incubation, and on a
differential sensitivity between neurogenesis and heart formation to low temperature
incubation.
Depending upon the length of exposure to the low temperature, morphologically
different blastoderms occur. The primitive streak forms but subsequently dis-
perses in a posterio-anterior direction. Blastoderms containing partial streaks are
called the "diffuse streak" type. As dispersal progresses blastoderms retaining
only the node result. These are called "node" blastoderms. With continued in-
cubation dispersal of the node occurs to yield the "diffuse node" type and finally
blastoderms are formed which are devoid of axiation, the "anidian-like" blastoderms.
Interest in the present work resulted from a discrepancy in previous data
(Harrison, 1957) which reported the adaptation of in vitro culture methods to
the study of the low temperature blastoderms. Following incubation at 25° C.
for 10 to 14 days, the blastoderms were removed, transected and cultured on a
yolk-albumen medium. The percentage of explants developing normally after 10
days exposure was lower than that for explants exposed for 11 days. The sigmoid
curve was not expected and did not agree with data published in 1954 (Harrison
and Klein). A possible explanation seemed to be a variation in technique as the
experiments progressed. The original method of transecting blastoderms was that
followed by Spratt (1947b) in which he cut blastoderms approximately 0.2 mm.
behind the primitive pit. This procedure was followed in the early experiments
but very soon the level of the cut was shifted posteriorly near the junction of the
area pellucida and area opaca. The developmental capacity of explants which
retained more of the primitive streak area appeared to be better at that time and
the procedure was subsequently followed in the experiments.
The present work reports on the attempt to determine if there is a significant
difference in developmental potency of blastoderms transected at different levels.
Due to the higher developmental potency of the diffuse streak blastoderms as op-
posed to the other degenerate types, this form was used. Since transection re-
1 This investigation was supported by a research grant (B-789) from the National Institute
of Neurological Diseases and Blindness, Public Health Service.
- The author wishes to express his appreciation to Margaret Ann Berry and Carol
Crumbaker for their interest and contributions to the work as assistants on the project.
436
LOW TEMPERATURE CHICK BLASTODERMS 437
moves both epiblast and hypoblast, data are presented to show the effect of the
removal of posterior hypoblast.
METHODS AND RESULTS
General
Fertile eggs were obtained locally from a two-year-old flock of White Rock
chickens during the period June, 1956 through October, 1957. A total of 567
eggs was used and showed a fertility rate of 83%. All eggs were less than 24
hours old when placed in incubators, to minimize effects from storage. Incubation
was carried out in two identical forced-draft, thermostatically controlled incubators
having a relative humidity of 75 ± 5.0%. For low temperature incubation, the
incubator was kept in a walk-in cold room with a temperature of 18° C., which
permitted maintenance of an incubation temperature of 25° ± 0.5° C. The control
incubator was maintained at the normal incubation temperature of 37.5° ± 0.5° C.
In all instances the period of low-temperature incubation was 9 days. On the
basis of previous work (Harrison and Klein, 1954; Harrison, 1957), this particu-
lar period was selected as being one which would yield a high percentage of
blastoderms containing diffuse primitive streaks. Of 390 fertile eggs which were
incubated for 9 days at 25° C., 238 (61%) contained blastoderms with the diffuse
streak.
The Petri-dish technique of tissue culture developed by Fell and Robinson
(1929) and subsequently modified by Spratt (1947a) was used and is fully de-
scribed elsewhere (Spratt, 1947b; Harrison, 1957). For the culture medium a
yolk-albumen-saline-agar mixture was used. This was prepared under sterile
conditions by adding the contents of one unincubated egg to 100 cm.3 of Ringer
solution.3 This mixture was shaken thoroughly and decanted to centrifuge tubes
in such a manner as to leave the larger masses of albumen behind. The fluid was
centrifuged for one hour at 825 g. Twenty cubic centimeters of the yolk-albumen
supernatant were added to a sterilized mixture of 20 cm.3 of Ringer solution, 1 cm.3
of indicator solution (0.02 gm. phenol red/100 cm.3 solution), and 200 mgm. of
Bacto Agar which had been cooled to 30°-40° C. After mixing, CO2 was added
by blowing through a sterile pipette with a cotton plug. One cubic centimeter of
the medium was pipetted into each culture watch crystal.4 The pH of the final
medium varied from 7.4 to 8.1. Appropriate explants were placed on this medium
and incubated at 37.5° C. for two days. At the termination of incubation, the
morphology and degree of development of the blastoderms were recorded. They
were then fixed in Bouin's solution. All explants were sectioned, stained with
Delafield's hematoxylin, and counterstained with eosin.
In ovo controls
Two types of in ovo controls were used to determine the developmental potency
of the intact blastoderms as opposed to those removed from the yolk. In the first
type, 21 eggs were incubated for two days at 37.5° C. Of these, 16 were fertile
s NaCl, 0.9 ; KC1, 0.042 ; CaCl2, 0.024 gm. per 100 cm.3 solution.
4 The yolk-albumen medium was prepared fresh each morning of the day in which ex-
plantations were made. Storage of culture plates reduced the success of explantations.
438
JOHN R. HARRISON
and 15 or 93.8% produced normal embryos. From this particular flock of chickens,
fertile eggs develop normally for the first two days in better than 90% of the cases.
In the second type 107 fertile eggs were incubated for 9 days at 25° C. followed
by incubation for two days at 37.5° C. Fifty-one of these (47.7%) produced
normal embryos. This potency for normal development was not as high as pre-
viously reported (Harrison and Klein, 1954). In the earlier work one group of
10 eggs was pretreated for 10 days at 25° C. Ninety per cent of the eggs produced
normal embryos. In the present work, 1 1 groups of eggs were set, and the highest
percentage of normal development for any group was 83.3% and the lowest was
0.0% normal development. The average of 47.7% is comparable to that obtained
in 1957 (Harrison) and is more indicative of the true developmental potency of
eggs exposed at 25° C. and subsequently incubated at 37.5° C. in ovo. The
present control groups consisted of 5 to 26 eggs and represent a much better
sampling than did the 1954 group. Differences between the groups, in terms of
susceptibility to the low temperature exposure, seem to be the result of individual
variations in eggs.
AREA OPACA —
AREA
PELLUCIDA
VACUOLE
PRIMITIVE
STREAK
I mm.
FIGURE 1. Morphology of a typical diffuse streak blastoderm resulting from
incubation for 9 days at 25° C.
Variations in level of blastoderm transection
To determine whether or not the amount of streak which remained in a
transected blastoderm affected its in vitro development, a series of experiments
was performed transecting the blastoderm at different levels of the primitive streak
behind the node. In all instances the blastoderms used were those previously
classified as "diffuse streak." Figure 1 represents a typical diffuse streak blasto-
derm. In general, the area pellucida tends to be elliptical rather than narrow at
the posterior portion as in normal definitive streak blastoderms. The posterior
portions of the streak are no longer visible, indicating a posterio-anterior gradient
of streak dispersal. The anterior end of the primitive streak remains but does
not contain a clearly defined primitive pit area. Vacuolation has occurred in the
area pellucida and frequently is more pronounced than shown in the figure. How-
ever, the degree of vacuolation is not correlated with ability to continue normal
development.
LOW TEMPERATURE CHICK BLASTODERMS
439
In preparation for explantation, blastoderms were removed from eggs exposed
to the low temperature. The morphology was determined and diffuse streak area
was measured from the anterior rim of the primitive node posteriorly to the edge
of the area pellucida with an ocular micrometer. A transverse cut was made at
the desired level with two finely ground steel needles. Excess area opaca was
trimmed from the anterior piece following the method of Spratt (1947b). The
length of the streak in the anterior piece was re-measured, and it was then placed
on the culture medium using a wide-mouth medicine dropper. Excess Ringer
solution was removed with a fine pipette.
TABLE I
Summary of results with transected diffuse streak blastoderms
incubated in vitro for two days
In vitro development of the explants
Explants containing from node to
Explants containing from 50% to
49% of the primitive streak
100% of the primitive streak
Expt.
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32
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6
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39
9
2
6
1
2
4
3
7
Totals 13
106
2
10
18
23
35
18
Table I summarizes the results of these explants after two days of incubation
at 37.5° C. One hundred and six diffuse streak blastoderms were transected and
the anterior portions explanted to the yolk-albumen medium. Thirty of the ex-
plants included only the primitive node region or up to 49% of the primitive streak.
Seventy-six of the explants retained from 50% to 100% of the primitive streak
tissue. Of the explants retaining 49% or less of the primitive streak tissue only
6.7% developed normally and 60% showed no development during the period of
incubation. The remaining 33.3% were classified as abnormal embryos. These
are embryos which are normal in at least one morphological system, i.e., nervous,
circulatory (heart and/or vitelline vessels), closed gut or notochord, as revealed
440
JOHN R. HARRISON
by histological examination of serial sections. As opposed to these, explants which
had no discernible embryonic systems or which lacked at least one normal system
were classified as "no development."
In contrast, explants which retained from 50% to 100% of the streak tissue
developed normally in 30.3% of the cases. Forty-six per cent of the explants were
classified as abnormal and 23.7% showed no development. This developmental
difference proved to be significant when a coefficient of correlation was made for
variation between individual experiments as opposed to occurrence of develop-
mental types. Comparison of normals, abnormals and those showing no devel-
opment for explants with 49% or less of the primitive streak gave an F- value
significant at the 0.1% level (F2oA5', n\ — 2, n2 = 15). The same comparison
lOCH
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o:
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50-
AMOUNT OF PRIMITIVE STREAK TISSUE PRESENT
jjj NODE- 49%
;li; 50-100%
- Wff: 1
J-6.6%
-53.3%
NO
DEVELOP-
MENT
NORMAL
BRAIN
NORMAL
NEURAL
CORD
NORMAL
HEART
NORMAL
VITELLINE
VESSELS
FIGURE 2. Comparison of explant development in relation to amount of primitive
streak tissue present.
for explants retaining 50% to 100% of the primitive streak was not significant.
Differences in the percentages of the developmental types were a factor of chance
rather than the result of transecting the embryo. The developmental potency of
transected embryos is enhanced when the level of transection is such that the explants
retain more than 50% of the primitive streak tissue.
Figure 2 summarizes material contained in Table I. The trend in develop-
mental types shown by explants containing 49% or less of the primitive streak
tissue is significant at the 1% level (-F5.066; n\ = 4, w2 = 25) and the trend for
explants with 50-100% of the primitive streak is significant at the 0.1% level
(-^7.798; n\ = 4, w2 = 50). Explants which possess a normal brain are normal
with respect to all other systems. Consequently the percentage given is the same
LOW TEMPERATURE CHICK BLASTODERMS
441
for those classified normal. The difference between this percentage and the per-
centage of explants showing no development is 6.6% for explants containing
50-100% of the primitive streak. For explants retaining 49% or less of the primi-
tive streak, the same percentage difference is 53.3%. This striking contrast in
developmental potency between the two types of explants is significant, since both
of the over-all trends are significant, and illustrates the necessity of including pos-
terior portions of the primitive streak in explants.
Removal of posterior hypoblast
Establishment of the importance of the posterior portion of the primitive streak
region for development of explants posed the problem as to whether or not the
hypoblast of this region was the stimulating tissue. In an attempt to answer this
the following experiments were performed. Low-temperature blastoderms were
I mm.
FIGURE 3. Preparation of diffuse streak blastoderm for explantation in hypoblast removal
experiments. See text for discussion.
removed from eggs and diffuse streak blastoderms were selected for explantation.
The most successful operative steps were as follows. Excess area opaca was re-
moved from around the entire area pellucida. The length of the primitive streak
tissue was measured from the anterior edge of the primitive node posteriorly to
the edge of the area pellucida. The blastoderm was then inverted and the meas-
urement checked from the ventral surface. With finely ground steel needles the
hypoblast was cut at right angles to the streak axis approximately at a level which
would leave 37% of the primitive streak tissue anterior to the cut. This is shown
in Figure 3 by the anterior margin of the stippled area. This transverse cut was
continued posteriorly and parallel to the primitive streak axis at each edge to the
junction of the areas pellucida and opaca. This flap of hypoblast was peeled back
by gently teasing with the needles. Removal of the hypoblast was most difficult
442
JOHN R. HARRISON
in the streak region where it had an intimate fusion with the tissue of the epiblast.
The operation was completed by making a transverse cut across the entire tissue
at a level which left approximately 75% of the primitive streak epiblast intact.
The explant was transferred to the culture medium.
Figure 3 summarizes schematically the steps involved in preparing a blasto-
derm for explantation. The solid line indicates the method of trimming the blasto-
derm to remove the excess area opaca. The stippled area represents the portion
of the hypoblast that is removed. The cross-hatched area is discarded after tran-
section, leaving approximately 75 % of the primitive streak tissue in the anterior
blastoderm portion to be explanted. Essentially the explant retains 75 % of the
original streak area from which about half of the underlying hypoblast is removed
to leave approximately 37% of the original hypoblast intact.
TABLE II
Summary of results in the hypoblast removal experiments
In vitro development of explants during two
days of incubation at 37.5° C.
Explants with hypoblast
Explants with hypoblast
removed
intact: Controls
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93
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2
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3
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2
2
3
3
0
3
3
3
3
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Totals 9
83
13
13
7
23
16
11
The decision to transect the blastoderms at a level which would leave approxi-
mately 75% of the primitive streak area intact was based on the foregoing experi-
ments dealing with level of cut. And in this present series of experiments a control
group consisted of blastoderms with the excess area opaca removed and transected
so that about 75 % of the primitive streak area remained intact. The only difference
was that the hypoblast was left intact.
Eighty-three explantations were made. In 33 of these hypoblast was removed,
and the remaining 50 were controls. The nature of the development of these ex-
plants during two days of incubation at 37.5° C. is summarized in Table II. Ex-
amination of serial sections of explants from which the hypoblast was removed
LOW TEMPERATURE CHICK BLASTODERMS
443
showed that in all instances the hypoblast reconstituted during the two day incuba-
tion period.
The information in Table II is summarized in Figure 4. The information in
the graph was analyzed for coefficient of correlation to see if the trends in develop-
ment types for both kinds of explant were significant. The trend as seen in the
graph for development of explants with hypoblast removed is significant at the 0.1%
level (-F7. 121; % == 4, n^ -- 30). The F-value for analysis of the trend in develop-
ment of explants with hypoblast intact is significant at the 1% level (F3.5S7 ; MI == 4,
u2 = 40). Removal of the hypoblast results in a decrease in developmental potency.
Although there is a very slight (0.8%) difference in the numbers of embryos show-
ing no development in the two types of explants, there is a significant lowering in
the numbers of explants showing normality of the nervous system. The hypoblast
\ HYPOBLAST REMOVED
8$ HYPOBLAST INTACT
UJ
o
z
UJ
CL.
or
o
O
N?
50-
-22%
33.3%
NO NORMAL NORMAL NORMAL NORMAL
DEVELOP- BRAIN NEURAL HEART VITELLINE
MENT CORD VESSELS
FIGURE 4. Comparison of explant development in relation to presence or absence of hypoblast.
of the posterior primitive streak region has some influence on the normal develop-
ment of the central nervous system. In contrast, the development of the circulatory
system, heart and/or vitelline vessels, is not affected. This specificity of hy-
poblast influence would be more clearly indicated if it were possible to show signifi-
cance for the difference in percentage between explants possessing a normal heart
as opposed to those with a normal brain for control explants in contrast to explants
with hypoblast removed. However, a coefficient of correlation for this difference
shows that there is as much variation between individual experiments as between
explant types. This lack of significance can be explained on the basis of develop-
mental sequence of the two systems. The heart forms later in development than
does the brain. Explants which do not form a heart in the two days of in vitro
444 JOHN R. HARRISON
incubation may be expressing a slower developmental rate rather than abnormal
development.
DISCUSSION
In any series of experiments which by necessity disrupt the normal conditions
under which an embryo develops, it is important to determine the extent of the
disruption. The various types of controls maintained in the present experiments
provide an opportunity to determine if application of the in vitro culture technique
has influenced the results. Of the series of in ovo controls which were exposed only
to incubation at the normal 37.5° C. temperature for 2 days, 93.8% developed
normally. In contrast, fertile eggs which were first incubated for 9 days at the
lower temperature (25° C.) and then incubated in ovo at 37.5° C. contained em-
bryos which developed normally only 47.7% of the time. This decrease in develop-
mental potency can only be ascribed to the effect of low temperature exposure. The
point of interest is that blastoderms which were exposed in ovo at 25° C. for 9 days
and then cultured in vitro at 37.5° C. as transected blastoderms developed normally
in 46% of the cases. This percentage is for explants under optimal conditions, i.e.,
those containing 75% of the primitive streak material. The close correspondence
of developmental capacity of low temperature blastoderms in ovo and in vitro in-
dicates that application of the culture method is valid and does not affect the
developmental potency of blastoderms in terms of the first two days of development.
The drop in developmental potency when posterior portions of the primitive
streak region are removed by transection or dissection indicates that tissue of this
region is concerned with normal development of the central nervous system, pri-
marily with morphogenesis of the brain. Specifically those experiments involving
removal of the hypoblast show that the tissue which is related to this process is
the hypoblast. Transection at levels which remove the posterior half of the
primitive streak region results in a drop in developmental potency which can be
duplicated by removal of the hypoblast from that area while leaving the epiblast
intact. This does not imply that the drop in developmental potency is reflected in
a great increase in the numbers of explants which show no development. Rather
the striking change is in the numbers of embryos that are abnormal as a result of
mal-development of the central nervous system.
A corresponding influence, or organizer-like activity, of the posterior hypoblast
is not shown in work with normal definitive streak blastoderms. Fraser (1954)
investigated the role of the posterior hypoblast in normal primitive streak embryos
and concluded that this tissue is not essential. The difference in conclusions may
be explained on the basis that the low temperature blastoderm and the normal
definitive streak blastoderm are not physiologically and developmentally identical.
The ability of a diffuse streak blastoderm which has not undergone involution
(Harrison, 1957) to re-form a primitive streak indicates that it still retains develop-
mental capacities of a normal pre-streak blastoderm. It is conceivable that the
posterior hypoblast of the pre-streak blastoderm possesses an organizer-like activity
that has been expressed by the time the blastoderm attains the definitive streak
stage. This concept agrees with Waddington's work (1933) in which he showed
that pieces of posterior hypoblast were capable of inducing axiation in anterior
epiblast which normally gives rise to extra-embryonic material.
If an organizer activity by the posterior hypoblast is assumed, it is possible to
LOW TEMPERATURE CHICK BLASTODERMS 445
hypothesize that the effect of lowered temperature is upon the hypoblast. The
greater sensitivity of the nervous system to low-temperature incubation as opposed
to that of the heart and/or vitelline vessels would be an expression of hypoblast
sensitivity. This would explain the apparent residual effect of low-temperature
incubation, i.e., low temperature exerts its effect at a time in development before
the central nervous system begins to form.
SUMMARY AND CONCLUSIONS
1. This paper reports work showing the influence of posterior hypoblast on the
in vitro development of diffuse streak blastoderms produced by incubation at low
temperature (25° C.).
2. Normal in ovo controls incubated at 37.5° C. for two days show 93.8%
normal development. In ovo controls incubated at 25° C. for 9 days followed by
two days' incubation at 37.5° C. produced normal embryos in only 47.7% of the
cases. Transected blastoderms explanted to a yolk-albumen medium following
9 days' exposure in ovo at 25° C. developed normally in 46% of the cases. Thus
the drop in developmental potency is the result of low-temperature exposure rather
than transection.
3. Fertile eggs were incubated for 9 days at 25° C. The blastoderms were
removed from the yolk and only those containing a diffuse streak were chosen for
transection and explantation to a yolk-albumen medium. The results show that
explants which retain only the primitive node region or up to 49% of the primitive
streak tissue have a lower developmental potency than explants in which 50-100%
of the primitive streak region was retained.
4. A series of explants were transected in such a manner that approximately
75% of the original primitive streak epiblast and about 37% of the original hypoblast
beneath the streak remained. In comparison with explants which retained 75% of
both hypoblast and streak region, those with hypoblast removed show a significantly
lower developmental potency with respect to formation of normal brain.
5. The present results are discussed in correlation with previous results dealing
with the effect of low temperature incubation on morphogenetic processes.
LITERATURE CITED
FELL, H. B., AND R. ROBINSON, 1929. The growth, development and phosphatase activity of
embryonic avian femora and limb buds cultivated in vitro. Biochem. J ., 23 : 767-784.
ERASER, R., 1954. Studies on the hypoblast of the young chick embryo. /. Exp. Zool, 126:
349-392.
HARRISON, J. R., 1957. Morphogenesis of chick embryo in vitro after exposure to lowered
temperature in ovo. Physiol. Zool., 30: 187-197.
HARRISON, J. R., AND I. KLEIN, 1954. Effect of lowered incubation temperature on the growth
and differentiation of the chick embryo. Biol. Bull, 106: 48-59.
SPRATT, N. T., JR., 1947a. A simple method for explaining and cultivating early chick embryos
in vitro. Science, 106 : 452.
SPRATT, N. T., JR., 1947b. Development in vitro of the early chick blastoderm explanted on
yolk and albumen extract saline-agar substrata. /. Exp. Zool., 106 : 345-366.
WADDINGTON, C. H., 1933. Induction by the endoderm in birds. Arch. f. Entw. 128: 502-541.
A STUDY OF THE POLYSACCHARIDE HISTOCHEMISTRY OF THE
OVIDUCT OF THE NEWT, TRITURUS VIRIDESCENS x
A. A. HUMPHRIES, JR. AND W. N. HUGHES -
Department of Biology, Emory University, Atlanta 22, Georgia
The physiological importance of the jelly secreted by the oviducts of amphibia
has become increasingly apparent in recent years, especially so with regard to the
process of fertilization (Good and Daniel, 1943; Bernstein, 1952; Kambara, 1953;
Nadamitsu, 1953, 1957; Tchou-Su and Wang Yu-Lan, 1956; Hughes, 1957).
Previous work in this laboratory seems to show that the jelly is somehow involved
in the normal blockage of meiosis in the eggs of Triturus viridescens (Humphries,
1955, 1958, and unpublished) ; thus for our work, it had become almost essential to
know more of the nature of the oviducal secretions and something of the parts of
the oviduct which were actively involved.
The oviducts of Triturus viridescens, as well as those of other urodele am-
phibians, exhibit a gross and histological differentiation into rather well-defined
regions (Adams, 1940, 1950; von Wahlert, 1953). Adams (1940) described six
regions: (1) the infundibular region, (2) a transparent region with a watery
secretion, (3) an opaque region with eosinophilic granules, (4) a transparent, wider
section, similar to section 2, (5) an opaque, chalky-white region, similar to section 3,
and (6) a straight vaginal section with fewer folds and fewer secretory cells, which
leads to the cloaca. The first three lie anterior to the kidney, the remaining three
lie parallel to the kidney. For our purpose, however, it seemed desirable to designate
the divisions of the oviduct somewhat differently, as follows : an infundibular, or
ostial region, apparently non-secreting, at the extreme anterior end ; a region A, just
posterior, of gray color, ending abruptly in a white, or B region, of about the same
diameter, which gradually decreases in diameter and whiteness until it passes into
region C, a division of small diameter and gray color. Region D begins abruptly
as a much wider section of dull white coloration, and region E as a division with
about the same diameter as D, but with a brighter white color. The posterior part
of E is narrower and less convoluted than its anterior portion. Our region A
seems to correspond to region 2 of Adams, B and C to her region 3, D to her
region 4, and E apparently includes regions 5 and 6.
In view of the striking regional zonation of the oviduct and what is known of
the chemistry of amphibian egg jelly, it appeared that an investigation of the poly-
saccharide histochemistry of the secretory portions might provide useful information.
MATERIALS AND METHODS
All animals were collected in April from the vicinity of Franklin, North Caro-
lina, and maintained in a refrigerator at about 12° C. until used. Oviducts were,
1 Supported by an Institutional Grant from the American Cancer Society.
- Present address : Department of Biology, Lubbock Christian College, Lubbock, Texas.
446
HISTOCHEMISTRY OF THE OVIDUCT 447
in general, taken from animals which had been treated with one or two injections
of Antuitrin "S" (Parke, Davis) to induce ovulation, hut several animals were taken
directly from the refrigerator and immediately sacrificed without treatment. No
differences between the oviducts of treated and untreated animals were observed.
In either case, the oviducts were in the normal condition for animals in the breeding
season. Oviducts were, in one instance, taken from an animal which had com-
pleted her breeding activity, and in this animal the oviducts were considerably
smaller in diameter and less convoluted than in females in breeding condition. The
results presented here represent studies of the oviducts of thirteen animals, though
not all of these were used for all procedures.
Periodic Acid-Schiff Method : Portions of the oviduct were fixed in Bouin's
fixative, 10% formalin, or Carney's fixative, embedded in Tissuemat, and sectioned
at 10 microns. The PAS procedure was carried through on both untreated sec-
tions and sections treated with saliva or malt diastase for removal of glycogen.
Toluidine blue: Fixation was in Bouin's fixative, 10', < formalin, Carney's
fluid, or in 4% basic lead acetate followed by formalin. Sections were stained for
ten minutes in a 0.03% solution of toluidine blue O in a citrate buffer at pH 3.8,
after which they were dehydrated in alcohol, cleared in xylene, and mounted in
Clarite or Canada balsam.
Alcian blue: Fixation was in Carney's fluid or 10% formalin. Most of the
staining was done with Alcian Blue 8GX 300, kindly supplied by Arnold, Hoffman
and Company, Providence, Rhode Island. However, several slides from each
region were stained with alcian blue obtained from the Hartman Leddon Company.
For our purposes the dye from the former source was considerably superior, since
it appeared to have a greater differential specificity for the several regions of the
oviduct. Sections were stained for twenty minutes in a 0.3% solution of the dye
in 3% acetic acid (pH 2.5 to 2.6).
Hyaluronidase : Nearly all the tests with hyaluronidase involved material from
region A only. Testicular hyaluronidase, assayed at approximately 300 U. S. P.
units per mg., was obtained from Nutritional Biochemicals Corporation. Sections
were treated in solutions of enzyme in 0.3% NaCl at pH 5.7 or in a phosphate
buffer at pH 6.7, at 37° C. Several concentrations and periods of treatment were
used, ranging up to 60 U. S. P. units per ml. for as long as eighteen hours. Stain-
ing was done with toluidine blue, as described above. Controls consisted of
alternate sections placed in comparable solutions of boiled enzyme and in enzyme-
free solutions.
Ribonuclease : Ribonuclease (Worthington) was used in a 0.1% solution at
pH 5.9. Sections (chiefly from region A) were treated for periods of one hour or
six hours at 37° C. Following treatment, sections were stained for ten minutes in
toluidine blue, as described above, in 1% pyronin Y in aqueous solution at pH
3.4, or in 1% pyronin Y in a phosphate buffer at pH 6.7.
OBSERVATIONS
All the descriptions to follow refer to the secretory epithelium alone. In general,
regions A, B, and D showed reactions in common while regions C and E responded
differently but like each other.
448 A. A. HUMPHRIES, JR. AND W. N. HUGHES
The entire secretory portion of the oviduct stained intensely with the PAS
technique. The ostial region, which presumably is relatively non-secretory, and is
characterized by very low epithelium, stained pink. The A region stained a bright
purplish red, whereas regions B, C, D, and E stained a distinctly different shade
which might be called reddish purple. The staining of all portions remained un-
changed after treatment with saliva or malt diastase.
There was a marked difference in the response of the several regions to
toluidine blue. The staining of the ostial region was orthochromatic. Region A
showed violet to purple metachromasia in all cases. This region in the one post-
breeding animal also showed metachromasia after all fixatives ; however, the color
produced in lead acetate-fixed material was chiefly blue. The metachromasia of
region A is alcohol-fast. After destaining for periods up to one and one-half hours
in 70% alcohol, the major part of the stain was removed, yet the violet meta-
chromasia persisted. Region B usually stained an orthochromatic dark blue, but
in some cases strong tinges of purple were seen. There was, however, no difficulty
in distinguishing between regions A and B following this stain. Sections from
region C showed little or no cytoplasmic stain, and that which was present was
usually a very light blue. In a few cases there were traces of metachromasia. The
staining reaction of region C, then, was a striking contrast to that of either A or B.
Preparations from region D were variable in their response to toluidine blue, but,
for the most part, the cytoplasm stained a pale blue, and nearly all preparations
showed at least some violet metachromasia. The cytoplasm in cells of region E
stained a pale blue, with no trace of metachromasia.
The reaction of the several regions to alcian blue was also varied. The cyto-
plasm of the secretory cells of region A stained a bright sky blue, as did that of
region B. The cells of region C, however, stained only lightly. In many cases the
stain in this region was so light as to be only barely detectable. The cytoplasm of
the cells of region D stained sky blue, similar to the reaction of regions A and B,
while that of region E was extremely lightly stained, similar to the reaction of the
cells of region C.
Although only one slide from each region was used, it may be worthwhile to
mention that results with Kale's technique were essentially the same as those with
alcian blue, but gave promise of less clear differentiation between the regions.
Treatment with hyaluronidase failed to alter the strong violet metachromasia of
region A, or, in the few tests run, the staining reactions of regions B, C, or D.
Similarly, treatment with ribonuclease failed to alter the staining reaction of the
cytoplasm of the cells of either region A or region B. No other regions were tested.
DISCUSSION
These results show the secreting epithelium of the newt oviduct to be rich in
polysaccharide, apparently distributed qualitatively among the several regions. The
histochemical differences follow the zonation which is anatomically demonstrable.
It is clear that the epithelium contains polysaccharide other than glycogen, since the
PAS reaction of all regions remains unchanged after glycogen digestion. The
PAS technique allows but little differentiation between the regions, however, since
region A is the only one which stains in a fashion distinctly different from the
others.
HISTOCHEMISTRY OF THE OVIDUCT 449
A striking parallelism between metachromatic staining with toluidine blue and a
positive reaction with alcian blue has been previously shown (Vialli, 1951 ; Wagner
and Shapiro, 1957). This finding was clearly borne out in our material. Un-
fortunately, the metachromatic staining reaction has had a long history of confusion
as to application and interpretation, but "true" metachromasia, in the sense of Lison
(1953) or Kramer and Windrum (1955), generally is taken to indicate the presence
of sulphated mucopolysaccharides, though nucleoprotein has also been reported to
stain metachromatically at times (Wiame, 1946; Penney and Balfour, 1949; Kramer
and Windrum, 1955). The occurrence of alcohol-resistant beta (violet) meta-
chromasia, such as encountered in our material, is strong presumptive evidence of
nucleoprotein, according to Kramer and Windrum (1955). However, we found no
change in staining reaction with toluidine blue or with pyronine following treatment
with ribonuclease. Results of several workers (especially Vialli, 1951 ; Lison,
1954; Alowry, 1956; and Wagner and Shapiro, 1957) indicate that alcian blue
positivity is good evidence for the presence of acidic carbohydrates. Combining the
evidence, then, the distinctive staining reactions of region A, in particular, and
probably those of regions B and D, would seem most likely attributable to the
presence of acid polysaccharide. The results with hyaluronidase seem to rule out
the possibility that the distinctive reactions, at least of region A, are due to hyalu-
ronic acid or anything very closely related to it.
Similar findings with respect to the PAS reaction, toluidine blue metachro-
masia, and hyaluronidase treatment have been reported for the Japanese newt,
Tritnnts pyrrhogastcr, by Kambara (1956a, 1956b, 1957a, 1957b). Due to lack of
certainty as to corresponding regions in oviducts of the two species, it is not possible
to make more than a rough comparison of our results with those of Kambara, but
our results show strong general agreement with his.
The most probable conclusion to be drawn, at present, from the findings of
distinctive reactions along the oviduct is that the differential staining is due to the
presence of acid polysaccharide in regions A, B, and D only, with the positive PAS
reaction of regions C and E due perhaps to neutral polysaccharide. However,
quantitative differences alone might account for the results. Since PAS positivity
and strong metachromasia do not tend to coexist, it is worthwhile to note the sug-
gestion of Hale (1957), that the combination may be caused by the presence of two
distinct substances. Our evidence, coupled with evidence from studies on the
chemistry and physiology of egg jellies themselves (Immers and Vasseur, 1949;
Vasseur. 1952; Kelly, 1954; Minganti, 1955; Runnstrom and Immers, 1956),
leads to the conclusion that the metachromasia and alcian blue positivity of regions
A, B, and D are probably due to the presence of a heparin-like compound. If this
is the case, and if, as seems quite probable, the innermost layer secreted about the
egg contains the substance, it may very likely have significant effects upon the
physiology of the maturing oocyte. The effects of heparin and heparin-like com-
pounds on the physiology of cells, especially egg cells, have been studied for years,
particularly by Heilbrunn and his co-workers (see Heilbrunn, 1956, for references).
In addition, one of us (Humphries, 1955, 1958) has obtained results which have
led to the hypothesis that the oviducal jell)- plays an important role in the natural
blockage of the second meiotic division in the oocyte prior to fertilization. Oocytes
never exposed to oviducal jelly, such as coelomic eggs and eggs stopped experi-
450 A. A. HUMPHRIES, JR. AND W. N. HUGHES
mentally in the ostial (non-secreting) part of the oviduct, are capable of completing
meiosis, while eggs exposed to jelly have in no case been seen to advance beyond
the normal stage of blockage, metaphase II. It is perhaps significant that as the
oocyte enters the first secreting portion of the oviduct it is completing the first
meiotic division or beginning the second (Humphries, 1956). One of the possible
explanations of the meiotic blockage is that the oviduct secretes an antimitotic
substance. This possibility led to the present study of the histochemistry of the
oviduct, with the aim of gaining information concerning the secretions of particularly
the more anterior regions. Since heparin-like substances have been shown to act as
antimitotics (see especially Heilbrunn, 1956, and Heilbrunn ct al., 1957), the dis-
covery that oviducal region A apparently produces a heparin-like compound is in
good agreement with the hypothesis. If this type of antimitotic is involved, how-
ever, it is surprising that blockage of the division occurs at metaphase, rather than
prior to spindle formation.
SUMMARY
1 . Application of some techniques of polysaccharide histochemistry to the oviduct
of the newt showed a histochemical differentiation of the secretory epithelium cor-
responding to the grossly and histologically demonstrable zonation of the oviduct.
All regions responded positively to the PAS technique. No difference was detected
in sections previously exposed to glycogen digestion methods. Regions designated
A, B, and D were metachromatic with toluidine blue and reacted positively to alcian
blue.
2. The most probable explanation of the differences in staining reaction seems
to be the presence of an acid polysaccharide, probably a heparin-like compound, in
regions A, B. and D, and its absence (or much lower concentration) in regions C
and E.
3. The possible significance of the findings relative to the physiology of the
oocyte, particularly with regard to meiotic blockage, is discussed.
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ADAMS, A. ELIZABETH, 1940. Sexual conditions in Triturus viridesccns. III. The reproductive
cycle of the adult aquatic form of both sexes. Ainer. J. Anat., 66: 235-275.
ADAMS, A. ELIZABETH, 1950. Sexual conditions in Triturus viridesccns. V. The effect of the
administration of diethylstilbestrol on adult normal and castrated females. /. Exp.
Zoo/.. 113: 464-497.
BERNSTEIN, G. S., 1952. Sperm agglutinins in the egg jelly of the frogs Rana pipicns Schreber
and R. clamitans Latreille. Biol. Bull., 103: 285.
GOOD, G. MERLIN, AND J. FRANK DANIEL, 1943. Fertilization of coelomic eggs of Triturus
torosus. Univ. Calif. Publ. in Zoo/., 51: 149-158.
HALE, ARTHUR J., 1957. The histochemistry of polysaccharides. Int. Rev. CytoL, 6: 193-263.
HEILBRUNN, L. V., 1956. The Dynamics of Living Protoplasm. Academic Press, New York.
HEILBRUNN, L. V., W. L. WILSON, T. R. TOSTESON, E. DAVIDSON AND R. J. RUTMAN, 1957.
The antimitotic and carcinostatic action of ovarian extracts. Biol. Bull., 113:
129-134.
HUGHES, NORMAN, 1957. An investigation into the fertilizability of coelomic and oviducal
eggs of the newt, Triturus iriridescens. ASB Bull., 4 : 13.
HUMPHRIES, A. A., JR., 1955. Experimental removal of metaphase II inhibition in ova of
the newt, Triturus viridcscens. ASB Bull., 2 : 7.
HISTOCHEMISTRY OF THE OVIDUCT 451
HUMPHRIES, A. A., JR., 1956. A study of meiosis in coelomic and oviducal oocytes of Triturus
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HUMPHRIES, A. A., JR., 1958. The oviducal environment and normal blockage of meiosis in
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IMMERS, J., AND E. VASSEUR, 1949. Comparative studies on the coagulation process with
heparin and sea-urchin fertilizin. E.vpericntia. 5 : 124-125.
KAMBARA, SHUNICHI, 1953. Role of the jelly envelope of toad eggs in fertilization. Annot.
Zool. Japan., 26: 78-84.
KAMBARA, SHUNICHI, 1956a. Histochemical studies on the distribution of phosphatases in
the oviduct of the newt, Trltnrus pyrrhogaster. Annot. Zool. Japan., 29 : 86-90.
KAMBARA, SHUNICHI, 1956b. Histochemical studies on the distribution of mucopolysaccharides
in the oviduct of the newt, Triturus pyrrhogaster. Annot. Zool. Japon., 29: 146-150.
KAMBARA, SHUNICHI, 1957a. Histochemical studies on the distribution of phosphatases and
mucopolysaccharides in the urogenital organs of the newt, Triturus pyrrhogaster.
Annot. Zool. Japon., 30: 143-149.
KAMBARA, SHUNICHI, 1957b. Metachromatically staining material in epithelial cells of the
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KELLY, JOHN W., 1954. Metachromasy in eggs of fifteen lower animals. Protoplasma, 43:
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KRAMER, H., AND G. M. WINDRUM, 1955. The metachromatic staining reaction. /. Histochem.
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LISON, L., 1953. Histochimie et Cytochimie Animates. Gauthier-Villars, Paris.
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MINGANTI, A., 1955. Chemical investigations on amphibian egg jellies. E.rp. Cell Research,
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MOWRY, R. W., 1956. Alcian blue technics for the histochemical study of acidic carbohydrates.
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PENNEY, J. R., AND B. M. BALFOUR, 1949. The effect of vitamin C on mucopolysaccharide
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THE CHEMICAL CONTROL OF FEEDING IN THE PORTUGUESE
MAN-OF-WAR, PHYSALIA PHYSALIS L. AND ITS BEAR-
ING ON THE EVOLUTION OF THE CNIDARIA *
HOWARD M. LENHOFF AND HOWARD A. SCHNEIDERMAN
Marine Biological Laboratory, Woods Hole, Mass., Howard Hughes Medical Institute,
Miami, Fla., and Department of Zoology, Cornell University, Ithaca, N. Y.
The remarkable demonstration by Loomis (1955) that reduced glutathione
(GSH) induces a feeding response in Hydra led us to examine the feeding re-
sponses of other cniclarians. Our object was to see whether the chemical control
of feeding occurred in other hydrozoans and to identify the chemicals involved.
The first organism selected was the Portuguese man-of-war, Physalia physalis L.
Many different types of zooids suspend from the crested pneumatophore, or float,
of this colonial hydrozoan (Figs. 1-4). Among the most numerous are the gastrozo-
oids (Fig. 5), which are the only members of the colony capable of ingesting food.
These gastrozooids have the usual polyp form, but lack tentacles. In the feeding
process, the prey is drawn up to the gastrozooids which apply their mouths to the
surface of the prey ; the lips of the gastrozooids then spread out until they envelop
the prey (Fig. 4) and digestion proceeds. This process has been described briefly
by Wilson (1947). The present report describes the chemical control by GSH of
the behavior of both isolated and attached gastrozooids, and discusses the possible
evolutionary significance of these findings.
MATERIALS AND METHODS
Physalia were captured off the coast of Woods Hole, Massachusetts, in the
summers of 1957 and 1958 and were maintained in aquaria supplied with fresh
running sea water. A total of fifteen colonies were studied. Hydra littoralis were
grown by the methods of Loomis and Lenhoff (1956).
Experiments testing the effects of GSH and cysteine were performed in the
following manner :
1) Groups of gastrozooids were removed from the float with forceps and scis-
sors. Single polyps were separated, collected in a finger bowl, and rinsed several
times to remove any fluids that had oozed from the cut surface. This washing
prevented most of the spontaneous opening of the gastrozooid mouth which oc-
casionally occurred after the gastrozooids were isolated. Apparently this spon-
taneous mouth opening is a response to some substance released from either the cut
surface of the gastrozooid itself, or from recently ingested food in the cavity of the
gastrozooid.
2) The rinsed gastrozooids were distributed randomly in a series of finger
bowls, each containing 90 cc. of fresh sea water (non-aerated). Only newly iso-
lated gastrozooids with closed mouths were used.
iThis research was supported by a grant (H-1887) from the National Heart Institute,
U. S. Public Health Service.
452
FEEDING IN PHYSALIA
453
FIGURE 1. Physalia fishing tentacle captures small fish.
FIGURE 2. Fishing tentacle draws fish into colony of zooids.
FIGURE 3. Enlargement of Physalia zooids. Note the long coiled fishing tentacle, and the
many small finger-like, white tipped gastrozooids.
FIGURE 4. Gastrozooids envelop captured fish. Note, in the area of the caudal fin, the
mosaic pattern formed by the rims of the spreading gastrozooids in contact with each other.
Compare this arrangement with that of the isolated spreading gastrozooids shown in Figure 7.
3) After the gastrozooids were added to the finger bowls, 10 ml. of a freshly-
prepared, neutralized solution of GSH or cysteine were added to give final concen-
trations ranging from 10 3 to 10~8 M.
EXPERIMENTAL RESULTS
1. Observations of the "feeding response" of Physalia gastrozooids
In the experiments to be described, the gastrozooids exhibited a specific "feed-
ing response," which is described here in detail. Several of the stages are illus-
trated in Figures 5-8.
454
H. M. LENHOFF AND H. A. SCHNEIDERMAN
FIGURE 5. Isolated gastrozooid. The mouth is at the uppermost part at the end of the
narrow cylindrical neck.
FIGURE 6. Gastrozooid induced to spread by GSH. Note the many small batteries of
nematocysts along the rim of the gastrozooid lip.
FIGURE 7. Several GSH-induced spreading gastrozooids cover a large surface area.
FIGURE 8. Gastrozooids turned partially inside out by GSH.
FEEDING IN PHYSALIA 455
Upon addition of GSH or fresh fish blood, the isolated gastrozooids immediately
began to writhe. Within five minutes their mouths opened. Whenever a mouth
contacted a solid object, such as a fish or a glass surface, it attached itself and began
spreading (Fig. 6) as if to enclose the object in conjunction with the other spread-
ing gastrozooids (Fig. 7). The gastrozooids, normally about 1 to 2 mm. in di-
ameter, frequently spread to a diameter of 20-25 mm. By this process many
small gastrozooids could surround and digest a large fish (Fig. 4). The spreading
phenomenon usually took about one-half hour from initial contact with the solid
object until the maximum diameter was reached. This spreading of the gastrozo-
oids may persist for only a few minutes or last for two or more hours. The dura-
tion probably depends in part upon the nature of the surface to which the gastro-
zooid is attached (i.e., smoothness, etc.) and upon their nutritional state. Oc-
casionally spreading gastrozooids detached from the solid object and folded back
over themselves, sometimes completely inverting so that endoderm was on the
outside and ectoderm on the inside (Fig. 8). A similar phenomenon was described
by Loomis (1955) in Hydra.
Some outspread gastrozooids migrated slowly over the surface of the solid ob-
ject leaving a "mucous" trail. The polyp probably secreted extracellular proteolytic
enzyme in this mucus to partially break down its prey.
2. Feeding response of the intact animal
A small piece of filter paper, soaked in a 10~3 M solution of GSH, was placed
several centimeters from the gastrozooids of an intact Physalia. A typical feeding
response occurred, with active squirming of the gastrozooids and then a spreading
of their mouths on the paper and on the wall of the aquarium.
3. Demonstration of a chemical feeding mechanism using live prey
Ten gastrozooids and a small killifish (Fundulus sp. ) (8 cm.) were placed in a
finger bowl. After a half hour no feeding response of the gastrozooids was ob-
served. At this time a fragment of a fishing tentacle armed with a large number
of nematocysts (Fig. 3) was dropped onto the fish. The fish thrashed about for a
moment and became immobilized. Within 10 to 15 minutes most of the gastro-
zooids began spreading on the surface of the finger bowl, apparently in response to
some substance released from the pierced fish. Had the gastrozooids been close to
the fish (as occurs when the intact colony draws the prey up to the float) then
doubtless their mouths would have enveloped the fish (Fig. 4).
4. The effect of different concentrations of reduced glutathione
Ten gastrozooids were placed in several concentrations of GSH. The number
of gastrozooids spreading was counted at intervals. As shown in Table I, after
approximately two and one-half hours, 80-90% of the gastrozooids in 10~5 — 10~6
M GSH had spread their mouths over the surface of the glass bowl. The fact that
higher concentrations failed to elicit this response is not unusual, and finds a parallel
in Hydra where concentrations of GSH above optimum caused a tight closing of
the animal's mouth. Also, as in Hydra, concentrations of GSH less than 10"7 M
were ineffective.
456
H. M. LENHOFF AND H. A. SCHNEIDERMAN
TABLE I
Number of gastrozooids spreading in different concentrations of reduced
glutathione after various time intervals*
Time (min.)
10
35
70
105
155
Reduced
glutathione
concentration
Number spreading
10~3 M
0
0
0
0
0
10-4 M
0
0
0
0
0
ID"5 M
0
1
5
6
9
10-6 M
1
2
2
6
8
10-7 M
0
0
0
0
0
lO-8 M
0
0
1**
0
0
0 M
0
0
0
0
0
* Ten isolated gastrozooids were exposed to each concentration.
** Spread only slightly.
A delayed spreading phenomenon was often observed at high concentrations of
GSH. For example, 9 of 10 gastrozooids, which initially failed to respond to 10~3
M GSH, responded 7 hours after the start of the experiment. Occasionally a few
gastrozooids took as long as 24 hours to respond. Perhaps these delayed responses
occurred when the "excess" GSH had oxidized, giving the optimum concentration.
5. A comparison of the effects of cysteine and of reduced glutathione
The effects of another biological reducing agent, cysteine, were tested. The
results shown in Table II clearly demonstrate that cysteine failed to induce a feeding
response at concentrations at which GSH was active.
6. Differences in the feeding response of animals in different physiological states
In one series of experiments the effects of GSH were studied on gastrozooids
from two Physalia in different physiological states. The first (ca. 16-cm. float) was
maintained without food in the laboratory for one week, while the second (ca. 24-cm.
float) was kept under the same conditions for one day. The results in Table III
indicate that the one-day captive responded rapidly to 10~4 M GSH, while the
TABLE II
A comparison of the effects of cysteine and of reduced
elutalhione on the sbreadins of eastrozooids*
ompanson oj the effects oj cysteine ana oj rec
glutathione on the spreading of gastrozooids*
Concentration
10-4 M
ID"5 M
10-6 M
10-' M
Number spreading at 90 minutes
Cysteine Reduced glutathione
1** 0
0 8
0 4
0 2
* Ten isolated gastrozooids were exposed to each concentration.
** Spread only slightly.
FEEDING IN PHYSALIA
457
7-day captive did not. Also, the response was quicker in the one-day captive
animal at all concentrations of GSH. These differences may have been due to the
nutritional state or over-all well-being of the one-day captive animal as compared
to the 7-day captive animal.
Further studies of more than a dozen Physalia revealed striking differences in
the responsiveness of the gastrozooids to GSH. Sometimes newly-captured Phy-
salia exuded a copious mucus secretion for several days. Gastrozooids from these
mucus-covered animals were usually unresponsive to GSH. By contrast, in
captives which produced little mucus the gastrozooids always responded. In one
TABLE III
A comparison of the effect of different concentrations of reduced glutathione after various time
intervals on the gastrozooids of a 7-day captive and 1-day captive Physalia*
7-day (16-cm. float)
one-day (24-cm. float)
10
35
10
35
Concentration
Per cent spreading
10-4 M
0
0
66
83
10-5 M
10
50
33
83
1C-6 M
20
50
50
50-66
* Ten isolated gastrozooids of the 7-day captive and 6 of the one-day captive were used at
each concentration.
case an animal was kept in the laboratory for two weeks and its gastrozooids re-
sponded throughout this period.
DISCUSSION
1. Feeding response
In Physalia, the feeding response of the gastrozooid involves mouth-opening,
spreading, and food ingestion. The food is drawn to the gastrozooids by the
dactylozooids. Each gastrozooid then writhes and stretches until its mouth comes
in contact with a solid object, whether it be the fish that the colony has captured,
or the wall of the container in which the gastrozooids were placed. In Hydra the
tentacles writhe and sweep inward toward the mouth. The mouth then opens and
ingests the prey whenever the prey is brought in direct contact with it (Ewer, 1947) .
Thus in contrast to Physalia where the whole gastrozooid writhes, in Hydra only
the tentacles writhe. Moreover, in Hydra, the body tube and mouth do not spread
when exposed to GSH, but do so only when the food, or some other solid object,
is brought to the mouth by the writhing and contracting tentacles. For example,
when 10 Hydra were placed in each of a series of Petri dishes having 25 ml. of
GSH of 10"3 to 10~7 molarity, none of the Hydra exhibited the spreading phenome-
non, although their mouths opened. This failure to spread was not due to the
tentacles blocking the mouth, because removal of the tentacles did not increase
spreading. However, when 10 Hydra were crowded in a 0.5-ml. volume of 10~4
458 H. M. LENHOFF AND H. A. SCHNEIDERMAN
M GSH so that their mouths were forced to contact the wall of the container or the
surface of the water, 6 animals spread within 3 minutes.
It must be stressed that mere mouth opening or writhing does not indicate a
feeding response in Physalia or in Hydra. The only true index of a feeding re-
sponse is an actual attempt to feed — that is, to spread the mouth over or around
the prey. Many deleterious chemicals cause writhing or mouth opening. For
example, in Physalia, high concentrations of alloxan (which, besides inhibiting the
true feeding response, subsequently killed the gastrozooids) caused occasional
"mouth opening" but no characteristic feeding response. This "gaping" has also
been observed in Hydra by using Tween, and other harmful compounds (Lenhoff,
unpublished observations).
2. The action of reduced glutathione
The data leave little doubt that GSH induces a feeding response in Physalia
just as it does in Hydra. We cannot be certain that GSH is the only biological
compound that will induce the response in Pliysalia but the clear response of the
gastrozooids to low concentrations of GSH, coupled with Loomis' finding (1955)
that no other commonly occurring compound of many tested worked on Hydra,
make this likely. Since a gastrozooid of Physalia is four to six times as long as a
Hydra, and thicker, it should be practical to explore the detailed mechanism of the
feeding response with greater ease than in the case of Hydra. For example, it
should be possible to locate the receptor cells sensitive to GSH and to explain the
mechanics of the transformation of the cylindrical gastrozooid into a disc.
3. Phylogenetic considerations
The Siphonophora are commonly regarded as the most specialized order of the
Hydrozoa in that they attain the highest degree of polymorphism and present the
greatest number of medusoid and polypoid types. While there is some disagree-
ment regarding the phylogenetic relations within the group, there appears to be no
question but that they have clear hydrozoan characters (Hyman, 1940; Totten,
1954). Furthermore it is generally accepted that save for the Chondrophora (e.g.,
Velella) which are now thought to have close affinities with tubularian hydroids
(Totten, 1954; Rees, 1957), the Siphonophora proper, including Physalia, early
diverged from the cnidarian stem and evolved in directions quite different from
other hydrozoans (Hyman, 1940). On the other hand, Hydra itself is commonly
considered a highly specialized gynmoblastic hydroid, in all likelihood a fresh-water-
adapted tubularian (Hyman, 1940). Thus among the Hydrozoa it would be hard
to find two forms which diverged earlier from one another during evolution — two
forms which are very specialized and not generalized members of their class. Rec-
ognizing this, the demonstration of a GSH-induced feeding response in both forms
assumes special interest. It suggests that either (1) this GSH-induced response
is primitive (Loomis, 1955) and has persisted through the course of geological
time since these animals diverged because they both retain a primitive carnivorous
feeding habit, or (2) that this is simply convergence. The former suggestion is
not only more attractive but is more likely, and implies that GSH-stimulation of
feeding is a very ancient coordinating system and will be found among many of the
FEEDING IN PHYSALIA 459
Hydrozoa and possibly among Scyphomedusae and Anthozoa as well. This con-
clusion finds support in recent observations that Campanularia flexuosa, a calypto-
blastic hydroid unrelated to either Hydra or Pliysalia, gives a feeding response to
GSH. It must be emphasized, however, that the presence of a GSH response in
these three hydrozoans does not rule out the possibility that other small molecules
in the fluids released from captured prey may function in the feeding response of
other cnidarians. The essential feature of the primitive feeding mechanism is that
the prey must release fluid when pierced (see below).
These experiments support the opinion offered earlier (Schneiderman and
Gilbert, 1958) that the evolution of chemical control mechanisms has proceeded by
particular groups of animals adapting available and often ubiquitous molecules to
special tasks. Under this view the evolution of hormonal coordination involves
primarily the evolution of receptor systems sensitive to specific molecules rather
than any evolution of hormones as such.
The chemical similarities between the nematocyst-GSH feeding mechanisms of
Physalia and Hydra invite inquiry into the nature of their possible common ancestor.
Both of these organisms sting and capture their prey by means of nematocysts. It
is of interest in this connection that the nematocyst capsule of Hydra (Lenhoff
et al., 1957) and Physalia (Lane and Dodge, 1958; Lenhoff and Kline, 1958) are
composed of similar kinds of unusual hydroxyproline-rich, collagen-like proteins.
After the prey is penetrated, the GSH in the fluids flowing from the wound stimu-
lates the feeding response.
Since Hydra and Physalia both have a nearly identical chemical control of their
feeding behavior, and since their nematocysts are of a unique chemical composition,
it seems probable that both animals evolved from a common hydrozoan stem-form
which also possessed these chemical characteristics. It is noteworthy that the
nematocyst-GSH mechanism can only be used to capture prey which has sufficient
body fluids to release enough GSH on being stung to elicit the feeding response
(i.e., organisms which have either a pseudocoelom, or vascular system) (Loomis,
1955). Therefore, either (1) the primitive nematocyst-bearing hydrozoan stem-
form ate prey which was more highly evolved than itself, or alternatively, (2) the
primitive nematocyst-bearing hydrozoan stem-form fed on some presently unknown
lower form, perhaps a large protozoan, with a great deal of body fluid. We favor
the first alternative and prefer to believe that the hydrozoan stem-form fed on
animals with "vascular" fluids (i.e., nematodes, and members of higher phyla).
These facts permit us to speculate about the feeding habits of the ancestral
cnidarians, i.e., the forms from which the Hydrozoa and the other classes of Cnidaria
evolved. Ordinarily one hesitates to base phylogenetic schemes on feeding habits
which in most phyla are notoriously labile. However, the universal use of nemato-
cysts to capture prey by all contemporary cnidarians supports the view that the
feeding habits of members of this phylum are far more stable than those of other
animals. The ancestral cnidarians most probably fed on animals which were of a
lower grade of organization than themselves, and hence could not have employed
the nematocyst-GSH mechanism. Most likely they were filter feeders like many
present-day Anthozoa. Under this view, nematocyst-bearing cnidarians with a
GSH-mechanism evolved from filter-feeding ancestors at the same time as did
higher forms. Thus cnidarians with nematocysts (a diagnostic feature of the
460 H. M. LENHOFF AND H. A. SCHNEIDERMAN
phylum) may be removed from their position as the ancestors of higher metazoans.
Whether they are members of a regressive line of evolution which have degenerated
because of a sessile habit (Hadzi, 1953), or are an offshoot of a progressive line
of evolution, remains to be proven. Since all these events took place in the Pre-
Cambrian Era, none of these suggestions can be ruled out at present and doubtless
others will arise.
Our special thanks go to the Supply Department of the Marine Biological
Laboratory at Woods Hole who went to great efforts to capture the Physalia used
in this study; to Dr. Sears Crowell for his helpful comments on the typescript of
this paper and for his help in conducting experiments with Campa nularia; and to
Mr. Ernest Bay for his most valuable contribution in photographing our
observations.
SUMMARY
1. Physalia gastrozooids, both isolated and in situ, exhibit a feeding response
when exposed to low concentrations of reduced glutathione (10>5 — 10~6 M).
2. Cysteine did not elicit the response.
3. The feeding response of the gastrozooid consisted of opening of its mouth and
the subsequent spreading of its lips over a large area in an attempt to envelop the
prey. This response resembles a similar GSH-induced feeding response found in
Hydra.
4. The sensitivity to GSH depended upon the physiological state of the
Physalia.
5. The significance of this primitive chemical coordinating mechanism in relation
to the evolution of the Cnidaria and of the Metazoa as a whole is considered.
LITERATURE CITED
EWER, R. F., 1947. On the functions and mode of action of the nematocysts of hydra.
Proc. Zool. Soc., 117: 365-376.
HADZI, J., 1953. An attempt to reconstruct the system of animal classification. Syst. Zool.,
2: 145-154.
HYMAN, L. H., 1940. The Invertebrates: Protozoa through Ctenophora. McGraw-Hill Book
Co., N. Y.
LANE, C. E., AND E. DODGE, 1958. The toxicity of Physalia nematocysts. Biol. Bull, 115:
219-226.
LENHOFF, H. M., E. S. KLINE AND R. HURLEY, 1957. A hydroxyproline-rich, intracellular,
collagen-like protein of Hydra nematocysts. Biochim. Biophys. Ada, 26: 204-205.
LENHOFF, H. M., AND E. S. KLINE, 1958. The high imino acid content of the capsule from
Hydra nematocysts. Anat. Rec., 130: 425.
LOOMIS, W. F., 1955. Glutathione control of the specific feeding reactions of hydra. Ann.
N. Y. Acad. Sci., 62: 209-228.
LOOMIS, W. F., AND H. M. LENHOFF, 1956. Growth and sexual differentiation of hydra in
mass culture. /. Exp. Zool., 132 : 555-574.
REES, W. J., 1957. Evolutionary trends in the classification of capitate hydroids and medusae.
Brit. Mus. Nat. Hist. Bull., 4 : 229-235.
SCHNEIDERMAN, H. A., AND L. I. GILBERT, 1958. Substances with juvenile hormone activity
in hydroids and other invertebrates. Biol. Bull., 115: 530-535.
TOTTEN, A. K., 1954. Siphonophora of the Indian Ocean together with systematic notes and
biological notes on related species from other oceans. Discovery Reports, 27 : 162 pp.
WILSON, D. P., 1947. The Portuguese man-of-war, Physalia physalis in British and adjacent
seas. /. Mar. Biol. Assn., 27: 139-172.
OBSERVATIONS ON THE GROWTH OF DUNALIELLA
EUCHLORA IN CULTURE 1
JACK McLACHLAN 2 AND CHARLES S. YENTSCH
Woods Hole Oceanographic Institution, Woods Hole, Mass.
Under natural conditions, an intimate, but poorly understood relationship exists
between bacteria and phytoplankton. Some investigators have claimed that phyto-
plankton produce a substance which is inhibitory to the growth of bacteria ( Steemann
Nielsen, 1955a, 1955b; Steemann Nielsen and Jensen, 1957). Contrariwise, the
results of Waksman ct al. (1937) suggest a harmonious relationship between algae
and bacteria.
In experimental work, the use of bacteria-free cultures is customary. It has
not always been possible to obtain bacteria-free cultures, and the assumption has
been that the growth of the alga would have not been altered if axenic cultures
had been used (rf. Goldberg et al., 1951; Kain and Fogg, 1958). This assump-
tion seems correct when using media with no organic additions (McLachlan, un-
published). However, little is known of the effects of bacteria on the growth of
algae in cultures containing organic supplements.
In the present investigation, growth of the green flagellate Dunuliella euchlora
WHOI-1 in pure culture and contaminated cultures containing organic enrichments
was studied. Growth was estimated by cell numbers and chlorophyll a synthesis.
The production of algal and bacterial inhibitors was also investigated.
MATERIALS AND METHODS
A pure culture of Dunaliella euchlora Lerche strain WHOI-1 (McLachlan,
1959) was used in this study. The alga was grown in a modification of the ASP
medium of Provasoli et al. ( 1957) ; the composition of this medium is presented
in Table I. In some of the experiments, the ASP medium was enriched with
organic material by the addition of beef extract (0.3 g./l.) and bactopeptone (0.5
g./l.). At 16% nitrogen, this corresponds to an addition of about 9.1 mM of
organic nitrogen, or nine times as much as is available in the basic medium. The
cultures were incubated at 16° C. under 3,000 meter-candles of illumination pro-
vided by 40-watt fluorescent lights. Growth of the alga was determined by cell
counts made in duplicate with a total of eight replicate counts using a Levy hemo-
cytometer, and is expressed as Iog2 Nt/N0 where N0 is the concentration of cells at
inoculation and Nt the concentration at time t.
Chlorophyll a was measured spectrophotometrically in acetone extractions ac-
cording to the Richards with Thompson method (1952) as modified for use with
the millipore membrane filter procedure of Creitz and Richards (1955). The con-
1 Contribution no. 1010 from the Woods Hole Oceanographic Institution. This investiga-
tion was supported, in part, by a grant from the National Science Foundation.
- Public Health Service Research Fellow of the National Institutes of Health.
461
462
JACK McLACHLAN AND CHARLES S. YENTSCH
TABLE I
Composition of the modified ASP medium
NaCl
MgSO4-7H2O
MgCl2-6H2O
CaCl2-2H2O
KNO8
K2HPO4
Na2SiO3-9H2O
410 mM
24 mM
22 mM
10 mM
1 mM
100
100
FeCV6H2O
H3BO3
MnCl2-4H2O
ZnCl2
CoCl2-6H2O
CuCl2-2H2O
Na2EDTA
1.5
185.0
7.0
0.8
0.02
0.0002
30.0
centration of chlorophyll a was determined using the nomographs of Duxbury and
Yentsch (1956), and the organic nitrogen content of the cells was estimated by the
procedure of Yentsch and Vaccaro (1958).
RESULTS
1. Groivth in contaminated beef extract-peptone cultures. To determine the
effect of bacterial contamination on the growth of Dunaliella euchlora, the alga
was inoculated into autoclaved and unautoclaved ASP medium, and autoclaved and
8
ASP
ASP, NOT AUTOCLAVED
BEEF-EXT.+ PEPTONE
BEEF-EXT.+ PEPTONE,
NOT AUTOCLAVED.
0 2 4 6 8 10 12
DAYS AFTER INOCULATION
FIGURE 1. Growth of the alga in contaminated and non-contaminated ASP medium and
contaminated and non-contaminated ASP medium containing beef extract and peptone.
OBSERVATIONS ON DUNALIELLA
463
unautoclaved ASP cultures containing beef extract and peptone. In the unauto-
claved cultures with organic enrichments, approximately 2.5 times as many cells
were obtained as in the other cultures (Fig. 1). The growth rate and the final
number of cells in the other treatments were approximately the same. From these
results it can be seen that the addition of beef extract and peptone per se did not
increase the growth of the alga, but bacterial breakdown of these materials was of
considerable benefit.
The unautoclaved cultures with organic additions became very dense, and after
twelve days of growth contained approximately three times as much chlorophyll a
TABLE II
The size of the population of Dunaliella euchlora, and the chlorophyll a content
after growth in media treated as discussed in the text
Experiment
Treatment
Days of
growth
No. cells/ml.
X10<
Mg ChlVcell
XI 0-9
Beef extract-peptone no. 1
Enriched and contaminated
12
1,089
2.24
Enriched, not contaminated
12
397
0.74
ASP-autoclaved
12
384
—
ASP-not autoclaved
12
370
—
Nitrate-nitrogen
1.0 mM
14
444
0.41
2.0 mM
14
868
0.40
5.0 mM
14
839
0.44
8.0 mM
14
716
0.39
10.0 mM
14
617
0.51
Beef extract-peptone no. 2
-3
15
320
0.81
0
15
634
0.66
+2
15
924
0.47
+4
15
1,631
0.60
+ 7
15
1,198
1.11
+ 11
15
792
0.68
Control
15
497
0.79
Dunaliella nitrate
ASP
11
504
0.44
Autoclaved nitrate
11
527
0.93
Unautoclaved filtrate
11
491
1.27
Bacteria nitrate
ASP
12
477
0.43
Filtrate
12
958
0.80
per cell as the autoclaved enriched culture (Table II). Using the method of
Yentsch and Vaccaro (1958), it was found that in the enriched contaminated
cultures about 10 mM per liter of organic nitrogen was incorporated into the algal
cells, or approximately 0.91 X 10~6 //.M of nitrogen per cell. In contrast, the non-
contaminated enriched culture contained about 0.9 mM per liter of organic nitrogen
as algal cellular material, or approximately 0.23 X 10~6 ,uM of nitrogen per cell.
The chlorophyll content of the two cultures containing only inorganic additions was
not determined, but other estimates have indicated that at the end of exponential
growth, all of the added nitrate-nitrogen is organically incorporated. This suggests
464
JACK McLACHLAN AND CHARLES S. YENTSCH
that in the non-contaminated beef extract peptone culture all of the incorporated
organic nitrogen had been obtained from the added nitrate-nitrogen, and not from
the organic material.
2. Grozvth in various concentrations of nitrate-nitrogen. To determine if the
number of cells and the amount of chlorophyll a obtained in the contaminated beef
extract-peptone cultures could be obtained by the addition of inorganic nitrogen,,
sodium nitrate was added at the following concentrations: 1.0 (ASP level), 2.0,
5.0, 8.0 and 10.0 mM. The ASP concentration of potassium was maintained by
the addition of potassium chloride, and the phosphorus concentration was maintained
at 100 fj-M in all cultures. The two highest concentrations of nitrogen slightly in-
hibited the growth of the alga (Fig. 2), but the total number of cells in all cultures
0 2 4 6 8 10 12 14
DAYS AFTER INOCULATION
FIGURE 2. Growth of the alga in various concentrations of nitrate-nitrogen.
exceeded that of the 1.0 mM culture after fourteen days of growth. The maximum
density of cells developed in the 2.0 and 5.0 mM cultures had a final cell count of
almost twice that of the 1.0 mM culture (Table II), although the initial growth
rates of these three cultures were approximately the same.
Pigment analysis showed that the chlorophyll a content of the cells in all cultures
was about the same (Table II). The total number of cells obtained in the cultures
containing 2.0 and 5.0 mM of nitrate-nitrogen approached that obtained in the
contaminated beef extract-peptone culture of the previous experiment, but the
amount of chlorophyll a was considerably less. Since the maximum amount of
chlorophyll synthesized in these cultures was considerably less than was found
in the contaminated culture containing beef extract and peptone, it may be concluded
OBSERVATIONS ON DUNALIELLA
465
that available nitrogen alone is not the limiting factor. Perhaps release of other
nutrients in beef extract and peptone by bacterial activity or direct utilization of
organic breakdown products by the alga also stimulated chlorophyll synthesis.
3. Growth in beef extract-peptone cultures with periodic addition of bacteria.
To determine the effects of organic breakdown products on the growth of the alga
at different phases of growth, a mixed culture of marine bacteria was obtained from
the surface waters of Woods Hole Harbor, and uniform inocula were added period-
ically to cultures of Dunaliella containing beef extract and peptone. The original
bacterial inoculum was collected using a sterile container so only bacteria present
C\J
e>
o
4 6 8 10 12 14
DAYS AFTER INOCULATION
16
FIGURE 3. Growth of the alga in the ASP medium containing beef extract
and peptone to which bacteria were added periodically.
in the surface water were introduced into culture. The bacteria were grown in
the ASP medium with the addition of beef extract and peptone under the same
conditions as the algal cultures. Before the bacterial cultures were used in the
experimental work, they were carried through a number of transfers in order to
obtain a uniform population.
Beef extract and peptone were added to the ASP medium and inoculated period-
ically with 1 ml. of the bacterial culture. In all cases the bacteria inocula were from
three-day-old cultures when introduced into the experimental flasks. Inoculation
466
JACK McLACHLAN AND CHARLES S. YENTSCH
CM
o
o
0 +2 + 4 +7
TIME OF BACTERIA INOCULATION
O
o
i\>
o
^
10
-*
^
O
^
P
o
+ 11
FIGURE 4. A comparison of algal cell density and chlorophyll a in cultures
to which bacteria were added periodically.
o AUTOCLAVED
• NOT AUTOCLAVED
x CONTROL
024 6 8 10 12
DAYS AFTER INOCULATION
FIGURE 5. Growth of the alga in autoclaved and non-autoclaved algal filtrate.
OBSERVATIONS ON DUNALIELLA
467
took place at the following times : three days before inoculation with the alga, at
the same time as the alga, two days, four days, seven days, and eleven days after
inoculation with the alga.
Bacteria introduced three days before and at the time of inoculation with
Dunaliella inhibited the growth of the alga (Fig. 3). In all cultures, except the
one inoculated with bacteria three days before the alga, growth after fifteen days
exceeded that of the control. In Figure 4 cell density and chlorophyll a after
fifteen days of growth are presented. The greatest number of cells occurred in
the culture which was inoculated with bacteria four days after the alga, and those
cultures which were inoculated with bacteria prior to this time showed less growth.
Less growth was also obtained in the cultures which had been inoculated with
6.0
5.0
4.0
3.0
2.0
1.0
FILTRATE
'• • CONTROL
FIGURE 6.
0 4 8 12 16 20 24
HOURS AFTER INOCULATION
Growth of Woods Hole Harbor bacteria in the algal nitrate
containing beef extract and peptone.
bacteria seven and eleven days after inoculation with the alga. However, the cells
in the culture inoculated with bacteria seven days after the alga contained more
chlorophyll a than the other cultures after fifteen days of growth (Table II). These
results show that growth of the alga was directly related to the time at which the
bacteria were added. Under the conditions of this experiment, chlorophyll a
synthesis preceded cell division (Fig. 4). The highest concentration of chlorophyll
per cell, however, was not as great as that obtained in the previous beef extract-pep-
tone experiment.
4. Growth of the alga in the algal filtrate. To determine if auto-inhibitors
are produced by Dunaliella euchlora, growth of the alga in the ASP medium was
compared with growth in the filtrate of a Dunaliella culture which had reached
maximum density. Part of the filtrate was autoclaved and the other part was not
46 S
JACK McLACHLAN AND CHARLES S. YENTSCH
autoclaved as there has been a suggestion of heat-labile inhibitors (Lefevre et al.,
1952). Nitrogen, phosphorus, and iron were added to all cultures at ASP con-
centrations. Growth in all cultures was identical as shown by the data in Figure 5.
The chlorophyll a content per cell was greater in the filtrate cultures than in the
control whether or not the filtrate had been autoclaved (Table II). This indicates
that the filtrate contained something which enhanced chlorophyll synthesis, but did
not promote cell division.
5. Growth of the bacteria in the algal filtrate. To determine if Dunaliella
filtrate would inhibit the growth of bacteria, a filtrate was obtained from a five-day
8 -
CM
o
o
o FILTRATE
• CONTROL
2 4 6 8 10 12
DAYS AFTER INOCULATION
FIGURE 7. Growth of the alga in the Woods Hole Harbor bacteria filtrate.
culture of the alga. Beef extract and peptone were added to the filtrate and to the
control ASP medium, and inoculated with the previously isolated Woods Hole
Harbor bacteria. Growth of the bacteria was measured by optical density at 750 m/x
using a Beckman model DU spectrophotometer, and is expressed as Iog2 ODt/OD0.
The growth of the bacteria was approximately the same in the filtrate as in the
enriched ASP medium with no suggestion of a bacteriostatic substance produced
by the alga (Fig. 6).
OBSERVATIONS ON DUNALIELLA 469
6. Growth of the alga in the bacterial filtrate. To determine the effects of
the bacterial filtrate on the growth of Dunaliella, a culture of the Woods Hole
Harbor bacteria was grown in the ASP medium containing beef extract and pep-
tone. After three days of growth, the culture was passed through a HA millipore
filter resulting in a clear filtrate which was autoclaved and inoculated with the alga.
There was a considerable increase in the maximum cell density in the nitrate culture
as compared with the control (Fig. 7). The inhibition noted in the previous beef
extract-peptone experiment where the bacteria had been added to the culture three
days before the alga did not occur in this experiment. This suggests that the
inhibitory substance is volatile or heat-labile. Pigment analysis also showed the
cells in the filtrate culture contained considerably more chlorophyll a than those in
the control culture (Table II).
DISCUSSION
The addition of beef extract and peptone to bacteria-free cultures of Dunaliella
•euchlora did not stimulate the growth of the alga. In the presence of bacteria, how-
ever, a considerable increase in the algal population, as determined by cell counts
and chlorophyll analysis, was observed. This increase presumably resulted from
the bacterial hydrolysis of the added organic material. The organic additions
were not entirely inert to algal metabolism as the amount of chlorophyll a per cell
in the uncontaminated cultures always exceeded that obtained with only the ad-
dition of inorganic salts.
The large amount of synthesis of chlorophyll a suggested that the alga was able
to utilize nitrogenous breakdown products of the peptone and beef extract. A large
fraction of the utilizable nitrogen from the beef extract and peptone was probably
present as ammonia, nitrite, or simple organic compounds since the conversion of
these to nitrate-nitrogen by marine bacteria is a slow process (Harvey, 1955; Sver-
drup et al., 1942). Harvey (1940) and Gibor (1956) observed that several species of
Dunaliella were able to utilize some amino acids as nitrogen sources. There are
also numerous reports of other autotrophic algae capable of utilizing organic nitro-
gen compounds (Fogg, 1953; Ryther, 1954). In addition, Huzisige et al. (1957)
found that Euglena synthesized chlorophyll more rapidly when provided with casein
hydrolysate than when provided with inorganic nitrogen compounds.
Growth of the alga in cultures containing organic additions was inhibited if
bacteria were introduced before the alga began to grow. However, no inhibition
of the alga was observed in the bacterial filtrate which had been autoclaved. This
suggests a volatile or heat-labile inhibitor. Inhibition could have resulted from
ammonia which is known to be toxic at low concentrations (Fogg, 1953; Myers,
1951). Any ammonia present in the bacterial filtrate would have been lost during
autoclaving, and the inhibition noted in the second beef extract-peptone experiment
may have been due to toxic concentrations of ammonia.
j
In the cultures which developed a heavy suspension of cells and chlorophyll, light
and probably carbon dioxide may have been limiting, but organic carbon could have
been utilized by the alga. Organic carbon could have been incorporated by the
alga simultaneously with the utilization of organic nitrogen (Fogg, 1953; Krauss,
1958), or as separate compounds. If the alga is capable of heterotrophic growth,
light would not necessarily be a limiting factor. It seems probable that the very
470 JACK McLACHLAN AND CHARLES S. YENTSCH
dense cultures obtained in this study may have been due in part to heterotrophic
growth.
It was not possible in this study to demonstrate that Dunaliella inhibited the
growth of bacteria, nor was there any indication of auto-inhibitors produced by the
alga. The production of antibiotics by algae may not be a general phenomenon.
The authors wish to express their grateful appreciation to Dr. Bostwick H.
Ketchum for his many helpful suggestions in preparing the manuscript.
SUMMARY
1. Bacteria added to cultures of Dunaliella euchlora in the presence of nitrog-
enous organic matter stimulated the growth of the alga, and enhanced the synthesis
of chlorophyll a.
2. It was not possible to obtain comparable concentrations of chlorophyll a by the
addition of nitrate-nitrogen, although a comparable number of cells could be
obtained.
3. If the bacteria obtained a "head-start" in the enriched cultures, growth of
the alga was inhibited. This inhibition could be overcome by autoclaving the
filtrate from the bacteria cultures.
4. Growth of the bacteria was not inhibited in the algal filtrate, nor was the
growth of the alga inhibited in the algal filtrate.
LITERATURE CITED
CREITZ, G. I., AND F. A. RICHARDS, 1955. The estimation and characterization of plankton
populations by pigment analysis. III. A note on the use of "millipore" membrane
filters in the estimation of plankton pigments. J. Mar. Res., 14: 211-216.
DUXBURY, A. C, AND C. S. YENTSCH, 1956. Plankton pigment nomographs. /. Mar. Res.,
15: 92-101.
FOGG, G. E., 1953. The Metabolism of Algae. Methuen and Co., Ltd., London.
GIBOR, A., 1956. The culture of brine algae. Biol. Bull, 111: 223-229.
GOLDBERG, E. D., T. WALKER AND A. WHISENAND, 1951. Phosphate utilization by diatoms.
Biol. Bull, 101 : 274-284.
HARVEY, H. W., 1955. The Chemistry and Fertility of Sea Waters. Cambridge University
Press, Cambridge.
HARVEY, H. W., 1940. Nitrogen and phosphorus required for the growth of phytoplankton.
/. Mar. Biol. Assoc., 24: 115-123.
HUZISIGE, H., T. TERADA, M. NISHIMURA AND T. UEMURA, 1957. Effect of amino acids and
streptomycin on the chlorophyll formation in Euglena. Biol. J. Okayama Univ., 3 :
209-222.
KAIN, J. M., AND G. E. FOGG, 1958. Studies on the growth of marine phytoplankton.
I. Asterionella japonica Gran. /. Mar. Biol. Assoc., 37 : 397-413.
KRAUSS, R. W., 1958. Physiology of fresh-water algae. Ann. Rev. Plant Physiol, 9:
207-244.
LEFEVRE, M., H. JAKOB AND M. NISBET, 1952. Auto, et Heteroantagonisme chez les Algues
d'eau douce. Ann. dc la Stat. Cent. d'Hydrob., 4 : 5-198.
MCLACHLAN, J., 1959. The growth of unicellular algae in artificial and enriched sea water
media. Canad. J. Microbiol., 5 : 9-15.
MYERS, J., 1951. Physiology of the algae. Ann. Rev. Microbiol, 5: 157-180.
PROVASOLI, L., J. J. A. MCLAUGHLIN AND M. R. DROOP, 1957. The development of artificial
media for marine algae. Arch. Mikrobiol., 25: 392-428.
OBSERVATIONS ON DUNALIELLA
471
RICHARDS, F. A., WITH T. G. THOMPSON, 1952. The estimation and characterization of plank-
ton populations by pigment analysis. II. A spectrophotometric method for the estima-
tion of plankton pigments. /. Mar. Res., 11 : 156-172.
RYTHER, J. H., 1954. The ecology of the phytoplankton blooms of Moriches Bay and Great
South Bay, Long Island, New York. Biol. Bull, 106 : 198-209.
STEEMANN NIELSEN, E., 1955a. An effect of antibiotics produced by plankton algae. Nature,
176: 553.
STEEMANN NIELSEN, E., 1955b. The production of antibiotics by plankton algae and its effect
upon bacterial activities in the sea. Pap. Marine Biol. Oceanogr., Deep-Sea Res.
Suppl, 3: 281-286.
STEEMANN NIELSEN E., 1958. Experimental methods for measuring organic production in the
sea. Rap p. Proc. Verb. Cons. Internal. E.vplor. Mcr., 144 : 38-46.
STEEMANN NIELSEN, E., AND E. A. JENSEN, 1957. Primary oceanic production. Galathea
Report, 1: 49-136.
SVERDRUP, H. U., M. W. JOHNSON AND R. H. FLEMING, 1942. The Oceans, Their Physics,
Chemistry and General Biology. Prentice-Hall, Inc., N. Y.
WAKSMAN, S. A., J. L. STOKES AND M. R. BUTLER, 1937. Relation of bacteria to diatoms in
sea water. /. Mar. Biol. Assoc., 22: 359-373.
YENTSCH, C. S., AND R. F. VACCARO, 1958. Phytoplankton nitrogen in the oceans. Limnol.
Oceanogr., 3 : 443-448.
INHIBITION OF FERTILIZIN AGGLUTINATION OF SPERM BY THE
DERMAL SECRETION FROM ARBACIA1
CHARLES B. METZ
Oceanographic Institute, Florida State University, Tallahassee, Florida,
and Marine Biological Laboratory, Woods Hole, Mass.
Upon appropriate stimulation, Arbacia release a yellowish fluid from the integu-
ment. This fluid strongly inhibits fertilization (Oshima, 1921; Pequegnat, 1948)
and is probably the material responsible for the inhibitory action ascribed to
Arbacia blood by Lillie (1914). Current interest arose from the observation that
this "dermal secretion" not only inhibits fertilization but inhibits fertilizin aggluti-
nation of sperm as well. Fertilizin is the specific sperm isoagglutinin obtained
from eggs of the species. It is initially present as a jelly surrounding the sea
urchin egg, but on standing in sea water this jelly dissolves charging the water
with the agglutinin. The dual effect of the dermal secretion suggests a causal
relation between inhibition of fertilizin agglutination of sperm and of fertilization.
Demonstration of such a relation would support the view that fertilizin is essential
for fertilization. The experiments described here and reported briefly elsewhere
(Metz, 1958) mainly concern the action of the dermal secretion on sperm and the
fertilizin agglutination of sperm. The results are consistent with the view that
inhibition of fertilization by the dermal secretion is related to an inhibitory action
on fertilizin.
MATERIALS AND METHODS
Arbacia punctulata was used in all experiments except the specificity tests
outlined in the text. Most of this material was collected in the vicinity of the
Florida State University Marine Laboratory, Alligator Point, Florida, although a
few collections we/e made from a very large population at Panama City, Florida.
A few experiments were performed at the Marine Biological Laboratory, Woods
Hole, Mass., using the local material.
Gametes were obtained by electrical stimulation of the animals. Gametes ob-
tained by the KC1 injection method, especially during the winter months, proved
unsatisfactory in both fertilization and sperm agglutination tests (Metz, 1957a).
In fact, investigation of the cause of this failure revealed that KCl-treatment fre-
quently stimulated release of the dermal secretion (see also Harvey, 1956, page 57)
as well as the gametes.
Sperm oxygen consumption was measured using standard Warburg apparatus.
Single side arm vessels of 15-20 ml. capacity containing 3 ml. of fluid were run in
duplicate at 20° C. The vessels were shaken at a rate of 120 cycles per minute
with an amplitude of 3.5 cm.
1 Aided by a grant from the National Science Foundation. Contribution number 95 from
the Oceanographic Institute, Florida State University.
472
INHIBITION OF SPERM AGGLUTINATION 473
Solutions of the dermal secretion were prepared in a manner similar to that
described by Pequegnat (1948), and consisted of the following steps: The Arbacia
were immersed in tap water for 1-3 minutes. They were subsequently rinsed in
distilled water and finally in filtered sea water. The animals were then placed in
a dry funnel and the yellow fluid which drained off was collected. The pH of
such fluid is about 7.5. In experiments where this might be critical the pH was
raised to that of sea water (pH 8.0-8.2). To test for sperm agglutination one to
two drops of the test solution were mixed with an equal volume of a sperm sus-
pension prepared by diluting semen to 2% with sea water. The mixtures were
examined both macro- and microscopically.
RESULTS
A) Action on sperm
Pequegnat (1948) noted that the dermal secretion of Arbacia stimulated sperm
to increased activity. This effect was observed repeatedly in the current investiga-
tion. Even preparations of dermal secretion at the initial pH (7.5), and therefore
at a pH disadvantage as compared to sea water controls, had the stimulating action.
As Pequegnat was aware, this effect is not a transient one. The sperm remain
strongly active for a prolonged period of time.
The enhanced motility of the sperm in the presence of the dermal secretion is
associated with an increase in oxygen consumption as seen in Figure 1. Again
the effect is not a short term one for the inhibitor-treated sperms were still respir-
ing at approximately twice the rate of the controls even at the end of one hour.
The increased oxygen uptake is clearly the result of enhanced sperm respiration,
not to the oxidation of the pigment in the dermal secretion (see later section).
This follows from the fact that the increase in oxygen uptake does not appear
until the dermal secretion is tipped into the sperm suspension. Furthermore, in
independent experiments dermal secretion collected over nitrogen to prevent pre-
mature oxidation failed to consume appreciable amounts of oxygen in Warburg
vessels.
Nothing definite is as yet known regarding the chemical nature or properties
of the sperm-stimulating agent, except that it is gradually destroyed by heat
(Fig. 1).
B) Action on fertilisin agglutination of sperm
An inhibitory effect of the dermal secretion on fertilizin agglutination of sperm
was first noted by Pequegnat (1948). The present investigation was designed
to obtain information concerning the mechanism of action of the material in this
effect. Attention was directed especially to determine if the inhibitory agent
acted upon the sperm, the fertilizin or both constituents of the agglutinating system.
Possible inhibitory action on the sperm. One possible means whereby the
dermal secretion could inhibit agglutination would be by action on the sperm.
For example, the agent could destroy the antifertilizin receptor sites on the sperm
surface. Such destruction might be expected to be irreversible in which case
washing dermal secretion-treated sperm should not restore agglutinability. Such
experiments show, however, that treated sperm recover agglutinability upon wash-
474
C. B. METZ
ing in sea water. A typical experiment from a series of four is given in Table I.
From this experiment it is clear that the inhibiting agent in the dermal secretion
does not render the sperm irreversibly refractory to the agglutinating action of
fertilizin.
200 r
x DS(UNHEATED)
0D.S. (HEATED)
A S.W(UNHEATED)
SW (HEATED)
0
MINUTES
FIGURE 1. Effect of dermal secretion (D.S.) on oxygen uptake of Arbacia sperm. Heated
aliquots (3 ml.) were immersed in a boiling water bath for 5 minutes. The pH of both
heated and unheated dermal secretion solutions was adjusted to 7.9. Vessels contained 1.8 ml.
of Arbacia semen diluted to 10% with sea water. Center wells contained 0.2 ml. 20% KOH.
The 1-ml. samples of dermal secretion (or sea water) in side arms were tipped into vessels
at 48 minutes.
Likewise, the inhibitor is not used up or destroyed by sperm in the inhibiting
reaction as seen in Table II (one of five experiments). Accordingly, it appears
likely that the dermal secretion acts upon the fertilizin, not the sperm, to inhibit
agglutination. Experiments supporting this view are described below.
INHIBITION OF SPERM AGGLUTINATION
475
TABLE I
Fertilizin agglutination of Arbacia sperm after washing from dermal secretion
1
2
3
4
Sperm
D.S.
(Sperm
re-suspended
in S.W.)
Sperm
+
D.S.
(Sperm
re-suspended in
original super-
natant D.S.)
Sperm
+
S.W.
(Sperm
re-suspended
in S.W.)
Sperm
+
S.W.
(Sperm
re-suspended in
original super-
natant S.W.)
Agglutination of sperm on ad-
dition of fertilizin after one
+
—
+ + + +
+ + + +
centrifugation
Agglutination of sperm on ad-
dition of fertilizin after sec-
+ + +
—
+ + + +
+ + + +
ond centrifugation
Semen concentration in the four 0.75-ml. samples was 8%. After standing 30 minutes the
samples were centrifuged twice at 4° C. Sperm in 1 and 3 was re-suspended in sea water; the
sperm in 2 and 4 in the original supernatant after each centrifugation. Aliquots of sperm from
all re-suspended samples were tested for agglutination with fertilizin.
Inhibitory action on fertilizin. Tests for action of the inhibiting solution on
fertilizin are complicated by the fact that fertilizin itself can inhibit sperm agglutina-
tion when in the univalent form (Metz, 1942). Indeed, one possible mode of
action of the inhibitor would be that it converts fertilizin from the multivalent,
agglutinating form to the univalent, non-agglutinating condition. Another pos-
TABLE II
Agglutination inhibiting action of sperm absorbed dermal secretion
Absorbing mixtures
i
2
3
4
0.5 ml.
Sperm (33%)
S.W.
Sperm (33%)
S.W.
0.5 ml.
D.S.
D.S.
S.W.
S.W.
Agglutination inhibiting assay of supernatants from above mixtures
Dilution of absorption
supernatant
1
2
3
4
1
—
—
+ + + +
+ + + +
i
±
—
1
+++
+++++
After standing for 30 minutes the absorbing mixtures were centrifuged, supernatants were
removed and diluted serially in S.W. Constant amounts of fertilizin and test sperm suspension
were then added to each dilution. The slight difference between No. 1 and No. 2 is not considered
significant.
476
C. B. METZ
sibility is that the inhibitor destroys the reactive sites of the fertilizin which com-
bine with the sperm surface. This could occur with or without conversion to
the univalent form.
Ideally, to test for direct action on fertilizin, the inhibiting agent and fertilizin
should be mixed and subsequently separated before testing the fertilizin for activity.
Unfortunately, no simple means has yet been devised for separating the two mate-
rials. However, results obtained using two other procedures leave no doubt that
the agglutination-inhibiting agent does destroy not only the sperm-agglutinating
action of fertilizin, but also the ability of the fertilizin to combine with the anti-
fertilizin of the sperm.
In the first procedure advantage was taken of the fact that the agglutination-
inhibiting action of the dermal secretion is rapidly destroyed by heating to 100° C.
TABLE III
Effect of heating on fertilizin-dermal secretion mixtures
Heated mixtures
Unheated mixtures
1
2
3
4
5
6
7
8
0.5 ml.
D.S.
D.S.
S.W.
S.W.
D.S.
D.S.
S.W.
S.W.
0.5 ml.
fertilizin
S.W.
fertilizin
S.W.
fertilizin
S.W.
fertilizin
S.W.
A.
Agglutination on addition
of sperm to mixtures
"
~
+ + + +
+ + + +
B.
Inhibition tests (agglutina-
tion on addition of con-
+ + +
+++
—
+ + +
—
+ + +
trol fertilizin to samples
tested in A above)
The heated mixtures were held at 100° C. for four minutes. In A the mixtures were tested
for agglutinating action on sperm. The heating failed to restore agglutinating action to the D.S.-
fertilizin mixture (Al). In B the mixtures were tested for agglutination inhibiting action by add-
ing control fertilizin to the mixtures in A following spontaneous reversal of the initial agglutination
in A3 and A7 . The heated mixture fails to inhibit agglutination (Bl and B5). Both the inhibitor
in the dermal secretion and the fertilizin have been destroyed in the heated mixture.
whereas fertilizin is relatively stable to such heating. As seen in Table III (A)
when a non-agglutinating, fertilizin-inhibitor mixture was heated sufficiently to
destroy the inhibitor but not the fertilizin, the mixture still failed to agglutinate
sperm. Restoration of agglutinating action would be expected if the inhibitor in
the mixture acted exclusively on the sperm. Accordingly, the experiment is
explained by an inactivation of the fertilizin by the inhibitor and a subsequent
inactivation of any remaining inhibitor by the heating. Four other experiments
yielded similar results.
The question now arises whether the agent inactivates the sperm combining
sites of fertilizin or converts the fertilizin to the univalent form without combining
site destruction. Evidence concerning this was obtained by testing for sperm
INHIBITION OF SPERM AGGLUTINATION
477
agglutination inhibiting action of the heated fertilizin-inhibitor mixture. As seen
in Table III (B) control fertilizin agglutinates sperm that was pretreated with the
heated fertilizin-inhibitor mixture. This agglutination means that the heated mix-
ture contains insufficient fertilizin combining sites to block the sperm surface.
However, controls in the experiment show that fertilizin combining sites sufficient
to block the sperm surface were initially present in the mixtures. Therefore, it
must be concluded that combining site destruction occurred in the fertilizin-inhibitor
mixture. Inactivation of agglutinating action by dermal secretion does not result
simply from conversion of the fertilizin to the univalent form.
In a second series of experiments fertilizin was tested for its ability to combine
with the sperm surface in the presence of unheated dermal secretion. As seen
in Table IV sperm was mixed with a non-agglutinating inhibitor-fertilizin mixture
in proportions that assured sufficient fertilizin to saturate the sperm surface. The
sperm in the mixture was subsequently centrifuged free of the mixture and tested
TABLE IV
Fertilizin agglutination of Arbacia sperm washed from fertilizin-dermal secretion mixtures
1
2
3
4
Sperm washed from
mixture of
D.S.
D.S.
S.W.
S.W.
fertilizin
S.W.
fertilizin
S.W.
Agglutination on addition of
fertilizin to the washed
+ + +
++++
—
+++ +
sperm
The four mixtures were prepared in the following proportions: 0.5 ml. dermal secretion,
0.25 ml. fertilizin, 0.25 ml. approximately 47% semen. They were centrifuged at 4° C., and the
sperm were re-suspended in 0.5 ml. sea water. These sperm suspensions were then tested for
agglutinability with control fertilizin. The sperm suspensions were not agglutinated in sea water
alone. Test 3 shows that the original mixtures 1 and 3 contained sufficient fertilizin to block
the sperm surface. In the presence of dermal secretion, this amount of fertilizin failed to block
the sperm surface (test 1). Controls not listed show that the mixtures contained an excess of
dermal secretion.
for agglutinability by control fertilizin. As seen in the table the sperm washed
from the fertilizin-inhibitor mixture agglutinated upon addition of control fertilizin,
but sperm washed from fertilizin alone failed to agglutinate. In the latter case,
the fertilizin evidently saturated the sperm surface (as univalent fertilizin) to
prevent agglutination by the control fertilizin. Failure of fertilizin to block the
sperm surface in the fertilizin-inhibitor mixture is most readily explained by an
inactivation of combining sites of fertilizin by the inhibitor prior to addition of
sperm.
C) Action on antifertilizin agglutination of eggs
In view of its action on solutions of fertilizin the dermal secretion might be
expected to affect fertilizin in the gel form. The natural jelly surrounding the
eggs of sea urchins is largely, if not entirely, fertilizin (Tyler, 1949). Thus the
dermal secretion might be expected to precipitate, dissolve or otherwise alter this
478
C. B. METZ
jelly layer in some visible way. In any event it should affect the egg agglutination
and jelly precipitation reaction that results when antifertilizin from sperm is mixed
with eggs.
According to Pequegnat (1948) (p. 79) the dermal secretion "appeared to re-
move part of the egg's jelly layer, in proportion to concentration or to the duration
of exposure." Similar action was sometimes observed in the present investiga-
tion. However, the effect was not consistently found. It seems likely that this
jelly-dissolving action is related to the pH of the inhibitor solutions. Aside from
this possible dissolving action the dermal secretion has no visible effect on the
egg jelly. It certainly does not precipitate the egg jelly in the form of a mem-
brane as might be expected in view of its ability to inactivate the combining sites
of fertilizin.
In spite of its failure to have a direct precipitatory action on the egg jelly, the
dermal secretion was found to inhibit the jelly precipitation and egg agglutination
TABLE V
Effect of heated and unheated dermal secretion on A, fertilizin agglutination of
sperm and B, antifertilizin precipitation of egg jellies
A
B
D.S.
D.S.
S.W.
S.W.
D.S.
D.S.
S.W.
S.W.
fertili/in
S.W.
fertilizin
S.W.
anti-
fertilizin
S.W.
anti-
fertilizin
S.W.
sperm
sperm
sperm
sperm
eggs
eggs
eggs
eggs
Unheated
+ + + +
+ + + +
D.S.
Heated
+ + + +
—
+ + + +
+ + + +
D.S.
The heated dermal secretion was treated for 5 minutes at 100° C. Two other experiments
gave similar results. In one of these the dermal secretion was heated for 25 minutes.
that results when sperm antifertilizin is mixed with eggs. However, the inhibiting
action on egg jelly precipitation was found to be reversible. After washing the
eggs from the dermal secretion or dermal secretion-antifertilizin mixture into sea
water, and subsequently adding antifertilizin they formed satisfactory jelly precipi-
tation membranes. Clearly, then, the dermal secretion inhibits antifertilizin ag-
glutination of eggs. However, the mechanism of this action is apparently more
complicated than would have been predicted from the mode of action of the dermal
secretion on sperm agglutination. Results from the latter study (above) indicate
that the dermal secretion inactivates the combining sites of fertilizin. The inhibi-
tion of egg agglutination by antifertilizin, however, appears to result primarily from
action of the material on the egg agglutinating agent, antifertilizin, in the sperm
extracts. This conclusion is suggested by 1) the ready reversibility of the inhi-
bition upon washing eggs, 2) the fact that a precipitate forms upon mixing anti-
fertilizin and the dermal secretion and finally, 3) the fact that the egg agglutination
INHIBITION OF SPERM AGGLUTINATION 479
inhibitor is heat-stable, whereas the fertilizin agglutination inhibitor is heat-labile.
These last relations are demonstrated in the experiment summarized in Table V.
From these experiments it would appear that there are two inhibitors in the dermal
secretion. One of these inhibits the sperm agglutinating action of fertilizin by
inactivating the combining sites of the agglutinin. The second agent inhibits the
egg jelly precipitating and agglutinating action of sperm antifertilizin by inactivating
the antifertilizin.
Although these experiments adequately explain failure of antifertilizin to ag-
glutinate eggs in the presence of dermal secretion, they still do not directly answer
the question whether the heat-labile agent can combine with the reactive sites of
fertilizin when the latter is in the gel form. To test for such combination, eggs
were treated with an excess of dermal secretion for periods up to 55 minutes. The
eggs were subsequently washed in sea water and extracted for fertilizin in normal
or acid sea water. In each of three such experiments the dermal secretion-treated
eggs yielded sperm agglutinating fertilizin solutions. Indeed the sperm agglutinin
titers of fertilizin solutions prepared by acid extraction of dermal secretion-treated
and control eggs were not significantly different. Evidently, then, the reactive
sites of fertilizin in the gel form are not accessible to the heat-labile inhibitor of
the dermal secretion.
D) Some pJiysical and chemical properties of the dcnnal secretion
No systematic study of the physical and chemical properties of the dermal
secretion has yet been made. Nevertheless, some information has been obtained
concerning the secretion and the sperm agglutination inhibitor contained in it.
This information is recorded here.
Color changes. The freshly prepared dermal secretion is light yellow-green in
color. Upon standing in air the solution gradually darkens to a deep brown or
black color. This color change is evidently a rather direct oxidation by atmospheric
oxygen, for the color change fails to occur in an atmosphere of nitrogen. Further-
more, it appears unlikely that the oxidation is mediated by enzymes because both
heated and unheated preparations undergo the color changes. Finally, the oxida-
tion is not reversed by reducing agents such as hydrosulfite. The colored material
is a component of a large molecule since it precipitates with (NH4)2SO4 and fails
to diffuse through cellophane. The sperm agglutination inhibitor is associated
with the pigmented material to the extent that the inhibitor also precipitates with
(NH,),SO4 and fails to diffuse.
Antigenic composition. No serious serological study has been made of the
dermal secretion. Nevertheless, the dermal secretion lowered the sperm aggluti-
nating titer of unti-Arbacia sperm serum. In agar diffusion precipitin tests
(Ochterlony tests) the dermal secretion produced three precipitin bands both with
antisera prepared against Arbacia sperm and with antisera against jellyless Arbacia
eggs. One precipitin band formed when the dermal secretion was diffused against
antiserum prepared against Arbacia fertilizin. No precipitates formed with control
(pre-injection) serum. The antisera used in these experiments were prepared
with considerable care. The sperm and jellyless eggs used for injection were washed
to remove contaminating material. Likewise, the fertilizin used as immunizing
antigen was obtained by careful acid extraction of washed eggs (see Tyler, 1949).
480 C. B. METZ
In view of this, it appears likely that the immunizing antigens were largely free of
contaminating material including dermal secretion. Evidently, then, the dermal
secretion contains antigenic groups in common with sperm, eggs and fertilizin.
The antigenic relationships here have not yet been investigated, but it is evident
that the dermal secretion contains at least three separate and distinct materials.
DISCUSSION
The observations of Oshima (1921) and Pequegnat (1948) combined with the
present investigation show that the dermal secretion of Arbacia has a variety of
effects on sea urchin gametes and their interaction. Action on sperm includes
enhancement of motility and respiration. These are not pH effects but appear
to depend upon an agent or agents in the dermal secretion. The two effects may
be due to separate agents although it seems more likely that increase in motility
and respiration are related and result from action of a single agent. Little infor-
mation concerning the mechanism of action of the agent has been obtained. How-
ever, it is unlikely that the motility and respiration enhancing effects are due to a
metal-binding action of the dermal secretion. Metal-binding agents do increase
the motility of sea urchin sperm (Tyler and Atkinson, 1950), but they differ from
the dermal secretion in that they fail to increase the rate of oxygen uptake (Tyler
and Rothschild, 1951; Tyler, 1953). Metal-binding agents and the dermal secre-
tion of Arbacia also differ in their action on Asterias sperm. The former agents
have a spectacular stimulatory action on Asterias sperm motility (Metz and Birky,
1955) whereas the dermal secretion of Arbacia was observed to have no effect on
Asterias sperm motility. Evidently, the dermal secretion does not contain appre-
ciable amounts of substances which bind metals. Finally, contamination with the
dermal secretion is a hazard to be avoided in studies of stimulating action of egg
water and other extracts on sperm motility and respiration.
The dermal secretion also inhibits fertilizin agglutination of sperm, antifertilizin
precipitation of egg jelly and fertilization. From the sperm centrifugation experi-
ments it is evident that the dermal secretion does not inhibit agglutination by
combining with and blocking the sperm surface in irreversible fashion. In this
respect the agent differs from fertilizin. As is well known, sea urchin sperm
washed from an excess of fertilizin after reversal of agglutination fails to reag-
glutinate upon a second addition of fertilizin. Indeed, sperm remove the fertilizin
from solution (e.g., Monroy et al., 1954). Conceivably, the agent could inhibit
agglutination enzymatically by digesting the antifertilizin from the sperm surface
with sufficient rapidity to prevent agglutination. If the antifertilizin were ar-
ranged in layers at the sperm surface, sufficient of this material might remain
after addition and subsequent removal of the inhibitor to insure agglutination by
fertilizin. However, even granting this unlikely possibility it is clear from the
experiments with dermal secretion-fertilizin mixtures that the dermal secretion
destroys the agglutinating action of fertilizin. In the first series of these experi-
ments non-agglutinating mixtures were heated sufficiently to destroy the inhibiting
agent but not the fertilizin. Such heated mixtures failed to agglutinate sperm and
to inhibit agglutination. In the second series sperm were washed from unheated
inhibitor-fertilizin mixtures containing sufficient fertilizin to block the sperm sur-
INHIBITION OF SPERM AGGLUTINATION 481
face. These washed sperm agglutinated on addition of fertilizin. These experi-
ments show that the dermal secretion of Arbacia can inhibit agglutination by inac-
tivation of the agglutinin fertilizin. This inactivation is not simply a conversion
of the fertilizin molecule to a form similar to the univalent fertilizin produced by
some other agent (see Metz, 1957b, for review). The inhibiting agent in the
dermal secretion inactivates or blocks the combining sites of the fertilizin that
react with the sperm surface in agglutination.
In view of this action on fertilizin it is surprising that the dermal secretion has
no visible effect on the sea urchin egg jelly since this jelly consists of undissolved
fertilizin. It is even more surprising that eggs washed from dermal secretion
will agglutinate with antifertilizin, since the agent in the dermal secretion inac-
tivates the combining sites of fertilizin. These unexpected observations are ex-
plained by experiments showing that the combining sites of fertilizin are not inac-
tivated by dermal secretion when the fertilizin is in the gel form. Possibly the
inhibiting agent fails to diffuse through the egg jelly. However, it seems more
likely that the specific combining sites are inaccessible to the dermal secretion
because they are blocked by cross linkages in the gel structure. This concept has
been offered as a possible explanation of passage of sperm through the egg jelly
without saturation by fertilizin (Tyler, 1941). It is also consistent with the ob-
servation that fertilizin in solution and in the gel form differs in staining properties
with metachromatic dyes (Monroy et al., 1954). Even with this explanation
one difficulty remains. If the combining sites of fertilizin in the gel form are not
available for reaction with dermal secretion, then they might reasonably be expected
to be unavailable to antifertilizin as well. This difficulty is resolved by assuming
that egg jelly precipitation results from combination of antifertilizin with a part
of the fertilizin other than the specific combining sites involved in sperm
agglutination.
Finally, consideration of the effect of dermal secretion on the egg jelly pre-
cipitating action of antifertilizin leads to other interesting implications. In the
presence of dermal secretion antifertilizin fails to precipitate egg jellies. This
inhibition results from a second, heat-stable agent in the dermal secretion which
acts upon antifertilizin. This complicates the problem further because the first
experiments performed in this study show that the dermal secretion does not irre-
versibly inactivate the combining sites of the sperm surface antifertilizin. There-
fore, the heat-stable inhibitor must inactivate egg agglutinating antifertilizin prepa-
rations by combination with some other part of the molecule. A final possibility
that is not excluded is that the "antifertilizin" extracted from sperm is not related
to the sperm surface material that combines with fertilizin in the sperm agglutina-
tion reaction. The "antifertilizin" may be a fortuitous product of the extraction
procedure which, like some other proteins, precipitates sea urchin egg jelly non-
specifically.
One other question requires consideration ; namely, the relation of the inhibitors
described above to inhibition of fertilization. Like the sperm agglutination in-
hibitor the fertilization inhibitor is heat-labile. This is consistent with the view
that inhibition of agglutination and of fertilization result from action of the same
agent. Further investigation of this relationship may reveal that the fertilization
inhibiting action of dermal secretion results from inactivation of fertilizin.
482 C. B. METZ
Other agents are also known to inhibit fertilization in the sea urchin. Notable
among these are the preparations from the brown alga, Fucus, studied by Runn-
strom and co-workers. Two inhibitory agents are recognized. One is a phenolic
substance and has not been studied in great detail (Wicklund, 1954). The other,
named "fertilization inhibitor (Fucus)," is heat-stable, increases motility of sperm
(Wicklund, 1954) but does not inhibit fertilizin agglutination of sperm (Runnstrom
and Hagstrom, 1955). Clearly this agent has different properties than the dermal
secretion and the two preparations probably act in different fashion to inhibit
fertilization. Indeed, they may very well block different steps in the initial stages
of fertilization. Accordingly, a detailed analysis of the site and mechanism of
action of these and other inhibitors might provide interesting information con-
cerning the sequence of events in the initial stages of fertilization.
SUMMARY
1. As demonstrated previously by Oshima (1921) and Pequegnat (1948)
Arbacia release a yellowish secretion upon appropriate stimulation.
2. This dermal secretion increases the motility and oxygen consumption of
sperm. The effect is not short lived and does not appear to depend upon a metal
binding action.
3. The dermal secretion inhibits fertilizin agglutination of sperm. This action
results from an inactivation of the specific combining sites of fertilizin. The dermal
secretion does not act upon the sperm surface to inhibit agglutination. The agent
in dermal secretion that inactivates fertilizin is destroyed by heating to 100° C.
and fails to diffuse through cellophane.
4. The dermal secretion has no visible action on the Arbacia egg or egg jelly.
5. The dermal secretion inhibits antifertilizin precipitation of intact egg jellies.
This action depends upon a heat-stable agent which precipitates antifertilizin.
6. Some properties of the dermal secretion are described. The material con-
tains at least three distinct antigens.
7. The results are in agreement with the view that fertilizin is essential for
fertilization.
LITERATURE CITED
HARVEY, E. B., 1956. The American Arbacia and Other Sea Urchins. Princeton University
Press.
LILLIE, F. R., 1914. Studies on fertilization. VI. The mechanism of fertilization in Arbacia.
J. Exp. Zool, 16: 523-588.
METZ, C. B., 1942. The inactivation of fertilizin and its conversion to the "univalent" form
by x-rays and ultraviolet light. Biol. Bull., 82: 446-454.
METZ, C. B., 1957a. Mechanisms in fertilization. In: Physiological Triggers, T. H. Bullock,
editor, American Physiological Society, Washington, D. C.
METZ, C. B., 1957b. Specific egg and sperm substances and activation of the egg. In: Begin-
nings of Embryonic Development, A. Tyler, R. C. von Borstel, C. B. Metz, editors.
American Association for the Advancement of Science, Washington, D. C.
METZ, C. B., 1958. Fertilization and agglutination inhibitors from Arbacia. Biol. Bull., 115:
325.
METZ, C. B., AND C. W. BIRKY, JR., 1955. The action of some metal ions and metal chelating
agents on the motility and respiration of starfish sperm. Biol. Bull., 109: 365-366.
MONROY, A., L. Tosi, G. GIARDINA AND R. MAGGIO, 1954. Further investigations on the
interaction between sperm and jelly-coat in the fertilization of the sea urchin egg.
Biol. Bull, 106: 169-177.
INHIBITION OF SPERM AGGLUTINATION 483
OSHIMA, H., 1921. Inhibitiory effect of dermal secretion of the sea urchin upon the fertiliza-
bility of the egg. Science, 54: 578-580.
PEQUEGNAT, W., 1948. Inhibition of fertilization in Arbacia by blood extract. Biol. Bull.,
95: 69-82.
RUNNSTROM, J., AND B. HAGSTROM, 1955. Studies on the action of the "Fucus fertilization
inhibitor" on sea urchin egg and sperm. E.vp. Cell Res., 8 : 1-14.
TYLER, A., 1941. The role of fertilizin in the fertilization of eggs of the sea urchin and other
animals. Biol. Bull., 81 : 190-204.
TYLER, A., 1949. Properties of fertilizin and related substances of eggs and sperm of marine
animals. Amer. Natur., 83: 195-219.
TYLER, A., 1953. Prolongation of life-span of sea urchin spermatozoa, and improvement of the
fertilization-reaction, by treatment of spermatozoa and eggs with metal-chelating agents
(amino acids, Versene, DEDTC, oxine, cupron). Biol. Bull., 104: 228-239.
TYLER, A., AND E. ATKINSON, 1950. Prolongation of the fertilizing capacity of sea urchin
spermatozoa by amino acids. Science, 112: 783-785.
TYLER, A., AND LORD ROTHSCHILD, 1951. Metabolism of sea urchin spermatozoa and induced
anaerobic motility in solutions of amino acids. Proc. Soc. Exp. Biol. Med., 76 : 52-58.
WICKLUND, E., 1954. The influence of some inhibiting substances on fertilization in the sea
urchin egg. Ark. f. Zool. S.2, 6: 485-503.
BODY TEMPERATURES IN SOME AUSTRALIAN MAMMALS.1
I. CHIROPTERA
PETER MORRISON
School of Physiology, University of Queensland, Brisbane ~ Australia and the Departments
of Zoology and Physiology, University of Wisconsin, Madison 6, Wisconsin
The Microchiroptera are a group of mammals which show unique thermo-
regulatory behavior since they alone exhibit a daily reversal between homeothermic
and poikilothermic conditions. Their poikilothermic nature is without question
since their body temperature (TB) often closely follows the ambient temperature
(T4) (Hock, 1951; Eisentraut, 1956a). However, some question has been raised
as to the nature of their homeothermism at other times by the suggestion that they
must be physically active in order to maintain their temperature; the implication
being that the elevation is a passive concomitant of the heat produced during exercise,
analogous perhaps to the warming of a bumblebee or a tarpon, and thus may not
represent positive regulation. In the present study the relation between activity
and body temperature is examined in one microchiropteran to throw light on this
question.
The situation in the Megachiroptera is also in some question. Although a
limited number of measurements (about a dozen) have suggested considerable
regulation in this group of larger bats (Burbank and Young, 1934 ; Eisentraut, 1938)
the statement has still been made recently, that all bats exhibit a lack of thermo-
regulatory control when they become inactive. The following observations on a
representative of this group will allow us to compare it more closely in this regard
both to other mammals and to the suborder of smaller bats.
MATERIAL AND METHODS
The Microchiroptera were represented by the bent-wing bat, Miniopterus
blepotis. This genus of simple-nosed bats (Vespertilionidae) is distinguished by
the extended terminal joint of the longest digit which is bent up against the upper
part of the wing during rest, and by the tail length which is equal to the head
and body. This species ranges through most of Australia and is known for its
communal, cave-dwelling habit (Troughton, 1941). These individuals were be-
tween our little brown and big brown bats in size with a weight range from 6 to
12 grams. The animals were taken from a colony living in a cave formed by
fallen slabs of rock in a small stream about 50 miles north of Brisbane. Its depth
1 These studies were supported by the Guggenheim Foundation and the U. S. Educational
Foundation in Australia, with supplemental assistance from the Wisconsin Alumnae Research
Foundation. The loan and transport of certain equipment were effected under contract between
the University of Wisconsin and the Office of Naval Research.
2 These studies would not have been possible without the enthusiastic support of Prof.
W. V. Macfarlane who generously made available the resources of his Department.
484
BODY TEMPERATURES IN CHIROPTERA
485
was about 30 feet and access was through the falling water, although other smaller
openings were available to the bats. The animals were closely packed, hanging
from the ceiling over an area of several square yards. They became active quite
quickly on disturbance since the ambient temperature was not low in this first
month of spring (September). A subsequent visit in the fall (March) showed
only a few scattered individuals, but whether this was a seasonal change or a result
of the previous disturbance is not known. We are greatly indebted to Dr. Peter
Ulrich for his assistance in locating and procuring these Miniopterus.
The megachiropteran studied was the grey-headed fruit bat, Pteropus polio-
cephalus, a representative of the single Australian genus, which was kindly loaned
us from his collection by Mr. David Flea of West Burleigh. This individual, a
22
FIGURE 1. Relation between body temperature and activity in Miniopterus. Symbols indicate
hours after capture. Ambient temperature, 19-21°.
486
P. MORRISON
250-gram male, had been well adjusted to captivity but following transfer it showed
some progressive readjustment during the week it was studied. This species, which
like its fellows feeds on fruit and eucalyptus blossoms of various sorts, is found in
central-coastal, eastern Australia. It has been distinguished from the three other
mainland species by its longer coat on body and legs which give it a furry appearance
(Troughton, 1941). Specimens up to a kilogram in weight and with a 4-foot
wing spread have been reported.
Body temperature measurements were made with iron-constantan thermo-
couples and a Leeds and Northrup recording potentiometer. The Miniopterus
were flown in a large room, which, however, did not provide sufficient space to
fly the Pteropits. One set of observations was carried out on the former animals
at night within 8 hours of capture. The group was then stored in a refrigerator
at 5-10° and subsequent observations were made one day and three days later.
Metabolic measurements of oxygen consumption utilized a manometric apparatus
(Morrison, 1951) and were carried out with the assistance of Mr. Len Morris and
Miss Nancy Harvey.
RESULTS
Miniopterus
The relation between activity and body temperature is summarized in Figure 1
in animals observed within 8 hours of capture. Activity is expressed as resting
TABLE I
Body temperature and activity in Miniopterus
Activity level
Hours after
capture
Mean ± S.D.
No.
Range
Cannot walk
84
22.7 ± 0.36
23.0
7
8
22.1-23.3
22.1-25.7
Can walk
Cannot fly
84
32
30.0 ± 1.15
30.7 ± 0.59
6
5
27.8-31.6
29.8-31.2
Can fly-
84
32
31.7 ± 1.24
32.0 ± 0.10
9
4
30.2-33.7
31.9-32.2
Resting after activity
8
37.2 ± 0.40
6
36.7-37.7
Walking
8
38.7 ± 1.16
16
36.7-40.6
Stop flying
8
38.9 ± 0.61
5
38.2-39.8
Short flight
84
32
36.4 db 1.10
35.8 ± 1.55
39.6 ± 0.72
39.2 ± 1.08
8
3
8
11
34.3-38.8
33.9-37.1
38.7-40.7
37.4-40.7
Long flight
32
8
39.6 ± 1.07
41.1 ± 0.45
10
13
38.7-41.2
40.3-41.7
8
39.1 ± 1.0
49
36.7-41.7
BODY TEMPERATURES IN CHIROPTERA
487
0
20 40 60
TIME IN MINUTES
80
100
FIGURE 2. Course of temperature changes during awakening and extended flight. TA — 20°.
Upper solid curve indicates flight at ca. 600 ft./min., followed by rest.
(1 + ), walking or stops flying (2+), short flight (3+) and long flight (4+).
These symbols follow a scale of relative activity which has been used with other
mammals (Morrison and Ryser, 1959), and ranges from sleep (0+), through
awake (1 + ), moving (2+), active (3+) and very active (4 4-). A condition
of sleep (0 + ) was not seen in any of these initial observations which were carried
out in the evening when the bats are normally active. Further, these animals were
undoubtedly disturbed by their recent capture and travel. Actually, the majority
of these animals were actively walking or flying, so that only a few were in the
"resting" state. However, the observed range of activity was as wide as in most
animals since it is usually not possible to elicit maximum activity (4+).
A regular increase in body temperature with activity level is seen. Values given
in Table I show a mean increase of 1.3° per activity grade, or an over-all increase
of 3.9° (1 -I — >4 +). These 49 values may be averaged to give a mean for the
awake condition, although this is an artificial situation and so gives an artificial
distribution of activity. This mean is 39.1° ± 1.00 (49), at a mean activity of
2.6 + and with an over-all range of 5.0° from 36.7-41.7°.
The values taken 32 and 84 hours after capture are similar, but they are quite
distinct from the earlier observations. After these animals were taken from the
refrigerator and warmed to room temperature, many were loath to engage in any
activity. Below a body temperature of 26° the bats could not walk; at 28-31.5°
488
P. MORRISON
the bats could fly when launched, sometimes with difficulty, but would not take
off spontaneously. They would bite.
Some of these temperatures were transient values during warming, but others
represented a maintained level in an inactive animal (0+ activity level). Thus,
for example, one bat (A) which was followed during warming rose to 22.7°
(+9.3°) in 20 minutes, and to 30.3° (+ 12.2°) in another hour, but only to
30.6° (+0.3°) in another 15-minute period (Fig. 2). Another, warmed to 22.1°
(+ 7.5°) in 20 minutes, and to 28.2° (+6.1°) in a second 20-minute period. After
a third period the value rose to 31.2° (+ 3.0°), but with a fourth period a slight
fall to 30.8° (—0.4°) was observed. Accordingly, these quiet animals are
maintaining their body temperatures well above the ambient temperature of 20°
42
40
TB
IN
°C
38
36
MINIOPTERUS
_L
10
4
•RESTING
0
•MINUTES
4
•FLYING-
8
10
FIGURE 3. Changes in body temperature following the initiation or completion
of flight in Miniopterus.
(AT = + 10°) but at the same time, greatly below the level of more active states;
10° less than the actively flying animal.
Some animals become active spontaneously and others were forced to be active.
Under these conditions shorter flights were made and lower body temperatures
were recorded than before (36° vs. 39°). The animals which made prolonged
flights averaged 3° warmer, equivalent to the "short flight" (3+) condition in the
fresh bats, but still 1.5° below the "long flight" (4+) level in fresh bats.
Figure 3 presents successive measurements on individuals to show temperature
change in active bats. During flights an average increase of 0.85°/min. was seen
for the first two or three minutes. After this the temperature leveled out abruptly,
showing a quite positive regulation at this higher temperature. The decline in
body temperature following flight was more gradual (— 0.32°/min.) but could
continue for as long as 10 minutes without leveling off at a lower value. Figure 2
BODY TEMPERATURES IN CHIROPTERA
489
38
37
IN
°C
36
35
03/14-18/55
• 3/19-21/55
PTEROPUS
0 +
ACTIVITY
FIGURE 4. Relation between activity and body temperature in Pteropus. TA = 25°.
Average ± S.D. (#) : 0 (asleep) = 35.2 ± 0.05(3) ;+ (awake, not moving) = 36.5° ± 0.49(15) ;
open circles only = 36.8 ± 0.39(9) ; closed circles only = 36.1 ± 0.27(6) ; ++ (moving around
or feeding) =37.5 ±0.25(5). Slope of curve = 1.2V+. Crosses show values of Burbank and
Young (1934) for P. geddei, P. giganteus and P. cotinus at TA = 19°.
38
37
IB
IN
°C
36
35
03/14-18/55
• 3/19-21/55
oo
PTEROPUS
MID
8 NOON
HOUR OF DAY
16
20
MID
FIGURE 5. Body temperature in Pteropus as a function of the hour of day. TA = 25C
Average values for solid symbols: 7^ 10 = 35.5° (5) ; 22-^24 = 36.7° (7).
8 -» 14 = 36.9° (6) ; 16 -> 22 = 36.9° (4) . A = 0, O = 1+, d = 2+ activity.
Open symbols
490
P. MORRISON
shows a value 40 minutes after stopping activity which has returned to the level
of quiet, awake animals.
These changes in body temperature may be used to estimate heat production
during warming. Thus, a maximum increase of 1.03°/min. (11.5-^23.8° in
12 minutes) was observed in one individual. This is equivalent to 0.85 cal. g.-1min.~1
or an oxygen consumption of 10.7 cc. g.~1hrr1 (other cases (24.6^32.2° in 10
FIGURE 6. Body temperature in Pteropus after exposure at various ambient temperatures.
Values indicate hours of exposure. Circles, earlier data (3/14-18) and squares, later data
(3/19-21) at 1+ activity. Top and bottom curves are for 2+ and 0+ activity, respectively.
Crosses show values of Burbank and Young (1934). Stars and dotted line show range of
ambient and body temperatures reported by Eisentraut (1938) for Rousettus angolensis.
minutes; 22.2^28° in 12 minutes; 22— > 28.3° in 14 minutes) were equivalent to
metabolic levels of 7.9, 5.0 and 4.7 cc. O-2 grMir."1.) Of course, the first estimate
with body temperature below ambient temperature was high because of the heat
flow into the animal, and the latter three were low for the reverse reason. An
average maximum between these is equivalent to 9 cc. g.~1hr.~1. This is a sub-
stantial but not unreasonable rate for such a small animal. But when it is recalled
that the body temperature was only 15-25°, it is much more striking. Even with
BODY TEMPERATURES IN CHIROPTERA
491
a modest Q10 of 2.0, this would be equivalent to a rate of some 36 cc. g.~1hr.~1 at
40° or greater than the maximum observed even in the smallest mammal, the
long-tailed shrew (Morrison et al, 1953, 1959).
Pteropus
Data relating activity to body temperature in this form are presented in Figure 4.
Because of its size, it was not possible to fly it, and thus activity was limited to
moving about its cage. However, three observations during sleep (0 activity)
PTEROPUS
RUN 114:3/19/55
8
FIGURE 7. Oxygen consumption in Pteropus at three ambient temperatures. Representa-
tive experiment showing variation, particularly in the form of short reductions from the average
level at TA = I°.
were made. These animals had closed eyes and were distinctly less responsive on
handling. The average increment was 1.2°/+. The average temperature was
36.5 ± 0.77° (23) at a mean activity of 2.1 +.
Temperature values for Pteropus are plotted against the hour of day in Figure 5.
During the first four days no value less than 36° was observed and no daily cycle
was seen. During the last three days, after the animal had settled down, there was
a definite diurnal difference with the day-time values (7-10) averaging 35.5° and
the night-time values (22-24) averaging 36.7°.
492
P. MORRISON
The influence of ambient temperature on body temperature in Pteropus is
shown in Figure 6. Exposures of up to 4 hours at 10° had little effect on the
body temperature. At an ambient temperature of 40° the body temperature was
maintained roughly constant through a 6-hour exposure but the level was elevated
to between 40 and 41°. The average value of 40.5° provided a temperature dif-
ferential of only 0.5° for the dissipation of body heat. However, this small differ-
ential was apparently adequate because of the very large surface available in the
wings, and the ceaseless fanning activity during this heat stress (Robinson and
Morrison, 1957). During the later period after the bat had settled down pre-
sumably with a lower resting metabolic rate, the upper limit of thermal neutrality
appears to have been substantially raised.
cc.
LJ
0.
o
o
OJ
O
AVERAGES
MINIMUMS
10
20
30
40
FIGURE 8. Oxygen consumption in Pteropus at various ambient temperatures. Minimum
and average values from three runs ($100, 105, 114). Bottom pair of curves shows conductance
at low temperature ; top pair, at room temperature.
The metabolic response to cold exposure measured as oxygen consumption is
illustrated in Figure 7. The measurements at higher temperatures (28-34°) were
extremely regular, reflecting the behavior of the animal which hung quietly. Indeed,
for all measurements the averages for each temperature period (12) were only 10%
higher than the minimum values, and the maximum values were only 32% higher.
Minimum and average values are plotted in Figure 8 against ambient temperature.
There is some question as to which of these most accurately describes the minimum
conductance at each temperature. The average value may be too high since extra
heat (i.e., above maintenance) is dissipated during movement and activity. On the
BODY TEMPERATURES IN CHIROPTERA 493
other hand, if there are fluctuations in body temperature a minimum value may rep-
resent not equilibrium but a negative heat balance. Such a situation appears in the
series at TA — 1° in Figure 7, which is characterized by several periods of brief
duration and much lower rate. We interpret these as inadequate heat production.
The oxygen deficit and durations (ca. 1 cc. g.^hr."1 and 3 min.) correspond to
a temperature change of 0.3°, and it may be noted that each of these low periods
is followed by a period of increased, presumably compensatory, activity. We have
previously observed this type of response (a sharp, brief fall) only in hibernators
entering hibernation. These low values are excluded in our estimate of conductance
but their values are indicated in Figure 8 along with the average and minimum
values. We feel that the latter is preferable in calculating minimum conductance,
but in this species the difference is only 10%.
In the simplest model homeotherm the metabolism below thermal neutrality is
represented by a single curve, M = C (TB — TA). This is not possible in the
present case which requires separate curves for the cold and for room temperatures.
This is not an uncommon situation and simply means that at moderate tempera-
tures the animal does not make use of its maximum insulative potential. In this
instance, the higher conductance at 25-30° (0.13 cc. O2 g.^hr.-10 C."1) is about
twice the minimum value measured at 0-5° (0.068 cc. O.2 g.'Mir.-10 C.'1).
DISCUSSION
The behavior of Miniopterus shows that when awake these animals can act as
homeotherms with a regulated body temperature comparable to other mammals.
On the night following capture the group was all awake and, although some were
quite inactive, no temperature less than 36.7° (out of 49 values) was observed.
While the level of the temperature varied with the level of activity, this is a normal
feature in homeotherms. The activity increment of 1.3°/+ or 3.9° from "awake"
to "very active" is comparable to that in non-hibernating mammals ; and indeed is
less than that seen in some. The average temperature of 39.1° is more than a de-
gree above the average for mammals (Morrison and Ryser, 1952). Although the
mean activity represented by these values is high (2.6 + ) , the value corrected to a
level of 1 -f , 37.0°, is still a reasonable one. Although 1° below the mean for "all"
mammals it is within one standard deviation (for species) and is close to mean
values for other hibernators in the awake condition. And it is almost identical to
that of Sorex, a small representative of another primitive group of mammals, the
insectivores (Morrison et al., 1953, 1959).
The observed values are also in accord with values of 37.8 and 38.2° made on
two "fully awake" but not flying individuals of a related species, M. in flat us
(Eisentraut, 1956b), since the latter lie within our range for walking animals.
This range, 36.7-40.6°, is slightly higher, but quite comparable to those for 5
species of temperate bats (limits, 34.4-40.8°) cited by Eisentraut (1938). But
it is distinctly above the range given by him for 5 tropical species from Africa
(limits 28-39.2°). Swanson and Evans (1936) report values of 36.7 and 36.1°
for aroused Eptesicus in winter.
Values for animals following extended flight were the most uniform of any
activity level (41.1 =t 0.45° (13)) and this may reflect the more positive thermo-
regulation in response to the excess heat of flying, using the very effective dissipat-
494 P. MORRISON
ing potential of the wings. Burbank and Young (1934) reported a maximum
temperature of 41.0° after flight in the horseshoe bat (Rhinolophus} . In Myotis
yumanensis, Reeder and Cowles (1951) observed temperatures of 39.0-40.3 after
flight and reported that vasodilation of the wings took place at a body temperature
of about 40°. This species also appears to be similar in its critical temperature
for flight, 31°, which represents the dividing point for Miniopterus. Burbank and
Young also note 30° as the minimum temperature for flight in Rhinolophus, and
30-33° as the temperature for initiation of flight in the noctule bat, Nyc talus
noctula. Minimum temperatures for flight in hibernating birds are also somewhat
above 30°. Miller (1950) reported a temperature of 34.0° after a short flight in
a poor-will (Phalaenoptilus). And Marshall (1955) described an awakening
captive individual that could not fly at a body temperature of 34.4°.
Following refrigeration at 10° some of the animals appeared in a different con-
dition. On transfer into the room they warmed to a level well above room tempera-
ture (31°), but much less than before even though some walked. Eisentraut
(1956b) reported the M. in flat us did not warm above 30° after refrigeration. Other
Miniopterus in the present study flew and warmed to a higher level, though still
less than following short flights in fresh animals. However, with extended
(forced) flights, almost as high values were noted. But after activity the body
temperature again fell to near 30°. This approach to the same level from above
as from below (Fig. 2) provides rather convincing evidence of a maintained level
and it appears significant that this level is such as to just permit flight. This sug-
gests that the lower temperatures did not result from any energetic deficiency, but
possibly from a less disturbed condition than on the day of capture. Pearson
(1947) in metabolic measurements, observed a case of continuously elevated me-
tabolism during the day in a specimen of Eptesicus which was disturbed for some
reason. It may be that bats can maintain different levels of temperature, without
exercise, in relation to different excitatory levels.
In this regard it should be noted that although bats have been considered unique
in their daily transformation into poikilotherms, observations from another phase
of this study have revealed essentially this same behavior in one of the Dasyurid
marsupials, Chaetocercus. During the day this animal's temperature may fall close
to the ambient temperature even though this is as high as 28°. A related, but not
so pronounced effect has also been observed in the deer mouse (Peromyscus leu-
copus) in which certain individuals cool substantially beyond the ordinary range of
variation during enforced inactivity even at moderate temperatures (Morrison and
Ryser, 1959).
Slonim (1952) describes studies of body temperature and metabolism in rela-
tion to ambient temperature in four microchiropterans. Two species (Miniopterus
schreibersi and Myotis Myotis) showed clear temperature regulation between 13
and 24° with a maintained temperature level and a strong metabolic response of
17-20%/° C. Two species of horseshoe bats (Rhynolophus jcrrum equinum and
R. hipposideros) showed limited, irregular response (at 20-25°). However these
latter observations were made in March and in the fall, while the first mentioned
species were studied in May and June. In all these experiments (10 values) the
maintained body temperatures ranged between 29 and 33°. It may be of signifi-
cance that this level of regulation (31-33°) is also that found in our most primitive
homeotherm, the echidna (Wordlaw, 1915).
BODY TEMPERATURES IN CHIROPTERA 495
The rates of rewarming were comparable to the maximum rates observed in the
same genus (1.28, 0.93°/min.) by Eisentraut (1956b) and in Myotis (1.33°/imn.)
by Reeder and Cowles (1951). But our maximum rate of warming during flight
(ca. 1.0°/min.) was substantially less than a value for Myotis.
Pteropus
The influence of activity on body temperature in Pteropus appears quite similar
to Miniopterus with an activity increment of 1.2°/+, although the observed range
was more limited. If we correct the mean body temperature of 36.5° (2.1 +) to
a standard activity of 1 + , we have a value of 35.2°. This is definitely lower than
in Miniopterus and the average for mammals, but it is quite comparable to values
for two larger insectivores, the mole and the hedgehog, 35.5 and 35.8° (Morrison and
Ryser, 1952). Similarly, the variability of the temperature (a- = 0.77°) is reason-
able. The range, 35.2-37.9°, is somewhat less than that of 34.4-38.6° (TA = 22-
35°) reported in an African form, Rousettus angolcnsis by Eisentraut (1938), but
the midpoint of this range (36.5°) falls just at our mean value. The other observa-
tions on megachiropterans are by Burbank and Young (1934). They found an
average of 35.9 ± 1.4° (8) in measurements on three species of Pteropus. Exclud-
ing one value for an active and two for sleeping individuals gave an average of
35.8° or within the a of our second group of values. Their single value on an
actual individual (37.5°) checks well with our value at 2 + activity (37.6°), but
two values on a "large male hanging quietly" were well below our range of values
(33.0 and 34.0°). Possibly the lower ambient temperature (18°) in these studies
was only of influence at 0 + activity. Under these conditions they noted a response
of shivering and a maintenance of body temperature between 33.0 and 37.5°. This
is considerably more variation than we observed in our specimen, which maintained
a body temperature of 36.5 even after a 4-hour exposure at a lower temperature
of 10°. But if we limit Burbank and Young's values to the four at 1 + activity
(neither active nor asleep) corresponding to our specimen in the cold, a close
correspondence is seen (36.8 vs. 36.5°).
It might be suggested that conditions of captivity prevented the observation of
temperature lability which still might be present in nature. This indeed appeared
to be the case in several specimens of Chaetocercus referred to above. While this
suggestion cannot be disposed of completely in Pteropus, what evidence there is
appears against it. Field observations show that these animals are not sluggish and
lethargic during the day as are smaller bats. Rather, we found them alert and
able to fly off at once when their roosting place is approached or their roosting tree
disturbed, even at midday.
Metabolic measurements confirm observations on body temperature in the cold.
Pteropus can substantially increase its heat production, the average minimum value
at 0° being 2.5 cc. O2 g.^hrr1 or 3 times the B.M.R. This rate corresponds to a
thermal conductance, C, of 0.068 cc. O2 g.^hrr10 C.'1. This minimum value is
close to that observed in similar measurements on small mammals from Wisconsin
(Morrison and Ryser, 1951). This series could be described as C = 0.95 W"48
which function has a value of 0.071 cc. O2 g.^hrr10 C.-1 at 250 grams. Accordingly,
this subtropical megachiropteran has an insulation quite comparable to temperate
species. This contrasts to the tropical mammals examined by Scholander et al.
496 P. MORRISON
(1949) which had inferior insulation as compared to Arctic forms. We have
previously observed less effective regulation in several Central American species
than that shown here by Pteropus (Morrison, 1946). The minimum temperature
at Brisbane approaches, but does not go below 0°, so these bats had the capacity to
regulate at any time during the year.
The "slips" in metabolism at low temperatures below the maintenance level
(Figs. 7 and 8) might be interpreted as incipient hibernation since we have seen
this phenomenon only in animals entering hibernation. However, there is nothing
to confirm this view, and an alternative suggestion relates to short rests between
bouts of shivering. Such a phenomenon has been observed in the dog with rests
of one to fifteen minutes and a concomitant reduction of metabolism to the basal
level (Hammel and Hardy, 1957).
A final point of interest relates to the basal metabolic level. The observed mean
value of 0.84 cc. O2 g.-Mir."1 compared closely with the "standard" value for a 250-
gram mammal, 3.8 W~-27 = 0.87 cc. O2 grMir.'1. Or conversely we may describe
the metabolic rate in Pteropus as 3.7 W~-27 cc. O2 grMir.'1. There are no data
in the literature with which to directly compare this value. Hock (1951) reports
a value of 3.0 cc. O2 grMir.-1 in Myotis at TA = 37° or about 5 W-27 cc. O2
g.^hr.-1. But although the body temperature probably lay between 37 and 38°,
these measurements made over one-hour periods undoubtedly included some muscu-
lar activity and cannot be taken as minimum values. Accordingly, in the matter
of the B.M.R. as well, the Chiroptera appear to be closely comparable to other
mammals.
CONCLUSIONS
In summary we may conclude from these studies and other data that the
Megachiroptera (Pteropus) are not to be distinguished from other mammals on
the basis of thermoregulation as are the Microchiroptera. Their temperature is
quite typical in regard to variability, activity response and cold response ; and its
level, although somewhat lower than average, is still higher than some other
mammals of comparable size such as the insectivores.
Although the Microchiroptera (Miniopterus) are well developed hibernators
which can become poikilothermic when circumstances demand, when awake they
may show thermoregulatory behavior which is comparable to other mammals in
regard to variability, activity response and mean level. But they may also show an
intermediate maintained level, neither poikilothermic nor homeothermic in the usual
sense.
LITERATURE CITED
BURBANK, R. C, AND J. Z. YOUNG, 1934. Temperature changes and winter sleep of bats.
/. Physiol., 82: 459-467.
EISENTRAUT, M., 1938. Die Warmeregulation tropischer Fledermaiise. Sitzungsberichte d.
gesellschaft naturforschender Freunde (21 Juni), 86-90.
EISENTRAUT, M., 1956a. Der Wintersschlaf mit seinen okologischen und physiologischen
Begleiterscheinungen.
EISENTRAUT, M., 1956b. Korpertemperaturen bei tropischen Fledermausen und Schuppentieren.
Sonderdruck mis Sdugetierkundlische Mittcilungcn, 4 : 64-67.
HAMMEL, H. T., AND J. D. HARDY, 1957. Shivering only variable source of heat in intact un-
acclimatized dogs upon exposure to cold. Fed. Proc., 16 : 54.
HOCK, R. J., 1951. The metabolic rates and body temperatures of bats. Biol. Bull, 101 : 289-
299.
BODY TEMPERATURES IN CHIROPTERA 497
MARSHALL, J. T., 1955. Hibernation in captive goatsuckers. Condor, 57: 129-134.
MILLER, A. H., 1950. Temperatures of the Poorwill in the summer season. Condor, 52:
41-42.
MORRISON, P. R., 1946. Temperature regulation in three Central American mammals. /.
Cell. Comp. Physiol, 27: 125-138.
MORRISON, P. R., 1951. An automatic manometric respirometer. Rev. Sci. Instru., 22: 264-
267.
MORRISON, P. R., AND F. A. RYSER, 1951. Temperature and metabolism in some Wisconsin
mammals. Fed. Proc., 10: 93-94.
MORRISON, P. R., AND F. A. RYSER, 1952. Weight and body temperature in mammals. Science,
116: 231-232.
MORRISON, P. R., AND F. A. RYSER, 1959. Temperature regulation in the whitefooted mouse,.
Pcromyscus Icucopus. Physiol. Zoo!., in press.
MORRISON, P. R., F. A. RYSER AND A. R. DAWE, 1953. Physiological observations on a small
shrew. Fed. Proc., 12: 100-101.
MORRISON, P. R., F. A. RYSER AND A. R. DAWE, 1959. Studies on the physiology of the
masked shrew, Sorex cinereus. Physiol. Zobl., in press.
PEARSON, O. P., 1947. The rate of metabolism in some small mammals. Ecology, 28 : 127-145.
REEDER, W. G., AND R. B. COWLES, 1951. Aspects of thermoregulation in bats. /. Mammalogy,
32: 389-403.
ROBINSON, K. W., AND P. R. MORRISON, 1957. The reaction to hot atmospheres of various
species of Australian marsupial and placental animals. /. Cell. Comp. Physiol., 49 :
455-478.
SCHOLANDER, P. F., V. WALTER, R. HOCK AND L. IRVING, 1949. Body insulation of some
tropical mammals and birds. Biol. Bull., 99 : 225-236.
SLONIM, A. D., 1952. Animal heat and its regulation in the mammalian organism. Academy
of Sciences, U.S.S.R., Leningrad and Moscow.
SWANSON, G., AND C. EVANS, 1936. The hibernation of certain bats in Southern Minnesota.
/. Mammalogy, 17: 39-43.
TROUGHTON, E., 1941. Furred Animals of Australia. Angus & Robertson, Sydney.
WORDLAW, H. S. H., 1915. The temperature of Echidna aculcata. Proc. Linn. Soc. N.S.W.,
40: 231-258.
STUDIES ON THE EFFECTS OF IRRADIATION OF CELLULAR
PARTICULATES.1 III. THE EFFECT OF COMBINED RA-
DIATION TREATMENTS ON PHOSPHORYLATION
HENRY T. YOST, JR. AND HOPE H. ROBSON
Department of Biology, Amherst College, Amherst, Mass.
Previous work has established that particulate-linked, oxidative phosphorylation
is very sensitive to ionizing radiation. This is true whether the radiation is de-
livered to the whole organism (van Bekkum and Vos, 1955) or to the isolated par-
ticulates (Yost and Robson, 1957). These findings suggest that one of the
principal modes of action of radiation is the destruction of the cells' ability to carry
on their "energy metabolism." Interference with oxidative phosphorylation would
be expected to have profound effects upon all reactions of the cell which are de-
pendent upon a supply of ATP (the synthesis of proteins and nucleic acids being
the most obvious). This suggests that some of the observed damage to the
chromosomes resulting from radiation might be the result of extra-nuclear damage
to the phosphorylating mechanism, which suggestion is supported by the observa-
tion of many investigators (see Wolff and Luippold, 1955) that the number of
chromosomal aberrations recovered after radiation treatment is altered by known
inhibitors of oxidative phosphorylation.
The radiation literature contains many references to the effects of combined
radiations in producing alterations in chromosomal aberration (Giles, 1954). It
has been shown that pretreatment with ultraviolet radiation will cause a decrease
in the number of translocations recovered after exposure to ionizing radiations
(Swanson, 1943). On the other hand, pretreatment with near infrared radiation
causes an increase in the recovered aberrations resulting from exposure to ionizing
radiations (Swanson, 1949). To date, no completely satisfactory explanation has
been advanced for these results. Explanations of complimentary or antagonistic
action by radiations used in combination are complicated by the duration of these
effects. In the case of infrared treatment, there may be a time lapse of at least 96
hours between the application of the infrared and the x-rays with no decrease in
effectiveness. Thus, the condition induced is stable, although it may be altered by
drastic environmental changes (Swanson and Yost, 1951). However, when the
data from combined radiation studies are taken together with the data on the effects
of combined radiation and chemical treatments (Kihlman, Merz and Swanson,
1957), an hypothesis may be formulated. It is possible that the various combined
treatment effects are the result of activation or inactivation (depending upon the
radiations or chemicals used) on oxidative phosphorylation.
The data presented in this paper are the result of studies designed to test the
hypothesis that the observed effects of combined radiation treatments are the result
1 This work was supported by a grant from the National Institutes of Health (C-2154)
and a grant from the Smith Klein and French Foundation.
498
RADIATION OF CELLULAR PARTICULATES 499
of interference with the normal phosphorylation pathways. The effect of ultra-
violet radiation alone and in combination with gamma radiation, and the effect of
infrared radiation alone and in combination with gamma and ultraviolet radiations
was studied. Isolated particulates from rat liver were chosen since the effects of
gamma radiation and some ultraviolet effects were already known (Yost and
Robson, 1957; Yost, Robson and Spiegelman, 1956).
MATERIALS AND METHODS
White laboratory rats were starved overnight and sacrificed by a blow on the
head. The liver was removed and placed in cold 0.85% KC1, where much of the
blood is washed free. The liver was weighed and pressed through a bronze screen
to remove connective tissue. The resulting mash was then suspended in 50 ml. of
cold 8.5% sucrose containing 0.005 M disodium versenate and homogenized in a
glass homogenizer. The mitochondria were then separated from the rest of the
homogenate by differential centrifugation (Schneider, 1948). The mitochondrial
fraction alone was kept. The final centrifugate was re-suspended in 1 ml. 8.5%
sucrose per gram of original liver. All dilutions were made from this stock solution.
For treatment with gamma radiation the preparation was diluted 1 in 20 with
cold distilled water. This diluted suspension was radiated in a 25-ml. "Lusteroid"
centrifuge tube in the beam of a 440 curie Co60 source. The radiation was filtered
by a half-inch lucite shield to remove the beta radiation. The intensity of radiation
was 1000 r per minute. The controls were treated in the same manner, with the
exception of the exposure to radiation. After radiation, control and treated sus-
pensions were centrifuged, and the mitochondria re-suspended in one ml. This final
suspension was assayed for phosphorylation.
For treatment with ultraviolet radiation the preparation was not diluted. Two
and five-tenths ml. were placed in a quartz flask rotated in the ultraviolet beam by a
slow speed stirring motor ; 2.5 ml. were kept in a Pyrex tube in the cold as a con-
trol. During the period of irradiation, the quartz flask was partially submerged
in an ice- water bath maintaining a temperature under 5° C. This flask was kept
11.5 cm. from a germicidal lamp for all radiations. The intensity of the radiation
was 5500 ergs/cm2/sec. For most studies the radiations were filtered by a Corn-
ing No. 7910 filter which transmits principally those wave-lengths above 2400 A
(Filter I). In one case, to test the effect of longer wave-lengths, a Corning No.
9700 filter (transmits principally above 2700 A) was used (Filter II). To test the
effect of the flask rotation on phosphorylation, experiments were run in which the
mitochondria were treated in the same way that they would have been for ultraviolet
treatment with the exception that the ultraviolet source was not turned on.
For treatment with infrared radiation, the preparation was not diluted. Four
or five ml. of the suspension were placed in a small test tube which was rotated in
the infrared beam by a slow stirring motor. Temperature changes were eliminated
by radiating the suspension while immersed in a water bath cooled by a Blue-M
cooling coil. The bath was maintained at 9° C. for all studies. Controls were
kept in the rear of the water bath wrapped in aluminum foil to prevent exposure to
radiation ; in two cases they were rotated as were the treated. The infrared source
was 6 inches from the center of the exposed test tube. All radiation passed through
a Corning No. 2540 filter, the glass side of the water bath (window glass), and 1
500 H. T. YOST, JR. AND H. H. ROBSON
cm. of water. This arrangement provides intense radiation in the region 7600 to
15,000 A. The source of radiation was a 250- watt commercial heat lamp operated
at 110 volts. The usual exposure was for three hours. The intensity of the
source is not accurately determined, but this was capable of increasing the chrom-
osome aberrations in Tradescantia by at least 100% when used in combination with
gamma rays.
For studies of the effects of combined radiations, the procedures outlined above
were followed. The controls were taken through all the steps with the exception
of exposure to radiation.
Some attempts to reactivate the ultraviolet-damaged phosphorylation mecha-
nism were made. In attempts to photoreactivate the system, the suspension was
exposed to visible light from a 250-watt incandescent spot-light filtered to remove
the red wave-lengths. This treatment was carried out at 10° C. in the same water
bath used in the infrared studies, to prevent heat-inactivation of the system.
Treatments were of 20 minutes duration. In attempts to reactivate the system by
the addition of co-factors, the co-factor was added to the reaction flask prior to
assay of phosphorylation and oxygen uptake. In one case, 0.1 ml. of 0.01 M DPN
was added, and in the other, 0.1 ml. of 10'* M FAD.
Estimation of phosphorylation was conducted by a modification of the method
of Maley and Lardy (1954), using succinate as the substrate. The main compart-
ment of the Warburg vessel contained: 0.3 ml. (30 /xM) phosphate buffer (pH
7.4), 0.3 ml. 0.1 M sodium succinate, 0.8 ml. 8.5% sucrose, 0.1 ml. (0.3 /*M) cyto-
chrome-c, 0.3 ml. (6 MM) ATP (Schwartz, neutral), 0.1 ml. (30 ^M) MgSO4,
0.1 ml. (40 //M) KF, and 0.5 ml. of the mitochondrial suspension. The center
well contained 0.1 ml. 5 N KOH, and the side arm held 0.5 ml. (10 mg.) of
hexokinase (Pabst) dissolved in 0.15 M glucose. To assure that the initial pH
of the reaction would be 7.4 or higher, the pH of some of the more acid reactants
was adjusted with NaOH before addition to the flasks. Readings of the oxygen
uptake were taken for 30 minutes, after which the reactions were stopped with TCA
and the phosphate determined by the Lowry-Lopez method as presented by Click
(1949). All Warburg assays were carried out at 25° C. All assays were made
in duplicate. All experiments were repeated at least three times.
RESULTS
The data presented in Table I show the effectiveness of ultraviolet radiation in
the inactivation of oxidative phosphorylation. Although the phosphorylation mech-
anism is very labile and therefore shows variation from experiment to experiment
and from assay to assay, it is clear that ultraviolet radiation inactivates phosphoryla-
tion rather easily. These data cannot be used to construct an inactivation curve
because of the variability, but there is clear indication that the inactivation proceeds
in a non-linear manner. It is also clear that the inactivation is not the result of
either the time lag between the isolation of the mitochondria and the assay or the
rotation in the quartz flask. Thirty minutes' rotation causes no loss of phosphoryla-
tion. Further, it can be seen that the filter which transmits principally those wave-
lengths above 2700 A is not effective in bringing about this change. It would
appear that the effective wave-length lies somewhere between the minimum of
Filter I and the minimum of Filter II, that is, between about 2200 and 2700 A.
RADIATION OF CELLULAR PARTICULATES
TABLE I
Inactivation of phosphorylation by ultraviolet radiation
501
Phosphate uptake (/iM)
Treatment*
No. runs
Decrease
Decrease O2
Controls
Treated
5 mill. UV
3
11.1 ± 2.1
10.2 ± 2.6
8.1
6.2
10 min. UV
3
12.7 ± 1.7
11.1 db 2.0
12.6
3.5
20 min. UV
14
12.8 ± 1.1
7.4 ± 1.5
42.2
11.8
30 min. UV
3
15.7 ± 1.4
6.4 ± 0.8
59.2
3.0
30 min. rotation
3
13.8 db 1.7
13.8 ± 1.7
0.0
0.6
30 min. UV
3
10.2 ± 2.6
1.8 ± 1.5
82.4
21.7
Filter I
30 min. UV
3
10.2 db 3.1
11.7 ± 2.7
-14.7
-11.6
Filter II
* Particulates undiluted ; treated in rotating quartz flask,
above 2400 A; Filter II transmits principally above 2700 A.
Filter I transmits principally
One other thing should be noted. The inactivation of the phosphorylating mech-
anism bears no clear relationship to the inactivation of the cytochrome oxidase
system. Oxygen uptake is essentially unaffected in many cases in which there is
extreme damage to phosphorylation. This is in agreement with earlier findings
with gamma radiation (Yost and Robson, 1957).
The data in Table II show that the inactivated phosphorylation mechanism can-
not be easily revived. Addition of DPN to the system stimulates phosphorylation
but does not overcome the effect of the ultraviolet. As a matter of fact, when the
TABLE II
Failure to reactivate ultraviolet-damaged phosphorylation
Treatment*
No. runs
Phosphate uptake
0*M)
%
Decrease
%
Decrease Oz
Control
4
10.3 ± 1.1
—
—
Control -f- DPN
4
13.7 ± 0.9
-33.0
- 7.7
20 min. UV
4
6.7 ± 1.5
35.0
16.4
20 min. UV + DPN
4
7.3 ± 0.8
29.1
5.7
20 min. UV + DPN using DPN
46.7
11.7
as control
Control
3
21.2 ± 0.7
—
—
Control + FAD
3
20.2 ± 1.0
4.7
1.5
20 min. UV
3
16.2 ± 1.4
23.6
-10.4
20 min. UV + FAD
3
14.2 ± 1.0
33.0
-19.0
Control
5
15.7 ± 1.2
—
—
20 min. visible
5
15.1 ± 1.9
3.8
2.1
20 min. UV
5
11.1 ± 2.2
29.3
1.8
20 min. UV + 20 min. visible
5
10.1 ± 2.5
35.7
2.0
* Particulates undiluted; treated in a rotating quartz flask. Ultraviolet filtered by Filter I ;
visible light filtered to remove ultraviolet and infrared. DPH (0.1 ml. of 0.01 M) and FAD
(0.1 ml. of 10~4 M) added after exposure.
502
H. T. YOST, JR. AND H. H. ROBSON
DPN-treated series is used as a control for a series having both ultraviolet and
DPN, the DPN-treated suspensions show greater inactivation than untreated.
FAD has a more pronounced inhibitory effect. This may well result from the
competition between the normal electron transport pathway in the mitochondrion
and the FAD for excess electrons, when the system is partially damaged. Similarly,
the system was not reactivated by light under our conditions. Treatment with
visible light increased the damage in most cases even when filtered to remove far
red and ultraviolet components. No attempt was made to use a narrow region of
the visible near 3600 A in these studies ; however, the treatment used is similar to
that used to reactivate bacteria successfully. Addition of cytochrome-c to the
suspension did not result in reactivation by visible light.
Table III presents the data obtained in studies of the effect of infrared radiation
on phosphorylation. It is important to note that this process is temperature-sensitive
and that any rise in temperature during the infrared treatment results in inactivation
TABLE III
The effect of infrared radiation alone and in combination
Treatment*
No. runs
Phosphate uptake
(MM)
%
Decrease
07
/o
Decrease Oz
Control
3
13.8 ± 1.7
—
—
3 hrs. IR
3
14.0 ± 2.1
- 1.4
-2.2
20 min. UV
5
10.5 ± 1.5
23.9
4.2
3 hrs. IR + 20 min. UV
5
10.3 ± 2.0
25.4
5.1
Control
3
12.8 ± 1.5
—
—
20 min. UV
3
8.8 ± 1.0
31.2
6.7
20 min. UV + 3 hrs. IR
3
8.8 ± 1.0
31.2
5.9
Control
7
9.9 ± 2.5
—
—
2 X 104 r 7
10
5.4 ± 0.8
45.5
1.5
2 X 104 r 7 + 3 hrs. IR
10
5.2 ± 0.8
47.5
-1.4
* Particulates undiluted for ultraviolet and infrared treatment; ultraviolet exposure in a
rotating quartz flask; infrared exposure in a rotating 5-ml. test tube. Particulates diluted 1 in
20 for 7 ray exposure.
of the phosphorylation mechanism. For this reason, the data reported here are
those from studies in which the temperature was carefully controlled and kept below
10° C. Under these conditions, there is no effect of infrared treatment either alone
or in combination with ultraviolet or gamma radiation. This statement must have
one reservation, however; the variability between runs was rather high in these
experiments as a result of the extended time of treatment and the dilution necessary
for gamma radiation. As a result the averages may not present a completely
accurate picture. In the case of pretreatment with infrared and treatment with
ultraviolet radiation, three runs showed some increased inactivation and two
showed decreased inactivation. There is a suggestion in these data that the
infrared may have a slight effect on the phosphorylating mechanism, but nothing
of the order of magnitude of its other known effects.
In Table IV are presented the data from studies of the effect of combined
ultraviolet and gamma radiation. The figures in this table need explanation. The
wide discrepancy between the two types of experiment with regard to the inactiva-
RADIATION OF CELLULAR PARTICULATES
503
tion by ultraviolet alone is the result of the fact that to treat the mitochondria with
gamma radiation the mitochondria must be diluted. Consequently in studies of
the effects of post-treatment with ultraviolet the mitochondria had been diluted,
centrifuged and re-suspended before treatment with ultraviolet. In this condition
they seem more sensitive. Because of the difficulties of doing studies of this kind,
these data can only be taken as suggestive. It would appear that the gamma radia-
tion sensitizes the phosphorylation mechanism to ultraviolet treatment, but not the
converse. However, unless these studies can be carried out in some manner which
is not so drastic to the mitochondria, it will be impossible to be sure of this point.
Attempts are being made to do this at present.
The oxidative phosphorylation of isolated particulates is sensitive to both aging
and dilution of the preparation. However, in undiluted preparations kept in the
cold (below 10° C.), the aging effects are slight for the first 4 or 5 hours. There-
fore, most experiments reported in this paper were conducted under conditions in
which the mitochondria were in fresh condition at the time of assay. Even the
delay between isolation and assay of three hours, experienced with preparations
TABLE IV
The effect of combined ultraviolet and gamma, radiation
Treatment*
No. runs
Phosphate uptake
(MM)
%
Decrease
%
Decrease Oz
Control
3
11.8 ± 1.4
—
—
5 min. UV
3
9.2 ± 1.5
22.0
3.4
104 r 7 rays
3
11.2 ± 1.0
5.0
- 5.6
5 min. UV + 104 r 7 rays
3
9.2 ± 0.03
22.0
5.1
Control
3
8.7 ± 2.1
—
—
5 min. UV
3
3.3 ± 0.1
62.1
26.9
104 r 7 rays
3
4.1 ± 0.9
52.9
22.4
104 r 7 rays + 5 min. UV
3
2.0 ± 1.2
77.0
28.2
* Particulates undiluted for ultraviolet treatment; diluted 1 in 20 for 7 ray treatment.
treated with infrared radiation, caused no appreciable decrease in phosphorylative
ability due to aging. On the other hand, when a preparation is diluted, the phos-
phorylative ability is damaged. Therefore, the experiments done with gamma
radiation show the superimposition of two effects : dilution and exposure to radia-
tion. The degree of inhibition resulting from dilution varies from preparation
to preparation. We are unable to explain this at the present time. This double
effect in the gamma-treated series may be obscuring an effect that the radiation
would have on undiluted preparations. The part of oxidative phosphorylation
which is sensitive to dilution may be highly sensitive to radiation. However, this
only means that the effects reported are the minimum effects of radiation. It should
be emphasized that the effect of which we speak is a decrease in the total phosphate
uptake and not a serious change in the P:O ratio. Some uncoupling occurs in
highly diluted preparations but none occurs in undiluted preparations or in diluted
preparations which are not allowed to stand in the diluted condition.
All the data presented in these tables represent the average values for the
experiments, with standard errors. The problem of the variation from run to
504 H. T. YOST, JR. AND H. H. ROBSON
run cannot be eliminated at this time. The mitochondria for each run are obtained
from a different rat. Phosphate uptake and sensitivity to radiation varies from
rat to rat. Attempts to standardize the procedures by using rats of uniform age
and of denned diet reduce this variation only slightly. Therefore, all of these
data must be regarded as indicating relationships in a manner that is qualitative
rather than quantitative.
DISCUSSION
The data presented in Table I show quite clearly that ultraviolet radiation in
the wave-length region of 2400-2700 A (probably 2600 A) readily inactivates the
oxidative phosphorylation mechanism of isolated cellular particulates. Further-
more, it is clear that the inactivation of phosphorylation is unrelated to inactivation
of the oxidase system, since oxygen uptake may be very slightly affected in some
cases in which phosphorylation is quite low. The separation of phosphorylation
from oxidation is a common observation in mitochondrial preparations. Aging of
the preparation will cause a loss of phosphorylative ability without appreciable
damage to the oxidative system. Ultraviolet radiation merely accelerates this
process. The observations with ultraviolet radiation are in complete agreement
with those using gamma radiation (Yost and Robson, 1957). It appears that any
one of a variety of agents will uncouple phosphorylation and oxidation.
The use of ultraviolet radiation to uncouple the phosphorylation mechanism
presents some interesting approaches to the general problem of radiation effects and
to the general problem of the sequence of events in oxidative phosphorylation.
On this last point, Beyer (1958) has recently suggested that reversal of the
effects of ultraviolet radiation may be used as a tool to indicate the normal pathway
of electron flow in this system. His observation that addition of vitamin K is
necessary to reactivate the glutamate system may be taken as evidence that this
vitamin participates in the over-all process. Similar experiments may be done to
elucidate further the mechanisms of phosphorylation. However, the observations
of Beyer that photoreactivation can be achieved after treatment with ultraviolet
radiation are not substantiated by the data presented in this paper. Under the
conditions employed in these studies, it has been impossible to reactivate phos-
phorylation with visible light or the addition of several co-factors. Although the
inactivation of oxidative phosphorylation by ultraviolet radiation may play a large
part in the death of cells exposed to radiations, and although it is a well known
fact that cells exposed to lethal doses of ultraviolet radiation can be revived by
subsequent exposure to visible radiation (Kelner, 1949), the results reported in
Table I indicate that the isolated particulate system cannot be reactivated by the
same means that the whole cells can. Whether this inability to reactivate the
phosphorylation mechanism is the result of a failure to use the right wave-lengths
(3600 A) exclusively, or is the result of failure to combine co-factor addition with
light treatment can only be determined in the future. Whether the phosphorylation
mechanism is inactivated in whole cells by ultraviolet radiation and then revived bv
light treatments will be the subject of a new series of investigations.
Since the report of inactivation and photoreactivation of DPN (Wells, 1956),
it has seemed possible that any interference with oxidative metabolism might be the
result of inactivation of the nucleotide-containing co-enzymes. These were tried
RADIATION OF CELLULAR PARTICULATES 505
in this system in attempts to reactivate phosphorylation although they have no
known function in the succinate system. That they failed to reactivate the system
is not surprising. It does indicate that the decrease in phosphorylative ability is
not the result of inactivation of some other (nucleotide-requiring) part of the system.
Since ATP is a normal part of the reaction mixture, it is safe to assume that the
effect is not upon the adenosine nucleotides. This agrees with the findings of
Beyer (1958).
The data in Tables III and IV represent an attempt to find a mechanism of
action for combined radiation effects. The effects of combined radiations on
chromosomes must have their explanation either in the alteration of the ability of
the breaks to rejoin or in the alteration of the fragility of the chromosome to
radiation. Unless a mechanism of action can be discovered, it is unlikely that any
satisfactory explanation will be forthcoming. The many experiments indicating
that the damage to chromosomes by radiation is greatly influenced by known
inhibitors of phosphorylation (Wolff and Luippold, 1955) suggests that studies
on the combined effects of radiations on phosphorylation might give some important
clues to radiation mechanisms. The data gathered in these experiments indicate
that there is no pronounced effect of combined treatments with ultraviolet, infrared
and gamma radiations. The potentiating effect of infrared, so clearly demonstrated
in Tradescantia (Swanson, 1949), cannot be found in the mitochondrial systems;
nor is there any indication of interactions (other than an additive effect of the
radiations) between gamma rays and ultraviolet. These findings are not in accord
with the report by Gordon and Surrey (1958) that near-infrared radiation acceler-
ates the decay of phosphorylative ability in rat liver mitochondria. The techniques
employed in the two studies are quite different, and the difference in results is
undoubtedly attributable to the greater sensitivity of the technique of Gordon and
Surrey. Indeed this may explain the variability of some of our results. It is
possible that a slight infrared effect is present in our test materials, but that our
test is too insensitive to give a consistent measure of its magnitude. However,
there can be little comparison of an effect on phosphorylation of this small degree
with the enormous effect of infrared radiation on chromosome breakage. It seems
highly unlikely that an increase in x-ray breakage of chromosomes amounting to
100 to 200 per cent can be induced by alterations of the phosphorylation mechanism
too slight to be detected by the hexokinase method employed in this study.
SUMMARY
Data are presented which show that ultraviolet radiation in the 2600 A region
inactivates oxidative phosphorylation. The addition of co-factors and the exposure
to light of the preparations failed to reactivate the phosphorylation after treatment
with ultraviolet. Treatment with infrared radiation and treatment with combined
infrared and ultraviolet, infrared and gamma, and ultraviolet and gamma radiation
failed to show any alteration in the normal inactivation patterns found with treatment
with ultraviolet or gamma radiation alone. These data suggest that both ionizing
and non-ionizing ultraviolet radiations damage cells, at least in part, by inactivation
of oxidative phosphorylation. However, these data do not give any clue to the
mechanism of action of combined radiations in altering the chromosomal structure
of cells.
506 H. T. YOST, JR. AND H. H. ROBSON
LITERATURE CITED
BEYER, R. E., 1958. Ultraviolet irradiation as a tool in studies of oxidative phosphorylation.
Radiation Research, 9 : 92.
GILES, N. H., JR., 1954. Radiation-induced chromosome aberrations in Tradescantia. In:
Radiation Biology, A. Hollaender, Ed., McGraw-Hill Book Co., New York. Vol. I
pt. 2, chap. 10.
CLICK, D., 1949. Techniques of Histo- and Cytochemistry. Interscience Publishers, Inc., New
York.
GORDON, S. A., AND K. SURREY, 1958. A biochemical basis for the far-red potentiation of
X-ray induced chromosomal breaks. Radiation Research, 9: 121.
KELNER, A., 1949. Effect of visible light on the recovery of Streptomyces griseus conidia from
ultraviolet radiation injury. Proc. Nat. Acad. ScL, 35 : 73-79.
KIHLMAN, B. A., T. MERZ AND C. P. SWANSON, 1957. Experimentally induced chromosome
aberrations in plants. II. The effect of cyanide and other heavy-metal complexing
agents on the production of chromosome aberrations by X-rays. /. Biophys. Biochem.
Cytol., 3: 381-390.
MALEY, G. F., AND H. A. LARDY, 1954. Phosphorylation coupled with the oxidation of re-
duced cytochrome-c. /. Biol. Chem., 210 : 903-909.
SCHNEIDER, W., 1948. Intracellular distribution of enzymes. III. The oxidation of octanoic
acid by rat liver fractions. /. Biol. Chem., 176: 259-266.
SWANSON, C. P., 1943. X-ray and ultraviolet studies on pollen tube chromosomes. I. The
effect of ultraviolet (2537 A) on X-ray induced chromosomal aberrations. Genetics,
29: 61-68.
SWANSON, C. P., 1949. Further studies on the effect of infrared radiation on X-ray induced
chromatid aberration in Tradescantia. Proc. Nat. Acad. Sci., 35: 237-244.
SWANSON, C. P., AND H. T. YOST. JR., 1951. The induction of activated, stable states in the
chromosomes of Tradescantia by infrared and X-rays. Proc. Nat. Acad. Sci., 37 :
796-802.
VAN BEKKUM, D. W., AND O. Vos, 1955. The effects of X-rays on the oxidative phosphoryla-
tion of mitochrondria in relation to nuclear damage. Brit. J. Exp. Pathol., 36 : 432-438.
WELLS, P. H., 1956. Photoreactivation of ultraviolet-inactivated disphosphopyridine nucleotide.
Science, 124: 31.
WOLFF, S., AND H. E. LUIPPOLD, 1955. Metabolism and chromosome-break rejoining. Science,
122: 231-232.
YOST, H. T., JR., H. H. ROBSON AND I. M. SPIEGLEMAN, 1956. Studies on the effects of ir-
radiation of cellular particulates. I. Inhibition of cytochrome oxidase by ultraviolet.
Biol. Bull., 110: 96-106.
YOST, H. T., JR., AND H. H. ROBSON, 1957. Studies on the effects of irradiation of cellular
particulates. II. The effect of gamma radiation on oxygen uptake and phosphorylation.
Biol. Bull., 113: 198-206.
INDEX
^CROPORA, skeleton formation in, 59.
Activity of bats, 484.
Adult silkworm, corpora allata of, 323.
Agglutination, fertilizin, inhibition of by
Arbacia dermal secretion, 472.
Aggregation of slime molds, 304.
Alga, marine, growth of in culture, 461.
Allatectomized Philosamia, egg development
in, 88.
ALLEN, M. J. Embryological development of
the polychaetous annelid, Diopatra, 339.
Allocentrotus, biology of, 362.
Almyracuma from Cape Cod, 115.
Amicula, shell repair in, 318.
Amino acids in chicken egg yolk, 420.
Amino acids in slime mold, 265.
Ammonium concentrations in Uca, 76.
Amphibian oviduct, histochemistry of, 446.
Amphibian red blood cell form, 397.
Amphibian water-drive, 429.
Amphidinium, energetics of motion of, 285.
Amphineurans, shell repair in, 318.
Analysis of nutrients in chick yolk, 420.
Analysis of slime mold amino acids, 265.
Anatomy of Callinectes larvae, 373.
Anatomy of dinoflagellates, 195.
Anatomy of frozen intertidal animals, 258.
Anatomy of lobster gynandromorph, 226.
Anatomy of new cumacean, 115.
Anatomy of Proctoeces, 184.
Annelid, development of, 339.
Antherea, endocrine activity of, 323.
Antherea, respiratory enzymes of, 136.
Antibiotics, use of in study of Tubularian
regeneration, 232.
Antigens in Arbacia dermal secretion, 472.
Antimycin A, effects of on silkworm respiration,
136.
Antisera against fertilizin, effects of on sea
urchin egg respiration, 217.
Arbacia dermal secretion, inhibition of fertilizin
by, 472.
Arthropod, photoreception in, 204.
Arthropod retina, reflecting pigment from, 125.
Astasia, periodicity of mitosis in, 162.
Australian bats, body temperatures of, 484.
Avian flight muscles, structure and physiology
of, 239.
Azide, effects of on silkworm respiration, 136.
gACTERIA, role of in growth of Dunaliella
in culture, 461.
Bacteria, role of in Tubularia regeneration, 232.
BALECH, E. Two new genera of dinoflagellates
from California, 195.
BARR, L. See J. W. GREEN, 76.
Bats, Australian, body temperatures of, 484.
BEETON, A. M. Photoreception in the opossum
shrimp Mysis, 204.
Behavior of molluscs at low temperatures, 258.
Beta-glucosidase of silkworm midgut, 95.
Bicolor lobster gynandromorph, 226.
Biology of Allocentrotus, 362.
Bird flight muscles, structure and physiology
of, 239.
Bisexual lobster, 226.
Blastoderm, chick, in vitro culture of, 420.
Blastoderm, chick, development of at low
temperature, 436.
Blockage of meiosis in salamander eggs, 446.
Blood cell, red, of amphibians, 397.
Blood ion exchanges in Pachygrapsus, 248.
Body temperatures of Australian bats, 484.
Bombyx midgut, beta-glucosidase of, 95.
BOOKHOUT, C. G. See J. D. COSTLOW, JR.,
373.
BOOLOOTIAN, R. A., A. C. GIESE, J. S. TUCKER
AND A. FARMANFARMAIAN. A contribution
to the biology of a deep sea echinoid,
Allocentrotus, 362.
BOOLOOTIAN, R. See A. C. GIESE, 49.
Brachyuran crab, osmotic regulation in, 37.
Brackish water cumacean, 115.
Brain formation in chick embryo, 436.
BROOKBANK, J. W. The respiration of un-
fertilized sea urchin eggs in the presence of
antisera against fertilizin, 217.
BURBANCK, W. D. See N. S. JONES, 115.
QALCIUM, possible role of in UV-irradiated
sea urchin eggs, 294.
Calcium concentrations in Uca, 76.
Calcium deposition in coral skeletons, 59.
California dinoflagellates, new genera of, 195.
Callinectes, larval development of, 373.
Cape Cod cumacean, 115.
Carbohydrate metabolism of chick embryo,
420.
Carbohydrate metabolism of crustacean, 175.
507
508
INDEX
Carbohydrate metabolism of slime mold, 265.
Carbon dioxide, role of in skeleton formation of
corals, 59.
Carbon monoxide-insensitive respiration of
silkworm pupae, 136.
Carbonic anhydrase, effect of on coral skeleton
formation, 59.
Carcinides, hormonal control of metabolism in,
175.
Carp pituitary, prolactin in, 429.
Carteria, energetics of motion of, 285.
Cecropia silkworm, corpora allata of, 323.
Cell, initiator, role of in slime mold aggregation,
304.
Cell division, periodicity of, in euglenids, 162.
Cell division in UV-irradiated sea urchin eggs,
294.
Cell form of amphibian erythrocytes, 397.
Cells, flagellated, motility and power dissipa-
tion in, 285.
Cellular particulates, irradiation of, 498.
Centrifuged and UV-irradiated sea urchin eggs,
294.
Cercaria, new species of, 184.
CHACE, F. A., JR., AND G. M. MOORE. A bi-
colored gynandromorph of the lobster,
Homarus, 226.
CHANG, J. J., AND F. H. JOHNSON. The in-
fluence of pressure, temperature and
urethane on the luminescent flash of
Mnemiopsis, 1.
Characteristics of low temperature chick
blastoderms, 436.
Chemical control of feeding in Physalia, 452.
Chick blastoderms, development of at low
temperatures, 436.
Chick embryo somite formation, 420.
Chicken egg yolk, nutrients in, 420.
Chiroptera, body temperatures of, 484.
Chitons, shell repair in, 318.
Chlamydomonas, motility and power dissipa-
tion in, 285.
Chloride concentrations in Uca, 76.
Chloride ion, role of in histophysiology of crab
gill and kidney, 37.
Chlorophyll a synthesis of Dunaliella, 461.
Chromatographic analysis of slime mold, 265.
Chromatography of chicken egg yolk, 420.
Chromatography of crustacean retinas, 125.
Cladocora, skeleton formation in, 59.
Cleavage of Diopatra, 339.
Cleavage of UV-irradiated sea urchin eggs, 294.
Cnidaria, evolution of, 452.
Cobalt-60 irradiation of cellular particulates,
498.
Cockroach, control of reproduction in, 406.
Coelenterate, chemical control of feeding in,
452.
Coelenterate, inhibition of regeneration in, 232.
Colacium, periodicity of mitosis in, 162.
Cold, effect of on amphibian erythrocyte form,
397.
Cold, effect of on Mnemiopsis flash, 1.
Cold, effects of on intertidal animals, 25
Cold, response of bats to, 484.
Color variant of lobster, 226.
Comb jelly, luminescence of, 1.
Combined radiation of cellular particulates,
498.
Control of feeding in Physalia, 452.
Control of metabolism in crustaceans, 175.
Control of reproduction in Diploptera, 406.
Cooling, effect of on development of chick
blastoderm, 436.
Copepods, hatching of eggs of, 15.
Coral skeleton formation, 59.
Corpora allata, role of in reproduction of
Diploptera, 406.
Corpora allata of Cecropia silkworm, 323.
Corpus allatum of Philosamia, 88.
COSTLOW, J. D., JR., AND C. G. BOOKHOUT.
The larval development of Callinectes
reared in the laboratory, 373.
Cryptochiton, shell repair in, 318.
Cumacean, new genus and species of, 115.
Crab, hormonal control of metabolism in, 175.
Crab, larval development of, 373.
Crab, osmotic stress in, 248.
Crab gill and kidney, histophysiology of, 37.
Crabs, fiddler, regulation of salt and water by,
76.
Crassostrea, frozen, histology and metabolism
of, 258.
Crustacea, osmotic regulation in, 272.
Crustacea, reflecting pigment from retina of,
125.
Crustacean, histophysiology of gill and kidney
of, 37.
Crustacean, hormones of, 30.
Crustacean, larval development of, 373.
Crustacean, new genus and species of, 115.
Crustacean, osmotic stress in, 248
Crustacean, regulation of water and salt by, 76.
Crustacean gynandromorph, 226.
Crustaceans, hormonal control of metabolism
in, 175.
Cryptoglena, periodicity of mitosis in, 162.
Ctenophore, luminescence of, 1.
Culture of Chlamydomonas, 285.
Cultured Dunaliella, growth of, 461.
Cycle, reproductive, of sea urchin, 49.
Cyclic sexual activity of Diploptera, 406.
Cyclops, hatching of eggs of, 15.
Cynthia, endocrine activity of, 323.
Cyprinus pituitary, prolactin in, 429.
INDEX
509
Cysteine, effects of on feeding reaction of
Physalia, 452.
Cytochrome oxidase, role of in silkworm pupa
respiration, 136.
3, effects of on silkworm respiration, 136.
DPN, effects of on dephosphorylation of rat
liver cell particulates, 498.
Dark adaptation in Mysis, 204.
Dark-adapting hormones of Palaemonetes, 30.
Darkness, effect of on mitotic periodicity of
euglenids, 162.
DAVIS, C. C. Osmotic hatching in the eggs of
some fresh-water copepods, 15.
DAVISON, J. Studies on the form of the am-
phibian red blood cell, 397.
Decapod, osmotic stress in, 248.
Decapod Crustacea, osmotic regulation in, 76.
Decompression, effects of on Mnemiopsis
luminescent flash, 1.
Deep sea echinoid, biology of, 362.
Deposition of calcium in coral skeleton, 59.
Dermal secretion of Arbacia, inhibition of
fertilizin by, 472.
Description of new species of dinoflagellates
195.
Desiccation of Pachygrapsus, 248.
Developing slime mold, amino acids in, 265.
Development of Callinectes, 373.
Development of chick somites, 420.
Development of Diopatra, 339.
Development of silkworm, role of corpora allata
in, 323.
Development of slime mold aggregations, 304.
Development of UV-irradiated sea urchin eggs
294.
Developmental characteristics of low temper-
ature chick blastoderms, 436.
Diamox, effects of on coral skeleton formation
59.
Diapausing silkworm pupae, respiratory en-
zymes of, 136.
Diapausing silkworm pupae, role of corpus
allatum in, 88.
Diapausing silkworms, endocrine activity of
323.
Diaptomus, hatching of eggs of, 15.
Dictyostelium, aggregation of, 304.
Dictyostelium, amino acids in, 265.
Digenetic trematode, life-cycle of, 184.
Dinitrophenol, effects of on silkworm respira-
tion, 136.
Dinoflagellates, new genera of , 195.
Diopatra, development of, 339.
Distal retinal pigment migration of Palae-
monetes, 30.
Distigma, periodicity of mitosis in, 162.
Distribution of fibers in pigeon flight muscle,
239.
Division, cell, in UV-irradiated sea urchin eggs,
Dugesia regeneration, effects of thiourea on
106.
Dunaliella, energetics of motion of, 285.
Dunaliella, growth of in culture, 461.
gCDYSONE, role of in endocrine activity of
silkworm, 323.
Echinoderm, deep sea, biology of, 362
Echinoderm, productivity of, 49.
Echinoderm dermal secretion, inhibition of
fertilizin by, 472.
Echinoderm eggs, respiration of, 217.
Echinoderm eggs, ultraviolet irradiation of
294.
Echinoid, deep sea, biology of, 362.
Ecology of Allocentrotus, 362.
Ecology of chitons, 318.
Ecology of cumacean, 115.
Effect of osmotic stress on Pachygrapsus, 248.
Effects of irradiation of cellular particulates
498.
Effects of thiourea on planarians, 106.
Eft water-drive factor in teleost pituitaries, 429.
Egg maturation in Diploptera, 406.
Eggs, copepod, osmotic hatching of, 15.
Eggs, sea urchin, respiration of, 217.
Eggs, sea urchin, ultraviolet irradiation of, 294.
Electrical measurements of Mnemiopsis lumi-
nescence, 1.
Embryo, chick, somite formation in, 420.
Embryological development of Diopatra, 339.
Embryology of UV-irradiated sea urchin eggs
294.
Embryos, chick, development of at low temper-
ature, 436.
Endocrine activity of Diploptera, 406.
Endocrine activity of silkworm, 323.
Endocrine relations in Philosamia, 88.
Endocrine studies on crab, 175.
Endocrinology of amphibians, 429.
Endocrinology of crustaceans, 30.
Energetics of cell motion, 285.
ENGELMANN, F. The control of reproduction
in Diploptera, 406.
ENNIS, H. M. See M. SUSSMAN, 304.
Enzyme studies on silkworm midgut, 95.
Enzymes, respiratory, of diapausing silkworm
pupae, 136.
Eri-silkworm, corpus allatum of, 88.
Erythrocytes of amphibians, 397.
Euglenineae, periodicity of mitosis in, 162.
Eutreptia, periodicity of mitosis in, 162.
Evolution of Cnidaria, 452.
Exchanges, ionic, in Pachygrapsus, 248.
510
INDEX
Excretion of crab, 37.
Exogenous rhythm of mitotic periodicity in
euglenids, 162.
Explantation of chick embryos, 420, 436.
Eye formation in regenerating planarian, 106.
Eyes of Crustacea, reflecting pigment from, 125.
Eyestalk removal, effect of on metabolism of
crab, 175.
P ARMANFARMAIAN, A. See A. C. GIESE,
49 ; R. A. BOOLOOTIAN, 362.
Fat content of pigeon flight muscle, 239.
Feeding, chemical control of, in Physalia, 452.
Fertilization of Diopatra eggs, 339.
Fertilization reactions in UV-irradiated sea
urchin eggs, 294.
Fertilizin antisera, effects of on sea urchin egg
respiration, 217.
Fertilizin inhibition by Arbacia dermal secre-
tion, 472.
Fiber architecture of pigeon muscle, 239.
Fiddler crab, regulation of salt and water by, 76.
FlNGERMAN, M., M. E. LOWE AND B. I.
SUNDARARAJ. Dark-adapting and light-
adapting hormones controlling the distal
retinal pigment migration of the prawn,
Palaemonetes, 30.
Fish pituitaries, prolactin in, 429.
Flagellate, growth of in culture, 461.
Flagellated cells, motility and power dissipation
in, 285.
Flash, luminescent, of Mnemiopsis, 1.
FLEMISTER, S. C. Histophysiology of gill and
kidney of crab Ocypode, 37.
Flight muscles of birds, 239.
Fluorescent compounds in arthropod retinas,
125.
Food, role of in growth of Callinectes larvae,
373.
Form of amphibian red blood cell, 397.
Formation of skeletons in corals, 59.
Fragilidium, a new species, 195.
FRASER, R. C. Somite genesis in the chick.
II., 420.
Fresh-water copepods, osmotic hatching of eggs
of, 15.
Frozen intertidal animals,- histology and
metabolism of, 258.
Fruit bat, body temperatures of, 484.
FULTON, C. Re-examination of an inhibitor
of regeneration in Tubularia, 232.
Fundulus, prolactin in pituitary of, 429.
pAMETES of Diopatra, 339.
Gamma radiation, effects of on cellular particu-
lates, 498.
GEORGE, J. C., AND R. M. NAIK. Studies on
the structure and physiology of the flight
muscles of birds, 4., 239.
GIESE, A. C., el al. Organic productivity in
the reproductive cycle of the purple sea
urchin, 49.
GIESE, A. C. See R. A. BOOLOOTIAN, 362;
J. S. TUCKER, 318.
Gill of crab, histophysiology of, 37.
Gill fluid ion concentrations in Uca, 76.
Glucose as energy source in chick somite genesis,
420.
Glucosidase of silkworm midgut, 95.
Glutamic acid, role of in development of slime
mold, 265.
Glutathione, feeding response to, by Physalia,
452.
Glycogen content of pigeon flight muscle, 239.
Gnorimosphaeroma, osmoregulation in, 272.
Goitrogenic compounds, effects of on regenera-
tion of planarian, 106.
Gonad development of Allocentrotus, 362.
Gonad index of sea urchin, 49, 362.
GOREAU, T. F. The physiology of skeleton
formation in corals, I., 59.
GRANT, W. C., AND G. E. PICKFORD. Presence
of the red eft water-drive factor prolactin
in the pituitaries of teleosts, 429.
GREEN, J. W., M. HARSCH, L. BARR AND C. L.
PROSSER. Regulation of water and salt
by fiddler crabs, 76.
Greenfield, L. See A. C. GIESE, 49.
GROSS, W. J. The effect of osmotic stress on
the ionic exchanges of a shore crab, 248.
Growth of Callinectes larvae, 373.
Growth of Dunaliella in culture, 461.
Growth-promoting factor in teleost pituitaries,
429.
Growth rates of coral colony parts, 59.
Growth of regenerating planarians, effects of
thiourea on, 106.
Guanine from lobster eye, 125.
Gynandromorph of lobster, 226.
I_J ABITAT of Allocentrotus, 362.
Habitat in relation to osmoregulation of
isopods, 272.
HARRISON, J. R. Developmental characteris-
tics of low temperature chick blastoderms.
I. ,436.
HARSCH, M. See J. W. GREEN, 76.
Hatching of copepod eggs, 15.
Healing of planarians, effects of thiourea on,
106.
Heat, effect of on Mnemiopsis luminescent
flash, 1.
Heat, response of bats to, 484.
Heat transfer in frozen intertidal animals, 258.
Hermaphroditism in lobster, 226.
Hibernating bats, body temperatures of, 484.
High temperature, response of bats to, 484.
INDEX
511
Histochemistry of arthropod retina, 125.
Histochemistry of newt oviduct, 446.
Histology of Diploptera endocrine organs, 406.
Histology of frozen intertidal animals, 258.
Histophysiology of crab gill and kidney, 37.
Homarus, reflecting pigment from retina of,
125.
Homarus gynandromorph, 226.
Hormonal control of metabolism in crustaceans,
175.
Hormonal control of reproduction in Di-
ploptera, 406.
Hormone, juvenile, of silkworms, 323.
Hormones of Palaemonetes, 30.
Hormones of Philosamia, 88.
Hormones of teleost pituitaries, 429.
Horseshoe crab, reflecting pigment from retina
of, 125.
HUANG, H. See A. C. GIESE, 49.
HUGHES, W. N. See A. A. HUMPHRIES, JR.,
446.
HUMPHRIES, A. A., JR., AND W. N. HUGHES.
A study of the polysaccharide histo-
chemistry of the oviduct of the newt,
Triturus, 446.
Hyaline layer reaction in UV-irradiated sea
urchin eggs, 294.
Hyalophacus, periodicity of mitosis in, 162.
Hyalophora, endocrine activity of, 323.
Hyalophora, respiratory enzymes of, 136.
Hydroid regeneration, 232.
Hydrozoan, chemical control of feeding in, 452.
Hypertonic medium, effect of on Pachygrapsus,
248.
Hypoblast, role of in development of low
temperature chick blastoderms, 436.
T CE formation in intertidal animals, 258.
ICHIKAWA, M., AND J. NlSHIITSUTSUJI-UwO.
Studies on the role of the corpus allatum
in the Eri silkworm, Philosamia, 88.
Immunology of sea urchin egg fertilization, 217.
Immunology of Arbacia fertilization, 472.
In vitro culture of chick blastoderms, 420, 436.
In vitro growth of Dunaliella, 461.
Incubation temperature, effect of on develop-
ment of chick embryos, 436.
"Induction" of aggregation centers in slime
molds, 304.
Infrared irradiation of cellular particulates, 498.
Inhibition of fertilizin by Arbacia dermal
secretion, 472.
Inhibition of regeneration in Tubularia, 232.
Initiator cell in slime mold aggregation, 304.
Injury respiration of silkworm, 136.
Insemination of Diopatra eggs, 339.
Intermolt period of crustaceans, hormonal
control of metabolism during, 175.
Intertidal animals, frozen, histology and
metabolism of, 258.
Ionic exchanges in Pachygrapsus, 248.
Ionic regulation in Uca, 76.
Ionizing radiation, effects of on cellular
particulates, 498.
Irradiation, ultraviolet, of sea urchin eggs, 294.
Irradiation of cellular particulates, 498.
Isopods, osmoregulation in, 272.
ITO, T., AND M. TANAKA. Beta glucosidase of
the midgut of the silkworm, Bombyx, 95.
J
ELLY of amphibian eggs, importance of, 446.
JENKINS, M. M. The effects of thiourea and
some related compounds on regeneration
in planarians, 106.
JOHNSON, F. H. See J. J. CHANG, 1.
JONES, N. S., AND W. D. BURBANCK. Al-
myracuma from brackish water of Cape
Cod, 115.
Juvenile hormone of silkworms, 323.
J^ANWISHER, J. Histology and metabo-
" lism of frozen intertidal animals, 258.
Katherina, shell repair in, 318.
Kidney of crab, histophysiology of, 37.
Killifish, prolactin in pituitary of, 429.
KLEINHOLZ, L. H. Purines and pteridines from
the reflecting pigment of the arthropod
retina, 125.
Krebs cycle in slime mold, 265.
KRIVANEK, J. O., AND R. C. KRIVANEK.
Chromatographic analyses of amino acids
in the developing slime mold, Dictyo-
stelium, 265.
KURLAND, C. G., AND H. A. SCHNEIDERMAN.
The respiratory enzymes of diapausing
silkworm pupae: a new interpretation of
CO-insensitive respiration, 136.
T ABORATORY-RAISED Callinectes larvae,
-^ 373.
Lactogenic hormone in fish pituitaries, 429.
Larvae of Diopatra, 339.
Larval development of Callinectes, 373.
Larval stages of Bombyx, beta-glucosidase
activity of midgut in, 95.
LASKER, R. See A. C. GIESE, 49.
LEEDALE, G. F. Periodicity of mitosis and
cell division in the Euglenineae, 162.
LENHOFF, H. M., AND H. A. SCHNEIDERMAN.
The control of feeding in the Portuguese
man-of-war, Physalia, and its bearing on
the evolution of the Cnidaria, 452.
Lepidoptera, endocrine activity of, 323.
Lepocinclis, periodicity of mitosis in, 162.
Life-cycle of Protoeces, 184.
512
INDEX
Light, effect of on coral skeleton formation, 59.
Light, effect of on mitotic periodicity of
euglenids, 162.
Light-adapting hormone of Palaemonetes, 30.
Light emission by Mnemiopsis, 1.
Light reactions of Mysis, 204.
Limulus, reflecting pigment from retina of, 125.
Littorina, frozen, histology and metabolism of,
258.
Liver cell particulates, effects of radiation on,
498.
Lobster, reflecting pigment from retina of, 125.
Lobster gynandromorph, 226.
Locomotion of flagellated cells, 285.
Low temperature, effect of on amphibian
erythrocyte form, 397.
Low temperature, response of bats to, 484.
Low temperature chick blastoderms, 436.
Low temperatures, effects of on intertidal
animals, 258.
LOWE, M. E. See M. FINGERMAN, 30.
Luminescence of Mnemiopsis, 1.
Lytechinus eggs, respiration of, 217.
\/( ADREPORARIAN corals, reef -building in,
1 59.
Magnesium concentration in Uca, 76.
Madracis, skeleton formation in, 59.
Mammals, Australian, body temperatures of,
484.
Manicina, skeleton formation in, 59.
Marine alga, growth of in culture, 461.
Marine fish, prolactin in pituitaries of, 429.
Mating, influence of on egg maturation in
Diploptera, 406.
Maturation of eggs in Diploptera, 406.
McLACHLAN, J., AND C. S. YENTSCH. Obser-
vations on the growth of Dunaliella in
culture, 461.
Measurement of skeleton formation rate in
corals, 59.
Megalops of Callinectes, 373.
Meiotic blockage in salamander eggs, 446.
Membrane elevation in UV-irradiated sea
urchin eggs, 294.
Membranes of copepod eggs, 15.
Menoideum, periodicity of mitosis in, 162.
Mesocyclops, hatching of eggs of, 15.
Metabolism of Arbacia sperm, 472.
Metabolism of bats, 484.
Metabolism of Chlamydomonas, 285.
Metabolism of frozen intertidal animals, 258.
Metabolism of rat liver particulates, 498.
Metabolism of sea urchin eggs, 217.
Metabolism of silkworm pupae, 136.
Metabolite load of pigeon flight muscle, 239.
Metabolites, bacterial, role of in Tubularia
regeneration inhibition, 232.
Metachromasia in salamander oviduct, 446.
Metamorphosis of Callinectes, 373.
Metamorphosis of silkworm, 323.
Method for measuring coral skeleton formation,.
59.
METZ, C. B. Inhibition of fertilizin agglutina-
tion of sperm by the dermal secretion from
Arbacia, 472.
Microscopic anatomy of arthropod retina, 125.
Microscopic anatomy of crab gill and kidney,
37.
Migration of slime mold cells, 304.
Midgut of silkworm, beta-glucosidase of, 95.
Miniopterus, body temperatures of, 484.
Mitochondria, effects of radiation on, 498.
Mitotic abnormalities in UV-irradiated sea
urchin eggs, 294.
Mitotic periodicity in euglenids, 162.
Mnemiopsis, luminescent flash of, 1.
Model of amphibian erythrocyte, 397.
Modiolus, frozen, histology and metabolism of,
258.
Mold, slime, aggregation of, 304.
Mold, slime, amino acids in, 265.
Molluscs, frozen, histology and metabolism of,
258.
Molluscs, shell repair in, 318.
Molt-inducing hormone of Philosamia, 88.
Molting hormone in teleost pituitaries, 429.
Molting in relation to metabolism of crus-
taceans, 175.
Montipora, skeleton formation in, 59.
MOORE, G. M. See F. A. CHACE, JR., 226.
Mopalia, shell repair in, 318.
Morphogenesis of chick embryo at low temper-
ature, 436.
Morphogenesis of chick somites, 420.
Morphogenesis of UV-irradiated sea urchin
eggs, 294.
Morphology of Callinectes larvae, 373.
Morphology of dinoflagellates, 195.
Morphology of lobster gynandromorph, 226.
Morphology of new cumacean, 115.
Morphology of Proctoeces, 184.
Morphology of slime mold initiator cells, 394.
MORRISON, P. R. Body temperatures in some
Australian mammals. I., 484.
Motility of Arbacia sperm, effect of dermal
secretion on, 472.
Motility in flagellated cells, 285.
Motion of cells, energetics of, 285.
Muscles, flight, of birds, 239.
Mutant of Chlamydomonas, 285.
Mysis, photoreception in, 204.
Mytilus, frozen, histology and metabolism of,
258.
Myxamoebae, aggregation of, 304.
Myxamoebae, amino acids in, 265.
INDEX
513
X^AIK, R. M. See J. C. GEORGE, 239.
Nauplii of copepods, hatching of, 15.
Neurosecretory activity of Diploptera, 406.
Neurosecretory hormone in Philosamia, 88.
New genera of dinoflagellates, 195.
New genus and species of cumacean, 115.
Newt oviduct, histochemistry of, 446.
NiSHiiTsuxsuji-Uwo, J. See M. ICHIKAWA,
88.
Nitrate nitrogen and growth of Dunaliella, 461.
Nutrients in chicken egg yolk, 420.
Nutrition, role of in growth of Callinectes
larvae, 373.
Nutrition of Allocentrotus, 362.
Nutrition of Physalia, 452.
Nutrition of sea urchin, 49.
QBSERVATIONS on growth of Dunaliella,
461.
Oculina, skeleton formation in, 59.
Ocypode, histophysiology of gill and kidney of,
37.
Onuphid, development of, 339.
Opossum shrimp, photoreception in, 204.
Organic productivity of sea urchin, 49.
Osmotic hatching of copepod eggs, 15.
Osmotic relations of crab gill and kidney, 37.
Osmotic relations in Uca, 76.
Osmotic stress, effects of on Pachygrapsus, 248.
Ova, copepod, osmotic hatching of, 15.
Ova, maturation of in Diploptera, 406.
Ova, sea urchin, respiration of, 217.
Ova, sea urchin, ultraviolet irradiation of, 294.
Oviduct of newt, histochemistry of, 446.
Oxygen consumption of Arbacia sperm, 472.
Oxygen consumption of Chlamydomonas, 285.
Oxygen consumption of frozen animals, 258.
Oxygen consumption of rat liver cell particu-
lates, 498.
Oxygen consumption of sea urchin eggs, 217.
Oxygen consumption of silkworm pupae, 136.
Oyster, frozen, histology and metabolism of,
258.
pACHYGRAPSUS, osmotic stress in, 248.
Palaemonetes, hormones of, 30.
Parasite of sea urchin, 362.
Parasitic trematode, life-cycle of, 184.
Particulates, cell, irradiation of, 498.
Pattern of pigmentation in lobster gynandro-
morph, 226.
Pectoralis muscle of pigeon, structure and
physiology of, 239.
Peranema, periodicity of mitosis in, 162.
Periodicity of mitosis in euglenids, 162.
Permeability of copepod egg membranes, 15.
Phacus, periodicity of mitosis in, 162.
Phenylthiourea, effects of on planarian re-
generation, 106.
Philosamia, corpus allatum of, 88.
Philosamia, endocrine activity of, 323.
Phosphorylated intermediaries in chick carbo-
hydrate metabolism, 420.
Phosphorylation, effects of combined radiation
treatments on, 498.
Photoreception in Mysis, 204.
Phylogenetic aspects of Physalia feeding habits,
452.
Physalia, chemical control of feeding in, 452.
Physiology of bird flight muscles, 239.
Physiology of skeleton formation in corals, 59.
Phytoplankton, growth of in culture, 461.
PICKFORD, G. E. See W. C. GRANT, JR., 429.
Pigeon flight muscle, structure and physiology
of, 239.
Pigment, reflecting, of arthropod retina, 125.
Pigment, retinal, of Palaemonetes, 30.
Pigment variation in lobster, 226.
Pigmentation in regenerating planarians, effects
of thiourea on, 106.
Pituitaries of teleosts, prolactin in, 429.
Planarian regeneration, effects of thiourea on,
106.
Plankton samples, new dinoflagellates from,
195.
Platysamia, endocrine activity of, 323.
Platysamia, respiratory enzymes of, 136.
Pleurodeles, erythrocytes of, 397.
Pocillopora, skeleton formation in, 59.
Pollachius pituitary, prolactin in, 429.
Pollack pituitary, prolactin in, 329.
Polychaete, development of, 339.
Polyphemus, endocrine activity of, 323.
Polyphemus, respiratory enzymes of, 136.
Polysaccharide histochemistry of newt oviduct,
446.
Population size in relation to slime mold
aggregation, 304.
Porites, skeleton formation in, 59.
Porolithon, skeleton formation in, 59.
Portuguese man-of-war, chemical control of
feeding in, 452.
Potassium concentrations in Uca, 76.
Power dissipation in flagellated cells, 285.
Prawn, hormones of, 30.
Premolt period of crustaceans, hormonal
control of metabolism during, 175.
Pressure, effect of on Mnemiopsis luminescent
flash, 1.
Proctoeces, life-cycle of, 184.
Productivity of sea urchin, 49.
Prolactin in teleost pituitaries, 429.
PROSSER, C. L. See J. W. GREEN, 76.
Protein metabolism of slime molds, 265.
Prothoracic gland hormone of silkworm, 323.
514
INDEX
Protista, new genera of, 195.
Pteridines from arthropod retina, 125.
Pteropus, body temperatures of, 484.
Pupae, diapausing silkworm, respiratory en-
zymes of, 136.
Pupae of Philosamia, corpus allatum of, 88.
Purines from arthropod retina, 125.
Purple sea urchin, productivity of, 49.
DADIATION effects on sea urchin eggs, 294.
Radiation treatments of cellular particulates,
498.
Radiocalcium uptake by coral colonies, 59.
Radiosodium studies with Uca, 76.
Rate of calcium deposition in coral skeleton, 59.
Rat cellular particulates, effects of radiation on,
498.
Reaction times of mysids, 204.
Reconstitution in planarians, 106.
Red blood cell form, 397.
Red eft water-drive factor in teleost pituitaries,
429.
Reef corals, rate of formation of, 59.
Re-fertilization of fertilized UV-irradiated sea
urchin eggs, 294.
Reflecting pigment of arthropod retina, 125.
Regeneration inhibition in Tubularia, 232.
Regeneration in planarians, effects of thiourea
on, 106.
Regulation of ions in Pachygrapsus, 248.
Regulation of water and salt by Uca, 76.
Renal structure in Ocypode, 37.
Repair of shells in chitons, 318.
Reproduction, control of in Diploptera, 406.
Reproduction of Allocentrotus, 362.
Reproductive cycle of sea urchin, 49.
Respiration of Arbacia sperm, 472.
Respiration of Chlamydomonas, 285.
Respiration of frozen intertidal animals, 258.
Respiration of rat liver cell particulates, 498.
Respiration of sea urchin eggs, 217.
Respiratory enzymes of diapausing silkworm
pupae, 136.
Retina of arthropods, reflecting pigment from,
125.
Retinal pigment migration of Palaemonetes, 30.
Rhabdocoel parasite of sea urchin, 362.
Rhythm of mitotic activity in euglenids, 162.
RIEGEL, J. A. Some aspects of osmoregulation
in two species of sphaeromid isopod
Crustacea, 272.
ROBSON, H. H. See H. T. YOST, JR., 498.
Role of corpus allatum in Philosamia, 88.
Role of initiator cell in slime mold aggregation,
304.
RONKIN, R. R. Motility and power dissipation
in flagellated cells, especially Chlamy-
domonas, 285.
RUSTAD, R. C. Consequences of unilateral
ultraviolet irradiation of sea urchin eggs
294.
QALAMANDER oviduct, histochemistry of,
D 446.
Salinity, role of in growth of Callinectes larvae,
373.
Salinity relations of frozen intertidal animals,
258.
Salt balance in isopods, 272.
Salt content of crab gill and kidney, 37.
Salt pool in Pachygrapsus, 248.
Salt regulation by Uca, 76.
Samia, respiratory enzymes of, 136.
Saturniids, endocrine activity of, 323.
SCHEER, B. T. The hormonal control of
metabolism in crustaceans. IX., 175.
SCHNEIDERMAN, H. A. See C. G. KURLAND,
136; H. M. LENHOFF, 452.
Scrippsiella, a new genus, 195.
Sea urchin, deep sea, biology of, 263.
Sea urchin, productivity of, 49.
Sea urchin dermal secretion, inhibition of
fertilizin by, 472.
Sea urchin eggs, respiration of, 217.
Sea urchin eggs, ultraviolet irradiation of, 294.
Serum ion concentrations in Uca, 76.
Setae of Diopatra, development of, 339.
Setation of Callinectes larvae, 373.
Sexual activities of Diploptera, 406.
Sexual anatomy of lobster gynandromorph, 226.
Sexual maturation of silkworm, role of corpora
allata in, 323.
Shell repair in chitons, 318.
Shore crab, osmotic stress in, 248.
Shrimp, opossum, photoreception in, 204.
Silkworm, corpus allatum of, 88.
Silkworm, endocrine activity of, 323.
Silkworm midgut, beta-glucosidase of, 95.
Silkworm pupae, diapausing, respiratory en-
zymes of, 136.
Sinus gland hormones of Palaemonetes, 30.
Siphonophora, chemical control of feeding in,
452.
Size and power dissipation of flagellated cells,
285.
Skeleton formation in corals, 59.
Slime mold, amino acids in, 265.
Slime mold aggregation, 304.
Snail, frozen, histology and metabolism of, 258.
Sodium-24 levels in Uca, 76.
Somite genesis in the chick, 420.
Spawning, relation of to chemical composition
of sea urchin gonads, 49.
Spawning of Allocentrotus, 362.
Spawning of fish in relation to prolactin
content of pituitary, 429.
INDEX
515
Spectral sensitivity of Mysis, 204.
Sperm fertilizin agglutination inhibition by
Arbacia dermal secretion, 472.
Sphaeroma, osmoregulation in, 272.
Stomach fluid ion concentrations in Uca, 76.
Stress, osmotic, effects of on Pachygrapsus, 248.
Strongylocentrotus, productivity of, 49.
Strongylocentrotus eggs, respiration of, 217.
Strongylocentrotus eggs, ultraviolet irradiation
of, 294.
Structure of bird flight muscles, 239.
Studies of amphibian red blood cell, 397.
STUNKARD, H. W., AND J. R. UZMANN. The
life-cycle of the digenetic trematode
Proctoeces and description of new cercaria,
184.
Sucrose, effects of on hatching of copepod eggs,
15.
Sulfadiazine, use of in study of Tubularia
regeneration, 232.
Sulfate concentrations in Uca, 76.
SUNDARARAJ, B. I. See M. FlNGERMAN, 30.
Suppression of fertilization membrane elevation
in UV-irradiated sea urchin eggs, 294.
SUSSMAN, M., AND H. L. ENNis. The role of
the initiator cell in slime mold aggregation,
304.
Syndesmis as a parasite of Allocentrotus, 362.
'pACTIC responses in Mysis, 204.
TANAKA, M. See T. Ixo, 95.
Taxonomy of cumaceans, 115.
Taxonomy of flagellates, 195.
Taxonomy of Diopatra, 339.
Taxonomy of flatworms, 184.
Telea, endocrine activity of, 323.
Telea, respiratory enzymes of, 136.
Teleost, prolactin in pituitaries of, 429.
Temperature, effect of on amphibian erythro-
cyte form, 397.
Temperature, effect of on Mnemiopsis lumi-
nescent flash, 1.
Temperature, effect of on radiation damage in
sea urchin eggs, 294.
Temperature, role of in growth of Callinectes
larvae, 373.
Temperature of incubation, effect of on
development of chick blastoderms, 436.
Temperature relations of bats, 484.
Temperature-sensitivity of rat liver cell particu-
late phosphorylative metabolism, 498.
Temperatures, body, of Australian bats, 484.
Thawing of frozen intertidal animals, 258.
Thermoregulatory behavior of bats, 484.
Thiourea, effects of on planarians, 106.
Thiouracil, effects of on planarians, 106.
Tonicity relations of isopods, 272.
Trachelomonas, periodicity of mitosis in, 162.
Transitional period of crustaceans, hormonal
control of metabolism in, 174.
Trematode, life-cycle of, 184.
Triturus, erythrocytes of, 397.
Triturus, histochemistry of oviduct of, 446.
Tubularia, inhibition of regeneration in, 232.
TUCKER, J. S., AND A. C. GIESE. Shell repair
in chitons, 318.
TUCKER, J. S. See R. A. BOOLOOTIAN, 362.
TTCA, salt and water regulation in, 76.
Ultraviolet irradiation of sea urchin eggs, 294.
Ultraviolet irradiation, effects of on cellular
particulates, 498.
Unfertilized sea urchin eggs, respiration of, 217.
Unilateral ultraviolet irradiation of sea urchin
eggs, 294.
Urethane, effect of on luminescent flash of
Mnemiopsis, 1.
Uric acid in crustacean retinas, 125.
Urine ion concentrations in Uca, 76.
Urine ion exchanges in Pachygrapsus, 248.
UZMANN, J. R. See H. W. STUNKARD, 184.
y ASCULAR cells of amphibians, form of, 397.
Visual reactions of mysids, 204.
Viviparous cockroach, control of reproduction
in, 406.
VyATER balance in isopods, 272.
Water-drive factor in teleost pituitaries, 429.
Water regulation by Uca, 76.
Water relations of crab, 37.
Water relations in hatching of copepod eggs, 15.
WILLIAMS, C. M. The juvenile hormone. I.,
323.
yENTSCH, C. S. See J. MCLACHLAN, 461.
Yolk, chicken egg, nutrients in, 420.
YOST, H. T., JR., AND H. H. ROBSON. Studies
on the effects of irradiation of cellular
particulates. III., 498.
T'OEAE of Callinectes, 373.
Zooxanthellae, role of in coral skeleton forma-
tion, 59.
Volume 116
Number 1
THE
BIOLOGICAL BULLETIN
PUBLISHED BY
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CONTENTS
Page
CHANG, JOSEPH JIN, AND FRANK H. JOHNSON
The influence of pressure, temperature and urethane on the lumi-
nescent flash of Mnemiopsis leidyi 1
DAVIS, CHARLES C.
Osmotic hatching in the eggs of some fresh- water copepods 15
FINGERMAN, MILTON, MILDRED 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, R.
BOOLOOTIAN AND R. LASKERJ
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
measuring 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-UWO
Studies on the role of the corpus allatum in the Eri-silkworm, Philo-
samia cynthia ricini 88
ITO, TOSHIO, AND MOTOZO 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. BURBANCK
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 diapausing silkworm pupae : A new inter-
pretation of carbon monoxide-insensitive respiration 136
LEEDALE, GORDON F.
Periodicity of mitosis and cell division in the Euglenineae 162
SCHEER, BRADLEY T.
The hormonal control of metabolism in crustaceans. IX. Carbo-
hydrate metabolism in the transition from intermoult to premoult in
Carcinides maenas r 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
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