(navigation image)
Home American Libraries | Canadian Libraries | Universal Library | Community Texts | Project Gutenberg | Children's Library | Biodiversity Heritage Library | Additional Collections
Search: Advanced Search
Anonymous User (login or join us)
Upload
See other formats

Full text of "The Biological bulletin"

THE 



3/0 



BIOLOGICAL BULLETIN 



PUBLISHED BY 

THE MARINE BIOLOGICAL LABORATORY 



Editorial Board 

E. G. CONKLIN, Princeton University CARL R. MOORE, University of Chicago 

DONALD P. COSTELLO, University of North Carolina GEORGE T. MOORE, Missouri Botanical Garden 

E. N. HARVEY, Princeton University G. H. PARKER, Harvard University 

LEIGH HOADLEY, Harvard University A. C. REDFIELD, Harvard University 

L. IRVING, Swarthmore College F. SCHRADER, Columbia University 

M. H. JACOBS, University of Pennsylvania DOUGLAS WfflTAKER, Stanford University 

H. B. STEINBACH, University of Minnesota 
Managing Editor 



VOLUME 96 

FEBRUARY TO JUNE, 1949 



Printed and Issued by 

LANCASTER PRESS, Inc. 

PRINCE & LEMON STS. 

LANCASTER, PA. 



11 



THE BIOLOGICAL BULLETIN is issued six times a year at the 
Lancaster Press, Inc., Prince and Lemon Streets, Lancaster, Penn- 
sylvania. 

Subscriptions and similar matter should be addressed to The 
Biological Bulletin, Marine Biological Laboratory, Woods Hole, 
Massachusetts. Agent for Great Britain: Wheldon and Wesley, 
Limited, 2, 3 and 4 Arthur Street, New Oxford Street, London, 
W. C. 2. Single numbers, $1.75. Subscription per volume (three 
issues), $4. 50. 

Communications relative to manuscripts should be sent to the 
Managing Editor, Marine Biological Laboratory, Woods Hole, 
Massachusetts, between June 15 and September 1, and to the De- 
partment of Zoology, University of Minnesota, Minneapolis 14. 
Minnesota, during the remainder of the year. 



Entered as second-class matter May 17, 1930, at the post office at Lancaster, 
Pa., under the Act of August 24, 1912. 



LANCASTER PRESS, INC., LANCASTER, PA. 



CONTENTS 



No. 1. FEBRUARY, 1949 

PAGE 

BODINE, JOSEPH HALL AND LAURENCE ROCKWELL FITZGERALD 

Effect of urea, thiourea, phenylthiourea and thiouracil on the oxygen con- 
sumption of blocked and active embryonic cells 1 

WINGFIELD, R. TERRELL AND KARL M. WILBUR 

Tbe effects of electrolytes and sugars on tbe erytbrocytes of the turtle, 
Cbelydra serpentina 9 

REIN HARD, EDWARD G. 

Experiments on the determination and differentiation of sex in the bopyrid 
Stegophryxus hyptius Thompson 17 

ROGICK, MARY D. AND HANNAH CROASDALE 

Studies on marine bryozoa, III. Woods Hole region bryozoa associated 
with algae 32 

CHENEY, RALPH HOLT 

Stratification and deformation of Arbacia punctulata eggs centrifuged in 
caffeine solutions 70 

KRAUSS, MAX 

A mucin clot reaction with sea-urchin fertilizin 74 

Ris, HANS 

The anaphase movement of chromosomes in the spermatocytes of the 
grasshopper 90 

No. 2. APRIL, 1949 

BOREI, HANS AND SIGVAR LYBING 

Temperature coefficients of respiration in Psammechinus eggs 107 

BOREI, HANS 

Independence of post-fertilization respiration in the sea-urchin egg from the 
level of respiration before fertilization 117 

BOVEE, EUGENE CLEVELAND 

Studies on the thermal death of Hyalella azteca Saussure 123 

GATES, G. E. 

Regeneration in an earthworm, Eisenia foetida (Savigny) 1826. I. 

Anterior regeneration 129 

SZENT-GYORGYI, A. 

Free-energy relations and contraction of actomyosin 140 

BORBIRO, M. AND A. SZENT-GYORGYI 

On the relation between tension and ATP in cross-striated muscle 162 



in 



63397 



iv t CONTENTS 

ANDERSON, WILLIAM W., JOSEPH E. KING, AND MILTON J. LINDNER 

Early stages in the life history of the common marine shrimp, Penaeus 
setiferus (Linnaeus) 168 

MACKLIN, CHARLES C. 

Mitochonclrial arrangement in alveolar epicytes and foam cells of mouse 
lungs, particularly as induced hy the vacuoloids 173 

WOOD, R. D. 

The Characeae of the Woods Hole region, Massachusetts 179 

No. 3. JUNE, 1949 

ABELSON, PHILIP H. AND WILLIAM R. DURYEE 

Radioactive sodium permeahility and exchange in frog eggs 205 

KLEINHOLZ, L. H. AND BARBARA CHASE LITTLE 

Studies in the regulation of blood-sugar concentration in crustaceans. I. 

Normal values and experimental hyperglycemia in Libinia emarginata 218 

BROWN, FRANK A., JR. AND GWEN M. JONES 

Ovarian inhibition by a sinus-gland principle in the fiddler crab 228 

SMITH, MARSHALL E. AND LYNWOOD B. SMITH 

Piperazine dihydrochloride and glycylglycine as non-toxic buffers in dis- 
tilled water and in sea water 233 

BOWMAN, THOMAS E. 

Chromatophorotropins in the central nervous organs of the crab, Hemi- 
grapsus oregonensis 238 

BEAMS, H. W. 

Some effects of centrifuging upon protoplasmic streaming in Elodea 246 

SEAMAN, GERALD R. 

The presence of the tricarboxylic acid cycle in the ciliate Colpidium 
campylum 257 

BUTLER, PHILIP A. 

Gametogenesis in the oyster under conditions of depressed salinity 263 

CHAMBERS, ROBERT AND EDWARD L. CHAMBERS 

Nuclear and cytoplasmic interrelations in the fertilization of the Asterias 

' egg 270 

BERRILL, N. J. 

Form and growth in the development of a Scyphomedusa 283 

MICHAELIS, L. 

Fundamental principles in oxidation-reduction 293 

THIMANN, KENNETH V. 

Plant hormones, growth and respiration 296 



OLIVER J. SMITH 

1878-1948 

Vice President, Lancaster Press, Inc. 

Associated with THE BIOLOGICAL BULLETIN 

1902-1948 



Vol. 96, No. 1 February, 1949 

THE 

BIOLOGICAL BULLETIN 

PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY 




. A 
i-l- 

EFFECT OF UREA, THIOUREA, PHENYLTHIOUREA AND 

THIOURACIL ON THE OXYGEN CONSUMPTION OF 

BLOCKED AND ACTIVE EMBRYONIC CELLS 1 

JOSEPH HALL BODINE AND LAURENCE ROCKWELL FITZGERALD 
Zoological Laboratory, State University of loiva, loiva City, loiva 

INTRODUCTION 

Considerable data on the action of urea, thiourea, thiouracil and related com- 
pounds on the intact organism are at hand, especially as regards their action in 
such phenomena as growth and thyroid function (Lynn, 1948). That thiourea 
and thiouracil are important in the activity of the thyroid gland has been widely 
demonstrated both clinically and otherwise. The exact mechanism of their ac- 

j 

tion on cells, however, seems to be less well understood. The fact that certain 
oxidations, involving cytochrome-cytochrome oxidase or the peroxidase systems. 
are significant in the reactions of thiourea and thiouracil seems well established 
(Sadhu, 1948). It becomes of some interest, therefore, to compare the effects of 
such chemicals upon the respiratory mechanisms of embryonic cells, both in the 
mitotically active as well as in the resting or blocked condition. The present 
paper has to do with results of studies on the effects of urea, thiourea, phenyl- 
thiourea and thiouracil upon the oxygen uptake of mitotically active and blocked 
cells of the embryo of the grasshopper, Mclanoplns diffcrcntialis. 

MATERIALS AND METHODS 

Eggs of the grasshopper, Mclanoplns diffcrcntialis, were obtained and dis- 
section of embryos carried out as previously noted (Bodine and Boell, 1934). 
Sterile phosphate-buffered Ringer solution (pH 6.8) was used as the suspending 
medium for the embryos. All solutions of chemicals (c.p. urea, thiourea, phenyl- 
thiourea, thiouracil 2 ) were made up in this buffered Ringer solution. Diapause 
(blocked) and post-diapause (active) embryos were, from all external appearances, 
morphologically identical (Bodine and Boell, 1936). Only embryos from eggs 

1 Aided by grant from the National Institute of Health. 

2 Samples of thiouracil were generously donated by the Lederle Laboratory of Pearl 
River, N. Y. 

1 



JOSEPH HALL BODINE AND LAURENCE ROCKWELL FITZGERALD 

of known temperature and developmental histories were employed. Diapause eggs 
were those kept constantly at 25 C. from the time of laying, and which were then 
confirmed by CX uptake determination to be in the blocked state. Post-diapause 
eggs were those diapause eggs previously kept at 5 C. for periods long enough to 
remove the block and which were also confirmed by further (X consumption tests 
to be in the active state. 

Oxygen determinations were carried out at 25 C. with standard Warburg 
equipment, using 5 cc. flasks. Control or blank runs on all embryos were for 30 
minutes, after which the reagents were added from the side arms and the oxygen 
consumption rates followed for periods of no less than one hour. Three banks of 
manometers, 18 in number, were used in individual runs and general averages cal- 
culated. Each concentration of reagent was tested many times, so that over-all 
reading totals for each point represent several hundred determinations. One hun- 
dred embryos were used in each flask. In comparing reactions of blocked and ac- 
tive embryos, all determinations on a single lot were carried out simultaneously, so 
that experimental conditions were kept similar throughout. 

Concentrations of reagents were calculated from the quantities added to the 
vessels and it was found that the degree of effect shown by 50 embryos compared 
favorably with that for 100 embryos, indicating that no appreciable amounts of the 
reagents were taken up by the embryos. 



RESULTS 



Urea 



The results of the effects of urea upon the oxygen uptake of both active and 
blocked cells of embryos are graphically depicted in Figure 1. An inspection of 




0.50 



1.00 



1.50 



2.00 



2.50 



3.00 



FIGURE 1. Effect of concentration of urea on the percentage stimulation or inhibition of 
the oxygen consumption of blocked and active embryos. Ordinate, percentage stimulation (top) 
or inhibition (bottom). Abscissa, molar concentration of urea. Solid curve is average for 
all experiments on blocked and active embryos for low concentrations and for active embryos 
in high concentrations. Results for a typical experiment are indicated solid circles for 
blocked, open circles for active embryos. 



COMPOUNDS OF UREA AND O 2 UPTAKE 3 

this figure shows a marked stimulation of the oxygen consumption with low doses 
of urea and a corresponding inhibition with higher doses. The general shape 
of the curve is similar to that found for ethyl carbamate (Bodine and Fitzgerald, 
1948), and is perhaps more or less typical of the reaction of this material to many 
such reagents. No significant differences in the response of blocked and active 
cells are apparent for the stimulating effects of low concentrations of urea. For 
higher, inhibitory doses, however, a marked resistance to the reagent is shown by 
the blocked cells, the mitotically active cells being much more affected bv such ex- 

JO ./ 

posures. Up to concentrations of approximately 2 molar, the inhibitory effects of 
the urea on the oxygen uptake of blocked cells are small and completely reversible. 
For the active cells at concentrations of 1.25 molar, one gets about 50 per cent 
recovery in oxygen consumption rates, while at 2.5 molar the maximum average 
recovery amounts to but 25 per cent. 




.6 1.7 
LOG I0 2 (CONC.) 



0.05 



0.15 



0.25 



0.35 



0.45 



FIGURE 2. Lower left, effect of concentration of thiourea on the percentage stimulation or 
inhibition of the oxygen consumption of blocked and active embryos. Ordinate, percentage stim- 
ulation (top) or inhibition (bottom). Abscissa, molar concentration of thiourea. Solid curve 
is average for all experiments. Vertical lines through points represent extent of variation for 
specific concentrations. Upper right, a log-log plot showing relation between ratio of unin- 
hibited (U) and inhibited (I) respiration and concentration of thiourea. Ordinate, log 10 2 
U/I. Abscissa, log 10 2 concentration of thiourea. 



A point of some interest as regards the reaction of the embryos, both blocked and 
active, to urea is that after exposure to the reagent and subsequent washing and sus- 
pension in Ringer solution, a decided swelling is observed. The intensity of this 
swelling seems dependent upon the concentration of urea employed. Such a re- 
action has been observed only after exposure to urea and no other compound. Fur- 
ther details concerning this reaction will be dealt with in a subsequent report. 



JOSEPH HALL BODINK AND LAURENCE ROCKWELL FITZGERALD 



Thiourea 

The effects of thiourea on the oxygen consumption of cells are qualitatively 
similar to those found for urea. Results are shown graphically in Figure 2. 
Marked stimulation of oxygen uptake by low concentrations and inhibition by 
higher concentrations are invariably produced. No significant differences in re- 
sponse to any concentration of the thiourea by the blocked and active cells are 
noted. Practically complete recovery in oxygen uptake occurs, except for the 
highest concentrations of reagent employed. The relative effective concentrations 
of thiourea, as will be noted from Figure 2, are much lower than for urea. 

60 

40 
^ 

^ 20 



m 







20 



O O 

o 



0.05 



0.15 



0.25 



0.35 



0.45 



FIGURE 3. Effect of concentration of phenylthiourea on the percentage stimulation or 
inhibition of the oxygen consumption of blocked and active embryos. Ordinate, percentage 
stimulation (top) or inhibition (bottom). Abscissa, molar concentration X 10 2 of phenylthio- 
urea. Results for typical experiments indicated. Solid circles for blocked, open circles for 
active embryos. 



140 
to 
* 20 






0.050 



0.100 



0.150 



0200 0250 0.300 



FIGURE 4. Same as Figure 3, but for thiouracil. Solid curve is average for all experiments. 

Results for typical experiment shown. 

Phenylthiourea 

The action of phenylthiourea on the oxygen uptake of both blocked and active 
cells is quite similar to that for urea and thiourea. As indicated in Figure 3. only 
stimulation of the oxygen uptake has been found for the concentrations employed. 
Such a result is doubtless due to its low solubility and to the low concentration of 
the compound used. Complete recovery is found for all concentrations of the 
reagent. 

Thiouracil 

Thiouracil, like phenylthiourea, is but slightly soluble and hence only low 
concentrations of this reagent are available. A marked increase in oxygen uptake 



COMPOUNDS OF UREA AND O, UPTAKE 5 

for both blocked and active cells is found (Fig. 4). No significant differences in 
response for blocked or active cells are noted. When washed and resuspended in 
Ringer solution, complete recovery in oxygen uptake occurs. 

DISCUSSION OF RESULTS 

The rather marked and similar effects of urea, thiourea, phenylthiourea and 
thiouracil upon the oxygen uptake of both blocked and active embryonic cells are 
indeed striking, and several points of general interest in respect to their funda- 
mental action arise. The fact that thiourea and thiouracil have been shown to be 
involved in the respiratory mechanisms of cells (Sadhu, 1948) would lead one to 
suspect similar actions on the oxygen uptake mechanisms of the type of cells em- 
ployed in these investigations. 

The oxygen consumption rates of blocked and active embryos (those mitotically 
blocked or active) are markedly different (Bodine and Boell, 1934). For morpho- 
logically similar embryos, the rates of oxygen uptake of active embryos are at least 
three to four times that of blocked ones. It seems important, therefore, in any 
discussion of the effects of various reagents upon the oxygen consumption of these 
forms, to keep in mind these basic differences in rates of oxygen uptake associated 
with their cellular behavior. Urea is the only compound found to differentially 
affect the oxygen consumption of the embryos, and then in such high concentra- 
tions as to be rather toxic for the active ones. Blocked cells are but little affected by 
relatively high concentrations of urea, and then in a completely reversible manner. 
For all other compounds employed, no significant differences in the response of 
blocked and active cells are found. Any consideration of the effects of these com- 
pounds, therefore, will refer equally to both blocked and active cells. 

The characteristic curve of response to different concentrations of these re- 
agentsa marked stimulation of oxygen uptake in low concentrations and a sim- 
ilarly marked inhibition in higher concentrations compares favorably with results 
found for ethyl carbamate (urethane) and related compounds (Bodine and Fitz- 
gerald, 1948). Urethanes are thought to have marked effects upon the dehydro- 
genases or carriers of the respiratory mechanisms of cells. Thiourea and thiouracil 
inhibit certain enzyme systems, probably the oxidase and peroxidase activity of 
cells (Sadhu, 1948). 

Many invertebrates, as is well known, use copper in functions normally taken 
over in higher forms by iron, for example haemocyanin. Grasshopper embryos, at 
the stages used in the present work, contain appreciable amounts of copper (ap- 
proximately 0.025 ^gm. per embryo 3 ), and it is reasonable to assume that it func- 
tions for them in enzyme systems in a manner comparable to other cations for 
higher forms. It is also well known that copper has marked action upon many 
enzyme systems, and especially sulfhydryl-containing enzymes (Barren and Singer, 
1945). As a specific example, it has been rather clearly shown that copper mark- 
edly inhibits the hemolysis of red blood cells in isosmotic glycerol solutions (Jacobs 
and Carson, 1934). Explanations of such phenomena suggest the action of copper 
on a sulfhydryl-containing enzyme system located in the red blood cell (LeFevre, 
1948). It becomes of some interest, therefore, to examine the possibility of the part 
played by the contained copper of grasshopper embryos in their reactions to the 
reagents employed. 

3 Unpublished data from this laboratory. 



JOSEPH HALL BODINE AND LAURENCE ROCKWELL FITZGERALD 



It can readily be shown that tyrosinase, a copper-containing enzyme, isolated 
from the grasshopper egg, is markedly inhibited by thiourea. 4 Such a reaction for 
this enzyme is well known, and the explanations based upon a copper-thiourea 
reaction seem well founded (Denny, 1943). The possibility of such copper combi- 
nations with the other reagents employed seems worthy of comment. 



A. 



H P N C S 




S C NH. 



B. 



0=C N H 



HC HC S 



HC-NH 




C. 



NH. 



.NH, 




C Gu C 



NH 



FIGURE 5. Scheme for possible combinations of reagents with copper. A thiourea, 

B = thiouracil, C = urea. 

For thiourea and thiouracil (A and B of Fig. 5 5 ), there is some evidence for 
the existence of the copper combination as indicated. Theoretically, there is no 
reason for which a complex copper-urea ion could not exist for urea (C of Fig. 5) in 
a form essentially as shown. Recently it has been pointed out by LeFevre (1948) 
that the molecular concentration of reactivating substances (containing SH groups) 
had to equal, or exceed by one to two times, the concentration of the inhibitor (cop- 
per) used in the case of hemolysis of red blood cells. Experiments carried out in 

4 Unpublished data from this laboratory. 

5 Acknowledgment is made to Professors George Glockler and Ralph L. Schriner of the 
Department of Chemistry for their suggestions in this problem. 



COMPOUNDS OF UREA AND O. UPTAKE 7 

connection with the present investigations, using concentration of copper and of 
thiourea in such proportions, corroborate the findings of LeFevre. 

The effects of thiourea and thiouracil on cell respiration and cell division have 
been variously reported ; in some cases inhibition, while in others stimulating re- 
sponses, are described (Fearon, 1942). Embryos of the grasshopper while in the 
blocked condition have been shown to lack mitotic activity and to have rates of 
oxygen uptake at a low constant level. When active, mitosis is always present and 
rates of oxygen uptake are increasingly higher than for the blocked or inactive 
states. Such naturally-occurring cellular conditions, therefore, make possible 
checks on the parts they play in reactions to various reagents. Urea is the only 
compound with which a marked and significant difference in response between 
blocked and active cells is noted. Blocked cells are extremely resistant to this 
compound while active ones are markedly affected, and usually in an irreversible 
manner. No explanation for such a basic difference in response is at hand. 

Published data concerning the effects of urea, etc., on the respiration of cells 
are fragmentary or almost completely lacking, so that comparisons between copper- 
containing cells like those used in the present experiments, and those for higher 
forms, seem practically impossible. The bacteriostatic action of urea alone and in 
combinations with the sulfonamides seems well established (Kirby, 1943). Alkyl 
ureas have been shown to depress certain respiratory enzymes (dehydrogenases, 
coenzymes) (Grant and Krantz, 1942). The effects of thiourea as well as of 
thiouracil upon such basic phenomena as mitosis, however, seem at present rather 
confused (Paschkis, Cantarow, Rakoff and Rothenberg, 1945). The effects of 
thiourea and thiouracil upon the growth of amphibians, as well as upon the thyroid 
gland itself, are rather clear and well defined (Lynn, 1948). 

The stimulating effects of low concentrations of all reagents, although typical, are 
for the present not readily explained. Permeability and other factors have been 
suggested for similar phenomena in other forms (Lillie, 1916). Lack of inhibition 
of oxygen uptake in the case of phenylthiourea and thiouracil appears to be the re- 
sult of the low solubility of these compounds, and thus for them solubility becomes 
the limiting factor. 

The most regular and consistent responses for any reagent used are those for 
thiourea, and even here solubility of the compound becomes the limiting factor in a 
complete analysis of its effect on oxygen consumption. Responses to it for blocked 
and active cells are not significantly different. An analysis of its inhibitory effects 
according to the law of mass action, as pointed out by Fisher and others (Fisher and 
Henry, 1943), would suggest that its action may be similar to that for urethane 
and that a single respiratory mechanism may be involved (Fig. 2). 

SUMMARY AND CONCLUSIONS 

1. The action of urea, thiourea, phenylthiourea and thiouracil on the oxygen up- 
take of the blocked and active cells of the embryo of the grasshopper, Melanoplns 
diffcrcntialis, has been studied. 

2. In general, low concentrations of these reagents produce stimulation, while 
higher concentrations produce inhibition of the oxygen consumption of both blocked 
and active cells. 



JOSEPH HALL BODINE AND LAURENCE ROCKWELL FITZGERALD 

3. Urea alone produces a differential effect, in that blocked embryos are little 
affected by high concentrations while active embryos are irreversibly inhibited. 

4. The relative effectiveness of the compounds upon the oxygen consumption of 
blocked and active cells is : thiouracil > phenylthiourea > thiourea > urea ; re- 
covery seems to work in the opposite order. 

5. Suggestions are made as to the possible significance of copper in determining 
the basic reaction of these compounds. 

ACKNOWLEDGMENT 

Acknowledgment is gratefully made to Etta Andrews, John Johnston, and Herman Tharp 
for technical assistance in carrying out these experiments. 

LITERATURE CITED 

BARRON, E. S. G., AND T. P. SINGER, 1945. Studies on biological oxidation. XIX. Sulfhydryl 
enzyme in carbohydrate metabolism. XX. Sulfhydryl enzymes in fat and protein 
metabolism. Jour. Biol. Chan., 157 : 221-253. 

BODINE, J. H., AND E. J. BOELL, 1934. Respiratory mechanisms of normally developing and 
blocked embryonic cells (Orthoptera). Jour. Cell. Cotnp. Phys'wl., 5: 97-113. 

BODINE, J. H., AND E. J. BOELL, 1936. Respiration of embryo versus egg (Orthoptera). 
Jour. Cell. Comp. Physiol., 8: 357-366. 

BODINE, J. H., AND L. R. FITZGERALD, 1948. The action of ethyl carbamate (urethane) on the 
respiration of active and blocked embryonic cells. Phys'wl. Zoo/., Oct. (In press.) 

DENNY, F. E., 1943. Inactivation of the browning system in dried apples. Contr. Boyce 
Thompson lust., 13 : 57-63. 

FEARON, W. R., 1942. Thiourea and wound repair. Brit. Mcd. Jour., Part II : 95. 

FISHER, K. C, AND R. J. HENRY, 1943. The effect of urethane and chloral hydrate on oxygen 
consumption and cell division in the egg of the sea urchin, Arbacia punctulata. Jour. 
Gen. Physiol., 27: 469-481. 

GRANT, W. C., AND J. C. KRANTZ, JR., 1942. The mechanism of action of certain urea deriva- 
tives on normal and tumor tissue. Cancer Res., 2 : 833-836. 

JACOBS, M. H., AND S. A. CARSON, 1934. The influence of minute traces of copper on certain 
hemolytic processes. Biol. Bull., 67 : 325-326. 

KIRBY, W. M., 1943. In vitro action of urea-sulfonamide mixtures. Proc. Soc. E.rp. Biol. 
and Med., 53: 109-111. 

LEFEVRE, P. G., 1948. Evidence of active transfer of certain non-electrolytes across the human 
red cell membrane. Jour. Gen. Phvsiol., 31 : 505-527. 

LILLIE, R. S., 1916. The theory of anesthesia. Biol. Bull., 30: 311-366. 

LYNN, W. G., 1948. The effect of thiourea and phenylthiourea upon the development of 
Eleutherodectyles recordii. Biol. Bull., 94: 1-15. 

PASCHKIS, K. E., A. CANTAROW, A. E. RAKOFF, AND M. S. ROTHENBERG, 1945. Mitosis stimu- 
lation in the thyroid gland induced by thiouracil. Endocrinology, 37 : 133-135. 

SADHU, D. P., 1948. Physiological mechanism of experimental goitrogenesis. Anicr. Jour. 
Physiol., 152: 150-156. 







THE EFFECTS OF ELECTROLYTES AND SUGARS ON THE 
ERYTHROCYTES OF THE TURTLE, CHELYDRA 

SERPENTINA x 

R. TERRELL WINGFIELD AND KARL M. WILBUR 
Department of Zoology, Duke University, Durham, N. C. 

The erythrocytes of the snapping turtle, Chclydra scrpenthia, exhibit a particu- 
lar sensitivity to the lack of calcium and will hemolyze in electrolyte solutions 
which do not contain this ion, though not in pure glucose (Lyman, 1945). The 
absence of hemolysis reported for glucose is presumed to be due to impermeability 
to the glucose molecule because of its size. If this is true, sugars of smaller molecu- 
lar volume might prove hemolytic. Two factors other than size may also have an 
influence. From Gibbs-Donnan equilibrium relationships, changes in membrane 
structure may be expected when a non-electrolyte replaces an electrolyte medium, 
all sugars having an equivalent action in this respect. In addition, sugars might 
affect the cell membrane by direct chemical action, and such effects may be charac- 
teristic for the individual sugars. With regard to the hemolytic action of calcium- 
free electrolyte solutions on Chelydra erythrocytes, it is not known to what extent 
electrolytes differ in their action, nor in what manner an alteration of the hydro- 
gen ion concentration of any given solution may influence the hemolytic process. 
These various factors have been considered in the present study. 

The results show that hemolysis occurring in calcium-free electrolyte solutions 
is influenced by the ionic composition of the medium. Moreover, by suitable ad- 
justment of the hydrogen ion concentration, the integrity of the cell membrane may 
be maintained for short periods even in the absence of calcium. In an examination 
of the effects of isosmotic solutions of various sugars, striking differences were 
found, some being hemolytic and others not. Agglutination and hemolytic reactions 
observed in certain sugars indicate that these compounds are not inert, but produce 
a definite alteration of the cell membrane. 

MATERIAL AND METHODS 

Blood (0.5 -- 1.0 ml.) of C. scrpcntina drawn without anticoagulant was washed 
twice in 40 ml. of frog Ringer and suspended in Ringer. In determining the rate 
of hemolysis, washed cells were centrifuged briefly ; the supernate was very care- 
fully removed, and 5.0 ml. of experimental solution added to give a cell concentra- 
tion of approximately 1 : 100. The optical density was measured within the first 
minute and at intervals with a Fisher electrophotometer (Wilbur and Collier, 1943). 
A Beckman spectrophotometer, made available through the kindness of Dr. W. J. 
Dann, was used for determinations of the optical density of hemoglobin solutions and 
for the hemoglobin spectrum of turtle blood. 

1 Aided by a grant from the Duke University Research Council. 

9 



10 



R. TERRELL WINGFIELD AND KARL M. WILBUR 



Hematocrit tests showed frog Ringer to be approximately isotonic with slight 
variations between individuals. Experimental solutions were made isosmotic with 
0.125 molal NaCl. Double distilled water was used throughout. Galactose, 
xylose and arabinose were Pfanstiehl brand and were free of calcium. NaCl was 
Merck Reagent For Biological Work. Other chemicals were reagent grade and 
were not further purified. 

We wish to thank Dr. M. H. Jacobs and Dr. H. B. Collier for their helpful sug- 
gestions and Mr. N. G. Anderson and Mr. R. L. Rigsbee for photographic work. 



RESULTS 



Electrolytes 



The observation of Lyman (1945) that erythrocytes of C. serpentina will hemo- 
lyze in isotonic Ca-free salt solutions was readily confirmed. Moreover the course 
of hemolysis in isosmotic NaCl, for example, could be arrested by the addition of a 
small amount of isosmotic CaCL solution to the hemolyzing suspension. However, 
in preliminary experiments it became apparent that the rate of hemolysis varies 
with the cation and anion employed and also with the hydrogen ion concentration. 

The effect of different cations was examined by following the course of hemoly- 
sis in buffered and unbuffered isosmotic solutions of NaCl, KC1, MgCl, and CaCl 2 . 
Hemolysis was always most rapid in KC1, followed by NaCl, slower in MgCl 2 
(Figs. 1 and 2), and completely absent in CaCL. 



66 



64 



16 20 24 28 32 




FIGURE 1. Hemolysis of Chelydra erythrocytes in 0.125 molal NaCl and 0.126 molal KC1. 
29 C. pH 7.41. Solutions buffered with corresponding isosmotic phosphate in the proportion 
9: 1. The initial rise in the curves indicates preliminary shrinkage probably due to slight hyper- 
tonicity. The final values for optical density represent complete hemolysis for both solutions, 
the difference apparently resulting from difference in opacity of the ghosts. 

The rate of hemolysis in any given electrolyte solution was always greater in an 
alkaline than in an acid solution. This could be shown by adjusting the pH with 
HC1 or the hydroxide of the cation being studied, or by use of phosphate buffers. 
Figure 3 illustrates the effect in the case of KC1 buffered with isosmotic phosphate. 



SALTS AND SUGARS ON ERYTHROCYTES 



11 



52 



50 



48 



in 

z 



046 



44 




12 16 

MINUTES 



20 



24 



28 



FIGURE 2. Hemolysis of Chelydra erythrocytes in 0.125 molal NaCl and 0.088 molal MgCL,. 
28 C. pH 7.41. Solutions buffered with 0.1 molal sodium phosphate 9:1. 

NaCl behaved similarly. The rate showed little change within the range pH 7.8 to 
pH 6.7 but was definitely decreased at pH 5.9 to pH 6.1. The inhibitory effect of 
acidity was demonstrated in another manner (Fig. 4). Cells were placed in NaCl 
buffered to pH 7.0 with a trace of phosphate. After hemolysis was well under way 
acidified NaCl was added. Hemolysis was quickly arrested. On the addition of 
alkaline NaCl hemolysis was resumed at the original rate, indicating reversibility of 
the inhibition. If cells remain in the acid NaCl longer than about 10 minutes, he- 
molysis will be resumed at a very slow rate. When the pH is restored to the alka- 
line range after 50 minutes the former rapid rate is regained. 

The effects of the cyanide and citrate of sodium and potassium were compared 
with the corresponding chloride at pH 7.25 or 7.4. Isosmotic mixtures of chloride 
and cyanide containing 0.019 molal cyanide exhibited a hemolytic action similar to 
that of isosmotic chloride. The same results were obtained with cells exposed to 



42 



4 I 
40 
39 

38J 



37 
g 

364 

35 
34 

33! 



32 




pH78 



8 12 16 20 24 28 32 36 40 

MINUTES 



FIGURE 3. Hemolysis of Chelydra erythrocytes in 0.126 molal KC1 buffered with 0.1 molal 

potassium phosphate 9:1. 28 C. 



12 



R. TERRELL WINGFIELD AND KARL M. WILBUR 



82 
80| 

78J 
76 
74 
724 



uj 504 
48- 
46 



36 
34 
32 

30J 



28 




Oil with ocid NoCI 



Oil, with bosic NoCI 




pH7.2 



2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 

MINUTES 



FIGURE 4. Effect of acidity on hemolysis of Chelydra erythrocytes in 0.125 molal NaCl. 
Ten minutes after the erythrocytes were mixed with 4.0 ml. NaCl solution (pH 7.0), and during 
the course of rapid hemolysis, 3.0 ml. of acid NaCl was added bringing the pH to 5.3. Hemoly- 
sis was arrested within one minute. Addition of 1.9 ml. of alkaline NaCl at 20 minutes raised 
the pH and hemolysis continued immediately. Dilution gave a marked change in optical 
density as indicated. The initial rise in the curve indicates shrinkage probably due to hyper- 
tonicity. 21 C. 

the same concentration of cyanide in Ringer for 30 minutes before adding the hemo- 
lytic solution. The absence of any marked effect is not surprising in view of pre- 
vious work with other electrolytes (Davson and Danielli, 1938; Hunter, 1947). 
Isosmotic sodium citrate gave slower hemolysis than NaCl, which may be the re- 
sult of impermeability to the citrate ion as compared with chloride (Jacobs, 1940). 

Sugars 

When Chelydra erythrocytes are suspended in unbuffered isosmotic dextrose 
solution, agglutination occurs followed by hemolysis. The amount of hemolysis 
can be determined by removing the cells by centrifugation and measuring the con- 
centration of hemoglobin in the solution. If the suspension is pipetted up and down, 
the masses of cells tend to break up and hemolysis is further increased. In dex- 
trose buffered at pH 7.5 with a small amount of sodium phosphate, agglutination 
was no longer apparent ; and, as might be expected from the effect of pH on he- 
molysis in- salt solutions, hemolysis was more pronounced. 2 A comparison of the 
hemolytic action of various sugars, including one disaccharide, two hexoses, and 
two pentoses (Table I), showed that the pentoses differed markedly from the other 
sugars, hemolysis being absent in arabinose and very slight in xylose. Dextrose 
gave complete hemolysis. On centrifugation of such hemolyzing suspensions, a 

- The buffer may well have an effect as an electrolyte. 



SALTS AND SUGARS ON ERYTHKOCYTES 



13 



jelly-like mass containing nuclei, many distorted and a few normal erythrocytes, 
was found. This contrasts with hemolysis in electrolytes in which normal ghosts 
were present, with about 50 per cent of the ghosts sphering in isosmotic MgCl 2 . 
Observations on agglutination indicate that differences exist between sugars in 
this respect as well as in their hemolytic action. Cells (0.07- -0.08 ml.) sus- 
pended in Ringer were centrifuged briefly; the supernate was carefully removed and 
8.5 ml. of isosmotic sugar solution was added. After stirring to give uniform cell 
distribution, the suspension was left undisturbed. 3 Results were similar for three 
individuals (Table I). \Yhile strict comparisons between certain sugars cannot 
be made because of differences in pH, it is apparent that all sugars cause agglutina- 
tion, but all are not equivalent. Moreover, the effects of agglutination do not 
parallel hemolytic actions. So, for example, sucrose is hemolytic and weakly ag- 
glutinating; arabinose. on the other hand, has a greater agglutinating action with- 
out producing hemolysis. 

TABLE I 

Hemolysis and agglutination in isosmotic sugar solutions 

Cells were removed from solutions after 3 hours and the optical density of the supernate 
was measured at 576 millimicrons.* pH 7.5-25 C. Blood from three turtles. 





Hemolysis 


Agglutination 




Sample 1 


Sample 2 




pH 


dextrose 


0.630 


0.510 


+ + + + 


5.4 


sucrose 


0.344 


0.378 


+ 


5.7 


d-galactose 


0.325 


0.163 


+ 


4.5 


d-xvlose 


0.087 


0.017 


+ + + + 


4.4 


1-arabinose 


0.007 


0.005 


+ + + 


5.7 


Ringer 


0.017 


0.014 





5.7 



* After initial mixing the suspensions were left undisturbed to minimize hemolysis and then 
centrifuged. Centrifugation may increase the hemolysis slightly. (Collier, 1948. Personal 
communication.) The absorption spectrum was determined for one sample of blood, and maxima 
were located at 543 and 576 millimicrons. These agree closely with values given for human blood. 



The influence of electrolytes on agglutination may be shown by adding one 
part of isosmotic NaCl, KC1, MgCL or CaCL to 14 parts of isosmotic dextrose. 
Agglutination was inhibited somewhat by the addition of electrolytes in all cases 
and was less pronounced in the presence of XaCl and MgCL than with the other 
salts (Fig. 5). But in no instance was agglutination completely prevented. Mi- 
croscopic observations indicate similar differences (Figs. 6 and 7). Quantitative 
aspects of this effect, previously studied on other cells (Radsma, 1918), have not 
been investigated. 

3 Sedimentation rate fails to give a measure of the extent of agglutination in this blood 
inasmuch as some cells do not agglutinate and therefore sediment relatively slowly, whereas 
the agglutinated masses in the same suspension fall rapidly or adhere to the wall of the tube. 
Adherence to the wall is observed in vertical tubes of 1.0 cm. or more in diameter. 



14 



R. TKKRKLL WINGFIELD AND KARL M. WILBUR 







FIGURE 5. 







r 






2 7 






FIGURE 6. 



/5k 

FIGURE 7. 



FIGURE 5. Effect of salts on agglutination of washed Chelydra erythrocytes in dextrose 
solution. Isosmotic salt solutions were added to unbuffered dextrose (0.23 molal) in the pro- 
portion 1:14. D dextrose; Na NaCl + dextrose ; K KC1 + dextrose ; Ca CaCL + dex- 
trose; Mg MgCL + dextrose; R Ringer (unbuffered). After initial mixing tubes were left 
undisturbed. 26.5 C. 

FIGURE 6. Chelydra erythrocytes in 0.23 molal unbuffered dextrose. Washed cells were 
mixed with dextrose solution and placed immediately on a slide without coverslip. Arrows 
indicate ghosts. 

FIGURE 7. Chelydra erythrocytes in 0.23 molal unbuffered dextrose with isosmotic NaCl 
14: 1. Shrinkage may be noted. 

DISCUSSION 

Consideration of the mode of action of electrolytes and non-electrolytes on 
Chelydra erythrocytes is complicated by the fact that related substances may be 
strikingly different in their actions. Thus, no single scheme will serve to explain 
completely the action of sugars, nor can cation effects be interpreted adequately in 



SALTS AND SUGARS ON ERYTHROCYTES 15 

terms of valence. The latter is in contrast to human erythrocytes, in which ions of 
the same valence are alike in preventing loss of salts from cells in sucrose solution 
(Wilbrandt, 1940). 

The effect of calcium in maintaining the normal permeability characteristics of 
the cell may be considered in relation to ( 1 ) the thickness of the ionic double layer 
and the adhesion of membrane components, and (2) crossbihding of anions within 
the membrane (Danielli, 1937, 1943). In both respects calcium bas a more pro- 
nounced action than sodium or potassium. Following the Gibbs-Donnan equi- 
librium, with a change of medium from Ringer to isosmotic NaCl or KC1 as was 
done in the present experiments, there will be a replacement of calcium in the sur- 
face layer. Compactness of the membrane will be decreased, which may in turn 
lead to an increase in permeability resulting in swelling and hemolysis. Such an 
increase in volume preceding hemolysis in these solutions may be readily observed 
under the microscope. Additional assumptions will be necessary to explain such 
differences as those found between the effects of calcium and magnesium on 
hemolysis and on sphering of ghost cells. 

With sugar solution as the medium, the salt concentration essentially zero, and 
a constant anion concentration in the cell surface, there is to be expected from the 
Gibbs-Donnan equilibrium a decrease in membrane concentration of mono- and 
divalent metal ions, and an increase in surface acidity, which has been thought to be 
of sufficient magnitude to alter the proteins of the membrane and accordingly cell 
permeability (Danielli, 1937; Wilbrandt, 1940). However, in this cell an increase 
in acidity of the medium stabilizes the membrane, as shown by the acid inhibition of 
hemolysis in both electrolytes and non-electrolytes. At the same time the loss of 
metal ions would result in increased repulsive forces within the membrane, giving 
greater distances between molecules and an increase in permeability (Danielli, 
1943). Even though it is assumed that the net effect favors an increased per- 
meability, the present results are not completely explained inasmuch as all sugars 
should behave similarly, whereas some have been shown to be hemolytic while 
others are not. 

Other factors to be considered are a differential permeability to sugars and 
effects of individual sugars on membrane structure. Any differences in per- 
meability which may exist must involve factors other than molecular volume since 
this does not correlate with hemolytic action. So, for example, sugars which were 
least hemolytic (pentoses) have the smallest molecular volume. (See also Ulrich, 
1934.) The agglutination of cells indicates an alteration of the cell surface by the 
sugar, the degree to which this occurs depending upon the particular sugar and 
ionic composition of the medium. Further, hemolysis in sugar, contrary to the 
results in electrolyte solutions, was characterized by disintegration of many of the 
cells, again pointing to a direct action on membrane structure. 

The effect of acidity in decreasing hemolysis obtained with electrolyte solutions 
suggests that molecular rearrangements within the membrane may in part com- 
pensate for the lack of calcium. The fact that the erythrocyte is stable in certain 
Ca-free non-electrolytes indicates that it is not the absence of calcium per se which 
causes hemolysis in electrolytes, but rather the effect of other cations which may 
replace the calcium of the cell membrane and so increase its permeability. The 
cell, then, would be sensitive to lack of calcium only because of the ready replace- 
ment of its calcium by other cations. 



16 R. TERRELL WINGFIELD AND KARL M. WILBUR 

SUMMARY 

1. The comparative hemolytic rates of Chclydra scrpcntina erythrocytes in 
isosmotic salt solutions as measured photometrically were. KC1 > NaCl > MgCL, 
and NaCl > Na 3 citrate. Hemolysis in cyanide (0.019 molal) was similar to that 
in chloride. No hemolysis occurred in isosmotic CaCL and the addition of CaCl 2 
to cells hemolyzing in Ca-free electrolyte solutions arrested hemolysis at once. 

'2. Hemolysis in sodium and potassium solutions was greatly retarded at about 
pH 6 and below. 

3. The hemolytic potency of isosmotic sugar solutions (pH 7.4) was found to 
be : dextrose > sucrose > d-galactose > d-xylose with complete hemolysis in dex- 
trose and none in 1-arabinose in three hours. 

4. Sugar hemolysis was accompanied by abnormal shape changes and disinte- 
gration of cells, whereas in Ca-free electrolyte solutions "normal" ghosts were 
found. 

5. Agglutination occurred in unbuffered isosmotic sugar solutions, the extent 
depending upon the particular sugar. Agglutinating action was not correlated with 
hemolytic potency. 

6. Results of experiments on the hemolytic and agglutinating properties of 
sugars indicate that certain sugars are not inert but have a definite action on the 
cell surface. 

LITERATURE CITED 

DANIELLI, J. F., 1937. The relations between surface pH, ion concentration and interfacial 
tension. Proc. Roy. Soc. London, B, 122: 155-174. 

DANIELLI, J. F., 1943. In DAVSON, H., AND DANIELLI, J. F., Permeability of Natural Mem- 
branes. The University Press, Cambridge, England, Chap. XXI. 

DAVSON, H., AND DANIELLI, J. F., 1938. Studies on the permeability of erythrocytes. V. 
Factors in cation permeability. Biochcm. Jour., 32: 991-1001. 

HUNTER, F. R., 1947. Further studies on the relationship between cell permeability and metab- 
olism. The effect of certain respiratory inhibitors on the permeability of erythrocytes 
to non-electrolytes. Jour. Cell. Comp. Physiol., 29: 301-312. 

JACOBS, M. H., 1940. Some aspects of cell permeability to weak electrolytes. Cold Spring 
Harbor Symposium, 8 : 30-39. 

LYMAN, R. A., JR., 1945. The anti-haemolytic function of Ca in the blood of the snapping 
turtle, Chelydra serpentina. Jour. Cell. Comp. Physiol., 25 : 65-73. 

RADSMA, W., 1918. Uber die Agglutination roter Blutkorperchen und die Hofmeisterschen 
Reihen. Biochcm. Zeitschr., 89: 211-219. 

ULRICH, H., 1934. Untersuchungen iiber die Permeation lipoidunloslicher Nichtleiter von 
Relativ grobem Molekularvolumen in die Erythrocyten von Saugetieren. Pflilgcr's 
Arch., 234: 42-50. 

WILBRANDT, W., 1940. Die lonenpermeabilitat der Erythrocyten in Nichtleiterlosugen. 
P finger's Arch., 243 : 537-556. 

WILBUR, K. M., AND COLLIER, H. B., 1943. A comparison of the hemolytic actions of lyso- 
lecithin and saponin. Jour. Cell. Comp. Physiol., 22: 233-249. 




EXPERIMENTS ON THE DETERMINATION AND DIFFERENTIA- 
TION OF SEX IN THE BOPYRID STEGOPHRYXUS 
HYPTIUS THOMPSON 1 

EDWARD G. REINHARD 

The Catholic University of America, Washington, D. C., and the Marine Biological Laboratory, 

Woods Hole, Mass. 

One of the great controversies related to the Bopyridae (a family of isopod 
crustaceans, suborder Epicaridea) , and one that has broader biological implications 
as well, is the question of sex-determination. Is sex already determined in the 
larval stage, or does the fate of the larva (i.e. whether it turns into a female or a 
male) depend on environmental influences? Can such external factors as the 
position the larva occupies or the nourishment it receives bring about sex reversal 
in an already sex-determined larva ? 

The chief views expressed in the past regarding this question are as follows : 

1. Giard and Bonnier (1887) maintained that in the Bopyridae all free-swim- 
ming larvae are males. The first larva, however, that invades a particular host 
undergoes sex reversal and transforms into a large female without ever having func- 
tioned as a male. The next to come settles on this female and metamorphoses into 
an adult dwarf male which fertilizes the former. 

2. Smith (1909) and Goldschmidt (1920) stated that all the species of Epi- 
caridea, Bopyrina as well as Cryptoniscina, are protandric hermaphrodites, each in- 
dividual being male while in a larval state, and then losing its male organization and 
becoming female as the parasitic habit is assumed. The females, therefore, result 
from males that have already functioned as males. 

3. Hiraiwa (1936) believed that the free-swimming larvae are not males but 
are sexually undifferentiated, although the sex is already predetermined. Differ- 
entiation follows fixation, but is probably not due to environmental factors. 

4. Recently, Caullery (1941), impressed by the influence of association on sexu- 
ality as exhibited in such animals as Bonellia, Crepidula, and Ophyotrocha,'made 
the suggestion that the sexes may not be fixed from the start, but that direct parasit- 
ism of a larva on a host entails differentiation into a female, and indirect parasitism, 
through the intermediary of a female on which it is stationed, entails differ- 
entiation into a male. Lacking direct evidence, however, he suggested an experi- 
mental approach to test the validity of this theory. He advised collecting the newly- 
arrived cryptoniscid larvae that can frequently be found in the brood pouch of a 
female bopyrid larvae which according to this view would evolve into typical males 
under the influence of the environment and placing them in contact with young 
crabs not yet parasitized. Caullery thought it probable that these larvae would fix 
to the crab and become females. 

These suggestions of Caullery moved the writer to undertake a series of experi- 
ments with the larvae of StegopJiry.rus Jiyptiits Thompson, an ectoparasite of the 

1 Supported in part by a grant from The Catholic University of America Research Fund. 

17 



18 



EDWARD G. REINHARD 



hermit crab Pagurus longicarpus Say. This work was carried on in the summer 
of 1946 at the Marine Biological Laboratory, Woods Hole, Mass. In addition to 
the experiment suggested by Caullery, reciprocal experiments were undertaken in 
which presumptive female larvae were taken from the host crab and transferred to 
the brood pouch of a female bopyrid to test the suspected masculinizing influence 
of the female on cryptonisci that attach to her body. 

The present paper reporting on this work was ready for publication when the 
writer's attention was drawn to an article in Italian by Reverberi and Pitotti, which, 
although it appeared in 1942, had not been mentioned in the abstracting journals 
until 1947. This paper provides the first experimental verification of any of the 
proposed sex-determination theories with reference to the Bopyridae. The au- 
thors, working with lone thoracica Montagu, showed that the control of sex- 
determination is environmental rather than genetic. However, since there are 
several points of difference between the biological cycles and sex phenomena of lone 
and Stegophryxus, it was decided not to alter the present paper as originally writ- 
ten, but in the discussion and footnotes to draw a comparison between the results 
reported by Reverberi and Pitotti and our own. 

LIFE CYCLE OF STEGOPHRYXUS 

Only about 1.5 per cent of Pagurus longicarpus at Woods Hole are parasitized 
by Stcgophry.viis hyptiits. Thompson (1901), in his original description of the 
species, gave an account of the morphology of the adult female, adult male and some 
of the immature forms, but the life cycle has heretofore not been discussed. 






B 



FIGURE 1. Adult female and epicaridium larva of Stegophryxus hyptius. 

A. Ventral view of adult female. The dwarf male, although not visible externally, is 
shown within the brood pouch by a dotted outline to indicate its position and relative size. X 5. 
B. The epicaridium or first larval stage shown in dorsal view. X 120. C. Lateral view of 
epicaridium larva. X 120. 

The female Stegophryxus, as is the case in all bopyrids, is much larger than 
the male (Fig. 1A). It occurs on the abdomen of the hermit crab, to which it is 
attached, back downward, by its mandibles and legs. Its thorax is concealed ven- 
trally by an enormous brood pouch, made up of five pairs of thin brood plates. 



SEX DETERMINATION IN STEGOPHRYXUS 19 

Within this brood pouch lies the slender dwarf male, whose function is not that of 
inseminating the female and then quitting her, but of remaining in readiness to fer- 
tilize the successive batches of eggs that are released into the brood pouch during 
the female's productive life. These eggs, within two weeks after fertilization, 
develop into first stage larvae that leave the mother and swim off. After an in- 
terval of about five days, the marsupium is again filled with eggs and another brood 
begins embryonic development. 

The first larval stage is known as the epicaridium. It is a short, broad, semi- 
barrel-shaped larva (Fig. IB) with sub-chelate pereiopods for clinging and with 
pleopods in the form of swimming organs. The epicaridium of Stegophryxus meas- 
ures about 270 fj. in length, 150 /x in breadth, and 120 ^ in depth (not including the 
appendages). In this stage the young of Stegophryxus escape from the brood 
pouch and swim off as plankton organisms. In the laboratory, they quickly rise to 
the surface of the water and remain there floating or swimming about for days. 

The subsequent history of the epicarid larva has not been investigated in 
Stegophryxus. It may attach to a pelagic copepod, undergo a molt, and become a 
microniscus larva, which, after feeding on the copepod, will eventually transform 
into a new larval stage known as the cryptoniscus that swims off to seek the defini- 
tive host. This type of development is known to occur in some of the Epicaridea 
(Sars, 1899; Caullery, 1907; Caroli, 1928; Reverberi and Pitotti, 1942). Or the 
epicarid larva may develop directly into a cryptoniscus larva, an abbreviated type 
of development which Hiraiwa (1936) believes is the case in most Bopyridae. We 
postulate the first alternative in the case of Stegophryxus because of the great dif- 
ference in size between its epicaridium and cryptoniscus stages, a difference which 
can only be accounted for by assuming the existence of an intervening stage. 

At any rate, however arrived at, the earliest larval stage of Stegophryxus that 
we find on the crab is the cryptoniscus. In this stage (Fig. 2) the parasite is 
typically isopod in its characteristics. It has an elongated body, dorsoventrally 
compressed, segmented and well chitinized, There are seven pairs of thoracic ap- 
pendages (as compared with six pairs in the epicaridium) all similar in form, six 
pairs of uniramous natatory pleopods, and one pair of biramous uropods. The 
cryptoniscus measures about 680 p in length, being therefore about two and one- 
half times longer than the epicaridium. 

We have been able to distinguish three phases in the life of the cryptoniscus 
larva on the basis of color pattern which we shall designate as (1) the brown 
chromatophore phase, (2) the black chromatophore phase, and (3) the striped con- 
tracted phase. 

The youngest cryptonisci, those that have recently settled on a crab, have a 
profusion of dark brown expanded chromatophores that cover the dorsal surface of 
the body in such a way as to leave an uncolored portion that resembles a cross 
(Fig. 2A). These chromatophores are present laterally on the head and segments 
1, 4, 5, 6, 7, and 8; are present centrally as well as laterally on segments 12 and 13 
and on the uropods ; and are entirely absent from segments 2, 3, 9, 10, and 11. The 
general body color is pale yellowish and is due to another system of chromatophores, 
which are scattered over the integument without definite plan. The eyes are red- 
dish brown. 

In phase 2, the light colored cross-shaped pattern remains much as before, but 
most of the areas formerly occupied by brown chromatophores are now occupied 



20 



EDWARD G. REINHARD 





B 



FIGURE 2. The cryptoniscus larva of Stegophryxus hyptiits. 

A. Young larva showing color pattern when in phase 1. The light cross-shaped area is 
devoid of chromatophores. B. Older cryptoniscus in phase 3 with contracted melanophores 
forming an interrupted stripe on each side. 



by expanded black chromatophores. Their distribution is as follows : present 
laterally on the head and segments 1, 4, 5, 6, 7, and 8; present centrally on seg- 
ments 11 and 12; absent from segments 2, 3, 9, 10, 13 and the uropods. The eyes 
have also become black. The yellow chromatophores are now more noticeable and 
have become restricted to segments 1 to 11 inclusive where they are present 
laterally. 

In phase 3 (Fig. 2B) the black chromatophores are much fewer in number and 
are all in the contracted state. They form a broken chain on each side of the body 
about midway between the center and margin of the dorsal surface, reaching from 
segment 1 to segment 8 inclusive, but absent on segment 3. On segments 10 and 1 1 
there are a few black chromatophores centrally located. Yellow chromatophores 
are intermingled with the black in the same chain but extend from segment 1 to 
segment 11. The eyes are black. In this stage the cryptoniscus is ready for the 
molt which will transform it into a juvenile female of the first postlarval stage. 



SEX DETERMINATION IN STEGOPHRYXUS 



21 



No structural differences have been detected in these three cryptoniscid stages. 
Since neither the brown nor the black chromatophores lose their color in alcohol, 
they are no doubt melanophores which presumably differ only in the amount of 
melanin present. 





B 







FIGURE 3. Juvenile males of Stcgophry.rus hyptiits compared with juvenile females of the 
same species and same degree of development. 

A. Juvenile male in first post-cryptoniscid instar. Specimen drawn measured 0.85 mm. 
Dorsal view. B. Older juvenile male measuring 1.07 mm. in length. Dorsal view. C. More 
advanced juvenile male measuring 1.4 mm. in length. Ventral view. D. Juvenile female in 
first post-cryptoniscid instar. Specimen drawn measured 0.85 mm. Dorsal view. E. Older 
juvenile female, 1.01 mm. in length. Dorsal view. F. More advanced juvenile female measur- 
ing 1.3 mm. in length. Ventral view. 



EDWARD G. REINHARD 

The juvenile female into which the cryptoniscus transforms is shown in Figure 
3D. It is broader than the cryptoniscus and the pleon lacks uropods and possesses 
only stump-like rudiments of pleopods. The terminal segment is extended into a 
tail-like outgrowth ending in a shallow notch. The animal is whitish with color 
pattern differing little from that of the last stage cryptoniscus. More advanced ju- 
venile females are shown in Figures 3E and 3F. 

About ten per cent of the crabs examined in the summer of 1946 were infested 
with one or more cryptonisci. This was fortunate, insofar as experimental possi- 
bilities were concerned, since infective cryptonisci have rarely been reported. 
Bonnier, for example, having studied about eighty species, came across cryptonis- 
cus larvae that had recently attached to the host only twice. Hiraiwa never found 
them during five years' study of Epipenaeon japonica. The cryptonisci of Stego- 
phryxus may be found on almost any part of the crab, but only those attached to 
the pleopods have actually settled down. The others are transients or new arrivals 
that wander off at the slightest disturbance. Even those on the pleopods, while 
more permanent than the others, are apt to leave when disturbed. The older the 
cryptoniscus, the more fixed in position it is, and if found attached to the last pleo- 
pod (third abdominal appendage of males, fourth of females), the favorite resting 
site, one can presume that it will remain there, barring accidents, until it eventually 
transforms into a female. Only after the juvenile female stage is assumed does the 
parasite leave the pleopod to fix itself permanently on the abdomen proper of the 
host. 

Cryptonisci destined to become functional males are those found attached to a 
young or mature female. They are identical in form, size, and color pattern with 
those found on the crab. They too pass through the same three phases, but meta- 
morphose into juvenile males. The first male instar is shown in Figure 3A. It 
is narrower in form than the corresponding female instar and has a strikingly dif- 
ferent type of pleon which is tongue-shaped and rounded at the tip. More ad- 
vanced juvenile males are shown in Figures 3B and 3C. 

TRANSFORMATION OF PRESUMPTIVE FEMALES INTO MALES 

The first question to be answered experimentally was whether or not the cryp- 
tonisci found on normal crabs, that is on crabs not infested with a female Stego- 
phryxus, could be transformed into males. Such larvae in all likelihood would be 
presumptive females. If removed from the crab and transferred to the brood 
pouch of a female bopyrid, would these cryptonisci metamorphose into males? 
I. In the first series of experiments, cryptonisci taken at random from normal 
crabs were placed in a dish with a crab parasitized by a mature Stegophryxus. 
The male was first removed from the brood pouch of the female to prevent inter- 
ference. This was necessary because if the adult male is allowed to remain, al- 
though cryptonisci will attach as freely as when no male is present, they will enjoy 
only a relatively brief period of attachment before they are driven off. 

Four experiments of this type were conducted. In all cases the greater pro- 
portion of the cryptonisci attached to the female bopyrid and the greater proportion 
likewise entered the juvenile male phase. But, after varying lengths of residence 
in the brood pouch and correspondingly varied degrees of attainment of the male 
phase, all but one out of each lot eventually deserted the female. The one that re- 



SEX DETERMINATION IN STEGOPHRYXUS 23 

mained in unchallenged possession eventually became a mature male, and in cases 
where the experiment was continued long enough this male functioned as such 
and successfully fertilized the eggs of its consort which then developed normally 
into epicaridium larvae. 

It will be sufficient to cite one experiment of this series in detail. This experi- 
ment was begun July 13, 1946 with five cryptonisci taken at random from unpara- 
sitized crabs and placed in a dish with a crab having a mature Stegophryxus (male 
removed) whose brood pouch contained late embryos. 

July 14. Two cryptonisci have attached to the female Stegophryxus. 

July 15. Four cryptonisci now present on the bopyrid. Epicarids are hatching. 

July 16. Three cryptonisci remain within the now empty brood pouch. They 
have developed to the black pigment stage. 

July 17. Metamorphosis of cryptonisci continuing; one, at least, has molted. 

July 19. The three cryptonisci have entered the juvenile male phase and one 
is slightly more advanced than the others. 

July 24. The three juvenile males are still present and continuing their 
development. 

July 26. One of the juvenile males has disappeared. One of the two remain- 
ing ones is permanently removed for examination and drawings are made of it. 

August 9. The brood pouch of the female bopyrid is now filled with eggs. 
(This means that the male has reached maturity.) 

August 11. The male was removed for measuring and returned to the brood 
pouch. Its length is 2.28 mm. 

August 24. Development of the eggs has continued normally and today the 
epicarid larvae are released. 

August 26. The male now measures 2.37 mm. Experiment discontinued. 

Similar results were obtained when female bopyrids, found in nature with a 
retinue of cryptonisci present in the brood pouch, were kept under observation. 
In one case a female Stegophryxus, non-ovigerous and lacking a male, had 18 
cryptonisci attached to it. The daily count showed a reduction as follows : 18, 16, 
14, 11, 9, 6, 4, 2, 2, 2, 2, 2, 2, 1. The remaining one reached maturity two weeks 
later and fertilized the eggs of the female which were not released until that time. 
Another reduction from an initial natural retinue of eight cryptonisci occurred as 
follows : from 8 to 2 in four days, but these two persisted for 1 1 more days to become 
juvenile males, then one disappeared. The survivor became a mature male. 

These experiments and observations, while they shed some light on the problem 
at hand, are inconclusive evidence for or against any theory of the sexual nature of 
the cryptonisci. They show that cryptonisci that enter the brood pouch of a fe- 
male metamorphose in the male direction, but what of those that leave early or fail 
to enter? Could not they be predetermined females unresponsive to masculinizing 
influences? 

II. To settle this point, it was decided to experiment with single cryptonisci. 
Moreover, only cryptonisci found clinging to the posterior pleopod of a normal 
crab were used. Nine experiments were undertaken. In five of these the crypto- 
niscus selected for insertion in the brood pouch of a female was in the brown 
chromatophore stage; the four other cryptonisci were in the more advanced -black 
stage. 



24 EDWARD G. REINHARD 

Each of the five Stage 1 cryptonisci remained in the brood pouch and made no 
efforts to crawl out. One was removed after six days, one after eight, one after 
eleven, and two after twelve days. Each one had metamorphosed into a male, 
whose size and extent of development was proportional to the length of time spent 
in the brood pouch. Those that had been on the female for eleven or twelve days 
had reached a size of from 1.5 mm. to 1.8 mm. 

The experiments with Stage 2 cryptonisci gave different results. In three cases 
the cryptoniscus crawled out of the brood pouch within a day or two and was either 
lost or found clinging to the crab instead. One experiment yielded positive re- 
sults. This cryptoniscus refused repeatedly to attach to the female, but after each 
escape it was returned to the brood pouch. Finally it remained there, and, eventu- 
ally, 28 days later, had become a 2 mm. male. 

The positive results obtained with the five Stage 1 cryptonisci strongly indicate 
that cryptonisci that would ordinarily become females can readily be transformed 
into males through attachment to the body of the female bopyrid, provided the 
transfer is effected at an early age. Even the one success with a Stage 2 crypto- 
niscus confirms this. It must be concluded that after a certain period of parasitism 
on the crab the cryptoniscus becomes female-determined and the direction of its 
sex development can no longer be changed under ordinary conditions. Subsequent 
experiments, using juvenile females for transfer, instead of cryptonisci, support 
this view and will now be briefly recounted. 

III. Four attempts were made to transform juvenile females into males. All were 
completely unsuccessful. Juvenile females in the early post-cryptoniscid phase 
were used, before they had developed far enough to leave the pleopod of the host for 
permanent attachment on the abdomen. 

One female remained inside the brood pouch for one day, crawled to the ex- 
terior and remained there one day, then disappeared. 

One left the brood pouch the day after transfer and attached to the abdomen of 
the crab where it remained for eight days, when the crab died. 

One left the brood pouch on the second day and attached to the underside of the 
telson of the crab. It remained there until the experiment was discontinued 12 
days later, and grew from an initial size of .85 mm. to 1.4 mm. 

The fourth one was transferred to the brood pouch together with the pleopod to 
which it was attached. This female remained for five days, then disappeared and 
could not be recovered for examination. 

It would seem that juvenile females are averse to becoming ectoparasites of 
other more mature females. They leave such an unnatural situation to return to 
direct parasitism on the crab. 2 There is no evidence that any of the four were 
modified by their brief sojourn in the brood pouch of another female. 

2 Reverberi (1947) came to the same conclusion with regard to lone. However, he then 
placed two females together in vitro apart from the host, one being a juvenile female and the 
other an adult from which the juvenile would have to derive its nourishment. As often as the 
adult died, another of the same age would be substituted. By this ingenious method he was 
able to maintain a direct association between a juvenile female and an adult lone for several 
months. One case of definite sexual inversion resulted from many trials of this sort. This 
particular juvenile female underwent external changes and gradually took on the appearance 
of a male. When killed and sectioned after nearly four months under these experimental condi- 
tions, the individual was found to have normal testes partially filled with sperm. 



SEX DETERMINATION IN STEGOPHRYXUS 25 



ATTEMPTS TO TRANSFORM PRESUMPTIVE MALES INTO FEMALES 

If presumptive female cryptonisci can be turned into males by altering the en- 
vironment, the question naturally arises regarding the possibility of producing fe- 
males from presumptive male cryptonisci. The method of experimentation would 
be to remove cryptonisci from the brood pouch of a female bopyrid and transfer 
them to a crab instead. This is the type of experiment on which Caullery pinned 
his hopes of verifying the theory of sex determination in Epicaridea based upon the 
type of association with the host. 

When this was tried it invariably led to failure because the transferred crypto- 
nisci did not remain attached to the crab long enough to show either positive or 
negative results. This failure to remain attached need not necessarily be attributed 
to aversion on the part of the cryptonisci for a strange environment. Indeed, the 
hazards in the case of direct parasitism on the crab are great. When it is recalled 
that approximately 10 per cent of the normal crabs have cryptonisci on their sur- 
face and only 1.5 per cent of all crabs are infested with female Stegophryxi, it be- 
comes clear that many potential parasites are eliminated through environmental 
difficulties. Moreover, no success was achieved in numerous attempts to rear. to the 
juvenile female stage cryptonisci found naturally attached to crabs. When crabs 
bearing cryptonisci are isolated in a dish and examined after a day or two, one 
finds that the cryptonisci have disappeared. Apparently they are eaten by the 
crab, since cryptonisci kept in dishes without crabs will remain alive for as long as 
two weeks. It may be remarked in passing that although isolated cryptonisci sur- 
vive, they do not develop, nor pass from the brown to the black phase. It is signifi- 
cant that the juvenile female Stegophryxi naturally occurring on crabs can be 
reared without difficulty in the laboratory. They are actually fixed to the crab and 
have lost their ability to swim, whereas the cryptonisci, as explained before, are still 
active and only perch on the crab without fastening themselves to it. Should they 
disengage themselves even momentarily, they are in danger of being caught up by 
the currents passing through the gill chambers of the crab and swept in the direction 
of the crab's mouth. The mouth parts of the crab are in constant motion and any 
particle that comes in contact with them is trapped and masticated. 

The failure of these experiments with cryptonisci removed from the brood 
pouch of a female and transfered to a crab may, therefore, with considerable as- 
surance, be laid to experimental difficulties. 3 When the proper technique is worked 
out for Stegophryxus, which will eliminate the hazards facing cryptonisci that at- 
tach to crabs, we feel confident that presumptive male cryptonisci can be transformed 
into females. 

3 This is especially reasonable in view of the fact that Reverberi and Pitotti (1942) ex- 
perienced a similar lack of success when they tried to implant the cryptoniscus larvae of lone 
on the gills of Callianassa. The cryptonisci invariably failed to remain on the host (pp. 148- 
149). But when they used post-cryptoniscid stages they were successful in bringing about the 
transformation of juvenile males into females. lone, unlike Stegophryxus, is a branchial para- 
site. When juvenile males of lone, removed from adult females, were placed in the branchial 
cavity of the host, they soon attached to the branchiae, began to feed, and in the majority of 
cases remained there more or less permanently. Such males gradually became females. 



26 EDWARD G. REINHARD 

THE FATE OF SUPERNUMERARY MALES AND FEMALES 

The first series of experiments reported above furnish evidence that although any 
number of cryptonisci may attach to the same female and develop into juvenile 
males, only one male is allowed to reach maturity. This point has not been realized 
by most previous investigators except Caullery (1941) and Reverberi and Pitotti 
(1942), and hence, in the older literature, several cases of supernumerary males as- 
sociated with one female bopyrid are mentioned, with the inference that they are 
highly unusual or evidence of polyandry. Perez (1924), for example, reports three 
instances of this from his own observations involving Pleurocrypta porcellanae 
Hesse, P. galatheae Hesse, and Athelges lorijcra Hesse. In the light of recent 
work, these must be interpreted as cases of extra males in process of development 
before they have realized the full adult state, since all cases, when analyzed, resolve 
themselves into the stegophryxoid pattern, namely, one adult functional male ac- 
companied by one or more smaller juvenile males or cryptonisci. 

It must be concluded, therefore, that it is a general rule in the bopyrids that 
only one functional male is permitted at a time. Other potential males, temporarily 
tolerated, are expelled sooner or later. Whether the legitimate male, by virtue of 
its larger size, actually drives off the others, or whether they are repelled in some 
more refined manner is a question still to be answered. 

Our observations on the question of excess females show that they, like the 
supernumerary males, are eliminated sooner or later, usually as juveniles. Only one 
adult female is ever found on an individual host. 

One hermit crab, collected August 23, 1946, carried a large ovigerous Stego- 
phryxus and had in addition four juvenile females in various stages of development 
attached to the abdomen and pleopods. This crab was isolated for daily observa- 
tions and in less than a week's time the four juvenile females had disappeared. Two 
other cases of parasitized crabs, each with a juvenile female present in addition to 
the adult female Stegophryxus, were observed under laboratory conditions. One 
juvenile persisted from July 24 to August 10 and grew considerably in size before 
it was eliminated; the other lasted from July 29 to August 11. 

It is significant that the lost juvenile females could not be found in the dish in 
which the crab had been isolated. Perhaps they drop off and are eaten by the 
crab. It is more probable, however, that they are driven off by the mature male. 
One finds, on occasion, the mature male wandering about on the outside of the fe- 
male brood pouch, and it is not unlikely that the male engages in occasional forays 
over the abdomen of the crab and drives off or destroys the excess females before 
they reach maturity. 

HlSTOLOGICAL OBSERVATIONS 4 

Four cryptonisci and ten males, the latter selected to form a graded series of 
sizes ranging from 1.2 mm. to 2.7 mm., were sectioned and studied histologically 
to determine the sexual nature of the larvae and the organogenesis of the male 
gonads. 

4 The author gratefully acknowledges the assistance of Cornelius Sharbaugh, T.O.R., who, 
under our direction, prepared the slides and made the morphological studies on which this 
portion of the paper is based. 



SEX DETERMINATION IN STEGOPHRYXUS 



27 



It was found that males of 2 mm. -length and over could he termed "adult"' as 
judged by the length and development of the testes and the presence of spermatozoa 
in the vasa deferentia. In such males the reproductive organs are seen as a pair 
of long tubular masses, beginning in the first thoracic segment, and extending back 
into the seventh thoracic segment. The testes lie dorso-laterally adjoining the two 
liver tubes, one on each side of the animal, except for the anterior extremity of each, 
which occupies a ventro-lateral position in relation to the liver. In the sixth and 
seventh thoracic segments, the testes become vasa deferentia which open separately 
to the outside on the ventral surface of the seventh thoracic segment. The beginning 
of the vas deferens is often dilated to act as a temporary seminal vesicle. 

The cells that make up the testis in the anterior-most portion of the organ are 
all of one type and equally distributed throughout the cross section. Elsewhere, 
spermatocytes, spermatids, and" spermatozoa may be seen arranged in three distinct 
zones : spermatocytes in the inner zone next to the liver, spermatids in the middle, 
and spermatozoa in the outer zone (Fig. 4). The cells of the inner and middle 
zones are grouped into areas or patches, but those of the outer zone extend without 
interruption the entire length of the testis. 







TESTIS 



LIVER 



FIGURE 4. Right half of transverse section through the third thoracic segment of a male 
Stcgophryxus hyptius measuring 2.7 mm. in length. The testis, dorso-lateral to the liver, 
shows the characteristic zonal arrangement : spermatocytes in the inner zone closest to the 
liver, spermatids intermediate in position, and spermatozoa in the outer zone. X 400. 



EDWARD G. REINHARD 

Males of approximately 1.5 mm. -length are essentially similar to the larger 
males except that the testes are shorter, beginning in the second or third thoracic 
segment, and the three characteristic zones appear only in the posterior portion. 
Anteriorly, the testes contain spermatocytes and spermatids, but no spermatozoa. 

In the smallest male examined, length 1.2 mm., the testis on the left side was 
undeveloped ; that on the right was short and contained spermatocytes and sperma- 
tids only. These occupied a relatively short middle section, with anterior and 
posterior extremities appearing empty. 

None of the four cryptonisci examined, three in cross section and one in longi- 
tudinal section, showed either gonads or groups of cells that might be regarded as 
traces of gonads. 

DISCUSSION AND CONCLUSIONS 

The experiments reported on here seem to indicate that the cryptoniscus larvae 
of Stegophryxus can develop in either of two directions : into males under conditions 
of parasitism on a female bopyrid, or into females, under conditions of parasitism 
on a hermit crab. As in the case of Bonellia (Baltzer, 1914), the larvae are ap- 
parently indifferent, with both sex potencies. Which potency will be realized de- 
pends on the conditions of the environment. 

It is evident that the female bopyrid exercises a masculinizing influence on the 
cryptonisci directly attached to it. This influence does not extend to cryptonisci 
which are merely in the neighborhood of the female. They receive no male stimu- 
lus. To be affected, the cryptoniscus must be in actual contact with the female and 
perhaps even imbibe her body fluids. Certainly they and the juvenile males receive 
nourishment from the female, or how else could they grow to increase as much as 
threefold in size? 

Whether nutritive conditions alone provide the masculinizing stimulus, or 
whether the controlling influence is a specific substance of hormonal nature, is a 
question requiring further experimental study. Nourishment, as pointed out by 
Zimmer (1927), is probably the determining factor in the production of females, 
but for the production of males it seems necessary to assume, as in Bonellia, the 
transfer of an actual secretion from the body of the female to the larvae that are 
attached to her, which acts as a specific masculinizing substance. 

The sex-determination theory proposed by Giard and Bonnier (1887), namely, 
that the first larva that invades a particular host transforms into a large female, 
while the next to come settles on this female and metamorphoses into a dwarf male, 
is an explanation entirely too simple ; but the first assumption, at least, is supported 
by the results of our experiments. The fate of subsequent comers is less certain. 
Conceivably, a second cryptoniscus might arrive shortly after the first and also 
settle down to become an incipient female. There must obviously be a time interval 
of some clays before the first-comer has metamorphosed sufficiently to invite the 
attention of new arrivals. Let us say, therefore, that the fate of subsequent comers 
is in no way different from the fate of any cryptoniscus ; viz. those that settle di- 
rectly on a crab become female-determined, while those that settle on a female of 
their species become male-determined. 

One of the objections to the theory of Giard and Bonnier has been the fact that 
two females are sometimes found on the same host. Thus Hiraiwa (1936) says: 



SEX DETERMINATION IN STEGOPHRYXUS 29 

"If the female in the (branchial) cavity makes the later invader into male, why are 
two females found in one and the same cavity?" 

The answer to this objection is now clear. A female does not influence the 
sex of later invaders unless they settle directly on her body. Should a cryptoniscus 
settle on the crab, no matter how close in position to a large female, the cryptoniscus 
will not be affected by this proximity so far 'as its sex-determination is concerned. 

The sex determination theory of Smith (1909) and Goldschmidt (1920) with 
reference to the Bopyridae requires no discussion. It is eliminated by the facts 
presented in describing the life cycle of Stegophryxus and has previously been 
sufficiently criticized by Hiraiwa (1936). 

Hiraiwa's own theory, disclaiming as it does differentiation due to environmental 
factors, is not in harmony with the results of the experiments reported here. His 
assumption, however, that the free-swimming larvae are not males but are sexually 
undifferentiated is in agreement with our findings. 

Caullery's theory (1941) finds ample confirmation in the results of our experi- 
ments. Although the exact experimental verification he hoped for has not yet been 
realized by us, the converse experiment of transferring cryptonisci from the host 
to the female bopyrid has yielded satisfactory evidence that the sexes are not 
fixed from the start. 

Coming now to the studies on lone tlwntcica made by Reverberi and Pitotti 
(1942) and Reverberi (1947), and their relation to the observations and deductions 
reported here on Stegophryxus hyptius, we find when we tabulate the two for com- 
parison (Table 1) that the same general pattern runs through both. Some, per- 
haps most, of the differences that do exist are modifications to be expected when 
comparing species of different genera that differ also in habits and habitat. Thus, 
since lone is a branchial parasite, the larvae have the opportunity of settling on the 
gills of the host (to become female-determined), on the female parasite itself (to 
become male-determined), or on the skin of the host (likewise to become male-de- 
termined). Stegophryxus presents a simpler condition since the female is attached 
not to the branchiostegite but to the abdomen of the host. Therefore only two 
substrates are selected for attachment by the larvae : the abdomen of the host or the 
female herself. If abundant nourishment is the factor that determines females and 
less abundant nourishment, as Reverberi and Pitotti at first thought, determines 
males, it is hard to understand why the abdomen of Pagurus should furnish abun- 
dant nourishment to the cryptonisci of Stegophryxus while the abdomen of Cal- 
lianassa should not likewise yield abundant nourishment to cryptonisci of lone. 
Later (1947), Reverberi, as a result of further experiments, came to the conclusion 
that the larvae that attach to the female parasite are masculinized by a sex-determin- 
ing substance produced by the female rather than by "less abundant nourishment" ; 
but the problem of the so-called "complementary males" on the skin of Callianassa 
is still very puzzling. 

Reverberi's experiments on lone were made almost exclusively with the older 
post-cryptoniscid larvae ; ours dealt almost entirely with earlier larvae in the 
cryptoniscus stage. Since the larvae on the body of Callianassa were already 
presumptive males, his chief experiments were to make females out of them. In 
Stegophryxus, on the other hand, the cryptonisci on the body of the host are pre- 
sumptive females, hence our main experiments were to make males out of them. 
All in all, the two studies complement and illuminate each other. Together they 



30 



EDWARD G. REINHARD 



TABLE 1 

A comparison between lone thoracica and Stegophryxus hyptius with respect to sex-determination. 
Data for lone compiled from the papers of Reverberi and Pitotti (1942) and Reverberi (1947) 



lone thoracica 



Stegophryxus hyptius 



1. Adult female lives in the branchial cavity of 
Callianassa. 

2. Females result from cryptonisci that settle 
on the gill of the host. 

3. Females secrete a diffusible substance ca- 
pable of attracting free cryptoniscus larvae. 

4. Cryptonisci that attach to the female be- 
come males. 

5. Only one adult male is retained. 

6. Cryptonisci that attach to the skin of the 
host became complementary males, capable 
of replacing the lost male of an adult pair. 

7. Juvenile males, removed from adult females, 
can be transformed into females by placing 
them on the gills of the host. 



8. A widowed male can become female if it 
succeeds in attaching to the host gill and 
getting abundant nourishment. 

9. The few comparable experiments reported 
did not yield results. 

10. Evidence supplied in 1947 that differen- 
tiated females can undergo sex reversal. 

11. Larvae that engorge host blood directly be- 
come females if nourishment is abundant 
(taken from gills), but become complemen- 
tary males if nourishment is less abundant 
(taken from skin of host). 

12. Reverberi first attributed maleness, when 
larvae are attached to a female, to "less 
abundant food," but later (1947) explained 
it as due to masculinizing substance. 

13. Gonad tissue first appears in older post- 
cryptoniscid forms and the earliest gonad is 
indifferent. 

14. Sex-determination is environmental, com- 
parable (with modifications) to that de- 
scribed for Ophryotrocha. 



1. Adult female lives on the abdomen of 
Pagurus. 

2. Females result from cryptonisci that settle 
on the abdomen of the host. 

3. Same. 

4. Same. 

5. Same. 

6. Cryptonisci that attach to the skin of the 
host become juvenile females. Replace- 
ment of lost males occurs from free cryp- 
tonisci only. 

7. No evidence that juvenile males can under- 
go sex reversal, but presumptive male 
cryptonisci could probably be transformed 
into females if they left the female and ob- 
tained nourishment from the host instead. 

8. It is unlikely that males, once differen- 
tiated, could undergo sex reversal. 

9. Presumptive female cryptonisci become 
males when removed from the host and 
placed on a female parasite. 

10. No evidence that differentiated females 
have the capacity for reversal to male. 

11. All larvae in a position to feed on the host 
directly become females. Only larvae that 
attach to the female become males. 



12. Data favors hypothesis of a masculinizing 
substance produced by the female. 



13. Cryptonisci lack gonads. The earliest 
gonad in juvenile males is a testis. No 
data presented on the earliest type of 
gonad in a juvenile female. 

14. Sex-determination is environmental, com- 
parable (with modifications) to that de- 
scribed for Bonellia. 



fulfill the expectations of Caullery and reveal a fundamental plan of sex-determina- 
tion and sex-differentiation, a plan, however, that can be expected to exhibit minor 
variations when utilized by different genera of Bopyridae. 

SUMMARY 

Stegophryxus hyptius Thompson, an ectoparasite on the abdomen of the hermit 
crab Pagurus longicarpus Say, seeks the definitive host as a cryptoniscus larva. 



SEX DETERMINATION IN STEGOPHRYXUS 31 

The free-swimming cryptonisci are sexually undifferentiated and sexually unde- 
termined. ' Differentiation follows fixation and is dependent on environmental fac- 
tors. These conclusions are justified on the basis of the following observations and 
experiments. 

1. Cryptonisci that settle directly on the host develop into females; those that 
attach to a female bopyrid develop into males. 

2. Changes in the color pattern of the cryptonisci following fixation furnish 
criteria for judging the length of time they have been subjected to a particular 
environment. 

3. Histological examination of the cryptoniscus shows that the gonads are not 
yet present as recognizable structures. 

4. Presumptive female cryptonisci, if removed from the host at an early age and 
transferred to the brood pouch of a female Stegophryxus, will transform into males. 

5. The failure of the converse experiment involving transfer of presumptive 
male cryptonisci from the female parasite to the host can definitely be laid to ex- 
perimental difficulties. 

6. The factor that determines maleness is a masculinizing substance imbibed 
with food from the female., but this substance does not act at a distance. 

7. Attachment of supernumerary females as well as excess differentiating males 
is terminated sooner or later so that a particular crab is host to only a single adult 
female paired with one functional male. 

LITERATURE CITED 

BALTZER, F., 1914. Die Bestimmung des Geschlechts nebst einer Analyse des Geschlechtsdi- 

morphismus bei Bonellia. Mittcil. Zool. Station Neapel, 22 : 1-44. 
CAROLI, E., 1928. La fase "microniscus" di lone thoracica Montagu ottenuta par allevamento 

sui Copepodi. Rend. Ace. Lined, 8 : 321-326. 
CAULLERY, M., 1907. Sur les phases du developpement des fipicarides ; verification experi- 

mentale de la nature des Microniscidae. C. R. Acad. Sci. (Paris), 145: 596-598. 
CAULLERY, M., 1941. Sur la determination du sexe chez les Isopodes fipicarides. C. R. Acad. 

Sci. (Paris), 212: 108-112. 
GIARD, A., AND J. BONNIER, 1887. Contribution a I'fitude des Bopyriens. Trav. Sta. Zool. 

Wimereux, vol. 5 : 1-250. 

GOLDSCHMIDT, R., 1920. Mechanismus und Physiologic der Geschlechtsbestimmung. Berlin. 
HIRAIWA, Y. K., 1936. Studies on a Bopyrid, Epipenaeon japonica Thielemann. III. Devel- 
opment and life-cycle, with special reference to the sex differentiation in the Bopyrid. 

Jour. Sci. Hiroshima L'niv. (Zool), 4: 101-141. 

PEREZ, C., 1924. Sur la transformation des formes Cryptonisciennes en males chez les Bo- 
pyriens. Assoc. Frangaise Avanc. des Sciences. Liege. 472-473. 
REVERBERI, G., 1947. Ancora sulla trasformazione sperimentale del sesso nei Bopiridi. La 

trasformazione delle femmine giovanili in maschi. Pubbl Staz. Zool. Napoli, 21 : 83-93. 
REVERBERI, G., AND M. PITOTTI, 1942. II ciclo biologico e la determinazione fenotipica del 

sesso di lone thoracica Montagu, Bopiride parassita di Callianassa laticauda Otto. 

Pubbl Stas. Zool Napoli, 19: 111-184. 
SARS, G. O., 1899. An account of the Crustacea of Norway. Vol. 2, Isopoda. Bergen 

Museum. 
SMITH, G., 1909. Crustacea. In: Crustacea and Arachnids. The Cambridge Natural History, 

vol. 4. London. 
THOMPSON, M. T., 1901. A new Isopod parasitic on the hermit crab. U. S. Fish. Coiuni. 

Bull., 21 : 53-56. 
ZIMMER, C., 1927. Isopoda. In : Kukenthal und Krumbach's Handbuch der Zoologie. Bd. 3. 

Berlin und Leipzig. 

/cNv-rr- 4 /x 

*;, $ '^ 

; -^l 

.-J R 



-')> 
"i\ .>. 



STUDIES ON MARINE BRYOZOA, III. WOODS HOLE REGION 
BRYOZOA ASSOCIATED WITH ALGAE 

MARY D. ROGICK 

College of New Rochelle, New Rochelle, N. Y. 
AND 

HANNAH CROASDALE 
Dartinontli College, Hanover, N. II. 

INTRODUCTION 

Heretofore there has been no extensive study made of the association between 
bryozoa and algae, except for the studies by Joliet (1877) and Prenant and Teissier 
(1924, 1927, 1932), although incidental association notes are scattered throughout 
taxonomic papers. 

The purposes of the present study are several: (1) to note any association that 
may exist between algae and certain bryozoa (bryozoa likely to be encountered in 
the Woods Hole region) ; (2) to aid collectors of bryozoa, since in some instances by 
collecting specified algae one is almost sure to find a number of desired bryozoa ; 

(3) to make any observations possible on the tentacle number, the occurrence of 
embryos, larvae and ovicells in specimens collected during the summer months, and 

(4) to report any additional species from the collection area. 

COLLECTION DATA 

The materials used in this study were marine algae of three general groups: (1) 
freshly collected specimens; (2) dry, pressed specimens mounted at various times 
in the past on herbarium sheets; and (3) specimens preserved in the Botany 
Course Stock Collection of the Marine Biological Laboratory (M. B. L.) of Woods 
Hole, Mass. More species were examined than are here recorded but only those 
37 algal species which had bryozoa growing on them are here listed. 

Some of the fresh material was obtained in the intertidal zone by shore collect- 
ing ; some had drifted in from deeper waters some distance from shore ; and some 
had been dredged with a scallop dredge from waters about 20 to 60 feet in depth. 

Some of the material was collected by the authors, some by the M. B. L. In- 
vertebrate Zoology and Botany classes on their field trips, some by the M. B. L. 
Supply Department and Collecting Crew, and one algal species by Dr. Maxwell 
Doty of Northwestern University. To all these the authors wish to express their 
most sincere appreciation, and especially to Dr. William Randolph Taylor of the 
University of Michigan for very helpful suggestions, continued kindly interest, and 
for specimens of Mcmbranipora tubcrculata and their algal hosts from his own col- 
lection. 

32 



MARINE BRYOZOA, III 



33 



The Lammaria longicruris was brought in by Dr. Doty from Race Point, near 
Provincetown, Mass, on VIII-18-1947. The Rye Ledge, Rye, New Hampshire 
specimens of Chondrus crispus, Phycodrys rubens and Phyllophora meinbranijolia 
were collected by the junior author on IX-30-1945. The New Rochelle, New 
York specimens of Ascophyllum nodosum, Ascophyllmn Mackaii, Chondrus crispus 
and Lammaria Agardhii were observed on IX-16 and 22-1945, and on X-3-1946 
by the senior author. The remainder of the specimens were collected in the waters 
around and between North Falmouth, Martha's Vineyard, Woods Hole, Vineyard 
Sound, Buzzards Bay, Penikese Island and New Bedford, all in Massachusetts, 
during the summer months, between Tune 30 and August 31 approximately, over 
a period of several years (Sept. 1874, 1916, 1930, 1935, 1936, 1938, 1939, 1944 
through 1947). Specimens from the five earliest years came from the M. B. L. 
Botanical Collection, some from wet mounts and some from dried herbarium mounts. 
Exact records, dates and collection numbers of all these algal specimens are on file, 
but only a very condensed amount of these data is included in Tables I to IV, to save 
space. 

The senior author identified the bryozoa, the junior author classified the algae 
and collected many of them. 

TABLE I 

Collection sites and bryozoa associated with green algae 





Cladophora 
gracilis 
f. tenuis 


Enteromor pha 
intestinalis 


L'lva Lacluca 
var. 
rigida 


Total No. green 
algae having this 
bryozoan species 


Bowerbankia gracilis 




X 


X 


2 


Bugula turrita 


X 


X 


X 


3 


Cryptosida pallasiana 




X 


X 


2 


Flustrella hispida 






X 


1 


Pedicellina cernua 


X 






1 


Total No. bryozoan spp. on 


2 


3 


4 




this alga 










Collected at Woods Hole, 


X 


X 


X 




Mass. 











To date there have been reported 84 species of marine bryozoa from the Woods 
Hole region, by Osburn (1912) mostly, and by Rogick (1945a, 1948). Some of 
the Woods Hole species have been previously reported from such widely separated 
regions as the coast of Africa, Australia, the Azores, Brazil, Denmark, Great 
Britain, Japan, New Zealand, Panama, Zanzibar, and the Pacific coast of North 
America. These were reported from various substrates as shells, rocks, hydroids, 
algae, various animals, piles, and other submerged objects. The present study was 
mainly directed toward finding the exact algal species on which bryozoa grow. 
Previous reports from the Woods Hole region occasionally did indicate the algal 
genera but rarely the species on which the bryozoa occurred. The following lists 
of algae and bryozoa are of species collected or examined for the present paper. 

Below follows a list of 37 algae on which were commonly found various bryo- 
zoans. Taylor's (1937) classification is used. 



34 



MARY D. ROGICK AND HANNAH CROASDALE 



TABLE II 

Collection sites and bryozoa associated with brown algae 

























,0 






o 
















Z 


-* 


to 
** 


2 


5 




-<s 

a 
**, 


to 


o 






"5 

^ 


<0 


2 


'1, 


1 


It 

.Vj 


1 


C* 




3 ' 


s; 


^ 




VJ 

a 


5 


o 


10 


o 

Ofl 


bo 









j 


2; 


s 


to 




to 


"^* 


2 


^ 


_5 


s 


bts a 




3 


a 


~ 


j~ to 
*~- 2 


'^2 


=: 


.^ 


to 

_o 


_a 


f <3 




'u o 




^ 


-s; 


o 


Q.-3 

^_O 

to ' * 


tft 

<u 
^ 
a 


1 


S 


"s 

^-J 


a 


3 


1 


ill 




o 1 


*. 

o 


g 


|.S 




2 


s 


b; 

g 


s; 


'5 


o 

CtQ 


~s j:> 




1 


1 


o 

-s: 
O 


G 1 


<s 


ft, 


"O 


ft! 




3 


V 


[" 'fl *> 


Aetea sica 




X 


X 


X 






X 


X 


X 




X 


7 


Aeverrillia armata 


















X 






1 


A everrillia setigera 




X 














X 




X 


3 


Alcyonidium polyoum 


















X 


X 




2 


Bowerbankia gracilis 




X 


X 


X 






X 


X 






X 


6 


Bowerbankia imbricata 














X 




X 




X 


3 


Bugula cucullifera 












X 








X 




2 


Bugula flabellata 














X 










1 


Bugula turrita 




X 


X 








X 


X 


X 




X 


6 


Callopora aurita 


















X 






1 


Cribrilina punctata 


















X 






1 


Crisia eburnea 




X 






X 








X 




X 


4 


Cryptosula pallasiana 


X 


X 












X 


X 






4 


Electra hastingsae 
















X 


X 






2 


Electra pilosa 




X 






X 


X 




X 


X 


X 


X 


7 


Flustrella hispida 




X 










X 










2 


Hippothoa hyalina 




X 












X 


X 




X 


4 


Membranipora lacroixii(?) 


X 






















1 


Microporella cilia ta 


















X 






1 


Pedicellina cernua 




X 










X 




X 






3 


Schizoporella biaperta 










X 








X 






2 


Schizoporella unicornis 
















X 


X 






2 


Scruparia ambigua 










X 


X 






X 






3 


Scruparia clavata 


















X 






1 


Smittina trispinosa 


















X 






1 


Total No. bryozoan spp. found 


2 


10 


3 


2 


4 


3 


7 


8 


20 


3 


8 




on this alga 


























Collected at Woods Hole 




X 


X 


X 






X 


X 


X 




X 




Collected at North Falmouth 




X 












X 






X 




Collected in Vineyard Sound 






X 




X 


X 










X 




Collected at Martha's Vine- 










X 


X 


X 




X 








yard 


























Collected at Penikese Island 
















X 










Collected at Provincetown 




















X 






Collected at New Rochelle 


X 


X 














X 









MARINE BRYOZOA, III 



35 



4? 
a 



- 

-S 



w 
j 

PQ 

< 
H 



>j 
8 



dds 



UEozoAjq 
a^B 
-ONJ 



vysnfqns 



snpunjos 



V33U3S 



ds 



X X X X 



X X 



X X X X X X X X X X X X X X X X X X 



X X X X X X XX 



X X X X X X 



XX 



X X 



ds 



sntfsuy stupuot/3 



uin.iqn,t 



atujoftjqtij uim 



XXX X 



X X X X 



X X X X X 



XX 
























8 1 

-S^ 
-j.. ^ 












-g 





4 












ill 

O *S .S 

^ a ^g g 



36 



MARY D. ROGICK AND HANNAH CROASDALE 



S 

8 




w 
h-1 

m 
< 
H 



UBOZoXjq 
sun TO M pajcpossB 
dds \v.S\v paj 'ON i^l"! 


*- -^ l-^ t^ rt 1 OO re 






vjVMjvif viuawKpoiftf 


X X 


i>. 


X X 


vysnfqns vjauwpotj-^j 




rt< 


X X 


vjvSnJva vtuoiftftstCjoj 


* X 


00 


X X 


SU31S3.lStU OtUOtflflSlCjOJ 


X X 


t-^. 


X XX 


v.<Siu miioti^tsfjoj 


X 


ro 


X 


V)v3noiJ viuoytfts/CjOfj 




- 


X 


snpunjo4 saptitfoj 




tN 


X 


V9JIA.3S VUVUDIJJ 


X 


<D 


X 


vtjofiuu.iqM2ui vMtitfoiiKiiJ 


X X X X X 


(T> 
<N 


XX XX 


l3DlpO.tg DJOljCfOflXljJ 


XXX X 


ID 


X X 


suaqn* sfcjpojfcqj 


X 


o 


X X 


ds wnmutvijioijjtj 




- 


X 


V.I9fftJOf Vt.Wll3V.lf) 


X 


ro 


X 


sapwiuafuoy vi.ivtfyv.i{) 




* 


X 


wnsoifAAiy 
utn3M<].tn4 uinnwpojsfj 


X X X X 


^H 


X X 


ds o.m?i<foj(fiCJ3 


X 


^H 


X 


sijvuryfo VUIJIVMJ 


X 


^ 


X 


sncfsiM srupuoijj 


X X X X X X 


O 
<N 


XX XX XX 


VptlMVtf DttflUVIf^) 




fO 


X 


uiiuqiu mntuiDMj 


X 


UO 


XXX 


aiujofuqn.i uiniiuvMj 


X 


1 I 


X 


wnasojt uotniuvi t )tjjD^) 


X 


^-H 


X 


V43U3} VlptljpMSy 




* * 


X X 




mbranipora tllberculata 
-.roporella ciliata 
icellina cernua 
izoporella biaperta 
izoporella unicornis 
uparia ambigua 
ittina trispinosa 


tn 

IS 

-M 

c 
o 

o. 
a 

tn 

C 
rt 
O 
N 
O 
*> 

L 

JO 

O 
^ 

- ^ 

3^ 


lected at Woods Hole 
lected at Martha's Vineyard 
lected in Vineyard Sound 
lected at North Falmouth 
lected on Penikese Island 
lected at New Bedford 
lected at New Rochelle 
lected at Rye, N. H. 
m Puerto de la Paloma, Uru- 

y* 




<u .5 "c ^ jg t ~ 
^^c^^^c^co 




H 


oooooooo2 
UUUUUUUUfc So 



c* 

^ 

Q 

JD 
C 



c/) 



OJ 

a 



MARINE BRYOZOA, III 



37 





a 
a 

o 

I 



-H ^ 

3 1 

W s 



spooA\ 

J-BAJBJ 

3upnpO-id-oAjquia 
' 



sjat[4o 'pa-ueis 
-un suopBAJasqo JEUOS 
jaj -jaquinu api3:juaj_ 



uoissnosip sapads 
ucozoAjq i[3Ba japun aag 
sja^joA\ jgq^o Aq pajjodaj 
SBA\ uEozoA'jq sit]; 
UIDJJ BjauaS 10 -dds 
jo -ox aqi ;o ;si[ 



III 'II 'I saiq^x ui pa^od 

-3J SBA\ UBOZOAjq lpU[A\ 
UIOJJ "dds [ESjB JO 'OJs{ 



BJ ap ojjanj 



-H 



> 

3 






= c ^ 

3 s 3 



-H 



e 



* 

O 



I 

O\ 



punog pjEAauiy\ x 



XX X 



XXX 



XXX 



XX 



XX X X X X 



ssep\; ' 



A\aj\j 



XX 



X X X X 



asa^jiuaj 



2 e a 



38 



MARY D. ROGICK AND HANNAH CROASDALE 



8 

^> 

-<, 

8 



W 
J 

CQ 



JB aSE}S 8UISEapJ-EAJt;i 

jo 3upnpojd-oAjquia 
'HajjAO-pajiy uj 


-H -H-H 

hi) bfl bi) 

3 33 

< -ti_ << 

r~t fe/D ***** "**'' 

'*3 ^ *3 *3 




pajjBjs sjaijio 'pajjB}s 
-un suoijEAJasqo JBUOS 
-jaj uaquinu apEiuajL 


* 
* * <"[' oo 

'Y'-o^)*T H < T'ooc s )-<-'O 
^!, - 1 - 1 * - 1 * ..^ 3 

' 7 rt 

00 




uoissnosip sapads 
UBOzoAjq ipBa japun aag 
sja?]JOA\ jaqio Aq pajjodaj 

SBA\ UBOZOAjq SltH tpHJM 

uiojj EjauaS jo -dds [B3[E 

JO 'O]^ aq} JO }ST[ JBpJBfJ 


^^ OO ^O ^O ro ^O I O ^O 




III 'II 'I S3 IQ"1 ln paiJOd 

UIOJJ 'ddS JB3[B (O - Ofy[ 


-------- .o-jo^ 


^. 


'BiuoiBj B[ ap oi-ianj 


X 




SSBJ^ 'UMOlaDUIAOJJ 


X 




aju.sdu^H Ma N 'aA H 


X XX 




^(jo^\ A\a^ 'a|[ai^Do>{ A\a^ 


XX XX 




'SSBJ^ 'ajo|^{ spoo^\ 


xxxxx xxxx 


X 


'SSB]/^ 'pJBAaiIIy\ S $ BIpJBp^ 


X X XXXXX 


X X 


SSBJAJ; 'punog pjBAauiy\ 


XX X 




SSBJ^; 'tpnouiiBj in-iofvj 


X X XXXX 


X 


SSBJ\; 'pjojpag A\a[s[ 


XX XX 




SSBN - P UB IS1 asa,,ua d 


X X 






5 Si a -a 

MO* !JU 

^ '~.|-a : s-2l 'S'-" ~ 

tO .^T ^ f^i t^J J*-* g* i,^ IS. ^ 

^o ^v ^o ^; *S ^ c3 <o ^ "^ ** r ^> 

.S.*e^^^aeeS 

c^. ^ F^;* * v "^^ "^^ ^^ --* '*** "^^ ^ 

S e'| i's S|H. 

""^cL^liS ^^^-S.-a^ 

r> ^ TT 1 tS Si *- v-CX^^^^e- 

11^ ^^.|"i 1 .. f 

CLI ^j S: ."^ .v^ ^o ^j &i ^ "^ ^ f^x: 
r~^r-s2>C'~'*-! L ^-i'^r? i ^ ^ ^ 
bqbqti,K^.l;^^^^c^cococo 


Scruparia clavata 
Smittina trispinosa 




\O t^ 1 * OO O^ ^> " ' CN <*O ^ *O MD l"^ CO 
^HT-IT i^-tCNCNCNCNCNCNCNCNCN 


^^\ ^^ 

CN rO 



a 
m 



4) 



C 03 



O 

OJ 



tn 

<v 



. 

tn O. 



O 

rt 



qj l>l 

c ^ 

3 

a >, 

"^ QJ 

o > 



03 ~ 

<-> u 

C C 

P tn 



C rt 
4) <U 



3 SJ 
C -C 

4^ 

* 

c c 

' 



C C 
bo C 



-Z 3 P 



to -T3 
0) is 

2 5 

03 



45 

>- 



0) 



H ~ 



o 



MARINE BRYOZOA, III 39 

LIST OF COLLECTED ALGAE 
CHLOROPHYCEAE (green algae) 

1. Cladophora gracilis (Griffiths) Kutzing, forma tennis Farlow 

2. Enteromorpha intestinalis (Linnaeus) Link (a proliferous form) 

3. Ulva Lactuca Linnaeus var. rigida (C. Agardh) Lejolis 

PHAEOPHYCEAE (brown algae) 

4. Ascophyllum Mackaii (Turner) Holmes et Batters 

5. Ascophyllum nodosinn (Linnaeus) Lejolis 

6. Chorda Filum (Linnaeus) Lamouroux 

7. Cladostephus vcrticillatus (Lightfoot) C. Agardh 

8. Desmarcstia aculcata (Linnaeus) Lamouroux 

9. Fucus evanescent C. Agardh 

10. Fucus vesiculosus Linnaeus 

11. Fucus vesiculosus var. spiralis Farlow 

12. Laminaria Agardh li Kjellmann 

13. Laminaria longicruris De la Pylaie 

14. Sargassum Filipendula C. Agardh 

RHODOPHYCEAE (red algae) 

15. Agardhiella tcncra (]. Agardh) Schmitz 

16. Callithamnion roscum (Roth) Harvey 

17. Ceramium rubriforme Kylin 

18. Ceramium rubruui (Hudson) C. Agardh 

19. Champia parvuhi (C. Agardh) Harvey 

20. Chondrus crispus (Linnaeus) Stack-house 

21. Corallina officinalis Linnaeus 

22. Cryptopleura sp. 

23. Cystoclonium purpnrcum (Hudson) Batters var. cirrhosum Harvey 

24. Gracilaria confcrvoidcs (Linnaeus) Greville 

25. Gracilaria -foliif era (Forsskal) BpYgesen 

26. Lithothamnium sp. 

27. Phycodrys rubens (Hudson) Batters 

28. Phyllophora Brodiaci (Turner) J. Agardh 

29. Phyllophora membranifolia (Goodenough et Woodward) J. Agardh 

30. Plnmaria scricca (Harvey) Ruprecht 

31. Polyidcs rotund us (Gmelin) Greville 

32. Polysiphonia cloiujata (Hudson) Harvey 

33. Polysiphonia nigra (Hudson) Batters 

34. Polysiphonia nigrcsccns (Hudson) Greville 

35. Polysiphonia varicgata (C. Agardh) Zanardini 

36. Rhodonicla subfusca (\\ r oodward) C. Agardh 

37. Rhodymcnia palmata (Linnaeus) Greville 

Below is a list of the 30 bryozoan species which were found growing on the 
various algae examined by the authors. 



40 MARY D. ROGICK AND HANNAH CROASDALE 

LIST OF COLLECTED BRYOZOA 
ENTOPROCTA 

1. Pedicellma cernua (Pallas) 1771 
ECTOPROCTA 

Cyclostomata or Stenolaemata 

2. Crisia cburnca (Linnaeus) 1758 

3. Lichcnopora hispida (Fleming) 1828 

Ctenostomata 

4. Acvcrrillia armata (Verrill) 1873 

5. Acvcrrillia sctigcra (Hincks) 1887 

6. Alcyonidiwn poly own (Hassall) 1841 

7. Bozvcrbankia gracilis Leidy 1855 

8. Bozvcrbankia imbricata (Adams ) 1800 

9. Flnstrclla liispida (Fabricitis) 1780 

Cheilostomata 

10. Aetca sic a (Couch) 1844 

11. Buynla CHcnllifcra Osburn 1912 

12. Bugula flabcllata (Thompson) 1848 

13. Bugula tnrrita (Desor) 1848 

14. Callopora anrita (Hincks) 1877 

15. Ccllcpora diclwtoma Hincks 1862 

16. Cribrilina aiinulata (Fabricius) 1780 

17. Cribrilina punctata (Hassall) 1841 

18. Cryptosula pallasiana (Moll) 1803 

19. Electro liastingsac Marcus 1938 

20. Elcctra pilosa (Linnaeus) 1767 

21. Hippoporina contracta (Waters) 1899 

22. Hippothoa hyalina (Linnaeus) 1767 

23. Membranipora lacroi.rii ( ?) 

24. Membranipora titbcrcnlata (Bosc) 1802 

25. Microporclla ciliata (Pallas) 1766 

26. Schizoporella biapcria (Michelin) 1842 

27. Schizoporella ntiicornis (Johnston) 1847 

28. Scrnparia amb'njua (d'Orbigny) 1841 

29. Scrnparia clavata Hincks 1857 

30. Sinittiiia trispinosa (Johnston) 1825 

BRYOZOAN GROWTHS ON ALGAE 

The bryozoa form white, grey, yellow, salmon-pink, or brown growths on the 
algae. Some bryozoan colonies are thin, flat, encrusting and closely adherent. 
Others are dendritic, arborescent, or may form a fuzzy mass of tiny vesicles. Still 
others coat the algae with a gelatinous, rubbery, or leathery film. The calcareous 



MARINE BRYOZOA, III 41 

bryozoa often retain their zooecial patterns and specific characteristics pretty well 
even though the algal host specimens have been dried and pressed in the normal 
course of herbarium sheet mounting. It was no harder to identify Hippothoa 
liyalina from a dry 1874 herbarium mount of Pliycodrys rub ens than from a 
freshly collected alga. 

Bryozoa grow on various parts of the algal plant. The holdfast processes of 
Laminaria and related forms are excellent sites for attachment of at least 21 hard, 
horny, or soft bryozoan species. Bryozoa grow on and between the holdfast proc- 
esses as well as on the rocks to which the holdfasts adhere. Laminaria and Rhody- 
menia blades are favorite attachment sites for Electro, pilosa which is very common 
and especially abundant on these algae, sometimes coating both sides of the entire 
blade for an area of several inches with a thin, frosty-white, single-layered cover of 
contiguous bryozoan colonies. Membranipora titbcrcitlata has the same habit of 
extensively encasing its algal hosts with the fine bryozoan mesh. 

The basal or most proximal parts of Clwndnts crispus and Phyllophora are 
encrusted by many bryozoans like Aeverrillia, Bowerbankia, Cellepora dichotoma 
and Hippothoa hyalina, while the most distal tips are somewhat less often utilized 
for bryozoan attachment. Sometimes, if the bryozoan growth is especially rich or 
dense on these two algal genera, the whole blade may be covered. Alcyonidium 
may encase a whole blade and sometimes extend even beyond the tips of the plant. 
The two Schizoporellae also may grow so readily as to produce shelf-like exten- 
sions of the colony beyond the plant thallus. 

The basal parts of Ascophyllum and Fucus are generally favored by the bryo- 
zoan colonies, as are the crevices and depressions around the airbladders and 
where branches originate. Flustrella hispida and Bowerbankia particularly favor 
these plants. 

The few zoaria (bryozoan colonies) found on the green algae generally were 
small, consisting of only a few zoids, and did not produce such luxuriant and 
extensive growths as did the species which grew on the browns and reds. 

The zoaria, as a rule, were one layer in thickness on the algae, with the excep- 
tion of occasional specimens of Schizoporella biapcrta, S. itnicornis and Sinittlna 
trispinosa, which might be laminate. The laminate condition is more common on 
the firmer substrates (rocks) than on algae. Hippothoa liyalina and Electro 
pilosa were always single-layered on the plants. 

Of the six most frequently encountered bryozoa (Actca sica, Bowerbankia 
gracilis and Crisia ebitniea each on 18 algal species. Electro pilosa and Hippothoa 
hyalina each on 17, and Biiynla tnrrita on 16 algal species) the least conspicuous 
is Aetea. It readily escapes detection unless the alga is examined microscopically. 
Because of their very characteristic growth habit and general appearance, Electra, 
Bugula, Bowerbankia and Crisia can be recognized with the unaided eye. Hip- 
pothoa, w r ith a little practice, becomes recognizable because it forms small, short, 
calcareous, white sheaths around the thin algal stalks and filaments. Hippothoa 
especially accommodates itself readily to the smallest filaments and branches. 

The bryozoa occurred in close association on the same algal thallus with many 
other animal forms. Numerous shells of Spirorbis sp. grew alongside the 
Lichenopora hispida from Rye, N. H. Sponges, hydroids, annelid worm tubes, 
Botryllus schlosseri, Molgida inanhattcnsis, Styela, Foraminifera, and several spe- 
cies of bryozoa were sometimes found on a heavily populated alga. Hydroids, 



42 



MARY D. ROGICK AND HANNAH CROASDALE 

PLATE I 




MARINE BRYOZOA, III 43 

Foraminifera, and several bryozoan species often were found on the same blade 
of Ascophyllum, Chondrus, Laminaria. Phycodrys, or Phyllophora. Aeverrillia 
and Aetea would sometimes grow on Bugula and hydroid colonies as well as on 
algal thalli. 

The tentacle number and the time of larval production were obtained for 
some species but not for all because sometimes the colonies died before they could 
be examined, and sometimes the organisms were so exasperatingly slow in extend- 
ing their tentacles for a count. Such data as could be obtained are listed in Table 
IV and also in the species' descriptive section which follows. 

AETEA SICA 
(Figures 1-3) 

Aetea sica is fairly common, although not reported from this region previously. 
It forms a thin, white, bristly tracery on 18 different algal species. The zoids 
resemble fine upright tubes just big enough to be seen with the unaided eye. 
Slender stolons connect the bases of the upright zoids and adhere closely to the 
substratum (Fig. 3). Nine to eleven tentacles were counted in a few zoids. 
Ovicells were filled with live developing pinkish larvae from at least July 31 
through August 6. 

The feature by which Marcus (1937. p. 29) distinguishes Aetca sica from 
the previously reported Actea anguina is the ratio of the aperture (opesium) 
length to opesium width. The opesial ratio for A. anguina is between 1.7: 1 and 
2:1. For A. sica it is between 2.6: 1 and 4:1. In Figure 1, one zoid has a 
4 : 1 ratio. If the ratio is a valid characteristic of the two species, then some of 
the previously reported Aetea anguina from the Woods Hole area must belong to 
Aetea sica. 

PLATE I * 

FIGURE 1. Actca sica. Upright zoid (Z) growing from a punctate stolon (S) enlarge- 
ment. The opesium (A) of this zoid is about three times as long as wide (a 3: 1 ratio). 
The scale above applies to this figure. Hadley Harbor specimens, VII-28-1939. 

FIGURE 2. Actea sica. Detail of a broken stalk. 

FIGURE 3. Actca sica. A colony of five zoids (Z) arising from stolons (S). The upper 
right zoid has an opesium (A) about four times longer than wide (4: 1 opesial ratio). The 
scale above applies to this figure. 

FIGURE 4. Aeverrillia armata. A sprig of a colony collected from Lagoon Pond, Martha's 
Vineyard, VIII-17-1945. 

FIGURE 5. Alcyouidiuin polyoinn. A polypide torn out of the colony, in its natural with- 
drawn position. It consists of tentacles (T), esophagus (E), caecum (C) and rectum which 
in this sketch contains a large dark fecal pellet (F). Collected off Davenport Park, New 
Rochelle, N. Y., on IX-22-1945. 

FIGURE 6. Alcyonidium polyoitm. Part of a young, fairly transparent colony most of 
whose zooecia contain a sketchily outlined withdrawn polypide (P). Of the same date and 
collecting locality as specimens of the preceding figure. 

FIGURE 7. Alcyonidium polyoum. Somewhat thicker-walled colony, with slightly raised 
orifices (A). Collected at Black Rock, New Bedford Harbor, on VIII-8-1945. 

* Figures on all plates, with the exception of Figures 13, 14 and 28, were drawn with the 
aid of a camera lucida. The species are alphabetically arranged except for Figure 14. 



44 



MARY D. ROGICK AND HANNAH CROASDALE 

PLATE II 
I "Z s?*3?&^ I I 

I ^B ' ' -~~ ?**S - 'r-^lS ' 

^J $SSs&t^Ssi^&N3i>i I 1 1 




HA- 




MARINE BRYOZOA, III 45 

AEVERRILLIA ARMATA 
(Figure 4) 

Aeverrillia aruiata is transparent, yellowish, and horny, and occurs on 
Laminaria Agardhii and Phyllophora incinbranifolia. The latter alga was heavily 
encrusted with ten other bryozoan species and several algal species. Aeverrillia 
annata consists of numerous slender, paired autozoids arising from narrow stolons 
which cling closely to the plant but which can be pulled off as slender threads. 
This species is very similar to A. sctiyera which was discussed very fully in a 
previous study (Rogick, 1945a), except that it lacks the basal clasping processes 
of A. setigcra. The polypides have eight tentacles in both species of the genus. 

AEVERRILLIA SETIGERA 

This delicate bryozoan was found growing inconspicuously on eight algal 
species. It clings closely to the plant thallus. It was pictured adequately in the 
previous study (Rogick, 1945a), so no figure of it is here included. The resem- 
blance between it and A. annata is so close that one could easily mistake the one 
for the other. 

ALCYONIDIUM POLYOUM 
(Figures 5-7) 

The various Alcyonidia are difficult to tell apart. The present Alcyanidium 
polyoinn forms a firm gray or sometimes slightly yellowish crust around the hold- 

PLATE II ' 

FIGURE 8. Boit'erbankia gracilis. An uncrowded stolonate colony of nine full-grown 
zoids (Z) and five smaller buds (B), growing on an algal filament (A). Other structures 
shown are : (C) caudal process; (G) gizzard; (M) parieto-vaginal musculature; (O) squared 
orifice; (P) polypide ; (S) stolon; (SC) setigerous collar. Collection site and date same as 
for Figure 7. 

FIGURE 9. Bowerbankia iinbricata. Upper part of an extruded polypide showing ten 
tentacles (T) which upon retraction can be withdrawn into the tentacular sheath (TS). 
Around that is a stiff transparent setigerous collar (SC) which in turn can be withdrawn into 
the vestibular sheath (VS). Some debris has accumulated on the edge of the squared orifice 
(O). From Glen Island, New Rochelle, N. Y. on IX-16-1945. 

FIGURE 10. Boii'crbankia iinbricata. A crowded colony which was scraped from Chondrus 
crispus. Collection date and site the same as for the preceding figure. Three of the long zoids 
have their tentacles extended. Three smaller ones have their setigerous collars partly ex- 
truded. Three zoids are shown with the polypides within them. The following parts are 
labelled: (E) esophagus; (G) gizzard; (I) intestine; (S) stolon; (T) tentacles. 

FIGURE 11. Bugula cucullifcra. Four zooecia, each provided with four spines (S). The 
upper three zoids show, at the side of the opesium (A), the remains of the short peduncle 
which had borne an avicularium. From Provincetown, Mass., on VIII-18-1947 ; Dr. M. Doty 
collector. 

FIGURE 12. Bugula cucullifcra. Three fertile zooecia topped with ovicells (O). The 
middle zooecium bears an avicularium (V), the other two have lost theirs. (A) is the 
opesium. The second row of zooecia which normally would be at the side of these zooecia 
was incomplete and was therefore not shown here. Same collection date and site and drawn 
to the same scale as the preceding figure. 

FIGURE 13. Bugula flabcllata. A freehand sketch, showing the close tuft-like mode of 
colony growth. About natural size. 

FIGURE 14. Bugula turrita. A freehand sketch, showing the dainty, open spiral mode of 
colony growth. About natural size. 



MARY D. ROGICK AND HANNAH CROASDALE 




w 



PH 



MARINE BRYOZOA, III 47 

fasts, stalks, and blades of at least five algal species. Prenant and Teissier (1924, 
pp. 23, 27) reported Alcyonidium from Ascophyllum, Chondrus, Fucus, Himan- 
thalia, Laminaria (saccharina?) and Saccorhiza bulbosa. Sometimes it coats the 
entire alga, using the various branches as cores around which to grow. The col- 
ony is rubbery to the touch. 

The polypides had 16 tentacles. Measurements for 18 zoids ranged thus: 
zoid length 0.36-0.648 mm. and zoid width 0.240.504 mm. These are similar 
in range to figures given by Harmer (1915, pp. 37-38). The extremes in tentacle 
number given by various authors are 12 (Harmer, 1915, p. 38) to 20 (Silen, 
1942, p. 11). 

BOWERBANKIA GRACILIS 

(Figure 8) 

Bowerbankia gracilis is very common. It forms a soft grayish furry mass on 
18 algal species. It consists of a number of transparent tubes clustered along a 
stolon, sometimes so densely that the stolon is scarcely visible. Caudal processes 
appear on some zooecia. The eight tentacles can be counted only when the animal 
is alive and in the expanded state. The zoids in Figure 8 are in the retracted 
state with the tentacles and gut (collectively called "polypide") withdrawn into 
the body cavity. Under such conditions the squared orifices show nicely. 

BOWERBANKIA IMBRICATA 
(Figures 9-10) 

Bowerbankia imbricata was found on only five algal species by the authors. 
Additional species on which it has been reported are : Ascophyllnm no do sum 
(Adams, 1800, p. 11), Corallina officinalis (Hincks, 1880, p. 521), Cystoseira 
fibrosa, Fucus terrains (Joliet, 1877, p. 294), Desmarestia aculeata, and Fur- 
cellaria jastigiata (Thompson, 1840, p. 252). Colonies may cover extensive areas 
of several inches, coating the "stems" and thalli of Chondrus. They do not ex- 
clude other forms from growing on the alga but may grow among hydroids, 
sponges, and other encrusting forms. 

Superficially, dense growths of Bowerbankia imbricata and B. gracilis are 
indistinguishable. Imbricata colonies whose zoids were filled with large ciliated 

PLATE III 

FIGURE 15. Bugula flabcllata. The upper parts of three fertile ovicell-bearing zooecia (F) 
and an ordinary zooecium (R). Other structures shown are: (A) opesium, (O) ovicell, (S) 
spine, (V) avicularium. These same labels apply to the other figures on this plate. 

FIGURE 16. Bugula flabcllata. Broad flabellate branches with up to 6 rows of zooecia 
(Z) per branch. Some have ovicells, some avicularia, or both, and others have neither, at 
the moment. Drawn to the same scale as Figure 18. 

FIGURE 17. Bugula turrita. Four fertile zooecia, each topped by a very shallow, fragile 
ovicell, set at an angle on the upper edge of the zooecium. Spines are well developed in this 
colony and one avicularium is shown. Drawn to the same scale as Figure 15. Collected at 
Woods Hole, VI-30-1938. 

FIGURE 18. Bugula turrita. Branches showing biserial arrangement of zooecia (Z). 
Some zooecia bear ovicells (O). 



48 



MARY D. ROGICK AND HANNAH CROASDALE 

PLATE IV 



2H 



23 




MARINE BRYOZOA, III 49 

globular larvae were salmon pink in color because the red pigment of the larvae 
showed through the parent zoid walls. Such embryos were especially abundant 
in colonies collected during the first ten days in August (1947). Some embryos 
were found in colonies collected as late as August 31. Joliet (1877, p. 295) 
observed larvae during the month of July and reported that sexual reproduction 
took place from the end of June to early August. 

Somewhat reniform larvae were released in great numbers on the morning of 
August 9, 1947. After a free-swimming period they attached to the substratum. 
Metamorphosis proceeded speedily, taking less than five minutes in some cases. 
The red color became concentrated at one end of the metamorphosing larva. 

Adult zoids generally have ten tentacles and are square-topped when retracted. 
Measurements of seven retracted zoids were as follows: zoid length 0.925-1.374 
mm.; zoid width 0.178-0.291 mm.; stolon diameter 0.040-0.101 mm. 

Some very young colonies consisting of only one or two developing zoids had 
nine tentacles but their development could not be followed beyond a few days, so 
it could not be determined if these in time would increase their tentacular number 
to ten. 

BUGULA CUCULLIFERA 

(Figures 11-12) 

Small fragments of this Bugula were found on Fiicus evancsccns and Rhody- 
uieuia pahnata from Vineyard Sound on VIII-1-1945, on Laminana longicrnris 
from Provincetown, Mass., on VIII-18-1947 and on Pliyllophora ineiiibranifolia, 
along with much Crisia eburnea and Aetca sica from New Bedford Harbor, on 
VIII-8-1945. 

PLATE IV 

FIGURE 19. Callopora aurita. Nine zooecia (Z) each capped by a rounded ovicell (O) 
which is decorated by a raised triangular ridge. Spines (S) and avicularia (V) are present 
near the large opesia (A). The same letters apply to the other figures on this plate. A cal- 
cined specimen from which all the soft tissues have been burned away. Drawn to the same 
scale as Figure 24. 

FIGURE 20. Cellepora dichotoiiui. Part of a very lightly calcined specimen showing the 
shape of the aperture and the position of the avicularium. From Nobska Beach driftweed, 
Woods Hole, VII-25-1944. Drawn to the same scale as Figure 21. 

FIGURE 21. Cellepora dichotoma. Aperture of a very young zooecium. 

FIGURE 22. Cellepora dichotoma. Portion of a moderately calcified colony. An avicu- 
larium is borne on the side of the umbo (U) and faces toward a sinus (SI) in the peristome 
or raised shelf encircling the front of the aperture. The ovicells have pores (P). 

FIGURE 23. Cribrilina aninilata. Seven zooecia. Drawn to the same scale as Figure 24. 

FIGURE 24. Cribrilina punctata. Seven zooecia, three of which are capped by ovicells. 
One or two avicularia border the wide aperture. From Penikese Island, Mass., VIII-3-1947, 
on Chondrus crispits. 

FIGURE 25. Cribrilina punctata. A more heavily calcified zooecium with 4 spines above 
the aperture. From Gay Head, Martha's Vineyard, VII-30-1946. Drawn to same scale as 
Figure 21. 

FIGURE 26. Crisia eburnea. Four internodes, separated by dark yellow horny joints or 
nodes (N), bear a number of tubular autozoids (Z). One internode bears the greatly swollen 
ovicell or ooecium. The zooecia have numerous pseudopores (PS). From Black Rock, New 
Bedford Harbor, VIII-8-1945. Drawn to same scale as Figure 24. 



50 



MARY D. ROGICK AND HANNAH CROASDALE 

PLATE V 

Ifcf.fc /t. 




MARINE BRYOZOA, III 51 

Some embryo-filled ovicells were present. Very long rhizoid processes grew 
from the basal part of some of the colonies. Thirteen tentacles were counted on 
one zoid. 

BUGULA FLABELLATA 
(Figures 13. 15, 16) 

A small colony of B. flabcllata was found on Fucits vesiculosus. It was far 
less common than B. titrrita. Also, it seemed to prefer attachment to piles, live 
cars, and other submerged wooden objects rather than to algae. It is a very 
sturdy form, growing in thick, fan-shaped, yellow-orange tufts (Fig. 13) which 
are about a half inch tall. 

Glass slides submerged in Eel Pond at Woods Hole from August 13 to 
August 31, 1945, were heavily overgrown with various animal forms, including 
Bugula flabellata. Colonies of the latter were by then about a /4 inch tall and con- 
tained hundreds of zoids. 

According to Grave (1933, p. 384) its breeding season is between June 1 
and November 15. 

BUGULA TURRITA 
(Figures 14, 17, 18) 

Bugula titrrita is very common, growing on at least 16 algal species. In gen- 
eral appearance it is more plant-like than animal-like. It is of yellow-orange color 
and has a soft, fluffy, but firm texture. It has a beautifully spiralling manner of 
growth (Fig. 14). The colony branches into a number of spiralling "turrets." 
Some of the colonies may be P/4 inches tall. 

The tentacle number is about 14. 

Ovicells were seen in colonies collected from the end of June through mid- 
August (Fig. 17). Many young colonies developed from released larvae during 
that time. 

PLATE V 

FIGURE 27. Electro liastingsae. Fifteen zooecia from the central part of a colony. One 
zooecium has lost all the spines around its opesium. The others have retained a varying 
number. Calcined specimen. 

FIGURE 28. Electro hastingsac. A freehand sketch showing the flat, spray-like mode of 
growth which is so characteristic of this species_. About natural size. 

FIGURE 29. Electro pilosa, long-spined form. Tip of an alga, Dcsinarcstia aciilcata, com- 
pletely encased by a bryozoan colony some of whose zooecia show an unusually long median 
spine. Shown in silhouette. Collected off Gay Head, Martha's Vineyard, VII-30-1946. 
Drawn to the same scale as Figure 33. 

FIGURE 30. Electro pilosa, short-spined form. Four zooecia whose lowest, median spine is 
heavier and longer than the other opesial spines but not so long as the spines pictured in Figure 
29. The two upper zoids show the crescent-shaped operculum rim in the upper part of the 
opesial area. The lower frontal wall of the zooecium is marked by numerous tremopores. 
From Devil's Foot, Woods Hole, VII-9-1945. 

FIGURE 31. Flustrella hispida. A very young, spineless zoid. From Woods Hole, VIII- 
15-1939. Drawn to same scale as Figure 32. 

FIGURE 32. Flustrella hispida. Two old zoids showing heavy "chitinization" of spines 
and lips of the orifice. The left zoid shows only circumoral spines while the right shows those 
and also additional spines located lower down on the zoid. From same colony as Figure 31. 

.FIGURE 33. Flustrella hispida. Twelve spine-encircled zoids from a less heavily "chitin- 
ized" part of the same colony as Figure 32. 



52 



MARY D. ROGICK AND HANNAH CROASDALE 

PLATE VI 

H3 

A. 
O 




MARINE BRYOZOA, III 53 

CALLOPORA AURITA 
(Figure 19) 

Callopora aitrita was not abundant on algal material, being found more com- 
monly and in more extensive patches on rocks. Very small white colonies were 
found on specimens of PJiycodrys rubens collected from Rye Ledge, Rye, New 
Hampshire, on IX-30-1945 ; on Pliyllophora ineinbranijolia dredged from Great 
Harbor, Woods Hole, Mass., on VIII-8-1946, and on holdfasts of Laminaria 
AgardJiii. The colonies form a fine encrusting calcareous mesh on the algal 
thallus. 

Ovicells were present in the colonies, but it was not possible to determine 
whether they were tenanted by larvae at the time of collection. Twelve tentacles 
were counted on one zoid. 

CELLEPORA DICHOTOMA 
(Figures 20-22) 

Its small, white, calcareous zoaria grow on Clwndrus crispus, Gracilaria con- 
jervoides, Pliyllopliora Brodiaei and P. membranifoHa. Its zoids are crowded 

PLATE VI 

FIGURE 34. Hippoporina conlracta. Portion of a young, uncrowded colony showing nine 
zooecia (Z), four of which are without avicularia (V) and three of which have a small 
rounded avicularium and two of which have spatulate avicularia (V). The distinctive serrate 
aperture (A) is readily distinguishable in these not heavily calcified zoids. The peristome 
( PR) is prolonged into a small bump or mucro below the aperture in the central zoid. Areolae 
(L) border each zooecium. The same labels apply to the other figures on this plate. Speci- 
mens dredged from Great Harbor, Woods Hole, on VIII-8-1946. 

FIGURE 35. Hippoporina contracta. A small, slightly pointed avicularium with part of 
its aperture serrated. Drawn to same scale as Figure 43. 

FIGURE 36. Hippoporina contracta. A small rounded avicularium with part of its aper- 
ture serrated. Drawn to same scale as Figure 43. 

FIGURE 37. Hippoporina contracta. A more crowded and calcified colony than that of 
Figure 34. The upper two zoids show the typical serrate aperture. Seven ovicells (O) with 
large, comma-shaped pores (OP) are visible. Beneath each ovicell is a large peristomice (AC) 
or peristomeal opening (here shown in black), at the bottom of which lies the distinctive serrate 
aperture (invisible in this picture). Calcined specimen. 

FIGURE 38. Hippoporina contracta. The serrate aperture characteristic of this species. 
The aperture, black in this calcined specimen, in life is closed over by an operculum which is 
pictured in Figure 41. The aperture has 14 to 18 small rounded denticles (D) and two large 
bifid cardelles (C). 

FIGURE 39. Hippoporina contracta. A spatulate avicularium seen at an angle. Drawn to 
tfie same scale as Figure 43. 

FIGURE 40. Hippoporina contracta. Upper half of a zooecium showing areola (L), 
denticles, cardelles, peristome (PR) and three spines above the aperture (A). Drawn to 
same scale as Figure 43. 

FIGURE 41. Hippoporina contracla. Operculum which closes the apertufe of the zooecium. 
It has a stiffened rim and lateral sclerites (LS). Drawn to same scale as Figure 38. 

FIGURE 42. Hippothoa hyalina. A colony showing a number of ordinary zooecia (Z) and 
a dwarfed one topped by an ovicell (O). Drawn to same scale as Figure 34. 

FIGURE 43. Hippothoa hyalina. Another view of the punctate ovicell, its dwarfed zooecium 
and a normal sized zooecium. The latter shows the typical aperture, rounded and with a sinus. 
The transverse grooving normally found in the zooecia is faintly indicated in the larger zoid. 
Specimens dredged off Gay Head", Martha's Vineyard, VII-30-1946. 



54 MARY D. ROGICK AND HANNAH CROASDALE 

against each other. Embryo-filled ovicells were present at the time of collection 
(July 25, 1944). 

There is some question as to the classification of this species. Ccllcpora arneri- 
cana, Cellepora avicularis and Ccllepora dichotoma show such integradation that 
their exact status or validity needs critical review by some future worker. The 
species of the present study is identical with Marcus' illustration of C. dichotoma 
(Marcus, 1938, Plate XI, Fig. 26). 

The species characteristics are as follows : ( 1 ) peristome with a sinus next to 
a raised umbo on the side of which is an avicularium facing the sinus; (2) aper- 
ture rounded, with postral sinus; (3) ovicell with pores, rounded and somewhat 
flattened; and (4) a few small pores (areolae) around the frontal wall of the 
zooecium. 

A heavily calcified zoarium may show the ovicells almost completely immersed 
on all sides except the frontal in the secondarily calcified zooecial wall. The 
frontal of such ovicells is provided with good-sized pores and is at a lower level 
than the secondarily calcified outer zooecial wall. 

CRIBRILINA ANNULATA 
(Figure 23) 

This encrusting species was very uncommon. Only one white calcareous 
zoarium was found on Phycodrys rubcns, from Rye, N. H., on the reverse side 
of the thallus from the finer, more fragile Cribrilina punctata. Three or four 
spines were present around the aperture. Marcus (1940, p. 203) reported C. 
annulata from Laminaria. 

CRIBRILINA PUNCTATA 
(Figures 24-25) 

Small patches of this fragile white calcareous form were found encrusting seven 
algal species. The number of spines around the aperture varied from none to 
five. The frontal pores were somewhat irregular in size and position. This was 
not a very common form ; only a few colonies appeared in the collection. 

CRISIA EBURNEA 
(Figure 26) 

Crisia eburnca was exceedingly common on 18 algal species. It was especially 
abundant on Chondrus crispus, the two Phyllophorae, and Phycodrys rubcns. 
A very large amount of it was collected from the driftweed along the beaches at 
Nobska, Gay Head, and Cuttyhunk. Dried specimens were just as useful as wet 
ones for taxonomic purposes. 

Prenant and Teissier (1924, p. 18) reported C. eburnca from Halidrys and 
certain Cystoseiras. 

It forms brittle, white, openly dendritic tufts up to 7 mm. tall on the thalli of 
the small, and around the holdfasts of the large, algae. A Crisia colony consists 
of a number of calcareous tubular zooecia forming internodes which are separated 



MARINE BRYOZOA, III 55 

from other internodes by short, narrow, yellowish to brown chitinous joints. A 
branch which consists of ordinary tubular zooecia (autozoids) alone is called a 
sterile internode. Three such are pictured in Figure 26. A branch which con- 
sists of a number of autozoids and a long, very swollen brood chamber (ooecium 
or ovicell) is called a fertile internode. One is pictured in Figure 26. In the 
identification of different species of Crisiidae the number of zooecia in the fertile 
and sterile internodes is important. In Crisia eburnca the sterile internode has 
four to eleven zooecia, and a fertile internode seven to ten (Borg, 1944, p. 158). 
Ovicells were found on specimens collected throughout the summer months. 
Embryos were seen in some on August 8, 1946. 

CRYPTOSULA PALLASIANA 

Cryptosnla pallasiana forms a round, flat, regularly patterned, pale orange to 
white encrustation on rocks, shells, and algae. It occurs more commonly and 
forms larger colonies on the harder substrates than on the algae but is not uncom- 
mon on the latter. It was found on eleven algal species which came from a 
number of collecting sites between Martha's Vineyard, Woods Hole, North Fal- 
mouth, and New Bedford (all in Massachusetts). They grew on the thalli of 
algae and on the Laminaria holdfasts. Colonies attached to Enteromorplia intcs- 
tinalis and Uha Lactuca var. rigida were young and small, consisting of few (five 
or less) freshly formed zoids (as of VIII 13 1945). Submerged glass slides, left 
in Eel Pond at Woods Hole for the first two weeks in July and kept a week- 
longer in running sea water in the laboratory, were well covered with many animal 
forms including Bugiila turrita, Pcdicellina cernna and Cryptosnla pallasiana. The 
Cryptosula colonies had from one to thirty zoids on these slides. Their polypides 
had 16 tentacles. 

Barrois (1877, p. 139) reported larvae in August and September. The Woods 
Hole specimens produced larvae in those months as wellas during June and July. 

Joliet (1877, p. 291) reported this bryozoan on Callothri.v pannorum. Prenant 
and Teissier (1924, p. 23) reported Cryptosula from other Laminariae, Himan- 
thalia, and Saccorhiza bulbosa. 

No drawings of Cryptosula are here included because the species was pre- 
viously figured (Rogick. 1945b, p. 3, Fig. 1). 

ELECTRA HASTINGSAR 
(Figures 27-28) 

A few small colonies of E. Jiastingsae encrusted the thalli of Fucus vesiculosiis 
var. spiralis and Laminaria Agardliii. Marcus (1938, p. 17) reported the bryo- 
zoan from Zostera. Sometimes it grows on the gill chamber of Libinia crabs. 
Generally, however, the bryozoan is found on hard substrates (rocks and shells) 
more often than on algae. 

Electra hastingsae is a fragile, white, calcareous species, forming completely 
adherent frond-like traceries on the substratum (Fig. 28). Some colonies lack- 
spines around the opesium. Other colonies have a variable number of very deli- 
cate ones, sometimes as many as 18. Some of the spines may break off (Fig. 27). 

A new zooecium may occasionally grow right out of the opesium of another 
empty one. Whether that is a case of regeneration or the settling of a new 
larva on an old colony, is not certain. Embryos were not observed. 



56 MARY D. ROGICK AND HANNAH CROASDALE 

ELECTRA PILOSA 
(Figures 29-30) 

Electra pilosa is an extremely common calcareous but fragile encrustation 
on 17 algal species. It has been reported previously from : Fucus scrratus 
(Joliet, 1877, p. 290) ; Ulva (Hutchins, 1945, p. 540) ; Laminaria saccharina 
(Leidy, 1855, p. 9); Furcellaria and Polyides (Marcus, 1940, p. 118); the 
Cystoseiras, Corallina (Prenant, 1927, p. 24) and Zostera (Prenant, 1932, p. 92). 

Electra pilosa forms grayish-white, single-layered colonies which spread like 
a fine, closely-woven mesh over large areas, sometimes a foot in length, of algal 
thalli. Laminaria and Rhodymenia thalli are particularly favored. Numerous 
colonies may grow toward and into each other to form an almost continuous thin 
crust over the thalli. The lacy Plumaria scricca fronds, in some instances, were 
completely encased in Electra pilosa. Many Foraminifera were scattered over 
the Electra. 

Great variation in degree of spination occurs. Several E. pilosa "forms" of 
dubious validity are mentioned in literature : forma t\pica, f. dent at a, f. la.ra and f . 
verticillata, differing slightly from each other, mainly in the presence or length 
of the principal median proximal spine. Borg (1930, p. 63) and others men- 
tioned that occasionally several of these growth forms may be found in a single 
E. pilosa colony, and therefore should not be considered valid varieties. 

The present writers found both long-spined (forma verticillata, Fig. 29) and 
short-spined (forma dentata, Fig. 30) growths in the collections, the latter be- 
ing far more common than the long-spined specimens. 

Tentacles numbered 12 to 14. 

FLUSTRELLA HISPIDA 
(Figures 31-33) 

Flustrclla Jiispida grows on five Woods Hole algal species: Ascophyllum 
nodoswn, Chondnis crispus, Fucus vesiculosus, Phyllophora membranijolia and 
Ulva Lactnca var. rigida. Also, the M. B. L. Collecting Crew has on numerous 
occasions brought in Ascophyllum covered with Flustrella from other localities. 
Additional algae from which it has been recorded are : Gigantina mamillosa 
(Hincks, 1880, vol. 1, p. 507) ; Fucus scrratus and Cystoseira (Joliet, 1877, p. 
292). It was far more common on Ascophyllum and Fucus than on the green 
or red algae in the Woods Hole region. 

Flustrclla Jiispida forms a brown, rubbery, and somewhat slimy crust over 
extensive areas of the algal thallus. The zoids are fairly soft and baggy (Figs. 
31, 32). Thirteen tentacles were counted on one specimen. Spines were lack- 
ing in the very youngest zoids (Fig. 31), but more mature ones show variation 
in distribution and number of spines (Figs. 32, 33). In the oldest parts of the 
colony the spines may become very thick and dark reddish brown, and appear 
mounted on horny pads (Fig. 32) about the zooecial orifices. In younger col- 
onies, and also in some older zooecia, as in the right zoid of Figure 32, spines 
appear elsewhere about the zoid than just around the orifice. The reinforced 
orifices are shaped like the top of a purse (Figs. 32, 33). 



MARINE BRYOZOA, III 57 

Barrois (1877, p. 214) found F. Jiispida colonies filled with embryos during 
the months of May, June and July. 

HlPPOPORINA CONTRACTA 

(Figures 34-41) 

White to buff-colored colonies of this species were found more often on rocks 
and shells than on algae. However, some did grow on Phyllophora Brodiaci and 
P. membranifolia, and were up to 2 cm. in diameter. The appearance of the 
colony varies greatly, depending upon the age of the colony, degree of calcifica- 
tion, the presence of ovicells and the nature of the substratum (compare Figs. 34 
and 37). 

The key character in identifying this species is the "beaded" aperture (Fig. 
38) whose circular outline, serrate antral border, and two bifid cardelles marking 
the postral border vary so little that they can be identified in either old or young 
colonies. The number of rounded denticles in the antral border ranges from 
14 to 18. In old. heavily calcified colonies (Fig. 37), the zooeciat wall and 
peristome around the aperture may increase in thickness so greatly that the pri- 
mary "beaded" aperture comes to lie considerably below the external body wall 
surface, at the bottom of a calcareous "well," the wall of which is formed by the 
peristome. The top opening of this calcareous "well" is called either the second- 
ary aperture or the peristomice (Fig. 37, AC). 

In younger, less calcified colonies, two to six oral spines, sometimes measur- 
ing 0.12-0.13 mm., may appear on the peristome (Fig. 40). These break off 
and their bases may become completely overgrown in the process of increasing 
calcification of the body wall. 

Marcus reported 12 tentacles for this species (1937, p. 98). 

The ovicells are quite characteristic also. They are smooth, hemispherical, 
and provided with a large, comma-shaped membranous area or pore (Fig. 37, 
OP) on the frontal surface. 

Six to thirteen marginal pores or areolae (Fig. 34, L) can be seen in the 
zooecial body wall. 

HIPPOTHOA HYALINA 
(Figures 42-43) 

Hippothoa hyalina was extremely common on 17 algal species in the Woods 
Hole region. Borg (1930, p. 84) listed it from Laminaria saccharina; Prenant 
and Teissier (1924, p. 22) from the Florideae and Dictyota, and Prenant 
(1927, pp. 26-27) from Laminaria cloustoni and Saccorhiza bulbosa. A dry her- 
barium mount of a pressed Phycodrys rubcns (collected by Dudley at Marble 
Head in Sept. 1874) was examined by the writers and found to contain easily 
recognizable and uncrushed H. hyalina zoaria. 

Hippothoa hyalina forms glistening white or grey calcareous patches usually 
from 1 to 8 mm. in diameter, either on or encircling the thalli of most of the 
mentioned algae and on the holdfasts of Laminaria and Rhodymenia. ''Stems" 
or filaments of Cystocloniuin pnrpitrcmn var. cirrhosum were encased in rough 



58 



HH 



MARY D. ROGICK AND HANNAH CROASDALE 

PLATE VII 

lOOxt. 

H9 

V- 



iir 




H6 



MARINE BRYOZOA, III 

calcareous sheaths of H. Iivaliua sometimes an inch in length. Often the sheaths 
of colonies were- arranged in a linear series, the total series attaining a length of 
several inches. 

Embryo-filled ovicells were plentiful in specimens collected during July and 
August in the Woods Hole area. Many ancestrulae or the single individuals 
from which a colony begins were observed in collections made up to August 11, 
1945. These ancestrulae arise from sexually produced larvae. Barrois (1877, 
p. 164) remarked that at Roskoff the embryos were carried in transparent ovi- 
cells in the months of May and June, so apparently the breeding season is of 
considerable length. 

LlCHENOPORA HlSPIDA 

(Figures 44-46) 

One fertile and less than a dozen small immature colonies of this species were 
found growing on Ph\codr\>s rubcns and Phyllophora mcmbranijolia collected 
from Rye, N.H. on IX-30-1945. 

The fertile colony (Fig. 46) has a brood chamber provided with a thin, 
rounded aperture and many small pores. The autozoids (Fig. 44) terminate in 
jagged edges. They are partly surrounded by reticulate alveoli (Fig. 45) which 
are lined with small calcareous projections from the interalveolar septa whose 
thickness is variable. 

Borg (1926) gives a good account of the development of various Cyclosto- 
mata, including the Lichenoporae, and discusses the terminology of the group. 

MEMBRANIPORA LACROIXII ( ?) 

Membranipora lacroi.rii is a species whose identification and synonymy are 
exasperatingly confused in literature. Part of this is due to vague original 

PLATE VII 

FIGURE 44. Lichenopora hispida. A fairly young colony showing autozooecia (Z) sep- 
arated by large cavities or alveoli (E). Drawn to same scale as Figure 46. 

FIGURE 45. Lichenopora hispida. Detail of the center of an immature though fair-sized 
colony, showing about 17 alveolar spaces (E), the interalveolar septa (SP) between them and 
the projections (K) from the calcareous cryptocyst of the septal wall. The sides of two auto- 
zoids (Z). Drawn to same scale as Figure 49. 

FIGURE 46. Lichenopora hispida. A damaged, fertile colony, showing an irregular, punc- 
tate brood chamber (O), the brood chamber aperture (OE) and numerous short (immature 
or damaged?) and some normal autozoids (Z). 

FIGURE 47. Microporclla ciliata. Nine zooecia, four of which have well-developed ovicells 
(O) and three of which have shelf-like beginnings of ovicells distal to the aperture (A). Each 
zooecium has a crescent-shaped ascopore (AS) and smaller frontal pores. Oral spines occur 
on the upper three zoids. The crescent-shaped ascopore and the hemispherical aperture are key 
characters for this species. 

FIGURE 48. Microporclla ciliata. A zooecium topped by an ovicell. Both have pores but 
of different size. Drawn to same scale as Figure 49. 

FIGURE 49. Microporella ciliata. A zooecium showing oral spines and a pointed avicu- 
larium (V) in a characteristic position. The avicularium may develop on either the right or 
left side of the zoid. Same collection area and date as Figure 43. 

FIGURE 50. Pediccllina ccrnua. A single zoid consisting of a stalk (ST) and a calyx 
(CA) containing the polypide (D) and rolled in tentacles (T). A few spines (P) occur on 
the stalk and calyx. From Black Rock, New Bedford Harbor, VIII-8-1945. 



60 



MARY D. ROGICK AND HANNAH CROASDALE 

PLATE VIII 




MARINE BRYOZOA, III 61 

descriptions, and part due to the apparently great variation in spination and de- 
gree of calcification of the zooecia. Such will continue to be the state of affairs 
until someone takes the trouble to make a very elaborate study of the variations 
of this species. The Woods Hole and New Rochelle specimens of the present 
study resemble the Mcmbranipora lacroi.rii pictured by Osburn (1912, Plate 22, 
Fig. 22), the Conopcum lacroi.rii pictured by Canu and Bassler (1920, Plate 13, 
Fig. 9), the Conopeum rcticnhtm pictured by Harmer (1926, Plate 13. Fig. 12), 
the Bifliistra aciculata of MacGillivray (1891, Plate 9, Fig. 5) and the Membrani- 
pora cntstnlenta- of Osburn (1944, Fig. 20, p. 32). Our specimens differ from 
the Conopcum rcticulmn pictured by Marcus (1938, Plate 2, Fig. 5A), the 
Conopeum lacroi.rii figured by Canu and Bassler (1923, Plate 29, Fig. 4), the 
Membranipora rcticulmn f. lacroixii and M. crustitlcnta of Borg (1930, pp. 63- 
65). The present study specimens definitely are not the M. cnistnlcnta of Borg 
because that species is pictured with a calcified operculum, a character not present 
in our specimens. Until the status and limits of the species are fixed, the present 
authors will continue to call it M. lacroi.vii, as in Osburn's 1912 paper. 

Membranipora lacroi.rii was found encrusting rocks, shells, and less fre- 
quently the algae Ascophyllum Mackaii, Chondrus crispus and Phyllophora mcm- 
branifolia. It formed a delicate, gray-white tracery which adhered so closely to 
the substratum, especially rocks, that it was difficult to dislodge. No avicularia 
or ovicells were found. The conspicuous triangular spaces mentioned as charac- 
teristic by Harmer and Marcus were not observed on our specimens. Calcifica- 

PLATE VIII 

FIGURE 51. Schizoporella biapcrta. Nine regularly arranged, moderately calcified zooecia. 
one of which is without an avicularium. Drawn to the same scale as Figure 54. Calcined 
specimen. 

FIGURE 52. Schizoporclla biaperta. A small ellipsoidal avicularium. Dra\vn to same 
scale as Figure 55. 

FIGURE 53. Schizoporella biapcrta. A small oval or somewhat pointed avicularium. 
Drawn to same scale as Figure 55. 

FIGURE 54. Schizoporclla biapcrta. A fertile area of a colony showing six ovicells. The 
middle ovicell is most nearly typical in appearance. The frontal area of its zooecium is more 
highly calcined than that of the other three zooecia above it, and than that of the zooecia of 
Figure 51. 

FIGURE 55. Schizoporclla biapcrta'. A moderately calcined zooecium showing two avicu- 
laria, the two apertural teeth (cardelles) and the sinus between them. A typical specimen. 

FIGURE 56. Schizoporclla biapcrta. A heavily calcified zooecium topped by an ovicell. 
The zooecial shape is atypical and due to crowding in the colony and to excessive calcification. 
The avicularium is heavily calcified. The depressed rim of the ovicell frontal has been acci- 
dentally over-emphasized and should look less depressed (see Figure 54, middle ovicell). 
Drawn to same scale as Figure 55. 

FIGURE 57. Schizoporclla unicornis. A sharply pointed avicularium. Drawn to same 
scale as Figure 55. 

FIGURE 58. Schisoporclla unicornis. Three zooecia, two of which have ovicells. The 
bottom zooecium is twice as broad as the other two to which it gives rise. The zooecial frontal 
wall and the ovicells have" pores. The sharply pointed avicularia vary in size and are near 
the aperture. 

FIGURE 59. Schisoporella unicornis. Another unusually shaped and very broad zooecium 
which would give rise to two rows of zoids. The pores of its frontal area and of the ovicell 
are better shown. Drawn to the same scale as Figure 54. 

FIGURE 60. Schisoporella unicornis. A smaller pointed avicularium, drawn to the same 
scale as Figures 55 and 57. 



62 



MARY D. ROGICK AND HANNAH CROASDALE 

PLATE IX 
I 185^ 




MARINE BRYOZOA, III 63 

tion was not heavy. Some New Rochelle specimens had up to ten spines, while 
others had no spines around the aperture all in the same colony. Some zoids had 
11 tentacles. 

This species was pictured in an earlier paper (Rogick, 1940, p. 167, Figs. 6-9). 

MEMBRANIPORA TUBERCULATA 

A specimen of Mernbranipora tuberculata was found on fronds of Cryptopleura 
sp. and Sargassum sp. which were sent to the writers by Dr. William Randolph 
Taylor. The Cryptopleura had come from Puerto de la Paloma, Uruguay, from the 
collection of Carmen de Franco de Pimienta. The Sargassum sp. had been collected 
by Adrian Questel on April 21, 1944, from Guadeloupe, Antilles. This Membrani- 
poran has been previously reported by Marcus from Laminaria (1939, p. 126) and 
Fucus (1937, p. 34) ; by Hastings (1929, p. 706) from Padina ; and by Osburn 
(1912, p. 231) from Sargassum baccijcrum which had drifted into Vineyard Sound. 

The extensive colonies of M. tuberculata spread flatly over the algal fronds in 
an ivory-white lacework, reminiscent of Elcctra pilosa. The two or three prominent 
calcareous tubercles at the anterior end of each zooecium from which this species 
gets its name may project separately and distally or may coalesce, forming a some- 
what rounded ledge. 

Since M. tuberculata was adequately pictured in both Osburn's (1912, as M. 
tclniclca) and Marcus' (1937) papers, no figure of it was included in the present 
study. 

MlCROPORELLA ClLIATA 

(Figures 47-49) 

Small, flat, circular colonies, white to irridescent in color, calcareous though 
fragile, encrust shells, rocks, and five algal species in the Woods Hole region. 
Prenant and Teissier (1924, p. 23) found Microporella ciliata on three additional 
algae : Himanthalia, Laminaria saccliarina and Saccorhisa bulbosa. Hadley Har- 
bor specimens were found growing on the same thallus with Foraminifera, Aetea 
sica, Crisia eburnea, Hippothoa hyalina, and Schisoporella biaperta. 

PLATE IX 

FIGURE 61. Schizoporella itniconiis. A single zoid showing typical rounded aperture with 
its postral sinus and two pointed avicularia situated on either side of the aperture. The pres- 
ence of two avicularia is a less frequent condition than the presence of one avicularium. 
Frontal surface of zooecium has a number of closely set pores. Calcined specimen. 

FIGURE 62. Schisoporella unicornis. Three zooecia showing varying degrees of calcifica- 
tion. The uppermost square zooecium has a completely calcified aperture. The second squared 
zooecium has the aperture and avicularium openings completely calcified or plugged up. 
Around the right, left and lower sides of this second zoid are white septa, outgrowths from a 
newly overgrowing colony whose marginal zooecium is shown as the partial, third, bottom 
zooecium with black aperture. 

FIGURE 63. Schisoporella unicornis. Portion of a typical colony. Three rows of zooecia 
are at the bottom and four at the top of this colony fragment, showing how a colony may 
increase in width at the periphery. Five zooecia have the avicularium on one side of the 
aperture, four on the other and three are without avicularia. Apertures may be placed either 
in the middle or at one side of the distal part of the frontal surface. The frontal surface is 
rather flat in this colony. Calcined specimen. 

FIGURE 64. Scruparia ambigita. A zoid with a frontal and distal bud. The basal proxi- 
mal part of the frontal bud and of the zoid is slightly twisted, a typical condition. 



64 



MARY D. ROGICK AND HANNAH CROASDALE 

PLATE X 



69 




MARINE BRYOZOA, III 65 

Some zooecia are without oral spines, avicularia or ovicells. Others have 
them. Oral spines may number from 3 to 7 (Figs. 47, 49). Ovicells are globose, 
"pebbled" in texture, and provided with small pores (Fig. 48). One avicularium 
is placed at an angle on either the right or the left frontal side, one-third to one- 
half of the way down and laterad from the aperture. The aperture is hemi- 
spherical and placed above the small crescent-shaped ascopore (Fig. 49). Canu 
and Bassler (1930, p. 47) reported 13 to 14 tentacles for this species. 

Embryo-filled ovicells were collected on VIII-28-1939. Many very young 
colonies were found developing at that time also. 

PEDICELLINA CERNUA 
(Figure 50) 

PediccUina cennia though small, soft-bodied, and inconspicuous was reported 
on 11 algal species. . Also, Leidy (1855, p. 11) reported it on the "roots" of 
Lauiinaria saccharina; Prenant and Teissier (1924, p. 19) reported it on the 
Cystoseiras, Florideae and Dictyota ; and Joliet (1877, p. 296) on Corallina 
squammata and Cladophora rupestris. 

It has a creeping stolon from which arise flexible stalked zoids. Spines oc- 
curred on the calyx and stalk (Fig. 50) of a few zoids but most specimens were 
without them. The tentacle number in several very young zoids was 8 to 12. 
The number increases with age. Marcus (1939, p. 212) gives the tentacle range 
for this species as 8 to 24. 

PLATE X 

FIGURE 65. Scruparia ambigua. A row of four zooecia encrusting a thick algal filament. 
From these arise four branches of zoids. The opesial rim of all but possibly the terminal 
budless individuals is parallel to the back of its own zoid. Drawn to the same scale as 
Figure 68. 

FIGURE 66. Scruparia clavata. Two full-grown zooecia and two developing buds. The 
smaller, left bud belongs to a developing ovicell. The ovicelled individual originates below the 
other bud. Drawn to the scale above. 

FIGURE 67. Scruparia clavata. Two grown zooecia and a third smaller one topped by a 
globose ovicell which has several large pores. The ovicell faces in the opposite direction from 
the ordinary zooecium. Drawn to same scale as Figure 66. 

FIGURE 68. Smittina trispinosa. Eighteen ordinary zooecia growing in a very regular 
fashion. Some zooecia have rounded and some pointed avicularia while others have none at 
all. The flared peristome about the aperture is well developed in many. Some have spines, 
others do not. Calcined specimen. 

FIGURE 69. Smittina trispinosa. A zooecium, ovicell and two avicularia. The circular 
aperture with the two cardelles (lateral teeth) and the lyrula (broader, median tooth) is a key 
character. So are the ovicell with its pores and leaning avicularium and the row of marginal 
zooecial areolae. Avicularia may be found in various locations on the frontal zooecial surface 
(compare this figure with Figures 68 and 71). The zooecium shows secondary calcification 
about the lower avicularium and partly covering the lower areolae. Drawn to same scale as 
Figure 70. Calcined specimen. 

FIGURE 70. Smittina trispinosa. An ovicell topped by a triangular avicularium. The 
zooecial peristome is heavily calcified and forms a collar at the bottom of which can be seen 
the aperture, lyrula and cardelle. 

FIGURE 71. Smittina trispinosa. A young zoid showing the characteristic apertural fea- 
tures and a frontal triangular or pointed avicularium. The frontal surface is slightly "beaded" 
and marginal areolae outline the thin edge. Drawn to same scale as Figure 70. 



66 MARY D. KOGICK AND HANNAH CROASDALE 

SCHIZOPORELLA BlAPERTA 

(Figures 51-56) 

This white to reddish-orange bryozoan is fairly common on nine algal species. 
Its colonies attain a diameter of 2 cm. or more and appear fairly sturdy. They 
grow either flat on the thallus or may extend beyond the thallus, forming cal- 
careous "ruffles" which may be lamellate (several layers in thickness). 

The key characters of this species are : ( 1 ) rounded aperture with a postral 
sinus between the two cardelles (Fig. 55) ; (2) one or two small oval or ellip- 
soidal avicularia (Figs. 52, 53) mounted on mammillate prominences at the right 
or left or both sides of the sinus area (Figs. 51, 55) ; (3) frontal wall perforated 
by irregularly sized and spaced pores, and (4) hemispherical ovicells the edge of 
whose frontal area is slightly depressed and marked by faint calcareous ribs (Figs. 
54, 56). Heavy calcification obscures some of these characters, especially the 
porous frontal area of the zooecium (Figs. 54, 56). 

Twelve tentacles were counted on one zoid. 

In July and August, the ovicells contained red embryos or larvae. 

SCHIZOPORELLA UNICORNIS 
(Figures 57-63) 

Schizoporella unicornis is very common on rocks and shells, but less frequent 
on algae. It grew on six Woods Hole region algal species. Additional algal 
hosts mentioned by Prenant and Teissier (1924, p. 23) are the Florideae, 
Himanthalia, and Saccorhisa bulbosa. 

There is great variation in the appearance of the colonies. Their color ranges 
from white to reddish orange to a dull red. Some are smooth, flat, and shining, 
others rough and extended beyond the thalli. They may be lamellate, one colony 
growing over another. No description of the species is necessary because Figures 
58 and 59 show the ovicells, and Figures 61 and 63 show the typical zooecial 
appearance, growth habit, aperture shape, and disposition of the avicularia. The 
avicularia grade in size (Figs. 57-60), but are always sharply pointed. Calci- 
fication may sometimes obliterate them and the aperture (Fig. 62). 

In the Woods Hole area, larvae were found in ovicells in July and August 
(the times when collection was made) and undoubtedly occurred before and 
beyond these dates. At Beaufort, North Carolina, they are found the year round, 
according to McDougall. McDougall (1943, p. 340) observed that the times of 
greatest abundance of larvae (as judged by settlings on experimental substrata 
there at Beaufort) were in May, June, September, October and November. 

SCRUPARIA AMBIGUA 
(Figures 6465) 

Most of the specimens of this dainty little dendritic form were dredged off Gay 
Head, Martha's Vineyard, on VII-30-1946. Some were growing on Bugula 
turrlia, others on eleven algal species. It also grew in close association with 
hydroids and Hippothoa hyalina. 



MARINE BRYOZOA, III 67 



Scruparia ambigua zoids are yellowish, horny, transparent, and slender. They 
ranged from 0.345 to 0.495 mm. in length, the average of 12 specimens being 
0.431 mm. The branching of the colony is quite open. Tentacles numbered ten 
in each of two zoids. No ovicells or larvae were found in the present material. 
Barrois (1877, p. 194) found ovicells and larvae at Roskoff during the month 
of June. 

Hastings (1941) made a careful study of this species and differentiated 
Scruparia ambigua from vS". chelata on the basis of the opesial slant and encrusting 
zooecia. In .5\ ambigua the opesial rim is parallel to the basal wall of the 
zooecium, and the free zooecial branches arise from a series of encrusting zooecia, 
as in Figure 65. 

SCRUPARIA CLAVATA 
(Figures 6667) 

A few scraps of this delicate, horny, dendritic, transparent bryozoan were 
growing on Laminaria Agardhii which was dredged off Gay Head, Martha's 
Vineyard on VII-30-1946. Some ovicells were present. The zooecia bearing 
them were slightly smaller (Fig. 67) than the other zooecia. The zooecial orifice 
is much smaller than that of Scruparia ambigua. 

Marcus (1940, p. 208) created a new genus Haplota for S. clavata. 

SMITTINA TRISPINOSA 
(Figures 68-71) 

This species was found with great frequency on shells and rocks, sometimes 
many layers in thickness on the latter. However, its occurrence on algae was in- 
frequent. Cliondrus crispus (from North Falmouth, Mass.), Laminaria Agardhii, 
Phyllophora Brodiaei, and P. membranijolia from Woods Hole had a few colonies. 

Rock colonies or nodules often are a light mustard yellow color ; colonies on 
algae, however, were never that striking a color, but were ivory or iridescent. 

Colonies are very fine grained in general appearance. The species shows a 
great deal of variation, depending upon age, degree of calcification, and nature 
of the substratum. 

SUMMARY 

1. A total of 30 bryozoan species was reported from 37 species of marine algae. 

2. Five bryozoan species were reported from three species of green algae, 26 
bryozoan species from 11 species of brown algae, and 27 bryozoan species from 
23 species of red algae. 

3. Phyllophora membranijolia yielded the greatest number of bryozoan spe- 
cies (23), Chondrus crispus and Laminaria Agardhii each yielded 20, Phyllophora 
Brodiaei, 15, and Cystoclonium pnrpureum cirrhosum yielded 11 bryozoan species. 

4. Each of the three commonest bryozoa, Actea sica, Bowerbankia gracilis and 
Crisia eburnea, was found on 18 algal species. 

5. Each of the two next commonest bryozoa, Electra pilosa and Hippothoa 
hyalina, occurred on 17 red and brown algal species. 



68 MARY D. ROGICK^AND HANNAH CROASDALE 

6. Buyula turrita, the next most common form, was found on 16 algal species. 

7. Crisia cburnca and Hippothoa hyalina were common on over half the red 
algal species examined. 

8. Some bryozoa seemed to grow most frequently and abundantly on certain 
algal species, namely : 

a. Alcyonidium polyoum on Chondrus crispus, Phyllophora Brodiaei, and P. 
membranijolia 

b. Bowerbankia gracilis on Ascophyllum nodosum, Chondrus crispus, Fucus 
vesiculosus, F. vesiculosus spiralis, Phyllophora Brodiaei and P. membrani- 
folia 

c. Crisia eburnca on Chondrus crispus, Phyllophora Brodiaei and P. niembrani- 
jolia 

d. Electro pilosa on Laininaria Agordliii and Rhodymenia pahnata 

e. Flustrella hispida on Ascophyllum nodosiim. 

9. To the 84 known Woods Hole region bryozoan species can be added three 
more : Actea sica, Cellepora dichotoma and Scruparia ambigua. It is quite pos- 
sible that some of the previously reported Aetca anguina and Cellepora americana 
material may have included Aetea sica and Cellepora dichotoma, respectively. 

10. Algal collections from New Rochelle, N. Y., yielded some of the same 
bryozoan species as are found in Woods Hole, namely : Alcyonidium polyoum, 
Bowerbankia imbricata, Cryptosula pallasiana, Electro Jiastingsae, Membranipora 
lacroi.rii( ?), and Pcdicellina cerniia. 

11. Algal collections from Rye, N. H., yielded some of the same bryozoa as 
are found at Woods Hole, namely : Callopora aurita, Cribrilina annulata, Cribrilina 
punctata, Crisia eburnca, Elcctra pilosa, Hippothoa hyalina and also a form, 
Lichenopora hispida, which did not occur at Woods Hole. 

12. Twenty-six of the thirty bryozoan species were carefully illustrated. 

13. Tentacle number counts were made for 15 species. 

14. Three bryozoan species were collected, observed, or known to be in the 
larva-producing stage on algae in late June ; seven species in July ; ten in August ; 
two in September. These were chance observations and the number of species 
would have been greater if more exhaustive collections over a greater number of 
months could have been made. 

LITERATURE CITED 

ADAMS, J., 1800. II. Descriptions of some marine animals found on the coast of Wales by the 

late John Adams, Esq. Trans. Linn. Soc., 5 : 7-13. 

BARROIS, J., 1877. Recherches sur 1'embryologie des bryozoaires. Six-Horemans, Lille. 
BORG, F., 1926. Studies on recent cyclostomatous bryozoa. Zool. Bidrag jrdn Uppsala, 10: 

181-507. 
BORG, F., 1930. Moostierchen oder Bryozoen (Ectoprocten) . Die Ticnvelt Dcntschlands, 

Tell 17 : 24-142. 
BORG, F., 1944. The stenolaematous bryozoa. Further Results Sivcd. Antarct. Expcd. 1901- 

1903, 3 (5) : 1-276. Stockholm. 
CANU, F., AND R. BASSLER, 1920. North American early tertiary bryozoa. Smithsonian 

Institution, U. S. Nat. Mus. Bull, 106: 1-879. Text vol. 
CANU, F., AND R. BASSLER, 1923. North American later tertiary and quaternary bryozoa. 

Smithsonian Institution, U. S. Nat. Mus. Bull., 125: 1-302. 



MARINE BRYOZOA, III 69 

CANU, F., AND R. BASSLER, 1930. Bryozoaires marins de Tunisie. Station Oceanogr. de 

Salammbo, Annales, No. 5 : 1-92. July. 
GRAVE, B. H., 1933. Rate of growth, age at sexual maturity and duration of life of certain 

sessile organisms at Woods Hole, Mass. Blol. Bull., 65 : 375-386. 
HARMER, S. F., 1915. The polyzoa of the Siboga expedition, Part I. The Entoprocta, Cteno- 

stomata and Cyclostomata. Siboga-Expeditie, Monogr. 28a, Livr. 75 : 1-180. 
HARMER, S. F., 1926. The polyzoa of the Siboga expedition. Part II. Cheilostomata Anasca. 

Siboga-Expeditie, Monogr. 28b, Livr. 105. 
HASTINGS, A. B., 1929. Cheilostomatous polyzoa from the vicinity of the Panama Canal, 

collected by Dr. C. Crossland. . . . Proc. Zool. Soc. London, Feb. 13, 1930, No. 47, 

Part 4: 697-740. 
HASTINGS, A. B., 1941. The British species of Scruparia (polyzoa). Ann. Mag. Nat. Hist., 

ser. 11, 7: 465-472. 
HINCKS, T., 1880. A history of the British marine polyzoa. Vol. 1, Text, pp. 1-601 ; Vol. 2, 

Plates. John van Voorst, London. 
HUTCHINS, L. W., 1945. An annotated check-list of the salt-water bryozoa of Long Island 

Sound. Trans. Conn. Acad. Arts and Sci., 36: 533-551. 
JOLIET, L., 1877. Contributions a 1'histoire naturelle des bryozoaires des cotes de France. 

Arch, de Zool. E.\-pcr. ct gen., 6: 193-304. 
LEIDY, J., 1855. Contributions towards a knowledge of the invertebrate fauna of the coasts of 

Rhode Island and New Tersey. Jour. Acad. Nat. Sci., Phila., Ser. 2, 3: 3-20. Plates 

X, XL 
MACGILLIVRAY, P. H., 1891. Art. XIII. Descriptions of new or little-known polyzoa, Part 

XIV. Proc. Royal Soc. of Victoria, N. S., 3 : 77-83. 
MARCUS, E., 1937. Bryozoarios marinhos brasileiros, I. I'nii: SCio Paulo, Bol. Fac. Philos., 

Sci. e Lctr., 1, Zoologia, No. 1 : 5-224. 
MARCUS, E., 1938. Bryozoarios marinhos brasileiros, II. Univ. Sao Paulo, Bol. Fac. Philos., 

Sci. e Letr., IV, Zool., 2 : 1-196. 
MARCUS, E., 1939. Briozoarios marinhos brasileiros. III. Univ. Sao Paulo, Bol. Fac. Filos., 

Cicnc. c Lctr., A7/7. Zool, 3: 111-354. 
MARCUS, E., 1940. Mosdyr (Bryozoa eller Polyzoa). Danmarks Fauna. Dansk Natur- 

historisk Forening. K0benhavn. 
McDouGALL, K. D., 1943. Sessile marine invertebrates at Beaufort, North Carolina. Ecol. 

Monogr., 13: 321-374. 
OSBURN, R. C., 1912. The bryozoa of the Woods Hole region. Bull Bur. Fish., 30 (760) : 

205-266. 
OSBURN, R. C., 1944. A survey of the bryozoa of Chesapeake Bay. Piibl No. 63, Chesapeake 

Biol Lab., Dcpt. of Research and Educ., State of Md., Bd. of Nat. Resources. 59 pp. 
PRENANT, M., AND G. TEISSIER, 1924. Notes ethologiques sur la faune marine sessile des 

environs de Roscoff . Trar. dc la Stat. Biol de Roscoff , 30 Avril, Fasc. 2 : 1-49. 
PRE'NANT, M., 1927. Notes ethologiques sur la faune marine sessile des environs de Roscoff, II. 

Trav. dc la Stat. Biol. de Roscoff, 30 Sept., Fasc. 6 : 1-58. 
PRENANT, M., 1932. fitude de bionomie intercotidiale la Baie et la Pointe de Quiberon. Trav. 

Stat. Biol Roscoff, 10 : 37-103. 
ROGICK, M. D., 1940. An ecological effect of the New England hurricane. Ohio Jour. Sci., 

40: 163-167. 
ROGICK, M. D., 1945a. Studies on marine bryozoa, I. Aeverrillia setigera (Hincks) 1887. 

Biol. Bull, 89: 201-214. 

ROGICK, M. D., 1945b. "Calcining" specimens. Aincr. Biol. Tchr., 8: 66-70. 
ROGICK, M. D., 1948. Studies on marine bryozoa, II. Barentsia laxa Kirkpatrick 1890. Biol. 

Bull., 94: 128-142. 
SILEX, L., 1942. Carnosa and Stolonifera (bryozoa) collected by Prof. Dr. Sixten Bok's 

Expedit. . . . Arkiv for Zool, 34A : 1-33. 
TAYLOR, W. R., 1937. Marine algae of the Northeastern coast of North America. Univ. of 

Mich. Press, Ann Arbor, Mich. 

THOMPSON, W., 1840. No. 31 of the volume. Art. XXVIII, Additions to the fauna of 
Ireland. Ann. Mag. Nat. Hist., ser. 1, 5: 245-257. 



STRATIFICATION AND DEFORMATION OF ARBACIA PUNCTULATA 

EGGS CENTRIFUGED IN CAFFEINE SOLUTIONS 

\ 

RALPH HOLT CHENEY 

Brooklyn College of the City of New York and the Marine Biological Laboratory, 

Woods Hole, Massachusetts 

INTRODUCTION 

During an investigation of the effect of methylated purines upon cellular be- 
havior, the following problems arose : Does contact with the trimethylated purine, 
caffeine, which is known to influence cellular metabolism, affect the viscosity of 
the cytoplasm of the cell ; and is there any evidence that this alkaloid influences 
the forces at the cell surfaces? Heilbrunn's informative studies (1926, 1928, 
1943) on viscosity and surface forces, with reference to numerous chemical sub- 
stances, did not deal adequately either with alkaloids as a group, or with caffeine 
in particular. Centrifugation followed by a study of the degree of granule strati- 
fication within the cytoplasm, together with changes in form of the cell itself, 
offered a satisfactory method of approach to these questions. From the experi- 
ments described below, it will be seen that caffeine does not alter the viscosity of the 
unfertilized egg but acts upon the membrane and cortical tension forces, thereby 
influencing cleavage in the fertilized egg. In the higher concentrations, even 
sperm entry is prevented by caffeine. 

The temperature factor is not significant in the work reported here, since 
caffeinized eggs were centrifuged at the same time and temperature as control eggs 
from the same female. In this way, relative viscosity effects could be observed. 

METHODS AND MATERIALS 

The author (1945, 1946a, b, 1946) has demonstrated by studies on O. 2 con- 
sumption and comparative sensitivity of developmental stages that caffeine retards 
cleavage in Arbacia. In the present study, the unfertilized and fertilized eggs 
[unfertilized in sea water (SW), unfertilized in 0.10 per cent caffeine-in-sea-water 
(CSW) ; fertilized, i.e. normal egg (N $) X normal sperm (N J 1 ), in sea water, 
and fertilized N $ X N J 1 in 0.10 per cent caffeine-in-sea-water] were centrifuged 
at 10,000 X g for five, seven, and twelve minutes, and also at 3000 X g, 40 min- 
utes after fertilization or at the equivalent time interval after shedding in the case 
of unfertilized eggs. This 40 minute period was chosen because that is when the 
viscosity of the protoplasm approaches the increased state typical at the time of 
cleavage. Comparable series employing other concentrations were also run. 

Experiments were conducted at the temperature of running sea water. The 
appearance of uncentrifuged and centrifuged caffeinized eggs was compared with 
photomicrographs and descriptions by E. B. Harvey (1940). Differences in the 
degree of stratification (compactness) of the pigment granules and vacuoles and 
the height of the hyaloplasm zone after centrifugation were noted as evidence of 

70 



SEA URCHIN EGGS CENTRIFUGED IN CAFFEINE 71 

relative viscosities. To avoid error due to a time variable caused by the return 
of granules by Brownian movement, all photomicrographic records were made ten 
minutes after centrifugation. 

RESULTS AND DISCUSSION 

Cleavage abnormalities in eggs centrifuged in CSW were no greater than those 
observed in the same concentration of CSW without centrifugation. There was 
no evidence that caffeine induced any primary change in viscosity which would 
prevent cleavage. Clearly defined effects were reproducible and similar in both 
unfertilized and fertilized eggs, but those in the former were more convincing 
because the normal viscosity changes during mitosis made it impossible to assume 
that controls and experimentals would be in exactly the same state. 

Bank (1932), using Arbacia pnnctulata, reported stratification within the un- 
fertilized eggs without centrifugation if they were held in 1 per cent caffeine for 
48 hours. This is not surprising since caffeine \vas shown by the author (1945, 
1948) to retard the O. 2 uptake of the Arbacia cell. The facility with which the egg 
contents stratify due to such a factor as O 2 uptake cannot be determined by cen- 
trifugation. In the experiments described in this paper, an indication of a surface 
effect was the fact that Arbacia eggs cannot be fertilized when immersed in 1 per 
cent CSW. Both the eggs and sperm, however, survive for a considerable period 
in 1 per cent CSW, and the eggs can be fertilized and undergo partial develop- 
ment if transferred to sea water. Therefore, this concentration of caffeine does 
.not destroy the internal physiological potentialities of these gametes with respect 
to fertilization. Over as long a period as 48 hours, the physical effect noted by 
Bank can be understood on the basis of the biochemical inhibition of cellular 
respiration, and/or as a surface effect, without assuming a primary viscosity 
change due directly to caffeine. 

Among the results observed in the present series, the delay of deformation, 
reduction in actual fragmentation, and the sharper margins of the layers (apparent 
under the earlier conditions of the experiments) w r ere the most readily distin- 
guishable and clearly associated phenomena. De Vries (1947), in his studies on 
viscosity and tension at the surface in eggs of the fresh water snail, L'unnaea stag- 
nalis L., based the interpretation of his results primarily on the occurrence of 
vacuoles and granules in the hyaloplasm zone, as well as on the height of this 
zone. ' He pointed out that the height of the hyaloplasm zone depends on both 
viscosity and the degree of stretching, i.e. tension at the surface. Therefore, it 
occurred to the present writer that the closer packing of the pigment granules 
might be attributable to the fact that the pigment in the spherical cell had a 
shorter distance to fall than the pigment in the uncaffeinized normal cell, which 
is always elongated by centrifugal force to the degree applied in the experiments 
up to this time. It seemed desirable to eliminate the effect of the stretching factor 
and resulting deformation in order to clarify the significance of the degree of 
stratification in interpreting viscosity changes. Accordingly, unfertilized and 
fertilized eggs, both control and experimental, were centrifuged at only 3000 X g 
for intervals varying from one-half to three minutes. These shorter centrifuga- 
tions at 3000 X g did not change the shape of the cells in either the control or the 



72 RALPH HOLT CHENEY 

caffeinized eggs, but did allow stratification. Therefore, comparisons of stratifica- 
tion could be made without the deformation factor. 

In the absence of internal viscosity changes, the increased force required to 
break caffeinized eggs indicates a surface effect. Harvey (1931) estimated that 
the centrifugal force necessary to pull the Arbacia egg into two halves indicates 
that tension at the surface for a 25 per cent increase in area is less than 0.2 dyne 
per cm. with considerable variation in eggs. At 10,000 X g for 12 minutes, a 
count of five fields of each of the experimental caffeine series showed the per- 
centage of breaking in the unfertilized eggs to be as follows : Controls in SW w r ere 
100 per cent broken; eggs in 0.02 per cent CSW, 12 per cent broken; in 0.10 per 
cent CSW, 0.50 per cent were broken ; and in the 2.0 per cent CSW, only 0.08 
per cent were broken although slight elongation did occur. 

The apparent absence of any significant osmotic change (Cheney, 1948) as 
well as of demonstrable viscosity changes in the internal protoplasm, together with 
the delay in deformation reported here, would indicate that caffeine may initiate 
a change in the surface of the cell. Such an effect might involve both the mem- 
brane and the cortical protoplasm, which Harvey and Shapiro (1941) demon- 
strated to possess a considerably higher viscosity than the interior protoplasm in 
the eggs of Arbacia punctnlata and Asterias jorbesii. 

SUMMARY * 

1. Caffeine does not change the existing viscosity state of the egg. 

2. Egg fragmentation, under centrifugation, decreases with increased caffeine 
concentration. 

3. The "apparent" effect of greater stratification of the granules in Arbacia 
eggs centrifuged in caffeine does not occur if the centrifugal force to which the 
eggs are subjected is sufficient to produce sedimentation but insufficient to cause 
deformation. 

4. Evidence indicates that the delay of deformation in the caffeinized eggs, 
centrifuged at 10,000 X g or less, may be due to the action of caffeine (tri- 
methylated purine) upon the total tension forces at the surface areas of both unfer- 
tilized and fertilized Arbacia eggs. 

LITERATURE CITED 

BANK, O., 1932. Stratification des oeufs d'oursin sans centrifugation. Compt. Rend. Soc. 
Biol., 110: 389-390. 

CHENEY, R. H., 1945. The effects of caffeine on oxygen consumption and cell division in the 
fertilized egg of the sea urchin, Arbacia punctulata. Jour. Gen. Physiol., 29 : 63-72. 

CHENEY, R. H., 1946a. Effect of caffeine concentration upon retardation of Arbacia develop- 
ment. Biol. Bull, 91 : 226-227. 

CHENEY, R. H., 1946b. Sensitivity of Arbacia development to caffeine. Anat. Record, 96 : 
547-548. 

CHENEY, R. H., 1948. Caffeine effects on fertilization and development in Arbacia punctulata. 
Biol. Bull., 94: 16-24. 

* Centrifugation facilities and aids granted the author by Dr. E. B. Harvey during this 
study are deeply appreciated. 



SEA URCHIN EGGS CENTRIFUGED IN CAFFEINE 73 

, G. A., 1^47. The influence of lithium chloride and calcium chloride on viscosity and 

tension at the surface of uncleaved eggs of Limnaea stagnalis L. Proc. Kon. Ned. 

Akad. v. Wetensch, Amsterdam, 50: 1335-1342. 
HARVEY, E. B., 1940. A comparison of the development of nucleate and non-nucleate eggs of 

Arbacia punctulata. Biol. Bull., 79 : 166-187. 
HARVEY, E. N., 1931. The tension at the surface of marine eggs, especially those of the sea 

urchin, Arbacia punctulata. Biol. Bull, 61 : 273-279. 
HARVEY, E. N., AND H. SHAPIRO, 1941. The recovery period (relaxation) of marine eggs 

after deformation. Jour. Cell. Comp. Physiol., 17 : 135-144. 

HEILBRUNN, L. V., 1926. The absolute viscosity of protoplasm. Jour. E.vp. Zool., 44: 255-278. 
HEILBRUNN, L. V., 1928. The colloid chemistry of protoplasm. Monograph. Berlin. 
HEILBRUNN, L. V., 1943. An outline of general physiology. Ed. 2. Saunders Co. 



A MUCIN CLOT REACTION WITH SEA-URCHIN FERTILIZIN 

MAX KRAUSS 
Kerckhoff Laboratories of Biology, California Institute of Technology, Pasadena 

INTRODUCTION 

Recent work on the fertilizins (the sperm-agglutinating constituents of egg 
water) of the eggs of sea-urchins and other animals has shown them to be of the 
nature of mucoproteins. Tyler and Fox (1939, 1940) showed that the fertilizins 
of Strongylocentrotus and of Megathura possess protein characteristics, but are of 
low nitrogen content. Similar evidence has been obtained with Arbacia fertilizin 
by Kuhn and Wallenfels (1940) and with Psammechinus fertilizin by Runnstrom, 
Tiselius, and Vasseur (1942). The latter workers also obtained a positive carbo- 
hydrate test. Tyler (1948) reported the presence of reducing sugars to the 
extent of about 15 per cent in hydrolyzed, purified preparations of Strongylocen- 
trotus fertilizin and identified galactose as one of the constituents. As will be 
shown later in this paper, the present author has found hexosamine to be present 
in amounts equivalent to about 2 per cent of the original material. According to 
Runnstrom, Tiselius, and Vasseur (1942) and Tyler (1946) the sea-urchin 
fertilizins are of pronounced acidic character. 

Acidic mucopolysaccharides are known (cf. Meyer, 1945, and Stacey, 1946) 
to co-precipitate with proteins upon acidification of the native fluid or of the 
neutral extracts in the form of "mucin clots," stringy or granular precipitates, 
depending upon the conditions of precipitation. This reaction is given, for ex- 
ample, by hyaluronic acid (Meyer and Palmer, 1936) and has been used in the 
assay of the enzyme hyaluronidase (McClean, 1943). It was of interest to deter- 
mine whether or not fertilizin preparations would give the mucin clot reaction. 
As the work reported here shows, fertilizin preparations do give such a mucin clot 
reaction. A titration method, based upon this, was developed for these prepara- 
tions, and comparisons made with their sperm-agglutinating activity in untreated 
condition, after dialysis, and after exposure to heat and to ultra-violet irradiation. 

MATERIALS AND METHODS 

The sea-urchins Lytechinus pictus, Strongylocentrotus purpuratus and S. 
franciscanus were used in these experiments. Most of the work was done with 
>$". purpuratus. 

Two kinds of fertilizin preparations were employed. One, which will be 
termed "crude fertilizin," was prepared by acidifying a 20 per cent suspension of 
washed eggs to pH 3-3.5, removing the supernatant fluid after five or ten minutes 
and readjusting the pH of this solution to 7-7 .S. The other, which will be termed 
"purified fertilizin," was further subjected to alkali precipitation, dialysis against 
3.3 per cent acid saline (pH 3.5-4) and alcohol precipitation according to the 
method described by Tyler (1948). Material prepared in this manner has been 

74 



FERTILIZIN MUCIN CLOT REACTION 

found to be eleetrophoretically homogeneous (Tyler, unpul).). For the various 
tests the solutions were made up in 3.3 per cent NaCl at a pH of about 7. 

Sperm-agglutinating titer of the fertilizin preparations was determined using 
the drop method of preparing two-fold serial dilutions with sea water in Syracuse 
watch glasses. To two drops of each dilution of fertilizin solution one drop of 
a uniform sperm suspension, usually 1 per cent (calculated as 1 cc. dry sperm per 
100 cc. sea water suspension), was added. The highest dilution in which agglu- 
tination is observable under the microscope gives the titer of the preparation. 

Tests for univalent fertilizin were made according to the method described by 
Tyler (1941 ) and Metz (1942). Essentially, this method consists of first treating 
sperm with the solution containing univalent fertilizin, which does not agglutinate 
the sperm, and then adding an equal volume of strong normal fertilizin solution 
to the suspension of sperm. Failure of the sperm to be agglutinated by the normal 
fertilizin presumably indicates that the combining groups on the sperm surface 
have been occupied by univalent fertilizin groups and are no longer available to 
unite with the normal fertilizin. Univalent fertilizin was obtained by heating and 
by irradiation with ultra-violet light of normal fertilizin preparations (cf. Tyler, 
1941, and Metz. 1942). 

Bovine serum albumin prepared by the Armour laboratories was used in 1 per 
cent solution for the co-precipitation tests. 

Hyaluronic acid was obtained from human umbilical cords according to the 
method described by McClean (1943), whereby the distilled water extract of 
acetone-dried, ground cords, extracted with 90 per cent acetic acid according to 
the method of Meyer and Palmer (1936), was precipitated with 1.25 volumes of 
cold, potassium acetate-saturated 95 per cent alcohol. The precipitate was washed 
with alcohol, acetone, and ether and dried over P 2 O 5 . The dry product was dis- 
solved in distilled water as required; a solution of 0.10.2 per cent of the dry 
material was clear, viscous and did not form a precipitate upon the addition of 
acetic acid, but co-precipitated with serum albumin in the presence of acetic acid, 
forming a stringy clot. In higher dilutions the mixture of serum albumin, acetic 
acid and hyaluronic acid solution resulted in the formation of a fine precipitate or 
turbidity. The highest dilution in two-fold serial dilutions in which turbidity was 
perceptible by visual inspection was taken as the titer of the hyaluronic acid 
solution. 1 

EXPERIMENTS AND OBSERVATIONS 

Co-precipitation of fertilizin with scrum albumin in acid solution 

A viscous solution of crude fertilizin of Lytcchinns pictus was prepared as 
described above and combined with a 1 per cent solution of bovine serum albumin 
in 0.9 per cent NaCl and 2 N acetic acid according to the method described by 
McClean (1943) in the mucin clot test. With this solution a very large clot was 
formed similar in character and appearance to the clot formed by hyaluronic acid. 
Purified fertilizin preparations of Strongylocentrotus f>ur^uratus, S. frouciscaims, 
and L. pictus were tested in the same manner and in each case a clot or precipi- 

1 A quantity of pure potassium hyaluronate was later supplied to me by the Sobering 
Corporation, through the courtesy of Dr. W. Alan Wright and Dr. Erwin Sch\venk. 



76 



MAX KRAUSS 



tale formed, depending upon the concentration of the solution. In Figure 1 a 
series of photographs of the mucin clot reaction of S. [>nr[>nr<itns fertilizin is shown, 
with the reaction given by hyaluronic acid for comparison. 

By taking advantage of the biological activity of fertilizin, i.e., its sperm- 
agglutinating activity, a simple test was performed which provided definitive evi- 
dence that it is the fertilizin which is co-precipitated with serum albumin and not 
some hitherto undetected component of the material. 




FIGURE 1. Mucin clot reactions of Strongylocentrotus pitrpitnttns fertilizin and of hy- 
aluronic acid in two-fold dilutions, a. Purified S. purfitratus fertilizin. The first tube contains 
fertilizin solution and serum albumin, but distilled water instead of acetic acid. The opacity 
of the first tube is due to the opalescence of the mixture, b. Crude S. purpuratus fertilizin. 
The first tube contains fertilizin solution and acetic acid but 0.9 per cent saline instead of serum 
albumin, c. The same as b with the tubes shaken prior to being photographed, d. Hyaluronic 
acid. 

A solution of purified fertilizin in 3.3 per cent saline was mixed with serum 
albumin and the pH brought to about 3.5 with 2 N acetic acid. The resulting 
precipitate was thrown down by centrifugation, the supernatant withdrawn and 
its pH adjusted to 7. As shown in Table I, the supernatant exhibited no sperm- 
agglutinating activity. 



FERTILIZIN MUCIN CLOT REACTION 77 

TABLE I 

Sperm-agglutinating activity of the supernatant and of the precipitate recovered separately after the 
addition of bovine serum albumin to an acidified solution of purified fertilizin, 

and results of control tests 

Per cent of 
sperm-agglutinating 
Reaction mixture activity* 

'0.05 ml. distilled H 2 O 

0.2 ml. 0.9% saline 100 

0.05 ml. 2 N acetic acid Supernatant: 

0.2 ml. 1% bovine serum albumin in 0.9% saline Dissolved precipitate: 100 



0.5 ml. fertilizin 
in 3.3% saline 



0.05 ml. distilled H 2 O 

0.2 ml. 1% bovine serum albumin in 0.9% saline 100 



0.05 ml. 2 N acetic acid 

0.2 ml. 0.9% saline 100 

0.05 ml. distilled H 2 O 

.0.2 ml. 0.9% saline, acidified and neutralized 100 

0.5 ml. 3.3% fO.05 ml. 2 N acetic acid 

saline \0.2 ml. 1% bovine serum albumin in 0.9% saline 

* Sperm-agglutinating activity of fertilizin, distilled water and 0.9% saline mixture taken 
as 100%. 

It had previously been found that the precipitate formed by the addition of 
serum albumin to a fertilizin solution acidified to a pH of about 3.5 dissolves com- 
pletely at a pH of 5.6 or higher. After centrifugation and withdrawal of the 
supernatant of the material being tested, the precipitate was resuspended in 3.3 
per cent NaCl solution, its pH brought to about 7, and the volume made equal to 
that of the original mixture. Upon testing this solution it was found, as is shown 
in the table, that the sperm-agglutinating activity was equal to that of the original 
fertilizin solution, showing that the activity was recovered quantitatively from 
the precipitate. 

A number of control tests were made, the results of which are summarized in 
Table I. These showed that (1) co-precipitation of fertilizin and serum albumin 
does not occur in the absence of acid; (2) it does not occur in acidified solution in 
the absence of added protein; (3) the presence of albumin does not affect the 
sperm-agglutinating activity of the fertilizin; (4) the sperm-agglutinating activity 
is not affected by acidification and subsequent neutralization of the solution, and 
(5) sperm agglutination does not occur in saline solution in the absence of fertilizin, 
nor is a precipitate formed when albumin and acid are added to saline. 

Quantitative recovery of the sperm-agglutinating activity from co-precipitated 
fertilizin and serum albumin, which is achieved by the simple expedient of raising 
the pH to 5.6 or higher, shows that the specific combining groups of the fertilizin 
are not irreversibly altered by its reaction with the albumin. The significance of 
this phenomenon cannot be evaluated at the present time, however, since essen- 
tially nothing is as yet known of the structure of the fertilizin molecule. 



78 MAX KRAUSS 

Tit-ration by the mucin clot method 

The method employed for titration was as follows : Two-fold serial dilutions 
of fertilizin in 3.3 per cent NaCl at about pH 7 are made in 10 X 75 mm. test 
tubes in 0.5 ml. quantities. To each tube 0.05 ml. of 2 N acetic acid is added and 
the contents mixed. The tubes are inclined and 0.2 ml. of 1 per cent bovine 
serum albumin in 0.9 per cent saline is slowly pipetted down the side of each tube ; 
after this the tubes are carefully returned to a vertical position and in most cases 
a ring of precipitate forms immediately at the zone of contact between the albumin 
solution and the acidified fertilizin solution. The rings are easily detected at high 
dilutions in which a diffuse turbidity is difficult to detect and score visually. The 
reactions are read at once in good natural light, since the rings tend to disperse 
rapidly as the albumin diffuses through the mixture. The highest dilution at 
which a ring is observed is taken as the mucin clot titer of the preparation. 

Effect of pH, albumin concentration and salt concentration 

Numerous titrations with the same stock samples of purified fertilizin using 
the method described above have given consistently identical mucin clot titers in 
the course of routine testing. It was thought advisable, however, to carry out 
some controlled tests to determine in a more definitive manner the amount of 
variability in titer to be expected within a rather limited range of pH, albumin 
concentration, and salt concentration. 

A sample of a purified fertilizin solution from eggs of 6". purpuratus was cen- 
trifuged at 3000 r.p.m. for five minutes. A slight sediment was thrown down, the 
clarified supernatant was drawn off, and the pH adjusted to 7.0 with the glass 
electrode. To assure maximum uniformity of different samples of the super- 
natant, it was thoroughly mixed before removing an aliquot. Two-fold dilutions 
were made in 0.5 ml. quantities with 3.3 per cent NaCl solution. Serum albumin 
was made up in 1 per cent solutions, and the same stock solution of 2 N acetic acid 
was used throughout. Various amounts of the latter two solutions were added to 
different sets of tubes of the fertilizin dilutions. The total albumin and salt con- 
centration in the different sets was thus altered, as noted below. The pH was 
measured with a Beckman glass electrode pH meter. After each titration the pH 
of the final mixtures was measured in the first tube, in the tube giving the end- 
point, and in an intermediate tube. The maximum difference in pH observed 
within any set was 0.26 unit. The results obtained in a series of nine titrations 
are given in Table II. Each of the pH values listed represents the mean of three 
determinations in each set. The total salt concentration is expressed as per cent 
NaCl. The mucin clot titers Oast column of table) are the end points of visible 
precipitation as determined by the ring method described above. 

As the data in the table show, no marked difference in mucin clot titer occurs, 
for the most part, as a result of the differences in pH, total salt and albumin con- 
centrations employed. In titration 5, the observable end-point would probably 
have been at least one dilution higher had rings formed. The failure of rings to 
form is attributable to the relatively large quantity of serum albumin solution used ; 
as noted above, in high dilutions a diffuse turbidity such as was produced in this 
case is more difficult to detect visually than a ring at the same dilution. It has 



FERTILIZIN MUCIN CLOT REACTION 



79 



been found that 0.5 nil. of albumin, solution is about the largest quantity prac- 
ticable for obtaining consistent ring formation. Since there is always a tendency 
to pipette too rapidly when a large number of tests is being performed, it has 
proven more convenient to use 0.2 ml. of albumin solution. 

The evidence presented in Table II indicates that pH is not a critical factor with 
respect to observed titer within the range tested. The albumin concentration is 
not critical, nor is the total salt concentration of the system in the range from 2.0 
per cent to 3.1 per cent. Below 2.0 per cent the salt concentration may be a more 
important factor, as shown by the higher titer obtained in titration 6. Although 
the total salt concentration in titration 5 is the same as that in number 6 (1.6 per 
cent), the two sets are not comparable for the reason mentioned above. 



TABLE II 

Mean pH, total albumin concentration, total salt concentration and mucin clot liters in nine titrations 
using uniform samples of a homogeneous purified fertilizin preparation of S. purpuratus 









Ma PI 










Titration 
no. 


Ml. 2 N 

acetic acid 


Ml. 1% 
albumin 
solution 


IN dV_.l 

concentration 
of albumin 
solution, 
per cent 


Mean 
pH 


Total 
albumin 
concentration, 
per cent 


Total 
NaCl 
concentration, 
per cent 


Mucin 
clot 
titer 


1 


0.05 


0.1 


0.9 


3.09 


0.15 


2.7 


64 


2 


0.05 


0.2 


0.9 


3.30 


0.27 


2.4 


64 


3 


0.05 


0.3 


0.9 


3.33 


0.35 


2.3 


64 


4 


0.05 


0.5 


0.9 


3.54 


0.47 


2.0 


64 


5 


0.05 


1.0 


0.9 


3.74 


0.65 


1.6 


32* 


6 


0.05 


0.5 


0.0 


3.58 


0.47 


1.6 


128 


7 


0.05 


0.5 


3.3 


3.58 


0.47 


3.1 


64 


8 


0.01 


0.2 


0.9 


3.88 


0.28 


2.6 


64 


9 


0.02 


0.2 


0.9 


3.78 


0.28 


2.5 


64 



* Scored as turbidity, not as ring. 

In the next section it will be shown that the mucin clot and sperm-agglutinating 
titers of a fertilizin solution are both reduced after dialysis of the preparation against 
distilled water with consequent removal of salt (NaCl). The data to be presented 
indicate that the physical state of the fertilizin is reversibly modified in the absence 
of electrolytes, at least under certain conditions of temperature. It seems likely, 
therefore, that there may exist some optimum concentration of electrolytes between 
zero concentration and that represented by 2.0 per cent NaCl (equivalent to an 
ionic strength of 0.34) at which co-precipitation of fertilizin and serum albumin at- 
tains a maximum. Further analysis of the effect of salt concentration on the co- 
precipitation of fertilizin and serum albumin is under way and will be reported 
in a later communication. 

In the system as employed in the routine titration procedure in the present in- 
vestigation, however, the mean pH, total albumin and total salt concentration fall 
well within the range for each of these factors in which identical titers are obtained, 
using uniform samples of a homogeneous fertilizin preparation. 



80 MAX KRAUSS 

Mucin clot and sperm-agglutinating titrations of fertilisin in salt-free solution 

A sample (I) of a stock preparation of purified fertilizin in 3.3 per cent saline 
was made salt-free by dialyzing against distilled water at 1 C. until it no longer 
formed a precipitate with AgNO 3 . It was found that a white, flocculent material was 
present in the dialyzed solution, whereas no such flocculence was evident in the 
control in 3.3 per cent NaCl solution which was kept at 1 for the same period. A 
considerable reduction in both mucin clot and sperm-agglutinating titer of the salt- 
free sample as compared with the control was observed. In the mucin clot titration, 
the character of the precipitate formed in the salt-free preparation upon the addition 
of serum albumin in the presence of acid differed from that in the control in being 
of larger particle size and somewhat stringy. Difficulty was encountered in making 
sperm-agglutinating titrations (where the salt content of the preparation was adjusted 
just prior to titration to 3.3 per cent NaCl by adding an equal volume either of 6.6 
per cent NaCl or of double sea water to the salt-free solution) in cases where sus- 
pended material was present. The spermatozoa clumped about the participate 
matter, and it was impossible to score the dilutions satisfactorily. Where tests were 
made with samples of thq dialyzed solution in which the amount of suspended ma- 
terial was visibly less than was originally present, both the mucin clot precipitation 
and sperm agglutination occurred in the manner characteristic of the control solu- 
tions ; in these cases the two titers were also somewhat higher, although they did not 
necessarily equal the values obtained for the control solution. 

A second sample (II) of the same stock preparation of purified fertilizin was 
dialyzed against distilled water until salt-free. This time the first five changes of 
the water used for dialysis, totaling 3 liters, were saved, combined, and lyophilized to 
dryness. The residue was taken up in about 10 cc. of distilled water and the 
resulting solution was approximately isotonic with sea water, as shown by the fact 
that sperm of 5\ pnrpurattis remained active when placed in it. This "dialysate- 
concentrate" was 300 times more concentrated than the original dialysate, but proved 
to be negative for both mucin clot formation and sperm-agglutinating activity. 
Sample II behaved in all respects like sample I. The results of tests with the two 
samples are summarized in Table III. 

The evidence obtained from the present experiments indicates that at tempera- 
tures near the freezing point (1 C.) the physical state of fertilizin can be reversibly 
modified by the removal of electrolytes. Macroscopic aggregates may appear in a 
fertilizin preparation under these conditions, and there is a correlated decrease in 
the mucin clot and sperm-agglutinating titers. Under the influence of added salt 
and elevated temperature (up to 21.5 C.), either separately or combined, there oc- 
curs a correlated decrease in the amount of visible macroscopic material and increase 
in mucin clot and sperm-agglutinating titers to values approaching those obtained 
with control solutions. The negative results of tests with the "dialysate-concen- 
trate" show that there was no actual loss of fertilizin during dialysis. 

Analysis of a purified fertilisin preparation 

The co-precipitation of fertilizin with protein in acidic solution in a manner anal- 
ogous to the behavior of acid mucopolysaccharides suggests affinity of fertilizin with 
this class of substances. It has recently been claimed, moreover, that a preparation 



FERTILIZIN MfCIN CLOT REACTION 



81 



TABLE 111 

Results of titrations of salt-free fertilizin preparations of S. purpuratus after various treatments 



Test no. 


Fertilizin 
preparation 


Treatment after removal from dialysis 
bath at 1 C. 


Sperm-agglutinating 
titration 


Mucin clot 
titration 


pH 


Titer 


PH 


Titer 


1 


I 


None 






5.2 


8 


2 


I 


Salt content adjusted to 3.3% NaCl. 


7.2 


64 










pH adjusted 










3 


T 


Salt content adjusted to 3.3% NaCl 






5.2 


16 


4 


I 


2.5 hrs. at 21.5 C. 






5.2 


16 


5 


I 


2.5 hrs. at 21.5 C. pH adjusted 






7.2 


16 


6 


I 


Dialvzed vs. 3.3% NaCl at 1 C. 


7.0 


64 


7.0 


16 






2 hrs. at 21.5 C. pH adjusted 










7 


I 


Dialyzed vs. 3.3% NaCl at 1 C. 


7.45 


1024 


7.45 


32 






24 hrs. at 8 C. pH adjusted 










8 


I 


Control. Not dialyzed vs. distilled 


7.4 


512 


7.4 


64 






water. pH adjusted 










9 


II 


1 hr. at 21.5 C. pH adjusted 






6.9 


32 


10 


II 


Salt content adjusted to 3.3% NaCl. 


6.9 


512 










3 hrs. at 21.5 C. pH adjusted 










11 


II 


Salt content adjusted to 3.3% NaCl. 






6.9 


64 






1 hr. at 21.5 C. pH adjusted 










12 


II 


Control. Not dialyzed against dis- 


7.3 


1024 


7.3 


128 






tilled water. pH adjusted 











from bull testes, presumably containing the enzyme hyaluronidase, is capable of 
causing the jelly of intact sea-urchin eggs to swell (Ruffo and Monroy, 1946; Mon- 
roy and Ruffo. 1947). It was of interest, then, to attempt to determine the extent 
to which fertilizin may be chemically similar to hyaluronic acid. The few avail- 
able data, which have been reviewed briefly in the introductory section, indicate that 
fertilizin is by no means identical with hyaluronic acid. The present investigation 
has shown that fertilizin differs from hyaluronic acid to a marked degree with respect 
to the two chief constituents of the latter substance, hexosamine and glucuronic acid. 
A homogeneous sample of a purified fertilizin preparation in 3.3 per cent NaCl 
was prepared as described above. In the present instance the supernatant fluid was 
filtered through hardened filter paper. The pH of the filtrate was adjusted to 7 and 



TABLE IV 

Results of chemical analysis of a purified fertilizin preparation of S. purpuratus 



Filtrate 



Sperm- 
agglutinating 
titer 


Mucin 
clot 
titer 


Dry 

weight, 
mg./ml. 


Total 
nitrogen, 
per cent 


Glucuronic 
acid 


Hexosamine, 
per cent 


a-amino 
acids 


2048 


512 


8.95 


4.1 


none 


1.6 


positive 














Ninhydrin 



Hydrolysate 



82 



MAX KRAUSS 



mucin clot and sperm-agglutinating titers were obtained. Aliquots of the filtrate 
were taken for the various analyses, which included determinations of dry weight, 
total nitrogen, hexosamine, a-amino acids and glucuronic acid. The results of the 
analyses are presented in Table IV. Dry weight per ml. was calculated from the 
weight of material precipitated from an aliquot of the filtrate with 1.25 volumes of 
cold 95 per cent alcohol. According to Tyler (1948), precipitation of fertilizin is 
complete under these conditions. The precipitate was washed with alcohol and 
dried in an oven at 55 C. to constant weight. Total nitrogen was determined for 
duplicate samples of the dried precipitate by the micro-Kjeldahl method. Another 
portion of the dried material was hydrolyzed by boiling in a sealed tube with 4N 
HC1 for eight hours. Hexosamine was determined in an aliquot of the hydrolysate 
by the method of Palmer, Smyth and Meyer (1937). Another portion of the 
hydrolysate was treated with Ninhydrin reagent for the determination of a-amino 
acids. For the determination of glucuronic acid the colorimetric method recently 
described by Dische (1947b) was employed, using a sample of the original nitrate, 



.600r 



.500 



.400 



O 



.300 
to 

2 

a. .200 

I- 

o 

3 .100 

I 



Hyafuronic acid 




Feriilizin 



4-50 460 470 480 490 500 510 

WAVE LENGTH 



520 530 540 



FIGURE 2. Absorption curves of reaction mixtures of mannose-thioglycolic acid-fertilizin 
and mannose-thioglycolic acid-hyaluronic acid. Wave lengths in HIM- Fertilizin AE( B io-i8o) = 
-0.097; hyaluronic acid AE( 5 io-4so> = +0.115. 



and for comparative purposes, a solution of pure potassium hyaluronate (Sobering) 
was tested at the same time by the same method. According to Dische, the reaction 
of carbohydrates with -SH compounds in H 2 SO. ( , which differentiates between vari- 
ous classes of carbohydrates and individual hexoses and hexuronic acids, is highly 
characteristic for glucuronic acid when mannose is employed. This reaction is the 
basis of the test. The reaction mixture with glucuronic acid gives a typical absorp- 
tion curve in the range 450-540 m/*, and it was found by Dische that the curve for 
hyaluronic acid is almost identical with that of glucuronic acid. In practice, accord- 



FERTILJZIN MUCIN CLOT RKACTION 

ing to Disclie, it is only necessary to measure the intensity of the mannose reaction 
at 510 and 480 m//, and subtract the second value from the first. This difference is 
positive for glucuronic acid and polyglucuronides, and negative for the other hex- 
uronic acids. Figure 2 shows the absorption curves for fertilizin and for hyaluronic 
acid. The difference between the intensity of the mannose reaction with fertilizin 
at 510 and that at 480 m/* is negative, and hence it may be concluded that fertilizin 
does not contain glucuronic acid. This result is in agreement with previous results 
obtained by Tyler (unpub. ) using an earlier method of Dische's (1947a). As 
shown in Table IV, fertilizin does contain hexosamine, but in small amount, which 
is in agreement with earlier results obtained by the present author using Kunitz's 
(1939) method. The Ninhydrin reaction was very weak but probably positive. 
Total nitrogen (4.1 per cent) of this material is somewhat lower than has been re- 
ported previously for S. pitrpuratns fertilizin by Tyler and Fox (1940), who found 
an average total nitrogen content of 5.2 per cent with crude preparations. 

These data show that fertilizin differs markedly from hyaluronic acid in its 
chemical constitution. It is obvious, therefore, that the ability to give the mucin 
clot reaction does not by any means indicate close similarity between fertilizin and 
hyaluronic acid, even though it may be evidence that the former is related to the 
group of acidic mucopolysaccharides. 

At the suggestion of Dr. Albert Tyler, the mucin clot titration procedure was 
used in conjunction with sperm-agglutinating titrations to investigate the effects 
of various kinds of treatment on fertilizin. In the following sections the results of 
parallel titrations of preparations subjected to heat and to ultra-violet irradiation 
are presented. 

Parallel titrations with heat-treated fcrtilisin solutions 

According to Tyler and Fox (1940), the sperm-agglutinating activity of 
Strongylocentrotus purpuratus fertilizin is rapidly destroyed at 100 C. The rate 
of inactivation, according to these authors, is a function of the pH, the fertilizin be- 
ing most stable in the range from 4 to 7. Their data show that at pH 7.3 the ag- 
glutinin is 95-100 per cent inactivated in 20-30 minutes at 100. In the present 
experiments in which S. purpuratus purified fertilizin solutions were used, the prep- 
arations have proven to be considerably more heat-stable than the material used by 
Tyler and Fox. Since the solutions employed by Tyler and Fox corresponded to 
crude fertilizin as defined in this paper, it may well be that the relatively purer con- 
dition of the fertilizin in the present preparations accounts for its greater stability. 

Initial loss of agglutinating activity does not appear to involve complete destruc- 
tion of fertilizin. At first the agglutinating fertilizin is converted into a "univalent," 
non-agglutinating form (cf. Tyler, 1941, Metz, 1942). It was of interest, accord- 
ingly, to test samples of heat-treated fertilizin for their uni valence (inhibition) titer 
as well as for their sperm-agglutinating and mucin clot titers. The method em- 
ployed for detecting univalent fertilizin has been briefly described in an earlier sec- 
tion of this paper ; determination of inhibition titer consists in determining the 
greatest dilution in which no agglutination occurs upon the addition to the test dilu- 
tions of equal amounts of normal (untreated) fertilizin solution (Metz, 1942). 

In the first experiments 1.5 ml. samples of the stock purified fertilizin solutions 
were placed in 13 X 100 mm. test tubes and immersed in a boiling water bath. The 



84 



MAX KRAUSS 



exposed portions of the tubes were cooled by means of a stream of air so that heating 
could be continued for long periods without appreciable loss of fluid. Since sperm 
are quickly inactivated in even slightly hypertonic medium, the fact that the sperm 
remained active in the solutions that had been heated was assumed to indicate that 
evaporation of water from the tubes during heating was insignificant. A thermom- 
eter placed in the water bath with the tubes showed that the temperature of the 
bath fluctuated between 96 and 98 C. 

In later experiments a quantity of fertilizin solution large enough to permit 
the withdrawal of a number of 1.5 ml. samples was placed in a flask with a reflux 
condenser attached by means of a ground glass joint. The solution was refluxed 
and loss of water was thus kept to a minimum. The temperature of the boiling 
fluid in the flask could be assumed to be about 100 C. A considerable excess of 
solution was used so that its concentration would not be significantly affected by 
the slight amount of water that failed to run back down. Before withdrawing a 
sample, the neck of the flask and the lower part of the condenser were cooled with 
cold water from a wash bottle. In all of the experiments the pH of the fertilizin sol- 
utions was adjusted with the glass electrode just before heating was begun ; a con- 
trol sample was allowed to stand at room temperature throughout the total time of 
heating. As each sample was removed from the water bath or from the reflux flask, 



TABLE V 

Results of parallel titrations of heat-treated, purified fertilizin preparations of S. purpuratus 



Fertilizin 
preparation 


Initial 
PH 


Sample 


Tempera- 
ture, 
degrees 
Cent. 


Time in 
hours 


Final 
PH 


Titer 


Sperm 
agglutina- 
tion 


Mucin 
clot 


Inhibition 


I* 


7.1 


a 


96-98 


0.5 


** 


256 


64 


*# 






b 


96-98 


1.0 





64 


64 









Control 


room 


1.0 





512 


64 





II* 


7.85 


a 


96-98 


4.5 





16 


64 









Control 


room 


4.5 





256 


64 





III* 


7.4 


a 


96-98 


3.5 





128 


256 









b 


96-98 


5.5 





64 


64 


32 






Control 


room 


5.5 





1024 


256 





IV*** 


7.5 


a 


100 


2.0 


7.5 


4096 


512 









b 


100 


3.0 


7.2 


4096 


512 









c 


100 


5.0 


7.1 


2048 


256 









d 


100 


6.0 


7.1 


2048 


256 









e 


100 


7.5 


7.1 


1024- 


256 

















2048**** 










Control 


room 


7.5 


6.8 


4096 


512 






* Individual 1.5 ml. samples heated in water bath. 

** Dashes indicate not tested. Zero inhibition titer indicates tested but inhibition not 
detected. 

*** Samples (1.5 ml.) withdrawn from refluxed solution. 
**** A trace reaction probably present in the higher dilution. 



FERTILIZIN MUCIN CLOT REACTION 

it was placed immediately in the freezer. In the later experiments the pH of each 
heated sample was recorded before it was frozen. The titrations were performed as 
soon thereafter as possible. The results of the experiments are presented in Table 
V. 

As may be seen from the table, reduction of sperm-agglutinating titer by heating 
is not necessarily accompanied by parallel reduction in mucin clot titer. Thus, for 
example, samples Ha and Ilia, heated for 4.5 and 3.5 hours respectively, showed 
no significant reduction in mucin clot titer although the sperm-agglutinating titer 
of the former was reduced to about 6 per cent and that of the latter to about 12 per 
cent of the original values. Sample Illb was heated for 5.5 hours with a reduction 
of sperm-agglutinating titer to approximately 6 per cent of its original value. In 
this case the mucin clot titer was reduced to 25 per cent of the original value. 
Samples IVa-IVe show a more nearly parallel reduction of sperm-agglutinating 
and mucin clot titers than any of the others. Preparation IV was refluxed. After 
boiling for 7.5 hours the sperm-agglutinating titer was reduced to 25-50 per cent of 
the original value and the mucin clot titer was reduced to about the same per cent 
of the original value. Sample Illb was the only one which gave an inhibition 
(univalence) titer. In the samples which were tested for inhibition but in which 
none was detected (Ilia, IVd, IVe), it is probable that insufficient univalent fertili- 
zin was present in the high dilutions to permit detection. The inhibition test is 
unambiguous only in dilutions containing sufficient univalent fertilizin to react with 
most of the added sperm. In the high dilutions enough sperm remain uncombined 
to be agglutinated upon the addition of normal fertilizin anc ; hus obscure the slight 
amount of inhibition that may be present. In the present experiments, sample Illb 
was the only one in which sufficient univalent fertilizin was produced in the lower 
dilutions to give clear-cut evidence of inhibition. Since the inhibition titer of Illb 
was 32, while the mucin clot titer was 64, it appears that the mucin clot reaction of 
fertilizin does not depend upon maintenance of the multivalent condition. Stronger 
evidence to support this view was afforded by experiments in which fertilizin was 
irradiated with ultra-violet light. 

Parallel titrations with ultra-znolet irradiated purified fertilizin preparations 

Metz (1942) showed that univalent fertilizin is produced by irradiation of 
normal (multivalent) fertilizin by ultra-violet rays. In the present experiments 
ultra-violet irradiation was carried out in an apparatus consisting of glass tubing, 
150 X 35 mm., fitted on the mid-section of a 15 watt General Electric "Germicidal" 
lamp, the diameter of which is 25 mm. The major part of the output of this lamp 
is concentrated in the 2537 A wave-length band. The space between the outer wall 
of the lamp and the inner wall of the tubing is the irradiation chamber. The chamber 
and lamp assembly is mounted on a motor-driven rocker. An opening in the top 
of the chamber, which can be closed with a rubber stopper, permits the introduction 
and withdrawal of fluid. The chamber is cooled by means of a small electric fan 
mounted on the rocker platform ; when the fan is in operation the temperature of 
fluid inside the chamber does not rise above 35 C. during irradiation. 

In the first experiment, a purified fertilizin preparation of S. purpuratns, the pH 
of which was first adjusted to 7, was irradiated for a total of 2.5 hours. It was 
found, as shown in Table VI, that the sperm-agglutinating titer was reduced to 



MAX KRAI SS 



TABLE VI 

Results of ultra-violet irradiation of purified fertilizin 



Fertilizin 
preparation 


Sample 
no. 


Initial 
pH 


Time of 
irradiation 
in hours 


Final 
pH 


Sperm 
agglutination 
liter 


Mucin 
clot 
liter 


Inhibition 
liter 


I 


Control 


7.0 








256 


64 







1 


7.0 


2i 





4 


32 


* 


II 


Control 


7.67 





6.60 


1024 


1024 







1 


7.67 


4 


5.52 





1024 


128 




2 


7.67 


6 


5.49 





1024 


128 




3 


7.67 


71 


5.49 





512 


4 




4 


7.67 


9 


5.49 





256 


4 



* Univalence present in this sample by inhibition test but titer not obtained. 

about 2 per cent of the original value and the mucin clot titer was decreased to 50 
per cent of the original. Tested by the inhibition method, the irradiated prepara- 
tion was found to contain univalent fertilizin. The inhibition titer of this sample 
was not obtained. In a second experiment, a quantity of the fertilizin preparation 
which was found to be very heat-stable with respect to its sperm-agglutinating activ- 
ity (preparation IV of the preceding section) was irradiated. The pH of the solu- 
tion was first adjusted to 7.7. Small portions (1.5 ml.) were withdrawn at inter- 
vals up to nine hours; the first sample was removed after 4.3 hours of irradiation. 
A control sample was allowed to stand in natural light (filtered through window 
glass) at room temperature throughout the entire period of irradiation. Immedi- 
ately upon the removal of each sample from the irradiation chamber, its pH was 
measured with the glass electrode, and then it was placed in the freezer. All of 
the samples, including the control, were stored in the freezer until the titrations could 
be performed. As shown in Table VI, all of the irradiated samples showed com- 
plete loss of agglutinating activity. Tested by the inhibition method, all of them 
were found to contain univalent fertilizin. The inhibition titers showed a progres- 
sive decrease as time of irradiation was increased. The mucin clot titers also 
showed a progressive decrease with increased time of irradiation. After nine hours 
the mucin clot titer was reduced to 25 per cent of its original value, and the inhibition 
titer was reduced to about 6 per cent of the value found after 4.3 hours of irradiation. 
The results of these experiments demonstrate conclusively that the mucin clot 
reaction of fertilizin does not depend upon maintenance of the multivalent condition. 
They also show that ultra-violet irradiation is a more effective agent than heat in 
converting multivalent, purified fertilizin to the univalent condition. The progres- 
sive decrease in inhibition titer found in the second experiment indicates that degra- 
dation of the fertilizin by ultra-violet light proceeds beyond the stage in which it ex- 
hibits univalence. 

DISCUSSION 

In general it may be said that the mucin clot titer of untreated fertilizin prepara- 
rations parallels their sperm-agglutinating activity. Sperm agglutination is usually 
detectable in higher dilutions than is the mucin clot reaction where the latter is ob- 
served by the ring method used in the present experiments. 



FERTILIZIN MUC1N CLOT REACTION 

Destruction of the sperm-agglutinating activity of fertilizin is not necessarily ac- 
companied by a reduction of mucin clot titer. Conversely, however, it is clear that 
fertilizin which has been subjected to treatment that causes a reduction in mucin clot 
titer, for example heating or irradiation by ultra-violet light for extended periods, 
will invariably show at least a parallel decrease in sperm-agglutinating activity. It 
has been shown in the experiments with ultra-violet irradiation that the capacity of 
the fertilizin to agglutinate sperm may be completely destroyed with but little, if 
any, loss of its ability to give the mucin clot reaction. The evidence shows that when 
the agglutinating (multivalent) form is degraded to the non-agglutinating (uni- 
valent) form, the latter continues to co-precipitate with protein in the mucin clot 
reaction. If a preparation in which all of the fertilizin has been made univalent is 
subjected to continued irradiation by ultra-violet light, a progressive decrease in 
both mucin clot and inhibition titers occurs. 

The phenomenon of sperm agglutination by fertlizin has been interpreted by 
Tyler (1941, 1942, 1947, 1948) as an antigen-antibody type of reaction in which 
complementary combining groups of a substance (antifertilizin) on the surface of 
the sperm cells unite in "lock and key" fashion with the combining groups of 
fertilizin. Where a number of such combining groups are available on the surface of 
the fertilizin molecule, agglutination occurs as the result of the building up of a 
lattice, as postulated for analogous immunological reactions by Heidelberger (1938) 
and Marrack (1938). The formation of univalent fertilizin is brought about by 
various agents e.g. heat, ultra-violet light, x-rays which, according to Tyler 
(1941), split the molecule into fragments, each of which contains a single combining 
group. These fragments are still of large size, since they are non-dialyzable (Tyler, 
1941). They are also capable of co-precipitating with protein in the presence of 
acid, giving the mucin clot reaction. 

The ability to give the mucin clot reaction is, at least in the case of hyaluronic 
acid, presumably a function of the degree of polymerization of the molecule (Meyer, 
1947). Depolymerized molecules are incapable of giving the reaction. Although 
fertilizin has been shown to be very different from hyaluronic acid in its chemical 
composition, the fact that it co-precipitates with protein in acid solution in an analo- 
gous manner suggests that it may be similar in its physical structure. Thus, fertili- 
zin may normally exist in a polymerized condition. Sperm-agglutinating activity 
may, then, accompany a range of polymer size, and the univalent condition may rep- 
resent a state of polymerization with which but a single combining element is as- 
sociated. Degradation of multivalent fertilizin to the univalent form would then 
entail a progressive splitting off of relatively stable univalent units. The evidence 
from the experiments with ultra-violet irradiation indicates that the univalent form 
is in fact the more stable, since complete conversion to univalence was observed after 
4.3 hours of irradiation, whereas even after nine hours both the inhibition and 
mucin clot titers retained significant values. 

SUMMARY 

1 . Preparations of fertilizin of three species of sea-urchin have been found to give 
a mucin clot reaction similar to that given by hyaluronic acid. Upon the addition of 
bovine serum albumin to an acidified solution of fertilizin, a precipitate forms which 
dissolves at a pH of 5.6 or higher. All of the sperm-agglutinating activity accom- 



88 MAX KRAUSS 

panics the precipitate and it is recovered quantitatively when the precipitate is dis- 
solved. 

2. A method for the determination of mucin clot titer of fertilizin is described. 

3. At temperatures near the freezing point (1 C.) the physical state of fertilizin 
can he reversibly modified by the removal of electrolytes by dialysis. Macroscopic 
aggregates appear, accompanied by a parallel decrease in mucin clot and sperm- 
agglutinating titers. Disappearance of the aggregates is accompanied by an increase 
in both titers. 

4. Chemical analysis of fertilizin shows that it contains no glucuronic acid, about 
2 per cent hexosamine and amino acids. Fertilizin, therefore, differs greatly from 
hyaluronic acid, but its ability to give the mucin clot reaction suggests an affinity with 
the class of mucopolysaccharides. 

5. In general, mucin clot titer parallels sperm-agglutinating titer of the same 
untreated fertilizin preparation, although sperm agglutination is detectable in higher 
dilutions than is the mucin clot reaction where the latter is observed by the ring 
method used in the present experiments. 

6. Parallel mucin clot and sperm-agglutinating titrations were made with fertili- 
zin preparations in untreated condition and after exposure to heat and to ultra- 
violet irradiation. The purified preparations used in these experiments proved to 
be exceptionally heat-stable ; irradiation by ultra-violet light was found to be a more 
effective treatment in degrading the material. 

7. Destruction of the sperm-agglutinating activity of fertilizin by heat and by 
ultra-violet irradiation does not necessarily cause a parallel decrease in mucin 
clot titer. The mucin clot reaction continues to be given by preparations in 
which all of the fertilizin has been converted from the normal, agglutinating con- 
dition to the non-agglutinating, "univalent" form. Continued irradiation of the 
univalent fertilizin is accompanied by a progressive decrease in both inhibition and 
mucin clot titer. 

8. It is suggested that fertilizin may normally exist in a polymerized condition and 
that the non-agglutinating, "univalent" condition may represent a relatively more 
stable lower polymer of the native, agglutinating form. 

ACKNOWLEDGMENT 

I wish to express my thanks to Prof. Albert Tyler for his helpful advice and many valuable 
suggestions, and for his critical reading of the manuscript of this paper. 

LITERATURE CITED 

DISCHE, Z., 1947a. A new specific color reaction of hexuronic acids. Jour. Biol. Cltcm., 167: 

189-198. 
DISCHE, Z., 19471). A specific color reaction for glucuronic acid. Jour. Biol. Client., 171 : 

725-730. 
HEIDELBERGER, M., 1938. The chemistry of the amino acids and proteins. Chap. XVII, pp. 

953-974. Charles C. Thomas, Springfield. 
KUHN, R., AND K. WALLENFELS, 1940. Echinochrome als prosthetische Gruppen hochmolekularer 

Symplexe in den Eiern von Arbacia pustulosa. Bcr. dent, chcin. Gcst., 72 : 458-464. 
KUNITZ, M., 1939. Purification and concentration of enterokinase. Jour. Gen. Physiol., 22 : 

447-450. 
MARRACK, J. R., 1938. The chemistry of antigens and antibodies. Medical Research Council, 

Special Report Series, No. 230. London. 



FERTILIZIN MUCIN CLOT REACTION 89 

McCLEAN, D., 1943. Studies on diffusing factors. 2. Methods of assay of hyaluronidase and 
their correlation with skin diffusing activity. Biochem. Jour., 37 : 169-177. 

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. 

MEYER, K., 1945. Mucoids and glycoproteins. Adv. in Protein Chem., 2 : 249-275. 

MEYER, K., 1947. The biological significance of hyaluronic acid and hyaluronidase. Physiol. 
Rev.. 27: 335-359. 

MEYER, K., AND J. W. PALMER, 1936. On glycoproteins. II. The polysaccharides of vitreous 
humor and of umbilical cord. Jour. Biol. Chcm., 114: 689-703. 

MONROY, A., AND A. RuFFO, 1947. Hyaluronidase in sea-urchin sperm. Nature, 159: 603. 

PALMER, J. W., E. M. SMYTH, AND K. MEYER, 1937. On glycoproteins. IV. The estimation 
of hexosamine. Jour. Biol. Chcm., 119: 491-500. 

RUFFO, A., AND A. MONROY, 1946. Ricerche sulla fisiologia della fecondazione. Nota II. 
Presenza nello sperma di riccio di mare di un fattore enzimatico fluidificante. Pubbl. 
stas. sool. Napoli, 20: 253-269. 

RUNNSTROM, J., A. TISELIUS, AND E. VAssEUR, 1942. Zur Kenntnis der Gamonwirkungen bei 
Psammechinus miliaris und Echinocardium cordatum. Ark. f. Kemi (Stockholm), 15: 
No. 16, 1-18. 

STAGEY, M., 1946. The chemistry of mucopolysaccharides and mucoproteins. Adv. in Carbo- 
hydrate Chcm., 2: 161-201. 

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., 1942. Specific interacting substances of eggs and sperm. Western Jour. Surg., 
Obs.. and Gync.. 50: 126-138. 

TYLER, A., 1946. Egg and sperm extracts and fertilization. The Collecting Net, 18: 28-29. 

TYLER, A., 1947. An auto-antibody concept of cell structure, growth and differentiation. 
Growth, 10 (suppl.) : 7-19. 

TYLER, A., 1948. Fertilization and immunity. Physiol Rev., 28: 180-219. 

TYLER, A., AND S. W. Fox, 1939. Sperm agglutination in the keyhole limpet and the sea- 
urchin. Science, 90: 516-517. 

TYLER, A., AND S. W. Fox, 1940. Evidence for the protein nature of the sperm agglutinins 
of the keyhole limpet and the sea-urchin. Biol Bull, 79: 153-165. 



THE ANAPHASE MOVEMENT OF CHROMOSOMES IN THE 
SPERMATOCYTES OF THE GRASSHOPPER 

HANS RIS 
From the Laboratories of The Rockefeller Institute for Medical Research, Neiv York 21, N. Y. 1 

Among the many complex processes involved in the division of cells, the move- 
ment of the chromosomes at anaphase is most accessible to a causal analysis. The 
beautiful preciseness of the processes involved in the orderly separation of chromo- 
somes has for a long time enticed biologists to search for their physico-chemical 
basis. However, before an analysis on this level is possible, it is necessary, first, 
to know what structural differentiations of the cell are involved, and secondly, to 
have detailed quantitative descriptions of the processes based on a study of living 
cells. 

A previous analysis of chromosome movement in certain insects (Homoptera 
and Hemiptera) has shown that the structures involved in anaphase movement 
are the kinetochores on the chro'mosomes, the chromosomal fibers, which connect 
the kinetochores to the spindle, and the spindle body (Ris, 1943). The kineto- 
chore determines the nature of the chromosomal fibers, which in the case of these 
insects are broad and sheet-like and attached to the entire length of the chromosome 
(cf. Hughes-Schrader and Ris, 1941). The movement of the chromosomes con- 
sists of two separate processes : first, the shortening of the chromosomal fibers, which 
moves the chromosomes to the poles of the spindle ; and secondly, the elongation 
of the spindle body, which further separates the chromosomes. In the Homoptera 
and Hemiptera these two components of anaphase movement are separated in time, 
so that first the chromosomes move to the poles of the spindle and then, after a 
pause of a few minutes, the spindle body stretches and carries the chromosomes 
further apart. 

In most animals and plants the chromosomal fibers are narrow bundles attached 
to a definite, restricted region of the chromosome. In this paper the spermatocyte 
divisions of the grasshopper were chosen in order to analyze chromosome movement 
in an organism with localized kinetochore. 

MATERIAL AND METHODS 

The measurements recorded here were made on spermatocytes of Chorthophaga 
viridifasciata. A few measurements on Dissostcira Carolina, Mclanoplus jemur- 
rubnim, Arphia xanthoptera and Hippiscus spec, gave similar results. 

The spermatocytes of the grasshopper are classical material in the study of 
living cells in division (Chambers, 1914, 1924; Lewis and Robertson, 1916; Belar, 
1929; Baumgartner and Payne, 1931). The usual technique consisted in breaking 
the testis follicles and spreading the cells on a coverglass in Locke's or Ringer's solu- 
tion. Baumgartner and Payne (1931) showed that the follicles can be left intact 
and the cells studied with high powers. They pulled the testis through an opening 

1 Part of the work for this paper was done in the Department of Biology, Johns Hopkins 
University. 

90 



ANAPHASE MOVEMENT OF CHROMOSOMES 



91 



of the body wall into a little pool of salt solution, but left it attached to the vasa 
efferentia. Since they had to remove the follicular membrane which contains the 
trachae, the testes may as well be completely removed from the animal. In the 
present work the testes were dissected out, the follicular membrane removed, and the 
follicles spread intact on a coverglass into a drop of Belar's solution (Belar, 1929). 
The coverslip was inverted over a depression slide and sealed with paraffin. Aseptic 
technique was not attempted since only preparations made on the same day were 
used for measurements. The temperature was kept constant at 30 C. with an 
electric stage warmer. A liquid filter of ferrous ammonium sulphate prevented heat 
from the lamp from reaching the object. The cells, thus, were disturbed as little 
as possible. 





2 

FIGURES 1 and 2. Camera lucida drawings of living spermatocytes of Chorthophaga. Side view 
of primary and secondary spermatocyte metaphases. 

To measure the movement of chromosomes a metaphase in side view was selected, 
and as soon as the chromosomes began to separate, the distance between kineto- 
chores was recorded at regular intervals with a camera lucida. In primary spermato- 
cytes a bivalent with terminalized chiasmata near the spindle axis was chosen. In 
the secondary spermatocytes a chromosome in a median optical section of the spindle 
was selected. Though the spindle itself is hardly visible, it is clearly outlined by the 
chondriosomes (Figs. 1, 2). This makes it possible to measure the length and the 
equatorial diameter of the spindle during the entire anaphase. The various distances 
were then plotted against time, yielding a curve which describes the movement of the 
chromosomes and the changes in spindle length and diameter. All measurements 
were made with a 4 mm. Zeiss apochromat and 15 X ocular. 



OBSERVATIONS 

Anaphase movement in the first spermatocyte division 

In Figure 3, three out of thirteen measured cells are presented. 2 
between separating kinetochores is plotted against time. 



The distance 
The resulting- curve con- 



2 The curves from different cells, even coming from different individuals, agree remarkably 
well, especially in the beginning of chromosome movement before spindle stretching sets in. 
The three cells shown indicate the degree of variation. 



92 



HANS RIS 



sists of an initial slow movement, then a straight portion of maximum velocity and 
a less regular part of gradually decreasing movement. Finally, before the cleavage 
furrow appears, the chromosomes move together again for a short distance, ap- 
parently due to the shrinkage of the spindle. 



H- 

60 



_ Chopthophaga Meiosis I 

12 Spindle length 



Distance between 
11 kinetochores 

*17 




Mm. 



FIGURE 3. Chromosome movement and spindle behavior in the first meiotic division of 

Chorthophaga viridifasciata. See text. 

The spindle becomes visible in prometaphase through the alignment of the 
filamentous chondriosomes on its surface. In polar view their optical cross sections 
outline the spindle around its circumference (Fig. 4). In side view they appear 
lined up from the poles to the equator where they flow out into the equatorial plane 
(Fig. 1). Later, when the spindle elongates, the chondriosomes are stretched 
tightly on the spindle surface. 

In Figure 3 the length of the spindle and its equatorial diameter are plotted 
against time during anaphase. We see how the spindle begins to elongate a few 
minutes after the onset of chromosome movement, contributing to their separation. 
There are thus two simultaneous processes involved in the later part of anaphase, 
namely, (1) the movement of the chromosomes to the poles due to the shortening of 
the chromosomal fibers, and (2) the elongation of the spindle. The diameter of 
the spindle increases in the later part of the chromosome curve, when the movement 
becomes irregular and slows down. Then it progressively decreases until the 
cleavage furrow cuts the spindle body in half. The spindle, therefore, increases 



ANAPHASE MOVEMENT OF CHROMOSOMES 



93 



appreciably in volume during mid-anaphase. A comparison of the curves in 
Figure 3 shows that the spindle elongation varies more from cell to cell than the 
shortening of the chromosomal fibers. This indicates a greater sensitivity of that 
process to external conditions. 

Anaphase movement in the second spcrtnatocyte division 

In the first division the chromosomes are distributed through the spindle body 
(Fig. 4). In the second division, however, they are oriented with their kineto- 
chores at the periphery of a "hollow" spindle, the long arms pointing outwards 
(Fig. 5). Figure 6 gives the curves of three out of fifteen measured cells. 2 The 





FIGURES 4 and 5. Camera lucida drawings of living spermatocytes of Chorthophaga. Polar 

view of primary and secondary metaphases. 



movements of the chromosomes and the behavior of the spindle are much like those 
described for the first division. The chromosome curve is distinctly S-shaped. 
The spindle elongates a few minutes after the chromosomes have separated and in- 
creases in diameter in the later part of anaphase. Again there is thus a great in- 
crease in volume of the spindle. The rate of chromosome movement and spindle 
stretching is appreciably greater than in the first division. 

In both divisions then we find the same type of anaphase movement. It begins 
with a shortening of the chromosomal fibers moving the chromosomes towards the 
poles. While this is continuing, the spindle begins to stretch, adding to the chromo- 
some movement. Towards the end of anaphase the spindle begins to increase in 
width and then gradually shrinks until the cleavage furrow cuts it in half. 

Experimental separation of the factors of anaphase movement 

In the Hemiptera and Homoptera the action of chromosomal fibers and spindle 
elongation represent two distinct processes separated in time (Ris, 1943). In the 
grasshopper there is no such independence ; the two processes are simultaneous and 
so neatly interwoven that a smooth movement of the chromosomes ensues. Is it 
possible to separate them experimentally? Methods have long been known which 
inhibit or destroy the spindle, such as ether, chloralhydrate, colchicine, etc. Is 



94 



HANS RIS 



50 



Chopthophaga MeiosisII 

27 2? 



Spindle 
length 



Distance 

between 

kmetochores 




Mm. 



10 



15 



20 



25 



25 
20 

15 



FIGURE 6. Chromosome movement and spindle behavior in the second meiotic division of 

Chorthophaga viridifasciata. See text. 

there an agent which would inhibit one of the two processes without affecting the 
other? Colchicine, if added to the medium, either destroyed the spindle completely, 
or in lower concentrations had no effect on anaphase movement. Chloralhydrate, 
on the other hand, proved more useful. In concentrations higher than 0.1 per 
cent the spindle became shorter and narrower, and finally disappeared. The chro- 
mosomes were scattered irregularly through the center of the cell. The chondrio- 
somes lost their regular orientation and began to penetrate between the chromosomes. 
At a concentration of 0.08 per cent cells were found in which the chromosomes moved 
to the poles, but where spindle elongation was inhibited. This seems to happen 
only within narrow limits of concentration of chloralhydrate inside the cell. If there 
is too much, the spindle will break down ; if there is too little, it will elongate nor- 
mally. This critical concentration is usually obtained only in a few cells of one 
cyst. The distance between chromosomes, and the length of the spindle, were then 
recorded during anaphase in primary spermatocytes exposed to chloralhydrate. 
In Figure 7 two such curves are shown (36 and 37). The spindle remained the 
same length all through anaphase in cell 37 and became only slightly longer in cell 
36. The chromosomal fibers, on the other hand, must have remained active since 
the chromosomes had moved to the poles in a regular fashion. It can be shown that 
this action of the chromosomal fibers is normal. If we subtract the spindle elonga- 
tion from the chromosome curve of an untreated primary spermatocyte, we obtain 
a curve which represents the movement of the chromosomes due to the chromosomal 
fibers alone. Two such curves are plotted in Figure 7 (13a and 17a). Since they 
agree well with the experimental curves (36 and 37), we must conclude that the 



ANAPHASE MOVEMENT OF CHROMOSOMES 



95 



action of the chromosomal fibers was not affected by the chloralhydrate even though 
the spindle was prevented from elongating. 

In the Hemiptera and Homoptera the two factors of anaphase movement, con- 
traction of the chromosomal fibers and elongation of the spindle body, are separated 
in time. In the grasshopper they overlap in time, but their differing sensitivity to 
chloralhydrate has made it possible to separate them experimentally, inhibiting 



50 



_ Chorthophaga meiosis I Chloralhydrate 



Spindle length 

36 . 

Distance 

between 

kinetochores 




Mm. 



FIGURE 7. Chromosome movement and spindle elongation in Belar's solution with 0.08 
per cent chloralhydrate (curves 36 and 37). Curves 13a and 17a represent the normal chromo- 
some movement after the spindle elongation has been subtracted. The four curves are similar, 
showing that the movement to the poles in the absence of spindle stretching is normal. 

spindle elongation without affecting the contraction of the chromosomal fibers. The 
difference between the grasshopper and the Hemiptera and Homoptera lies mainly 
in the relative timing of the component processes. In the grasshopper, spindle 
elongation sets in before the chromosomes have reached the poles. In the Hemiptera 
and Homoptera the spindle does not stretch until a few minutes after the poleward 
movement of the chromosomes has been completed. 

Recently Callan (1941) described a case in which a separation of the components 
of anaphase movement occurs under natural conditions. In a trisomic grasshopper 
(Mecostethus) the unpaired extra chromosome sometimes moves into the equatorial 
plane during the first meiotic anaphase. In these cases the spindle does not elongate. 
The poleward movement of the chromosomes, however, does not seem to be 
disturbed. For reasons unknown, spindle elongation is inhibited under these con- 
ditions while the chromosomal fibers do not seem to be affected. Of course, we do 
not know here whether the rate of movement is normal as was shown in the 
chloralhydrate experiments. 



96 



HANS RIS 



The effect oj temperature on the anaphasc movement of chromosomes 

The effect of temperature on mitosis has been repeatedly investigated, in most 
cases, however, on over-all processes such as the length of the mitotic phases, the 
rate of cleavage, etc. (see Belehradek, 1935). Only little can be concluded from 
such studies unless the processes are broken down into their components and the 
effect of temperature on these components analyzed. The effect of temperature 
on chromosome movement in living cells was studied by Bucciante (1927) in chick 
fibroblasts and by Barber (1939) in Tradescantia stamen hair cells. They found 
an increase in the rate of chromosome movement with rising temperature. This in- 
crease was large at lower temperatures and small at higher temperatures. In 
chick fibroblasts there is a maximum rate at 40 C. Faure-Fremiet (1925) had 
earlier reported an optimum temperature (37 C.) for cell division in Ascaris. 
Measurements of chromosome movement in the secondary spermatocyte of the grass- 
hopper at 17, 23 and 30 C. agree with these findings (Fig. 8 and Table I). The 
data for Tradescantia (Barber, 1939) and grasshopper also suggest an optimum 
temperature, though the range of temperature was not wide enough to show the 
decrease at higher temperatures. In the grasshopper a temperature above 32 C. 
destroys the spindle and thus inhibits chromosome movement. 



_ Chorthophaga Heiosis II Effect of temperature 

Spindle elonqation 
9 



10 



Q 




Distance between 
kinetochopes 



Min. 



5 JO 15 20 25 30 35 

FIGURE 8. Chromosome movement and spindle elongation at 17, 23, and 30 C. 



ANAPHASE MOVEMENT OF CHROMOSOMES 



97 



TABLE I 

Effect of temperature on the rate of -chromosome movement (chromosomal fibers only), 
and on the rate of spindle elongation. Micro, /minute 



Temperature 

17 C. 

23 

30 



Maximum velocity 
chromosomes 

0.4 
1.2 
2.5 



Maximum rate 
spindle elongation 

1.4 
2.4 
3.6 



Exposure to low temperature (l-5 C.) destroys the mitotic spindle, as has 
been known since the experiments of O. Hertwig (1890) on sea urchins. If grass- 
hopper spermatocytes at metaphase are exposed to 1 C., the spindle disappears, the 
chondriosomes become arranged at random, and the chromosomes are dispersed 
through the former spindle area. The cells can remain in this state for hours. If 
they are again exposed to a higher temperature (30 C.) the spindle forms anew, the 
chondriosomes are lined up on its surface and the chromosomes arranged in the 
metaphase plate. This process can be repeated several times on the same cells. 

\ 

Abnormal spindle elongation 

The increase in volume during anaphase is a characteristic property of the 
spindle in most animal cells. This swelling manifests itself especially in a pro- 
nounced elongation which contributes to the anaphase separation of the chromosomes 
that are attached to it by means of chromosomal fibers. As was shown above, 





10 





11 



12 



FIGURES 9-12. Diagrams demonstrating the abnormal lateral stretching of the spindle in 
primary spermatocytes after X-ray-induced sticking of the chromosomes. 



98 HANS RIS 

the spindle also increases in width in mid-anaphase and then gradually shrinks un- 
til it gets pinched through by the cleavage furrow. This stretching ability of the 
spindle is especially impressive under certain abnormal conditions. If the first 
meiotic division is observed in living cells after X-raying, in hypertonic medium or 
at temperatures around 32 C, one finds that chiasmata have a tendency to stick so 
that bivalents can not separate at anaphase. When the spindle begins to stretch, its 
normal elongation in the polar axis is inhibited by the combination of chromosomal 
fibers and sticking chromosomes. The spindle then begins to bulge in the equator, 
opposite the sticking chromosomes (Figs. 9, 14). If several bivalents fail to sepa- 
rate, the spindle may bend outward in several places (Figs. 10, 16). With the 
further elongation of the spindle, these lateral bulges become long and narrow pro- 
jections which begin to push out the cell membrane. The poles of the spindle ap- 
proach each other during this process, probably because of the action of the chromo- 
somal fibers, which, instead of pulling the chromosomes to the poles, now draw the 
poles closer together (Figs. 11, 15). In fixed and stained preparations, the course 
of the continuous fibers shows clearly that the lateral projections are parts of the 
spindle bending outwards (Figs. 13-15). If chromosomes stick only on one side 
of the spindle, a very characteristic bent spindle results, looking like a spindle folded 
in the middle. Actually the origin is quite different, as described above. Some- 
times the sticking chromosomes separate in mid-anaphase. The spindle then is 
able to assume its normal shape. It elongates in the polar axis and the lateral 
bulges disappear. 

Spindle elongation and cleavage furrozv 

The relation of spindle elongation to cytoplasmic division, as demonstrated by 
these abnormal anaphases, is of special interest. When the spindle does not elongate 
normally, the cleavage furrow is always delayed or does not appear at all. More 
striking are the cells in which the spindle has been forced to elongate laterally in 
the equatorial plane. The lateral bulges of the spindle begin to push the cell out 
into long narrow processes (Figs. 11, 14). At the time when normally the cleavage 
furrow is formed, constrictions become visible around these cell projections. Often 
these constrictions develop into regular cleavage furrows and pinch off one or more 
small anuclear buds (Figs. 12, 17). In the cysts with secondary spermatocytes, 
one finds then cells with the diploid number of chromosomes which undergo the 
second division, and anuclear buds which do not divide any more. In grasshopper 
spermatocytes the cleavage furrow is therefore dependent on cell elongation caused 
by the stretching of the spindle. The location of the furrow is not predetermined, 
but can occur wherever the cell is pushed out. 

Time relations 

The main difficulty in the timing of the phases of mitosis, particularly in living 
cells, is the separation of the process into clearly delimited sections. The usual 
separation into prophase, metaphase, anaphase and telophase is not well suited for 
this purpose since the beginning or end of these phases is usually without sharp 
boundary. The duration of the following well-marked phases was measured in the 
spermatocytes of the grasshopper at 30 C. : First division: (1) Metakinesis, from 



ANAPHASE MOVEMENT OF CHROMOSOMES 



99 




\ 



i 




15 




* 



16 




tf 
/ < *., 

* r&G 




17 

FIGURES 13-15. Anaphase in primary spermatocytes of Chorthophaga after irradiation with 
X-rays (100 r). Note the sticking of chromosomes and the lateral expansion of the spindle. 
Fixation: Sanfelice; stain: Iron-hematoxylin. 4 mm. Zeiss Apochromat, 15 X ocular. Com- 
pare with Figures 9-12. 



the disappearance of the nuclear membrane to the formation of the metaphase plate. 
(2) Metaphase, from the establishment of the metaphase plate to the beginning of 
anaphase separation. (3) Ana-telophase, from the beginning of chromosome 
(kinetochore) movement to the appearance of the nuclear membrane. Interphase : 
from the formation of the nuclear membrane to its breakdown in the secondary 
spermatocyte. Second division : same phases as in the first division. The processes 
which mark these stages are clearly visible in the living cell. In the first division 
the asters are visible mainly due to the radial arrangement of the chondriosomes, 
but sometimes astral rays can be seen. The nuclear membrane, which was sharply 
outlined in prophase, becomes irregular and wrinkled, then disappears first near 
the asters. Wrinkled remnants can be seen for a few minutes before they vanish. 



100 



HANS RIS 



The metaphase spindle then slowly takes shape after the orientation of the 
asters. Its outline is marked by the chondriosomes. The spindle is at first 
rather narrow and short (cf. Belar, 1929, p 433). The chromosomes are thus 
crowded into the middle of the cell. Then, while the chromosomes hecome ar- 
ranged into the metaphase plate, the spindle increases in width and in length. 
During metaphase the spindle remains constant in size and varies little from cell 
to cell. In telophase the nuclear membrane appears around a light area contain- 
ing the chromosomes. The nucleus then enlarges until the regular interphase size 
is reached. In the second division the same processes are repeated, except that no 
asters can be seen in living cells. Table II gives the duration of these phases. Cells 1 
and 2 w-ere followed through the two divisions, cells 3 and 4 only through part of 
meiosis. Cells 3 and 4 are from a different individual than cells 1 and 2. At con- 
stant temperature the length of each phase varies only slightly from one individual 
to another. 

TABLE II 

Time relations in the meiotic divisions of the grasshopper Chorthophaga (30 C.) (hours and minutes) 





Cell 1 


Cell 2 


Cell 3 


Cell 4 


I. Metakinesis 


50 





43 





I. Metaphase 


2/30 


2/45 


2/40 





I. Anaphase 


1/25 


1/40 


1/25 





Telophase 










Interphase 


2/15 


2/40 








II. Metakinesis 


25 








25 


II. Metaphase 


1/25 


1/25 





1/35 


II. Anaphase 


1/45 


1/47 





1/45 


Telophase 











DISCUSSION 

The causal analysis of mitosis strives to dissect the complex process of cell divi- 
sion into its component factors and to elucidate their composition and their mode of 
action. Since Belar's classical study, the structures involved in the mitotic move- 
ments have again received deserved attention. In the grasshopper we can dis- 
tinguish the following mitotic organelles : center, kinetochores, chromosomal fibers, 
spindle body. 

The center 

Like most animal cells the grasshopper spermatocytes contain a pair of centrioles 
which move to opposite sides of the nucleus in prometaphase and form the poles of 
the developing spindle. The asters are rather inconspicuous as in other cells w r ith 
relatively little cytoplasm. In living cells they can be seen in prophase and occasion- 
ally in metaphase, especially in a hypertonic medium (cf. Belar, 1929). In second- 
ary spermatocytes asters are even less distinct. Little is known about the function 
of the centers, except that they are probably involved in the organization of the 
spindle and in the cytoplasmic streaming which goes on during metaphase and 
anaphase. 



ANAPHASE MOVEMENT OF CHROMOSOMES 101 

Kineto chores 

This specialized region of the chromosome is essential for the regular movements 
within the spindle. Fragments which are devoid of it lag behind and do not show 
any regular orientation, but may be moved passively by the stretching spindle or 
cytoplasmic currents on the surface of the spindle (White, 1935, 1937 ; Carlson, 1938). 
The main function of the kinetochores, perhaps in cooperation with the spindle or 
centriole, appears to be the formation of chromosomal fibers. Without kinetochores 
no chromosomal fibers can be formed. 

Chromosomal fibers 

Soon after the nuclear membrane has disappeared and the spindle begins to take 
shape, we can find in fixed and stained cells a distinct fibrous connection between 
the kinetochores and the spindle poles. These are the chromosomal fibers. They 
are usually not visible in living cells and some investigators therefore deny their 
existence. 3 Yet there is enough circumstantial evidence to show that they exist as 
differentiated structures within the spindle, and that they are the major factor in 
the anaphase movement of chromosomes (cf. Cormnan, 1944; Schrader, 1944). 

Belar (1929) emphasized the role of these '"traction fibers." He assumes that 
they originate as a fluid secretion by which the kinetochore attaches itself to a fiber 
of the spindle body ("Leitfaser") and which allows the chromosome to glide 
along this "Leitfaser" in anaphase. Schrader (1944) accepts this view of Belar and 
bases on it his classification of spindles. Yet, the present writer could find no evi- 
dence for this indirect formation of the chromosomal fibers. They appear in pro- 
metaphase even before the spindle is fully formed as direct connections to the cen- 
ters. They anchor the chromosomes to the poles of the spindle. So, when the 
spindle elongates, the chromosomes are carried along, the pull being transmitted 
through the chromosomal fibers to the kinetochores. If a chromosome sticks at ana- 
phase, it will prevent the spindle from elongating on that side. The combination 
chromosome-chromosomal fibers is thus stronger than the spindle, while the cell 
membrane, for instance, yields to its pushing force. The chromosomal fibers, 
when they contract at anaphase, can even pull the spindle poles together and force the 
spindle out to one side of the cell (Figs. 9-12). The chromosomal fibers must thus 
be of greater consistency than the spindle body. 

The spindle body 

In grasshopper spermatocytes the spindle body develops from nuclear material 
between the two centers. The area around the chromosomes remains distinct even 
after the nuclear membrane has disappeared, and stays free of cytoplasmic inclusions 
like chondriosomes. In metaphase the spindle is a viscous body which can be moved 
about and dissected out by microneedles (Chambers, 1924) . It appears homogenous 
in the living cell and fibrous after fixation. The spindle is essential for the orienta- 
tion of chromosomes and for the action of chromosomal fibers since they are 
anchored at its poles. The spindle can be destroyed by a number of agents : colchi- 

3 Chromosomal fibers are sometimes visible in forms with diffuse kinetochore, if the chromo- 
somes are viewed on end and the light therefore has to pass the entire length of the sheet-like 
chromosomal fibers (Hughes-Schrader and Ris, 1941; Ris, 1942). 



102 HANS RIS 

cine, chloralhydrate, cold, heat, hypertonic medium, etc. At the same time the regu- 
lar arrangement of the chromosomes disappears and all chromosome movements 
are stopped. 

The most striking action of the spindle is the elongation during anaphase. 
Belar (1929) found that in hypertonic media this elongation appears to be greatly 
exaggerated, and this led him to a Very detailed study of spindle stretching in hyper- 
tonic solutions. His conclusions are briefly: (1) The spindle has a tendency to 
stretch; this tendency is exaggerated in hypertonic media. (2) The spindle, by 
origin, is differentiated into two half spindles, the "Stemmkorper" (pushing body) 
developing at anaphase between the daughter plates. (3) At anaphase, it is the 
"Stemmkorper" in particular which elongates. 

As was shown above, abnormal spindle stretching occurs not only in dehydrated 
cells, but always when daughter chromosomes are made to stick together. A hyper- 
tonic medium is just one way of causing chromosomes to stick at anaphase. This 
effect of hypertonic solutions on chromosomes was described by Konopacki (1911) 
in cleavage divisions of echinoderm eggs, by Kostanecki (1898) in Myzostorna, and 
by Moellendorff (1938) in tissue cultures. Similar accidents are found at high tem- 
peratures (over 30 C.) and after exposure to X-rays. It is therefore not the hy- 
pertonic medium which induces the abnormal spindle stretching, but the resistance to 
elongation in the main axis, brought about by the sticking of chromosomes. From 
Belar's figures, it is obvious that in primary spermatocytes all the abnormal spindles 
are correlated with sticking chromosomes. The bent spindles in secondary sper- 
matocytes are of a different and less extreme kind (Fig. 45, Belar, 1929). Here 
it seems to be the cell membrane which offers resistance to the elongating spindle 
and causes it to bend. X-ray-induced bridges cause the same kind of abnormal 
spindles in secondary spermatocytes as Belar described in the first division. Lateral 
expansion of the spindle after X-ray-induced chromosome sticking was also figured 
by White (1937, Figs. 12, 13). 

But Belar figures some cells which show exaggerated elongation of the spindle 
without sticking of chromosomes. These cells had been treated in anaphase. 
During anaphase the spindle increases not only in length, but also in volume. Belar 
believed that the volume remained constant, though he did not commit himself 
definitely. His beautiful drawings, however, indicate quite clearly the swelling of 
the spindle which measurements have now substantiated. In hypertonic solutions 
the cell shrinks greatly, and as Belar pointed out, the cytoplasm more so than the 
spindle. The swelling of the spindle, then, encounters resistance, and it is probably 
this factor which causes the spindle to be longer, but narrower than normally. 
Even so, these spindles are only found in free floating cells. In this writer's prepara- 
tions where the cells remained in the follicles, they did not occur. Another case of 
spindle stretching without chromosome sticking is found if prometaphases are treated 
with hypertonic solutions (Belar, 1929, Fig. 55; Ris, 1942, in spermatocytes of the 
bearberry aphid). Here again the spindle increases in width during its formation. 
In dehydrated cells this lateral growth is interfered with, and the spindle becomes 
long and narrow. In this connection it is important to note that the volume of the 
abnormally stretched spindles appears to be not larger than in normal spindles. 
There is, therefore, only a distortion in shape, not an actual increase in the spindle 
material. 



ANAPHASE MOVEMENT OF CHROMOSOMES 103 

Two factors then cause abnormal spindles : interference with the increase in 
width in prometaphase and mid-anaphase ; and interference with normal stretching 
during anaphase through the sticking of chromosomes. 

From his studies of these abnormal spindles, Belar came to the conclusion that 
the part of the spindle between the separating chromosomes was mainly responsible 
for the stretching. He called it the "Stemmkorper" (pushing body) and distin- 
guished it from the two half spindles between the chromosomes and poles. This 
subdivision of the spindle is, however, artificial and unjustified. Belar himself 
points out the uniformity in the aspect of the entire spindle. Fibers and clefts are 
continuous. The only difference in anaphase is the presence of chromosomal fibers in 
the cone-shaped region between the chromosomes and the poles. This is responsible 
for the darker appearance after staining. In Belar's Figure 41 the chromosomal 
fibers are especially clear. The "Stemmkorper" concept originated in the obser- 
vation that the region between the daughter plates elongates more rapidly than the 
entire spindle. This appears so, not because this region is a special part of the 
spindle, but because the chromsomal fibers actually shorten during spindle elonga- 
tion, pulling the chromosomes to the poles. In this way the impression of a special 
stem body between the daughter plates is produced. 

Furthermore, Belar thought that the initial separation of the chromosomes 
through action of the traction fibers releases the tension in the spindle and originates 
the action of the "Stemmkorper." But actual timing has now shown that the 
chromosomes travel a good distance to the poles before the spindle elongates. Be- 
sides, spindle stretching can occur without any action of the chromosomal fibers as is 
shown in the first spermatocyte division of Tamalia. The chromosomal fibers act 
merely as passive anchors for the chromosomes (Ris, 1943). In the lepidopteran 
Orgyia the spindle elongates though the chromosomes have no chromosomal fibers 
at all (Cretschmar, 1928, Figs. 48-50). All the evidence then indicates that there 
is no differentiation into "half spindles" and "Stemmkorper." The only real 
differentiations are the chromosomal fibers and the spindle body. The chromosomal 
fibers pull the chromosomes to the poles. The stretching of the spindle has nothing 
to do with this phase. It can go on just as well without spindle elongation 
(chloralhydrate experiment). But spindle elongation has its important functions. 
It separates the daughter plates still further by pushing the poles apart and thus in- 
directly moves the chromosomes anchored to them. 

The picture of anaphase movement in grasshopper spermatocytes presented here 
is essentially in agreement with Belar's view. There is a "pulling action" of 
chromosomal fibers and the stretching of the spindle. But there are some modifica- 
tions. The subdivision into half spindles and "Stemmkorper" is found to be arti- 
ficial. The spindle as a whole elongates, at the same time increasing in volume. Its 
action on the chromosomes is indirect, through the chromosomal fibers which con- 
nect them to the spindle poles. 4 The chromosomal fibers are thought to connect 
the kinetochores directly to the poles without the intervention of a "Leitfaser." 
They shorten during anaphase and are alone responsible for moving the chromosomes 
to the spindle poles. 

4 Just how the chromosomal fibers are attached to the spindle is a very puzzling problem 
and nothing definite can be said about it at present. 



104 HANS RIS 

In addition to being a major factor in the movement of chromosomes, the spindle 
body also seems to play a role in the division of the cytoplasm. If the spindle does 
not elongate, as in the chloralhydrate experiments, no cleavage furrow is formed. 
When the spindle stretches laterally instead of in its long axis, a cleavage furrow 
does appear at a right angle to this elongation in a quite unorthodox position and 
produces an anuclear bud (Figs. 9-12). Bauer (1931) illustrates a similar situation 
in spermatocytes of Tipula with abnormal spindles. His Figure 23 h suggests that it 
originated in the same fashion. Many examples can be found in the literature 
which show how the failure of spindle stretching causes absence of the cleavage 
furrow (for instance Dobzhansky, 1934; Callan, 1941). In most plant cells there is 
little or no stretching of the spindle and the 'cytoplasm is divided by the formation of 
a cell plate. But in the pollen mother cells of some plants a cleavage furrow is 
formed, and it is then associated with elongation of the spindle (Guignard, 1897; 
Farr, 1918). It appears then that in dividing cells, elongation of the cell and cleav- 
age furrow are associated with spindle elongation (in contradiction to the un- 
warrantable generalization of Buchsbaum and Williamson, 1943). Dan has re- 
cently (1943) assembled convincing evidence that spindle elongation is the active 
agent in cell elongation and the following formation of a cleavage furrow. 5 On the 
other hand, in certain abnormal cases the spindle elongates and yet no cleavage fur- 
row appears. The formation of a cleavage furrow clearly depends on other factors 
in addition to spindle elongation. 

In a recent paper, Hughes and Swann (1948) published chromosome separation 
and spindle elongation curves for chick embryo cells in tissue culture. Chick 
chromosomes possess a localized kinetochore and the achromatic apparatus is 
similar to that of the grasshopper spermatocytes. The anaphase movement as 
described by the curves of Hughes and Swann is essentially the same as we found 
in the grasshopper. Their curves show spindle elongation to start right from the 
beginning of anaphase, while in the grasshopper it does not begin until the chromo- 
somes have moved a considerable distance. This is probably not a real difference 
but the result of the great difficulties involved in making measurements in early ana- 
phase on the small chromosomes and spindles of the chick embryo cells. 

We have set out to describe the movement of chromosomes during anaphase in 
terms of the mitotic organelles involved. The structures responsible for this chromo- 
some movement were found to be the chromosomal fibers and the spindle body. The 
chromosomal fibers move the chromosomes to the spindle poles by decreasing in 
length. The spindle body swells and stretches and moves the daughter chromo- 
somes further apart, since they are anchored to the spindle by the chromosomal 
fibers. If these are broad sheets attached to the entire length of the chromosome 
(diffuse kinetochore), the spindle does not elongate until the chromosomes have 
reached the spindle poles (hemipteran and homopteran insects). If the chromo- 
somal fibers are narrow bundles attached to a very short region of the chromosome 
(localized kinetochore), the spindle begins to stretch shortly after the chromosomes 
have begun to move. The two processes then act simultaneously producing a 
smooth unbroken chromosome separation curve.- Though we cannot see here 
directly how the two factors act on the chromosomes, we can separate them experi- 

5 I am indebted to Dr. D. Costello, University of North Carolina, for making this paper 
available to me. 



ANAPHASE MOVEMENT OF CHROMOSOMES 105 

mentally by inhibiting spindle elongation with chloralhydrate. It is then possible 
to study the action of the chromosomal fibers alone. 

Very little is known about the nature and mode of action of these organelles, and 
this aspect will not be discussed here. Many more exact data on the structure, 
composition, and behavior of spindle and spindle components under various condi- 
tions are needed before a fruitful hypothesis on the physico-chemical level can be 
brought forward. 

SUMMARY 

The movement of chromosomes and the changes in spindle size have been re- 
corded in living spermatocytes of the grasshopper during the meiotic divisions. 
Anaphase movement consists of two separate processes which are related to the ac- 
tion of distinct cellular organelles: (1) The shortening of chromosomal fibers moves 
the chromosomes to the poles. (2) The elongation of the spindle further separates 
the daughter plates. The two processes act simultaneously in the grasshopper. 
With chloralhydrate, spindle elongation can be inhibited without affecting the ac- 
tion of the chromosomal fibers. This demonstrates the independence of these two 
factors. 

The effect of temperature on chromosome movement is shown by measurements 
at 17, 23 and 30 C. Between 17 and 23 there is a greater increase in velocity 
of chromosome movement than from 23-30 C. Temperatures above 32 C. in- 
hibit mitosis through the destruction of the spindle. 

Abnormal spindle elongation is found whenever chromosomes stick at anaphase. 
The spindle, unable to elongate in its long axis, expands laterally into a disc-shaped 
body which later forms one or several finger-like processes, pushing out the cell mem- 
brane. These lateral elongations usually give rise to one or more cleavage furrows, 
pinching off one or, rarely, more anuclear buds. This demonstrates clearly the re- 
lationship between spindle elongation, cell elongation, and cleavage furrow. 

The role of the mitotic organelles in the anaphase movement of chromosomes is 
discussed. Indispensable for a regular anaphase are the kinetochore.s on the chromo- 
somes, the chromosomal fibers, and the spindle body. No evidence was found for 
a specialized region in the spindle acting as "Stemmkorper." The spindle is uni- 
form in structure and elongates uniformly. 

Distinct recognition of the structures involved in anaphase movement, and a 
quantitative description of their function, forms a basis for experimental analysis of 
their composition as well as their mode of action. 

LITERATURE CITED 

BARBER, H. N., 1939. The rate of movement of chromosomes on the spindle. Chromosoma, 

1 : 33-50. 
BAUER, H., 1931. Die Chromosomen von Tipula paludosa Meig. in Eibildung und Spermato- 

genese. Z. Zcllf., 14: 138-193. 
BAUMGARTNER, W. J., AND M. A. PAYNE, 1931. "Intravitam" technic used in studies on the 

living cells of grasshoppers. Jour. E.vp. Zoo/., 59 : 359-384. 
BELAR, K., 1929. Beitraege zur Kausalanalyse der Mitose. II. Untersuchungen an den Sper- 

matocyten von Chorthippus (Stenobothrus) lineatus Panz. Arch. f. Entwicklungs- 

mcch, 118: 359-484. 
BELEHRADEK, J., 1935. Temperature and living matter. Protoplasma Monographien 8, Born- 

traeger, Berlin. 



106 HANS RIS 

BUCCIANTE, L., 1927. Ulteriori ricerche sulla velocita della mitosi nella cellule coltivate "in 

vitro" in funzione della temperatura. Arch. Exp. Zellf., 5 : 1-24. 
BUCHSBAUM, R., AND R. R. WILLIAMSON, 1943. The rate of elongation and constriction of 

dividing sea urchin eggs as a test of a mathematical theory of cell division. Physiol. 

Zool., 16: 162-171. 

CALLAN, H. G., 1941. A trisomic grasshopper. Jour, of Hcrcd., 32: 296-298. 
CARLSON, J. G., 1938. Mitotic behavior of induced chromosomal fragments lacking spindle 

attachments in the neuroblasts of the grasshopper. Proc. Nat. Acad. Sci., 24: 500-507. 
CHAMBERS, R., 1914. Some physical properties of the cell nucleus. Science, 40: 824-827. 
CHAMBERS, R., 1924. The physical structure of protoplasm as determined by microdissection 

and injection. Cowdry's General Cytology, pp. 268-276. 
CORNMAN, J., 1944. A summary of evidence in favor of the traction fiber in mitosis. Am. 

Nat., 78: 410-422. 
CRETSCHMAR, M., 1928. Das Verhalten der Chromosomen bei der Spermatogenese von Orgyia 

thyellina BTL und antiqua L. sowie eines ihrer Bastarde. Z. Zellf., 7 : 290-309. 
DAN, K., 1943. Behavior of the cell-surface during cleavage. VI. On the mechanism of cell 

division. Jour, of the Facility of Science, Tokyo Imp. Univ. IV, vol. 6: 323-368. 
DOBZHANSKY, TH., 1934. Studies on hybrid sterility. I. Spermatogenesis in pure and hybrid 

Drosophila pseudoobscura. Z. Zellf., 21 : 169-224. 

FARR, C. H., 1918. Cell division by furrowing in Magnolia. Am. Jour. Bot., S: 379-395. 
FAURE-FREMIET, E., 1925. La cinetique du developpement. Presse Universitaire, Paris. 
GUIGNARD, L., 1897. Les centres cinetiques chez les vegetaux. Ann. Sci. Nat. Bot., 8 : 177-220. 
HERTWIG, O., 1890. Experimented Studien am tierischen Ei vor, waehrend und nach der 

Befruchtung. Jcnaische Zeitschr., 24: 268-313. 
HUGHES-SCHRADER, S., AND H. RIS, 1941. The diffuse spindle attachment of coccids, verified 

by the mitotic behavior of induced chromosome fragments. Jour. Exp. Zool., 87 : 

429-456. 
HUGHES, A. F., AND M. M. SWANN, 1948. Anaphase movements in the living cell. Jour. Exp. 

Bio!., 25 : 45-70. 
KONOPACKI, M., 1911. Ueber den Einfluss hypertonischer. Losungen auf befruchtete Echiniden- 

eier (Strongylocentrotus lividus und Echinus microtuberculatus) . Arch. Zellf., 7: 

139-183. 
KOSTANECKI, K., 1898. Die Befruchtung des Eies von Myzostoma glabrum. Arch. Mikr. 

Anat., 51 : 461-480. 

LEWIS, M. R., AND W. M. R. ROBERTSON, 1916. The mitochondria and other structures ob- 
served by the tissue culture method in the male germ cells of Chorthippus curtipennis 

Scudd. Biol. Bull, 30 : 99-125. 
MOELLENDORFF, W. VON, 1938. Zur Kenntnis der Mitose. IV. Der Einfluss von Hypo- und 

Hypertonie auf den Ablauf der Mitose sowie auf den Wachstumsrhythmus von 

Gewebekulturen. Z. Zellf., 28: 512-546. 
RIS, H., 1942. A cytological and experimental analysis of the meiotic behavior of the univalent 

X-chromosome in the bearberry aphid Tamalia (Phyllaphis) coweni (Ckll). Jour. 

Exp. Zool., 90: 267-330. 
RIS, H., 1943. A quantitative study of anaphase movement in the aphid Tamalia. Biol. Bull., 

85: 164-178. 

SCHRADER, F., 1944. Mitosis. Columbia University Press, New York. 
WHITE, M. J. D., 1935. The effect of X-rays on mitosis in the spermatogonial divisions of 

Locusta migratoria L. Proc. Roy. Soc. London, B119: 61-84. 
WHITE, M. J. D., 1937. The effect of X-rays on the first meiotic division in three species of 

Orthoptera. Proc. Roy. Soc. London, B124: 183-196. 



Vol. 96, No. 2 April, 1949 

THE 

BIOLOGICAL BULLETIN 

PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY 



TEMPERATURE COEFFICIENTS OF RESPIRATION IN 

PSAMMECHINUS EGGS 

HANS BOREI AND SIGVAR LYBING 

Wenncr-Gren's Institute for Experimental Biology, University of Stockholm 

INTRODUCTION 

In Arbacia punctnlata Rubenstein and Gerard (1934) using Warburg technique, 
found a Q UI of oxygen consumption for fertilized eggs of 1.8 between 13 and 30 C, 
whereas unfertilized eggs had a much higher value, viz., 4.1. These remarkable find- 
ings were principally confirmed by Korr (1937), who extended the experiments 
(Warburg technique) and discussed the results from biochemical and physiological 
points of view. 

On the other hand Tyler and Humason (1937), working on Strongylocentrotus 
purpuratus (Warburg technique), found no significant difference in respiration Q lf , 
between fertilized and unfertilized eggs in the temperature range 5-22 C. So, for 
example, the Q 10 values for 10-20 C. were 2.3 and 2.6 respectively. Similar results 
were reported for Dendraster, Ciona and Urechis. 

In earlier investigations (Warburg technique) by Ephrussi (1933) on Para- 
centrotus lividns the same Q 10 of oxygen uptake \vas found between 14.9 and 22.4 
for unfertilized eggs and gastrulae, viz., ' 2-2.5. At lower temperatures there was 
a tendency to slightly higher Q 10 values in the eggs. Loeb and \Vasteneys (1911) 
using Winkler technique reported very low values for unfertilized Arbacia eggs 
(Qio '' 1-3 between 5 and 25 C.), but normal values for fertilized eggs (Q 10 <- 
2-2.5 for the range 3-25 C.). 

In view of the probable significance of temperature coefficients of respiration for 
elucidating the different oxidative mechanisms of fertilized and of unfertilized sea- 
urchin eggs, it was thought of importance to investigate the matter in another species. 
Recently Borei (1948a) studied the respiration of eggs of Psammechinus tniliaris 
before and after fertilization. Because of the facts already known about this species, 
it was chosen for the present investigation. 

107 



108 



HANS BOREI AND SIGVAR LYBING 



EXPERIMENTAL DATA 

The experiments have been performed with Cartesian diver micro-respiration technique, 
suitable for measurement of the oxygen consumption of ' 100 eggs at a time. Concerning 
material and methods, corresponding -chapters (2.1-2.3 ; 3.111; 3.114) in Borei (1948a) should 
be consulted. Diver charge type I (Borei, 1948b) was used throughout. 

The temperature range was 10-21 C. The maximum temperature for normal larval 
development of Psammechinus inillaris has been studied by S. Runnstrom (1927), who found 
it to be 22 C. ; the minimum temperature was found by this author to be 8 C. For measure- 
ments at lower temperatures, the cooling coil of the diver apparatus thermostat was fed with 
refrigerated salt-\vater of approximately +5 C. The desired temperature was obtained by 
counteracting the cooling device by operating a thermostatically controlled electric heating bulb. 

In order to obtain more comparable values, measurements were only performed 
on the flatter part of the declining respiration curve of the unfertilized egg (cf. 
Borei, 1948a, Chapter 3.112.1). Thus the time of actual measurement usually ran 
from three to six hours after removal from the ovary. This means that the con- 
stant part of the respiration is dominant during the measurements, whereas the "rap- 
idly declining" part characterizes the preceding 2.5-3 hours, during which the eggs 
were kept at 16-18 C. The eggs were, on an average, placed in the diver 2.5 hours 
after removal from the ovary. The diver was then immediately placed in the thermo- 
stat at the experimental temperature and left there for a half hour for temperature 
equilibration before starting the measurements. Usually two diver thermostats were 
operated simultaneously, thus allowing measurements at two different tempera- 
tures. The time schedule of the experiments may be seen from Figure 1. 



LJ 



LJ 

52 

L.X 

00 

LJ 



01 




DIVER PLACED IN THERMOSTATE 
START OF MEASUREMENTS 



END OF MEA- 
I SUREMENTS 

f 18 C. 



IOC. 



01 234567 
HOURS AFTER REMOVAL 

FROM OVARY 

FIGURE 1. Average time schedule of experiments. 

An actual experiment with measurements at 10 C. and a control experiment at 18 C. is 
assumed in the figure. 

After completed diver measurements, the cells were washed out of the divers 
with sea-water, re-counted and then microscopically observed as to condition and 
fertilizability. Only those experiments were accepted in which the cells passed 
these post-diver measurement controls satisfactorily. 



TEMPERATURE AND EGG RESPIRATION 



109 



Previous authors have stated their results in rates of oxygen consumption per 
volume of cell matter. Great pains were taken by them to estimate the volume ac- 
curately. Probably the most correct way will be that adopted by Korr (1937) : 
egg volume obtained by multiplying the number of eggs by the average egg volume. 
Technical difficulties were met, however, in estimating the exact number of eggs. 
In the present investigation neither the counting of the eggs nor the measurement 
of the average size of the eggs will give any difficulties. In view of these facts 
and of the great variability of the cell diameter, it has been thought more advisable, 
even in this investigation, to state the rate of oxygen uptake on a cell volume basis. 
This procedure also permits of direct comparison with the results of the previous 
authors. 

In order to obtain a measure of the cell volume, the cell diameter was estimated of a number 
of eggs (> 20) from every female used, by means of a calibrated ocular micrometer (cf. Borei, 
1948a, Chapter 3.114). 

TABLE I 

Oxygen consumption at different temperatures of unfertilized Psammechinus iniliaris eggs 
Volume of oxygen, measured at C. and 760 mm. Hg, consumed per volume of cell matter 
and hour. All measurements are on egg material from S-form animals, except those marked *, 
which are on material of the Z-form. 



Temperature 

(C) 


Oxygen 

consumption 


Temperature 
PC) 


Oxygen 

consumption 


10 


0.024 
0.053 
0.069 




0.080 
0.082 
0.084 
099 


11 


0.062 
0.066 
0.069 


18 


0.107* 
0.132 
0.151* 
160 


1 ~) 


On^\7 






12. 


.uo/ 




086 


13 


0.056 
0.076 


19 


0.089 
0.140 
145 


14 


0.078 
0.083 




0.173 
0.188 


15 


0.060 
0.113 


20 


0.065 
0.111 
116 




0.033 
On i f. 




0.126 


16 


.U4O 

0.099 
0.112 


21 


0.090 
0.122* 
131 


17 


0.061 
0.076 
0.088 




0.172* 


18 


0.063 
0.065 







110 



HANS BOREI AND SIGVAR LYP.ING 



The oxygen consumption figures obtained are referred to C. and 760 mm. 
Hg, in order to render them intercomparable, irrespective of the actual temperature 
of measurement. Corrections are introduced according to Figure 2 of Borei 
(1948a) for deviations from the time schedule of the above Figure 1. 

The results of the experiments are given in Table I. 

The average cell volume, calculated from the separate figures used for the evaluations in 
Table I, is 5.89 X 10" 4 Ml. per egg (n = 44). (Corresponding value given by Borei, 1948a, 
= 5.84 X ICT 4 .) The average oxygen consumption rate at 18 C. was found in the experiments 
(n = 10) to be 0.51 X 10~ 4 Ml. per cell and hour. (Corresponding value given by Borei, 1948a, 
= 0.53 x 10" 4 .) 

DISCUSSION 

Rubenstein and Gerard (1934) expressed their results according to the van't 
Hoff-Arrhenius equation and thought that the critical thermal increments (/A) 
might indicate the nature of the oxidative processes of the unfertilized and fertilized 
egg. This view was criticized by Korr (1937), who stressed that biological scatter- 
ing and the narrow temperature limits within which respiration can be measured 
make the graphical evaluation of //.-values uncertain. Moreover, biological processes 
are governed by enzyme reactions. Such reactions have repeatedly been found not 
to give constant thermal increments. 



xlO 



O 

I 



-2 



(T 

LJ 



Ld 
O 

LJ 



O 



10 



NUMBER OF EXPTS 

I 04 

2 '. 

3 9 10 



PSAMMECHINUS 



10 15 

TEMPERATURE 



20 



FIGURE 2. Oxygen consumption at different temperatures of unfertilized Psammechinus 
miliaris eggs. 

Each dot is the mean value for the temperature in question. Diameter of dot indicates 
number of experiments. A standard curve according to Krogh (1914) is drawn in, passing 
through the value of 18 C. 



TEMPERATURE AND EGG RESPIRATION 111 

The mass plot, recommended by Korr (1937), is difficult to interpret owing to 
the extent of the biological scattering. The mean of the values at each temperature 
gives a better representation. Figure 2 is plotted in this manner. 

The figure shows that the standard curve of Krogh (1914) describes the ob- 
tained results quite well. This curve was originally obtained in basal metabolism 
experiments and found to be valid for a number of vertebrates. It was recently 
extended by Zeuthen (1947) to hold even for the respiration of a number of minute, 
chiefly marine invertebrate organisms. Thus Krogh's standard curve better de- 
scribes the response in respiration on increase of temperature than does the van't 
Hoff-Arrhenius equation. 

Formula ( / ) 

For the temperature range of the present investigation the curve indicates that the relative 
temperature increment is very closely proportional to the relative increment in respiration. 
Thus it may be expressed by (Rj/Ro) (t,/t,) = const., where R, and R, are the rates of respira- 
tion at the temperatures t, and t 2 . The constant is dependent on the chosen difference between 
t, and t,. For t, - t, = 2 C., it is = 0.93. 

In Figure 3 the results of this and previous investigations are given on a rela- 
tive scale. The curve according to Formula (1) represents the material of this 
investigation. With this curve coincide the values of Tyler and Humason (1937) 
on the respiration of Strongylocentrotus eggs. On the other hand the results 
with Arbacia differ markedly from those on Psammechinus or Strongylocentrotus. 

The temperature characteristics of the unfertilized sea-urchin egg thus represent 
two distinct classes: (1) the Arbacia type with high Q 10 values, and (2) the 
Strongylocentrotus-Psammechinus type with Q ]0 values in close concordance with 
the standard curve of Krogh. 

The temperature coefficients of the fertilized Psammechinus egg differ in no 
way from those found by previous authors for fertilized eggs of other sea-urchin 
species. Thus in the range 12-20 C. a Q 10 ^ 2-2.5 was found. 

A comparison of the Q 10 values found in different investigations further stresses 
that there are two classes in respect to temperature characteristics of respiration of 
the unfertilized eggs (see Table II). Strongylocentrotus and Psammechinus have 
for both fertilized and unfertilized eggs a Q 10 value at room temperature of approxi- 
mately 2.5. The fertilized Arbacia egg shows the same value, but the value of the 
unfertilized egg is higher. The unfertilized egg of Paracentrotus has, at lower 
temperatures, a tendency in the same direction as the Arbacia egg. 

Apparently Q, n as in most biological processes is higher at lower temperatures. The results 
of Tyler and Humason (1937) and of Korr (1937) seem, however, to contradict this conclusion, 
but the aberrations are probably to be attributed to experimental circumstances. (Lucke and 
co-workers, 1931, found that the Q 10 of the permeability of the Arbacia egg to water increased 
with temperature.) 

In the sea-urchin egg the rate of respiration is increased greatly by fertilization. 
This higher respiration is suppressed by cyanide, CO and other poisons of cyto- 
chrome oxidase. The oxidase in operation is an iron porphyrin, but probably not 
fully identical with the usual cytochrome oxidase. The respiration of the unferti- 
lized egg was found by Runnstrom (1930) and Korr (1937) to be comparatively 



112 



HANS BOREI AND SIGVAR LYBING 



o: 

o 



u 



_J 
u 
u 

LJ 

D 
_) 

O 



o 

I 
LJ 



u 

(T 



O TYLER 4 HUMASON (1937) 

(STRONGYLOCENTROTUSI 

RUBENSTEIN 8. GERARD U934) 

(ARBACiA) 

KROGH (I9i4) 

(STANDARD CURVE) 



KORR (1937) 
(ARBACIA) 




CURVE ACCORDING TO 

FORMULA (I) OF THIS 

INVESTIGATION 

(PSAMMECHINUS) 



10 15 

TEMPERATURE 



2O 



c 



FIGURE 3. Comparison between different authors' results concerning temperature depend- 
ency of oxygen consumption in unfertilized sea-urchin eggs. 

Rates of oxygen consumption given on a relative scale, putting the values at 18 C. alike. 
The curve for Korr's results is recalculated from this author's Figure 8. 



slightly affected by said oxidase poisons. Both oxidase and dehydrogenases can 
be brought to work as effectively in the unfertilized egg as in the fertilized one. 
This permits the conclusion that neither the oxidase nor the dehydrogenases are the 
limiting factors in the respiration of the unfertilized egg (Runnstrom, 1935, 1942; 
cf., however, Ballentine, 1940). Runnstrom thinks that the difference in respira- 
tion rate between unfertilized and fertilized eggs is caused in the unfertilized egg 
by a lack in substrate saturation of the oxidase. Korr interprets the difference as 
being dependent on a factor in the oxidase part of the carrier chain, inactive or held 
apart in the unfertilized egg, but put into operation on fertilization. He thinks 
that this link might be cytochrome c and furthermore that the unfertilized egg re- 
spires over an autoxidizable, non-ferrous carrier. For a fuller review see Need- 
ham (1942) and Borei (1948a). 



TEMPERATURE AND EGG RESPIRATION 



113 



TABLE II 

<2io values of oxygen consumption of sea-urchin eggs 



Author 



Loeb and Wasteneys 
(1911) 

t 



Rubenstein and Gerard 
(1934) 



Korr (1937) 



Unfertilized eggs 



(Arbacia punctulata 
5-25 C. Q, =1.31) 



Arbacia punctulata 
13-30 C. Qio = 4.1 



Arbacia punctulata 



13-23 C. 
18-28 



4.5 



Fertilized eggs 



Arbacia punctulata 

(round 1st mitosis) 

3-27 C. Qio~ 2-2.5 



Arbacia punctulata 

(up to 5 hours after fert.?) 

13-30C. Qio=l-8 



Arbacia. punctulata 

(up to 5 hours after fert.?) 

13-23 C. Qio=2.2,2.8 

18-28 2.5, 3.1 



Ephrussi (1933) 

* 



Paracentrotus Hindus 

10.4-22.4 C. Q 10 =3.85 

12.9-22.4 3.28 

14.9-22.4 2.18 

16.75-22.4 1.88 



Paracentrotus lividus 

(gastrulae) 

10.4-22.4 C. Qio = 2.36 
12.9-22.4 2.57 

14.9-22.4 2.32 

16.75-22.4 1.88 



Tyler (1936) 
* 



Strongylocentrotus purpuratus 

(round 1st mitosis) 

7. 5-20 C. Qio=2.54 

10 -20 2.30 

15 -20 1.85 

(25.th-26th hour after fert.) 

15 -20 C. 0,0=1.88 



Tyler and Humason 
(1937) 



Strongylocentrotus purpuratus 



7. 5-17 C. 

8 -18 
10 -20 
12 -22 

5 -20 



Q,o = 2.67 
2.58 
2.63 

2.54 
2.35 



Strongylocentrotus purpuratus 

(a few hours after fert. ) 

7. 5-17 C. Q 10 =2.79 

8 -18 2.69 

10 -20 2.26 

12 -22 2.33 

5 -20 2.62 



This investigation 

t 



Psammechinus tniliaris 
10-1 2 C. Qw = 3.52 
3.07 
2.81 
2.58 
2.40 



12-14 
14-16 
16-18 
18-20 
20-22 



Psa nimechin us m iliaris 

(a few hours after fert.) 

12-20 C. Q 10 ~2-2.5 



2.29 



t Winkler technique. 
* Warburg experiments. 



J Uiver technique. 

Only a single experiment. 



114 HANS BOREI AND SIGVAR LYBING 

Recent investigations on Arbacia eggs (Robbie, 1946) show that the respira- 
tion of the unfertilized egg can also be completely abolished by cyanide. A cyanide- 
stable respiration, catalyzed by formed CN -compounds, develops, however, after the 
initial inhibition. These findings confirm Lindahl's (1939, 1940 and 1941) re- 
sults, and are in full concordance with his opinion that the cyanide-resistant respira- 
tion develops under the influence of the cyanide and that it has nothing to do with 
normal respiration. Robbie's findings show that the respiration of the unfertilized 
egg must proceed over iron porphyrins in the same manner as does that of the fer- 
tilized egg. Runnstrom (1930) had previously expressed the same opinion. 

Robbie's results show conclusively that the respiration is of principally the same 
type both before and after fertilization. Thus it is understandable that temperature 
characteristics of respiration are found, as in the Psammechinus and Strongylocen- 
trotus cases, that are the same before and after fertilization. On the other hand it 
is harder to understand how Q 10 can differ so widely before and after fertilization 
as it does in Arbacia. It may be that the respiratory system operating in the un- 
fertilized Arbacia egg is somehow unlike that of the just mentioned species. Any 
fundamental respiration pattern differences between the two classes of unfertilized 
sea-urchin eggs have, however, not been found. 

Ballentine (1940) thinks that the oxidation in the sea-urchin egg of dimethyl-p- 
phenylenediamine requires cytochrome c as a mediator to the echinoderm oxidase. 
If so, cytochrome c must already be available and ready to function in the unferti- 
lized egg, since Runnstrom (1932) and Orstrom (1932) found that dimethyl-p- 
phenylenediamine was oxidized at the same rate in unfertilized and fertilized eggs. 
Borei and Renvall (1949) could, however, not find that cytochrome c is essential 
for the cellular oxidation of dimethyl-p-phenylenediamine. Furthermore, hydro- 
quinone, which can be oxidized by cytochrome oxidase (Keilin and Hartree, 1938) 
or echinoderm oxidase (Krahl and co-workers, 1941 ; Borei, 1945, Chapter IV: Cl) 
only in the presence of a suitable mediator, has been shown by Runnstrom (1932) 
to be utilized in the unfertilized egg at a lower rate than in the fertilized one. This 
fact could possibly be taken as an indication that the carrier oxidized by the echino- 
derm oxidase has a lower concentration (or is less active or accessible) in the un- 
fertilized egg than in the fertilized. 

Many authors have doubted the existence of cytochrome c in the egg, as it has 
never been possible to find the cytochrome bands (cf. Krahl and co-workers, 1941). 
Thus Korr (1939), who originally (1937) thought that cytochrome c was released 
at fertilization, and Ballentine (1940) suppose that some other link, situated nearer 
the substrate, is put into operation at fertilization. It is, however, not unlikely that 
cytochrome c may have a sufficient carrier capacity in the sea-urchin egg, and yet not 
have a concentration high enough to permit spectroscopic detection. It may be 
pointed out (a) that the sea-urchin egg has a comparatively low Q 02 , and (b) that 
cytochrome c is far more catalytically effective when attached to the proper intra- 
cellular protein particles than when working in solution. It may' also be that an- 
other carrier has the same function in respect to echinoderm oxidase as has cyto- 
chrome c to cytochrome oxidase. (Concerning the differences between echinoderm 
oxidase and cytochrome oxidase, cf. Borei, 1945, and Krahl and co-workers, 1941). 

The surplus in respiration induced by fertilization is either merely an addition to 
the respiration of the unfertilized egg, or caused by the fact that the induced respira- 



TEMPERATURE AND EGG RESPIRATION 115 

tion might compete with and depress the latter. The smooth increase in post- 
fertilization respiration from the level of the unfertilized egg in Asterias speaks in 
favor of the addition possibility (cf. Borei, 1948a and Borei and Lybing, 1949), 
provided the respiratory mechanisms in eggs of starfishes and sea-urchins may be 
freely compared. The facts concerning temperature characteristics of oxygen con- 
sumption rates revealed in Arbacia, do not in themselves distinguish between the 
two possibilities, nor do any facts gained in this investigation concerning Psam- 
mechinus material. Concerning the declining pre-fertilization respiration see, how- 
ever, Borei (1949), where it is shown that this respiration part does not influence 
post-fertilization respiration. 

Parallels have been drawn between the respiration of the unfertilized sea-urchin egg and 
the diapause egg of the grasshopper. In respect to temperature characteristics, the grasshopper 
diapause egg has a very low Q, n in comparison with that of the active stages (Bodine and 
Evans, 1932), which is in contrast to the state in the sea-urchin egg. Too much stress may 
thus not be laid on such comparisons. 

SUMMARY 

With Cartesian diver micro-respiration technique the temperature characteristics 
of the respiration of Psammechinus iniliaris eggs were investigated : 

1. Between 10 and 21 C. the gradual rise in oxygen consumption rate of the 
unfertilized egg is best represented by Krogh's standard curve. Q 10 at 18 C. is 
around 2.5, at 10 C. around 3.5. 

2. Fertilized eggs have the same temperature characteristics as unfertilized 
ones. 

3. In respect to temperature characteristics just before and just after fertili- 
zation, two classes are distinguishable among the sea-urchins: The Strongylocen- 
trotus-Psammechinus group with equal O 10 values before and after fertilization, 
and the Arbacia group with higher Q 10 values before fertilization. 

4. The significance of temperature characteristics for the biochemical processes 
involved in respiration of eggs before and after fertilization is discussed. 

The authors wish to express their deep gratitude to the Kristineberg Zoological Station of 
the Royal Swedish Academy of Science for laboratory facilities and for the great pains taken 
by the station Staff in supplying materials. 

LITERATURE CITED 

BALLENTINE, R., 1940. Jour. Cellular Comp. P!i\siol., 15: 21". 

BODINE, J. H., AND T. C. EVANS, 1932. Biol. Ru'll.. 63: 235. 

BOREI, H., 1945. Arkiv Kcmi, Mineral. GcoL. 20A : No. 8. 

BOREI, H., 1948a. Biol. Bull, 95 : 124. 

BOREI, H., 1948b. Arkiv Zbol. 40A : No. 13. 

BOREI, H., 1949. Biol. Bull., 96: 117. 

BOREI H., AND S. LYBING, 1949. Nature, 163: 451. 

BOREI, H., AND S. RENVALL, 1949. Arkiv Kcmi, Mineral. GcoL, 26A : No. 28. 

EPHRUSSI, B., 1933. Arch. biol. (Liege), 44: 1. 

KEILIN, D, AND E. F. HARTREE, 1938. Proc. Roy. Soc. (London), B125: 171. 

KORR, I. M., 1937. Jour. Cell. Conif. Physinl.. 10: 461. 

KORR, I. M., 1939. Cold Spring Harbor 'Symposia Quant. Biol., 7: 419. 



116 HANS BOREI AND SIGVAR LYBING 

KRAHL, M. E., A. K. KELTCH, C. K. NEUBECK, AND G. H. A. CLOWES, 1941. Jour. Gen. 

Physiol., 24: 597. 

KROGH, A., 1914. Intern. Z. physik. chem. BioL, 1: 491. 
LINDAHL, P. E., 1939. Z. vcrgleich. Physiol, 27 : 136 and 233. 
LINDAHL, P. E., 1940. Arkiv Kcmi, Mineral. Gcol, 14A : No. 12. 
LINDAHL, P. E., 1941. Science, 93: 332. 
LOEB, J., AND H. WASTENEYS, 1911. Biochcm. Z., 36: 345. 

LucKE 1 , B., H. K. HARTLINE, AND M. McCurcHEON, 1931. Jour. Gen. Physiol., 14: 405. 
NEEDHAM, J., 1942. Biochemistry and Morphogenesis. Camhride Univ. Press. 
ORSTROM, A., 1932. Protoplasma, 15 : 566. 
ROBBIE, W. A., 1946. Jour. Cell. Comp. Physiol.. 28: 305. 
RUBENSTEIN, B. B., AND R. W. GERARD, 1934. Jour. Gen. Physiol., 17 : 677. 
RUNNSTROM, J., 1930. Protoplasma, 10: 106. 
RUNNSTROM, J., 1932. Protoplasma, 15: 532. 
RUNNSTROM, J., 1935. Biol. Bull., 68: 327. 
RUNNSTROM, J., 1942. Scientia (Milan), 71: 149. 

RUNNSTROM, S., 1927. Bergens Museums Arbok, Natnrv. Rekkc, No. 2. 
TYLER, A., 1936. Biol. Bull, 71 : 82. 

TYLER, A., AND W. D. HUMASON, 1937. Biol. Bull., 73: 261. 
ZEUTHEN, E., 1947. Cowpt. rend. trav. lab. Carlsberg. Ser. chim., 26: 17. 



INDEPENDENCE OF POST-FERTILIZATION RESPIRATION IN 
THE SEA-URCHIN EGG FROM THE LEVEL OF RESPIRA- 
TION BEFORE FERTILIZATION 

HANS BOREI 

W enner-Gren's Institute for Experimental Biology, University o] Stockholm 

INTRODUCTION 

The unfertilized sea-urchin egg just removed from the ovary respires at an 
oxygen consumption rate comparable with that of the newly fertilized egg (Borei, 
1948a). After a few hours the respiration gradually drops, however, to a low. 
fairly constant level. This level represents the low respiration value of the unfer- 
tilized sea-urchin egg previously recorded in the literature. From this level there is 
a sudden jump at fertilization to the respiration rate of the newly fertilized egg. 
In Psammechinus the rate of the latter is usually three to four times that of the un- 
fertilized egg at the constant level. For further details see Borei (1948a). 

Now the question arises whether the increase in respiration after fertilization is 
merely an addition to the pre-fertilization respiration, or whether it competes with 
the existing respiration, eventually suppressing it completely. 

In view of the concept (Runnstrom, 1930, Robbie, 1946) that the oxidase sys- 
tem in operation in the egg is the same before and after fertilization, and if parallels 
to events in the Asterias egg are considered (Borei and Lybing, 1949), the addi- 
tion possibility is not unlikely. The difficulties arising in trying to interpret in this 
manner results from cyanide-inhibition experiments on fertilized eggs (Korr, 1937) 
could be avoided if Lindahl's (1940) findings are taken into account. He states 
that the cyanide-stable respiration arises under the influence of the inhibitor, and 
his view is strongly supported by Robbie's (1946) experiments. 

On the other hand, Ballentine's (1940) concept that at fertilization a link in the 
dehydrogenase part of the oxidative chain is introduced, thus inducing an aug- 
mented respiration, offers a possibility that post-fertilization respiration, compet- 
ing with that of pre-fertilization, may successfully suppress the latter, in spite of the 
fact (Robbie, 1946) that the same oxidase system is in operation both before and 
after fertilization. Korr, who first (1937) favored the view that cytochrome c is 
the link put into operation at fertilization, later (1939) abandons this and also turns 
to a concept that new ultimate substrate is released from precursors. 

Runnstrom's (1930, 1932 and 1935) view that the oxidase is unsaturated with 
its substrate in the unfertilized egg, is equally consistent with both possibilities. 

Borei and Lybing (1949) find that results from experiments on the temperature 
characteristics of sea-urchin eggs before and after fertilization can not decide be- 
tween the two possibilities. It was stated by them that all facts at present known 
about sea-urchin egg respiration still leave the question open : It is just as possible 
to assume a simple addition as to believe in a competition. 

It was thought that measurements of post-fertilization respiration in such ex- 
periments, where the eggs were fertilized at times corresponding to different points 

117 



118 HANS BOREI 

on the decreasing respiration curve of the unfertilized egg, would help to elucidate 
the matter. If the rate of oxygen consumption before fertilization influenced the 
respiration of the fertilized egg, the addition possibility would have to be strongly 
considered. If not, one would be inclined to think that post-fertilization respiration 
is, as a whole, different from that before fertilization. 

For a fuller discussion of previous literature on the biochemical aspects con- 
cerning sea-urchin egg respiration changes at fertilization, see Borei and Lybing 
(1949). 

MATERIAL AND METHODS 

The egg respiration of Psammechinus miliaris has recently (Borei, 1948a, Borei and 
Lybing, 1949) been studied in some detail. This species (phenotype S) was therefore chosen 
also in this investigation. For particulars concerning the material see Borei (1948a), Chapters 
2.1, 3.111 and 3.21. 

In order to make measurements possible on several lots of eggs taken from the same 
female and fertilized at subsequent times from the moment of removal from the ovary, one must 
use a method on the /4. scale. Thus Cartesian diver micro-respiration technique was employed. 
Concerning technical points see Borei (1948a), Chapters 2.2 and 2.3. Diver charge Type I 
(Borei, 1948b) was used throughout. 

All experiments were performed at 18 C. After completed diver measurements, the cells 
were taken out of the divers and observed as to condition and fertilizability. The only experi- 
ments accepted were those in which these controls turned out satisfactorily. 

In the actual runs, the procedure was as follows : Eggs were removed from the ovary, a 
lot was immediately fertilized, then a diver was charged with unfertilized eggs and these were 
brought to measurement as rapidly as possible. ( The first respiration values could thus be 
obtained 20 mins. after egg removal from ovary.) This control diver was then continuously 
followed during all subsequent measurements on fertilized eggs from the same animal. From 
the first fertilized lot a diver was now charged. Subsequently new lots were fertilized and 
corresponding divers charged. In the experiments with fertilized eggs, the respiration rate at 
120 minutes after fertilization, where the exponentially increasing respiration curve is still rather 
flat (see Borei, 1948a, Figure 3), was estimated and used for comparison. For unfertilized 
eggs a number of about 100, and for fertilized about 50, were found to be most suitable for 
charging the divers, which were of approximately 7 Ml. capacity. 

RESULTS AND INTERPRETATIONS 

The results from experiments on eggs from three females are represented in 
Figure 1. 

It is obvious that respiration after fertilization reaches exactly the same level, 
irrespective of the height of the prevailing respiration at the moment of fertilization. 
It is of no importance whether the fertilization sets in at a very early moment, when 
the egg has just been removed from the ovary and accordingly respires at a very 
high rate, or whether it is effected a very long time after the removal, when the egg 
has alrady reached a fairly constant, low-rate respiration. In both cases the post- 
fertilization respiration will be the same. 

In this connection it must be pointed out that the respiration of the fertilized egg in this 
investigation has been followed until more than nine hours after removal of the eggs from the 
ovary. This is considerably longer than in the cases reported by Borei (1948a), where the 
measurements were discontinued after about six hours. In no case, however, has there been 
observed any rise in the respiration rate at the end of the experiments, as might have been 
expected in consideration of the early findings of Warburg (1914) and Runnstrom (1928). 
Goldforb (referred to in Gerard and Rubenstein, 1934) found that the increase begins about 
five hours after shedding, and Tyler and Humason (1937) report a steady increase over the 



SEA-URCHIN EGG RESPIRATION 



119 



whole measurement period. Runnstrom (1928) thinks that the increase is an indication of the 
"aging" of the egg (cf., however, Borei, 1948a, Chapter 3.113, who finds that over-ripe eggs 
have lower respiration than ripe ones and still lower than under-ripe ones). Tyler, Ricci and 
Horowitz (1938) found that the increase is avoided if the experiments are carried out under 
sterile conditions. It must be stated that all previous investigators have worked with Warburg 
technique. It might be that the dense packing of the eggs and the shaking conditions in this 
procedure support the growth of bacteria, whereas the Cartesian diver technique is more favor- 
able in this respect. However, in this investigation no particular measures have been taken 
against bacterial contamination. 



xlO 



O 
or 



^. 

UJ 

OL 
O 



UJ 
O 



O 



-4 




UNFERTILIZED 



_ O IOO 2OO 3OO 4OO 5OO 

MINUTES AFTER REMOVAL 

FROM OVARY 

FIGURE 1. Oxygen consumption of unfertilized eggs and of fertilized eggs, fertilized at 
different times after the eggs' removal from the ovary. Psammechinus miliaris. 

Each dot represents the respiration rate at 120 mins. after fertilization and is marked at 
the time of fertilization. O, 6 and stand for the three females used. Temperature 18 C. 
Oxygen consumption rate of unfertilized eggs at 230 mins. after removal from ovary : O = 0.50, 
ft =0.58 and C = 0.47 X 10" 4 Ml./cell and hour (mean value found by Borei 1948a = 0.53) ; 
mean value of rates of fertilized eggs: O 1.85, & = 1.78 and C> := 1.79 X 10" 4 Ml./embryo and 
hour (Borei, 1948a = 1.84) . For the respiration of fertilized eggs, the best fit according to 
the method of least squares is indicated by a dotted line. 



Borei ( 1948a, Chapter 3.21) compares the quotient between respiration after 
fertilization and that before, and finds that the values from different sea-urchin 
species vary considerably, and moreover that for one and the same species, greatly 
differing values are reported. So for example for Arbacia punciulata quotients 
from 2.6 to 5.3 have been given. For Psammechinus the values 3.6 and 5.7 are 
recorded. On the other hand, if the conditions of measurement are well defined 
as to time from fertilization and from removal from ovary respectively, the quotient 
will become fairly constant, as the results of Borei (1948a, Chapters 3.112.2 and 
3.21 ) show. Considering the possible influence of values of pre-fertilization res- 
piration on the quotient, obtained at different points on the declining egg respiration 
curve, Borei ( 1948a) thinks that quotient values given in the literature are of minor 
importance for quantitative considerations concerning respiration changes at fertili- 



120 



HANS BOREI 



zation, but merely show that the oxygen consumption of the unfertilized egg some 
few hours after its removal from the ovary is considerably lower than that of the fer- 
tilized egg some few hours after fertilization. The present investigation supports 
this concept thoroughly. If the quotients from the experiments in Figure 1 are re- 
corded (Table I), it is clearly seen that the quotient value will become smaller and 
smaller the closer the time of fertilization lies to that of egg removal. It would 
rather seem that values < 1.0 could be obtained in the earliest experiments, i.e. 
that the respiration of the just removed egg is actually higher than that of the 
fertilized egg during the first hours of development (cf. Borei, 1948a). 



TABLE 1 

Quotients between oxygen consumption rate 120 wins, after fertilization and oxygen consumption rate 
of unfertilized eggs at the moment of fertilization. Psammechinus miliaris 

Same experiments and denotations as in Figure 1. Value given by Borei (1948a) for 230 
mins. after egg removal from ovary = 3.6. In quotients marked * respiration values of un- 
fertilized eggs are obtained graphically from extrapolated curves. 









C 


Mins. after 
egg removal 


Quotient 


Mins. after 
egg removal 


Quotient 


Mins. after 
egg removal 


Quotient 


6 


<1.6* 


8 


<1.0* 


2 


<1.0* 


52 


2.3 


40 


1.2 


21 


1.2 


120 


3.3 


100 


2.2 


41 


2.5 


185 


3.5 


170 


2.5 


60' 


2.8 


241 


4.3 


264 


3.2 


120 


3.1 


286 


3.8 


349 


3.5 


179 


3.6 






502 


3.4 


265 


3.9 



It would appear from the presented data, as the value of the pre-fertilization 
respiration rate seems to be of no importance for the oxygen consumption rate after 
fertilization, either that pre-fertilization respiration constitutes no integral part of 
the respiration after fertilization, or that fertilization brings about a release from 
inhibiting factors active on respiration in the unfertilized egg. As the oxidase sys- 
tem is probably the same both before and after fertilization (Runnstrom, 1930, Rob- 
bie, 1946; see also Introduction of the present paper), it is reasonable to suppose 
that changes occur at fertilization in those parts of the system which are situated be- 
tween the oxidase and the dehydrogenases. The dehydrogenases themselves, how- 
ever, are not likely to be affected. (Dimethyl-p-phenylenediamine experiments by 
Runnstrom, 1930 and 1932, Orstrom, 1932, Borei and Renvall, 1949; hydroquinone, 
Runnstrom. 1930; pyocyanine, Runnstrom, 1935 ; methylene blue, Runnstrom, 1930; 
cf., however, Ballentine, 1940, who claims that the dehydrogenases are not capable 
of maximum activity in the unfertilized egg.) (Cf. Korr, 1939: "release of sub- 
strate from precursors.") 

From experiments on the respiratory quotient of pre- and post-fertilization 
respiration in the sea-urchin egg, it appears very probable that different substrates 
are utilized before and after fertilization. These changes in RQ upon fertilization. 



SEA-URCHIN EGG RESPIRATION 



121 



which support the view that post-fertilization respiration competes with that of 
pre-fertilization, eventually suppressing it more or less completely, are seen from 
Table II. 

It must, however, be kept in mind that a constant respiration part is assumed for 
the unfertilized egg (Borei, 1948a, Chapter 3.112.1) as well as a decreasing part. 
The above-presented data do not indicate whether even the constant respiration part 
is abolished at fertilization. It is still an open question whether this part of pre- 
fertilization respiration survives fertilization or not. 

TABLE II 

RQ of sea-urchin egg respiration before and after fertilization 



Time in relation to fertilization 


RQ 


Species 


Author 


Before* 
0-30 mins. after 

30-40 mins. after 
40-50 mins. after 

35-50 mins. after 

1-2 hr. after 
7-8 hr. after 

2-cell stage-hatching 


1-1.2 


Psammechinus 


Borei (1934) 


0.84 


Laser and Rothschild (1939) 


0.78 
0.64 


Borei (1934) 


0.66 


Laser and Rothschild (1939) 


0.73 
0.85 


Paracentrotus 


Ohman (1940) 


0.8 


Ephrussi (1933) 





* Ashbel (1930) finds the value 1.06 before fertilization (Arbacia). 



SUMMARY 

Using Cartesian diver micro-respiration technique, it was found that in 
Psaimnechinus miliaris the rate of respiration of the newly fertilized egg is inde- 
pendent of the rate of respiration of the unfertilized egg at the moment of 
fertilization. 

The quotient (respiration after fertilization) /(respiration before fertilization) 
was found to decrease considerably (probably even to values < 1.0) if the time 
interval between egg removal from the ovary and fertilization was diminished. If 
the decreasing part of pre-fertilization respiration is given time to disappear before 
fertilization, the quotient lies between 3 and 4. 

It is thought possible that the decreasing respiration part of the unfertilized 
egg is abolished upon fertilization, due to probable changes in the function of mem- 
bers of the oxidizing system, situated between the oxidase and the dehydrogenases. 
It cannot be decided from the experiments whether the constant respiration part of 
the unfertilized egg still participates in the respiration of the fertilized egg or not. 

The author is much indebted to the Kristineberg Zoological Station of the Royal Swedish 
Academy of Science for working conditions and for great courtesy on the part of the Station 
Staff. 



122 HANS BOREI 

LITERATURE CITED 

ASHBEL, R., 1930. Boll. soc. ital. biol. spcr., 5 : 72. 

BALLENTINE, R., 1940. Jour. Cell. Comp. Phvsiol.. 15: 217. 

BOREI, H., 1934. Z. vergleich. Physio!., 20: 258. 

BOREI, H., 1948a. Biol. Bull.. 95: 124. 

BOREI, H., 1948b. Arkiv Zool, 40A : No. 13. 

BOREI, H., AND S. LYBING, 1949. Biol. Bull, 96: 107. 

BOREI, H., AND S. RENVALL, 1949. Arkiv Kemi, Mineral Geol,. 26A : No. 28. 

EPHRUSSI, B., 1933. Arch. biol. (Liege), 44: 1. 

GERARD, R. W., AND B. B. RUBENSTEIN, 1934. Jour. Gen. Phvsiol, 17: 375. 

KORR, I. M., 1937. Jour. Cell. Comp. Physiol.. 10: 461. 

KORR, I. M., 1939. Cold Spring Harbor Symposia Quant. Biol., 7 : 419. 

LASER, H., AND LORD ROTHSCHILD, 1939. Proc. Rny. Soc. (London), B126: 539. 

LINDAHL, P. E., 1940. Arkiv Kcmi, Mineral. Geol., 14A: No. 12. 

OHMAN, L. O., 1940. Arkiv Zool, 32A : No. 15. 

ORSTROM, A., 1932. Protoplasina, 15: 566. 

ROBBIE, W. A., 1946. Jour. Cell. Comp. Physiol, 28 : 305. 

RUNNSTROM, J., 1928. Acta cool. (Stockholm), 9: 445. 

RUNNSTROM, J., 1930. Protoplasina. 10: 106. 

RUNNSTROM, J., 1932. Protoplasina. 15: 532. 

RUNNSTROM, J., 1935. Biol. Bull., 68 : 327. 

TYLER, A., AND W. D. HUMASON, 1937. Biol. Bull., 73 : 261. 

TYLER, A., N. RICCI, AND N. H. HOROWITZ, 1938. Jour. Exp. Zool, 79: 129. 

WARBURG, O., 1914. Arch. ges. Physiol (Pfliigers), 158: 189. 



STUDIES ON THE THERMAL DEATH OF HYALELLA AZTECA 

SAUSSURE 1 

EUGENE CLEVELAND BOVEE 
Iowa State Teachers College - and the loi^a Lakeside Laboratory 

INTRODUCTION 

Much work has been done in the past half century concerning the effects of tem- 
perature on the survival of organisms. However, little has been done in that 
respect with the Crustacea, particularly the fresh \vater forms. 

A study was made from June 16, 1947, to August 15, 1947, concerning the 
effects of temperature on the survival and death rates of the amphipod Hyalella 
asteca Saussure (Hyalella knickerbockeri Bate, Hyalella dentata Smith). This 
amphipod is abundant in the waters of Little Miller's Bay in West Okoboji Lake. 
Dickinson County, Iowa, at the site of the Iowa Lakeside Laboratory, where the 
investigation was conducted. 

HISTORICAL SURVEY 

Geisler (1944) suggests that rate of development in Hyalella asteca is directly 
related to the temperature, but does not record the effects of higher temperatures. 
For marine copepods and decapods, Huntsman and Sparks (1924) report heat 
death at temperatures between 22 and 33 C. when the animals were exposed to 
temperatures rising at the average rate of 0.2 C. per minute. Brown (1928) re- 
ported a temperature characteristic, p., of 187,000 calories from 35 to 41 C. for 
thermal death in Daphnia magna. 

MATERIALS AND METHODS 

Numerous Hyalella were secured daily by placing fresh masses of the green 
alga, Cladophora fracta, in which they feed, in a pail of lake water. Amphipods 
which came to the surface of the water were transferred to a stock tank of fresh 
lake water at 20 to 22 C. by means of a tea strainer. This lake water was par- 
tially changed daily and replaced weekly, and was used in all experiments. The 
temperature of the tank water was about the same as that of the natural water where 
the animals were taken. 

The water baths used in the experiments were five-gallon containers heated 
electrically and controlled manually to 0.2 C. 

1 Undertaken in partial fulfillment of the degree of Master of Science at the State Univer- 
sity of Iowa. The author wishes to express his gratitude for helpful criticism of the initial 
and final drafts of the paper on the part of Dr. Theodore Louis Jahn of the University of Cali- 
fornia, Los Angeles, formerly of the University of Iowa, and Dr. Robert L. King of the 
University of Iowa, at whose suggestion the problem was begun. 

2 On leave of absence for research and study at the University of California. Los Angeles. 



124 EUGENE C. BOVEE 

In short exposures several hundred Hyalella were dipped from the stock tank 
with a tea strainer, over which a muslin square was then fastened and held taut with 
a rubber band. The strainer was immersed and oscillated in the water bath for the 
desired period. Then the Hyalella and the strainer were transferred quickly to 
enamelled basins of fresh lake water at 20 to 22 C. 

In exposures longer than thirty seconds small tin cans with tops and bottoms 
removed were employed. Muslin squares fastened over the open ends kept the 
amphipods confined, but exposed to the water. A number of these tins were placed 
in the water bath and removed as desired. 

The water bath was stirred between short exposures, and during exposures 
longer than thirty seconds was continually aerated with compressed air. The tins 
containing the Hyalella were placed on a wire platform as near the center of the 
water bath as possible, and at least six inches away from the source of heat. 

Exposed animals were allowed to remain in recovery basins four or more hours 
before counting. Counting was usually done six to eight hours after exposure. 
Check counts showed that injured animals which w ? ere alive four hours after ex- 
posure were always dead after sixteen hours. Therefore, injured animals were 
counted as dead. Control animals remained alive, except for rare exceptions, after 
twenty-four hours in the basins. 

Water from each basin was individually strained through muslin to collect the 
the amphipods for counting. The muslin was spread flat on a moist, concrete 
slab for counting under a sixty-watt electric bulb. Because of negative tropism 
to light (Phipps, 1915), the living amphipods crawled toward the periphery. 
A few cubic centimeters of water dropped at the center of the muslin hastened the 
outward movement. After thus separating the living and dead, the living were 
counted first. Those evidencing injury or feebleness when stimulated with water 
or the points of a pair of tweezers were counted as dead. 

Mature specimens were separated into two groups ; so also were immature speci- 
mens. Those longer than 7 mm. were considered very large adults ; those from 3*/> 
to 7 mm. as large adults ; those from 2% to 3 mm. as medium-sized adolescents ; 
and those still less as small and juvenile (Geisler, 1944). A separate count was 
taken according to sexes for each of the adult groups on the basis of salient char- 
acteristics (Geisler, 1944) as seen through a dissecting scope. 

Time intervals were determined with a watch calibrated in fifths of a second. 

EXPERIMENTAL RESULTS 
Survival in constant temperature baths 

Constant temperatures used included one degree intervals C. from 38 to 50. 
Tests were also run at 36.5, 35 and 33. 

Thermal death times and temperatures here shown are those at which fifty 
percent of the organisms survived. 

At 50, less than one second was sufficient time to cause thermal death. At 
lower temperatures the time increased gradually so that at 40 an exposure of 75 
seconds was needed. Below 40 the increase in time required to kill was very 
sharp, so that at 33 more than eleven hours (39,600 seconds) was necessary 
(Table 1). 



THERMAL DEATH OF HYALELLA 



125 



Size and survival in constant temperature baths 

Age and size are directly correlated in Hyalella; the larger the animal, the older 
it is (Geisler, 1944). Large adults showed the greatest resistance, usually higher 
than the average figure for the total of all simultaneously exposed. Very large 
adults varied in their resistance, but their rate of survival usually approached the 
average. Medium adolescents showed still less resistance ; and small juveniles were 
least resistant (Table 1). 

TABLE 1 

Heat death for Hyalella azteca in constant temperature baths 



Degrees 
C. 


Time 
exposed in 
seconds 


Per cent survivors 


Total 
number 
animals 
exposed 


Total 
animals 
exposed 


Very 
large 
adults 


Large 
adults 


Medium 
adolescents 


Small 
juveniles 


50.0 


1 


50.8 


52.1 


62.5 


45.6 


43.3 


240 


49.0 


4 


51.2 


64.8 


59.4 


47.5 


44.4 


416 


48.0 


7 


50.3 


48.7 


51.8 


52.0 


47.6 


320 


47.0 


8 


50.8 


58.3 


57.2 


48.2 


46.6 


114 


46.0 


12 


49.3 


31.7 


51.9 


54.3 


47 1 


162 


45.0 


14 


47.1 


52.2 


65.4 


36.3 


43.6 


318 


44.0 


15 


55.0 


40.1 


56.2 


56.7 


49.4 


623 


43.0 


24 


49.5 


58.5 


63.6 


43.0 


43.3 


656 


42.0 


39 


49.9 


52.8 


56.4 


49.1 


37.9 


204 


41.0 


60 


47.6 


33.6 


67.6 


51.3 


46.1 


432 


40.0 


75 


49.6 


49.9 


68.8 


47.6 


46.8 


626 


39.0 


135 


49.3 


49.3 


58.4 


44.6 


48.4 


636 


38.0 


240 


48.9 


49.2 


55.8 


45.0 


46.9 


547 


36.5 


1,800 


49.9 


44.8 


52.9 


51.1 


53.6 


210 


35.0 


6,300 


51.8 


50.2 


56.2 


46.1 


46.8 


583 


33.0 


39,600 


53.7 


51.2 


51.9 


51.5 


48.2 


146 



Sex and survival in constant temperature baths 

No valid evidence was found to indicate that sex affects resistance to heat in 
constant temperature baths. For all adult specimens, male survivors outnumbered 
females in sixteen out of thirtv-two cases. 



Survival in rising temperature baths 

Using the same equipment as that employed for the constant temperature baths, 
Hyalella were exposed to four average rates of temperature rise, beginning at 
20 to 22 C. Rates of rise were: 0.375 per minute; 0.261 per minute; 0.150 
per minute; and 0.036 per minute. At lower temperatures the rate of rise per 
degree C. was more rapid than at higher temperatures. The rates of rise are here 
expressed as average rates, in order to make them comparable to those of other in- 
vestigators who previously encountered the same difficulty (Huntsman and Sparks. 
1924). Within the rates of rise investigated, thermal death did not occur below 
39 C. and always was found above 41 C. (Table 2). 



126 



EUGENE C. BOVEE 



TABLE 2 

Heat death in Hyalella azteca in rising temperature baths 



Rate* of rise 
in degrees C. 
per minute 
from 20 to 22 
as a base 


Degrees 
C. 


Total 
animals 
exposed 


Per cent survivors 


Total 
animals 


Very 
large 
adults 


Large 
adults 


Medium 
adolescents 


Small 
juveniles 


0.375 


37.0 


178 


89.4 


94.6 


92.6 


88.8 


74.1 




38.0 


212 


84.4 


85.7 


77.8 


75.8 


75.0 




39.0 


189 


87.8 


93.7 


88.8 


82.7 


78.3 




40.0 


201 


51.7 


52.7 


50.2 


50.0 


51.8 




41.0 


170 


60.2 


69.7 


54.8 


52.6 


50.0 




42.0) 
















to ! 

45.0] 


#200(a>, 












0.261 


37.0 


462 


85.9 


95.5 


96.3 


89.1 


83.1 




38.0 


470 


81.1 


91.1 


83.7 


77.2 


76.7 




39.0 


456 


83.9 


93.1 


83.1 


83.7 


78.6 




40.0 


534 


68.1 


81.0 


79.9 


63.1 


64.6 




41.0 


340 


27.6 


31.2 


23.6 


26.5 


29.0 




42.01 
















to \ 
45. Oj 


#400 @ 












0.150 


37.0 


421 


93.5 


96.4 


95.8 


86.5 


87.5 




38.0 


492 


82.9 


92.3 


85.8 


76.8 


77.1 




39.0 


493 


77.4 


88.6 


76.9 


75.4 


66.6 




40.0 


430 


70.7 


75.0 


73.0 


65.3 


65.5 




41.01 
















.0 ! 

45. OJ 


#400 












0.036 


36.0 


464 


94.8 


97.6 


96.0 


89.6 


91.8 




37.0 


306 


86.9 


94.6 


95.6 


82.5 


78.7 




38.0 


493 


65.9 


73.5 


81.5 


59.8 


55.9 




39.0 
40.01 

to } 
45.0 J 


194 
#350 


29.3 


12.7 


39.0 


42.0 


25.6 



* Average rate of rise. 

# Approximate number at each degree of temperature within the bracketed limit- 
rate count taken since all were dead. 



no accu- 



Size and resistance to rising temperatures 

Very large adults demonstrated the highest resistance in rising temperature 
baths, except at the slowest rate of rise. Large adults, medium adolescents, and 
small juveniles showed, respectively, less resistance (Table 2). 

Sex and resistance to rising temperatures 

X<> evidence was found that sex causes any variance in resistance to rising tern- 



THERMAL DEATH OF HYALELLA 127 

peratures. Male adult survivors in some trials outnumbered females, and vice 
versa, but never in significant numbers. 

Adjustment to rising temperatures oj water 

A temporary adjustment to rising temperatures was noted. For example, or- 
ganisms plunged into and continuously exposed to a pre-heated constant tempera- 
ture bath at 38 C. readied thermal death in four minutes. Within the rates of 
rise investigated, thermal death was not found to occur at 38, 87.8 per cent 
still surviving at that temperature at the fastest rate of rise, and 65.9 per cent 
surviving at the slowest rate of rise (Table 2). 

Temperature coefficients for thermal death 

Adaptations of the v'ant Hoff-Arrhenius equation are often used to express the 
rate of progress in biological reactions, although it is possible that such character- 
istics are more descriptive than analytically accurate. 

Computation and comparison of Q in for a number of temperature ranges within 
the full range investigated revealed that although death occurs more quickly at 
higher temperatures, the rate at which the lethal effect progresses decreases as the 
temperature increases. The decrease in O 10 was very marked for intervals below 
40 C. Above that temperature there was a sharp break in the deceleration of 
the rate of progress and the O 1(1 variance was not so great (Table 3). 

TABLE 3 

Temperature characteristics for Hyalella azteca for 
thermal death in constant temperature baths 

Temperature intervals 

in degrees C. Qio 

33.0-35.0 9,768.00 

35.0-40.0 7,056.00 

38.0-43.0 100.00 

40.0-45.0 28.91 

43.0-48.0 11.75 

45.0-50.0 196.00* 

* Apparent divergence may be due to experimental inaccuracies. 

SUMMARY 

1. Thermal death occurs in Hyalella azteca at constant temperatures from 33 
to 50 C. The time required to produce thermal death varies from more than 
eleven hours (39,600 seconds) at 33, to less than one second at 50 C. 

2. Comparison of Q 10 values for narrow ranges within the broad range of tem- 
peratures investigated indicates a marked decrease of Q 10 values at higher tempera- 
tures in spite of a more rapid lethal effect. 

3. Thermal death occurred in rising temperatures, the slower the rate of rise, 
the lower the killing temperature, being not below 39 nor above 41 for the rates 
of rise investigated. 



128 EUGENE C. BOVEE 

4. A temporary adjustment was found to occur to rising temperatures, delaying 
thermal death at a given temperature for some time past the period necessary to kill 
on immersion in the constant temperature bath at the given temperature. 

5. Resistance to the effects of heat appears to he directly related to the size 
and age of the animal, the older and larger the animal the greater the resistance, ex- 
cept for the largest animals (which might have reached a state of senility). 

6. Resistance to the effects of heat does not appear to be related to sex in 
Hyalella azteca. 

LITERATURE CITED 

BROWN, L. A., 1928. Comparison of the rates of killing of the parthenogenetic and sexual 
forms of Daphnia magna at higher temperatures. Proc. Soc. Exp. Biol. Med., 25 : 
732-734. 

GEISLER, FRANCIS SOLANO, S.S.J., 1944. Studies on the post-embryonic development of Hyalella 
azteca (Saussure). Biol. Bull., 86: 6-22. 

HUNTSMAN, A. G., AND M. I. SPARKS, 1924. Limiting factors for marine animals. 3. Rela- 
tive resistance to higher temperatures. Contributions to Canadian Biology, 2: 95-114. 

PHIPPS, C. F., 1915. An experimental study of the behavior of amphipods with respect to 
light. Biol. Bull., 28 : 210-223. 



REGENERATION IN AN EARTHWORM, EISENIA FOETIDA 
(SAVIGNY) 1826. I. ANTERIOR REGENERATION 

G. E. GATES 

Colby College, IVaferi'Ulc, Maine 

These contributions present the results of an attempt to obtain for one particu- 
lar species of earthworm complete characterization of regenerative capacity with 
reference to exact levels. In this part anterior regeneration, by posterior substrates 
only, is considered. 

SUMMARY OF PREVIOUS WORK 

Information available as to the morphological nature and segmental constitu- 
tion of anterior regenerates on posterior substrates is summarized, with certain 
reservations, in Table I. 

In earlier work on E. foetida, as well as other species of earthworms, determina- 
tion of morphological nature of regenerates seemed unnecessary. Later, Michel 
(1898, p. 283), recalling Bonnet's heteromorphic tails in aquatic Oligochaeta, 
suggested that two of his own anterior regenerates, as well as some of those of 
Joest and Rievel, were caudal. Although anterior heteromorphosis was definitely 
confirmed by Morgan (1899) no attempt was made then or since to clarify the 
situation, and in particular to determine the limits of homomorphic head and 
heteromorphic tail regeneration. 

The consequent uncertainty as to morphological nature of regenerates at a con- 
siderable number of levels and even as to the levels (because of postregeneration 
estimation), as well as absence of data for numbers of levels and paucity at other 
levels, indicated the advisability of a systematic investigation of regeneration at 
each level from ^ posteriorly. 

MATERIALS AND METHODS 

Material was first secured from a heap of decaying leaves, later from manure 
heaps. Worms were kept in moist filter paper or paper towelling until the gut 
was cleared. Individuals with any indication of damage by collecting, disease, ab- 
normality, homoeosis or previous regeneration were rigorously rejected, and only 
those which were clitellate, or which had been clitellate when brought into the labora- 
tory, were used. Animals were kept throughout at ordinary room temperature, 
which in winter probably was never above 68 F. 

Anaesthesia was brought about in 0.2 per cent chloretone. Transections were 
made under a dissecting binocular microscope exactly across the animal on an inter- 
segmental furrow. 

After operation worms were placed in water until recovery from anaesthetic and 
were then transferred to filter paper, paper towelling, or cheesecloth. On several 

129 



130 



G. E. GATES 



TABLE I 

A nterior regeneration in Eisenia foctida 



Level 


Regenerate 


Comments 


Author 


Date 


Page 


Cephalic 
Number of segments 


'S 

u 
HI 

(J 

^ 

* 


Caudal 









2 


3 


4 


5 


6 


7 


? 


1/2 


6 


























See note (a) 






447 


2/3 










Morgan 


1895 


3/4 


5 
4 


9 

1 


5 
1 



















Morgan 
Michel 


1895 
1898 


447 
261 


4/5 


1 
3 

1 
1 


8 
1 
10 

2 
8 

5 

2 

4 

1 

1 

















Morgan 
Hescheler 
Michel 


1895 
1896 
1898 


448 
228 
262 

448 
228-31 
268 
453 


5/6 
EL 


3 
5 
1 

5 


1 
1 










See note (b) 
See notes (c) (d) 


Morgan 
Hescheler 
Michel 
Morgan 


1895 
1896 
1898 
1895 


6/7 
EL 


3 
6 

9 


1 


















Hescheler 
Morgan 


1896 
1895 


228 
453-6 


7/8 
EL 


1 








See note (e) 


Hescheler 
Michel 
Morgan 


1896 
1898 
1895 


228 
262 
453 


8/9 
EL 


1 


3 


1 

5 


3 
1 


1 














Michel 
Morgan 


1898 
1895 


262 

455 


9/10 
EL 


1 


1 




3 






2 










Morgan 


1895 


453-6 


10/11 
EL 

11/12 
EL 

12/13 
EL 


1 


1 
1 

1 


3 
2 

3 
1 


5 

1 
1 


3 
1 





1 






U = "Imp" 


Morgan 
Carpenter 
Morgan 


1895 
1948 
1895 


451 
625-6 

455 


1 






U = "2 (or three very imper- 
fect)" 


Michel 
Morgan 


1898 
1895 


263 
456 


2 
1 


1 




1 









U:S = indistinct 
100-0-0% 


Morgan 
Michel 
Dimon 
Morgan 


1895 
1898 
1904 
1895 


451-6 
263 
350 
456 


13/14 
EL 




1 




















Morgan 


1895 


456 


14/15 








1 






10 










100-0-0% 


Morgan 
Dimon 


1895 
1904 


451 
350 



REGENERATION IN AN EARTHWORM 



131 



TABLE I Continued 





Level 


Regenerate 


Comments 


Author 


Date 


Page 


Cephalic 
Number of segments 


* Uncertain 


Caudal 


V 

o 

z 


2 


3 


4 


5 


6 


7 


) 


15/16 

EL 














22 
3 


2# 
4 



1? 


5 


92-8-0% 
? = "possibly a new tail" 


Dimon 
Morgan 


1904 
1902 


350 
579 


16/17 
EL 














11 


1# 
2 







92-8-0% 
3-4 S, "not regenerated 
(mouth present)" 
(See note (f)) 


Dimon 
Morgan 


1904 
1895 


350 

455 


17/18 















12 


_1# 







92-8-0% 


Dimon 


1904 


350 


18/19 












26 


5# 


2 




79-15-6% 


Dimon 


1904 


350 


19/20 
















3 


1 

?# 


? 




U = "4or 5 S" 
See note (g) 


Morgan 
Dimon 


1895 
1904 

1899 


451 
350 


20/21 
EL 














1 




17 S See note (h) 


Morgan 


409 


? 












4 


7# 


12 




17-30.5-52.5% 
See note (g) 


Dimon 


1904 


350 


22/23 

















2 






Very imperfect 


Morgan 


1895 


452 


23/24 
EL 


















1 

1 






Very imperfect 
3 or 4 S, imperfect 


Morgan 
Morgan 


1895 
1895 


452 
456 

574 


24/25 






2 






See note (h) 


Morgan 


1897 


EL 25/26 














1 




1 




T=17 S 
See notes (h) and (i) 


Morgan 


1899 


40 


EL 30/31 
















1 




1 




H = 7 or 8 S 
See note (h) T = 21 S 


Morgan 


1899 


408 


EL 34/35 




1 













* 


Morgan 


1901 


7,Fig.G 
455 


EL 3 5/36 
















1 




(L 63 S) T=15 + S 
See notes (h) and (i) 


Morgan 


1895 




































L 75 S 












1 


35 S 


Morgan 


1899 


409 


EL 50/51 








1 








2 


14 


15 


T = 5-25 S 


Morgan 
Morgan 


1901 
1902 


7,Fig.H 
579 

































132 



G. E. GATES 



TABLE I Continued 



Level 


Regenerate 


Comments 


Author 


Date 




Page 


Cephalic 
Number of segments 


* Uncertain 


Caudal 


OJ 

a 
o 
Z 


2 


3 


4 


5 


6 


7 


) 


L20S 


















5 




Michel 


1898 


263 


L10S 


























5 




Michel 


1898 


263 


L5S 








5 




Michel 


1898 


263 


L12-7S 
















98 




Morgan 


1897 


575-6 



NOTES TO TABLE I 

(a) "Attempts made to cut off 1 and 2 segments" (Morgan, 1895, p. 449). As a result of 
confusion re numbering of containers there was but one case in which it was thought "one segment 
must have been cut off" and that specimen could have been a posterior homoeotic. 

(b) The last two specimens in Morgan's Table IV were homoeotic and are here excluded. 
The three specimens next above are assumed to have been normal. (Homoeotics are excluded 
here, as well as from author's operations, to obviate possibility of complications resulting from a 
previous regeneration and because gradients cannot be expected to be the same as in normal 
specimens.) 

(c) Amputations, apparently as a result of operating without anaesthesia, were often diagonal 
(Morgan, 1895, p. 457, also Korschelt), or if transverse then at an intra- rather than intersegmental 
level. In each case a portion of a segment is treated as if a whole segment, i.e. if 10 1 or 10 seg- 
ments were removed from the anterior end, the level of regeneration is still considered to be 10/11. 
In favor of this convention is Morgan's conclusion, after study of deliberately made very diagonal 
cuts, that simultaneous completion of missing parts of segments did not interfere with replacement 
of those metameres that had been completely amputated (1895, p. 457). 

(d) EL estimated level. Level of amputation in many operations was estimated after re- 
generation and from one of the following landmarks (Morgan, 1895, pp. 450 and 452): (1) Position 
of vasa deferentia, i.e. location of male pores. Subject to variation of six segmental levels 
(Morgan, 1895, p. 403). (2) Location of seminal receptacles, apparently thought to be three 
pairs. Eisenia foetida has only two pairs of spermathecae but four pairs of seminal vesicles. 
These landmarks are doubtful. [If three pairs of spermathecae were present another species was 
involved, possibly Dendrobaena octaedra (Savigny) 1826 or Allolobophora chlorotica (Savigny) 
1826, both of which are found in compost heaps and apparently have been confused with foetida. ~\ 
(3) Location of clitellum. That may begin on any of segments xxiv xxvii and end on xxxi xxxiv, 
a variation of three to four levels. Pre-clitellar amputation was variously listed as at 20/21, 
25/26, and postclitellar as at 25/26, 30/31, 35/36. (4) and (5) The middle and the end of the 
body, the former regarded as at 50/51 and the latter as the hundredth segment. Number of 
segments varies from 67-125. 

Actual variations, when recognizable, were: for (3) of seven segmental levels, i.e. 19/20-26/27 
and 31/32-38/39, for (4) and (5) to about 20 levels, 41/42-64/65, etc. 

Postregeneration determination of level of amputation would probably render unlikely 
detection of reorganization of substrate segments. Such reorganization, in some species, could 
affect the determination by one to three segmental levels. 

In certain of Morgan's cases it is not clear whether levels mentioned were determined or 
estimated. 

(e) Number of segments of other regenerates at this level "tres variable." 

(f) The regenerate segments were "very irregular." The characterization "not regenerated 



REGENERATION IN AN EARTHWORM 133 

occasions when the supply of cloth had been exhausted, worms were kept in large 
crystallizing dishes in water just sufficient to cover the bottom and keep the animals 
moist. Although E. joetida appeared to do as well in water, in cool weather, as in 
moist cloth, the method is not recommended, for in later work several long series of 
operated animals were completely lost over night. To prevent accumulation of 
metabolic wastes, water or paper was changed (or cloth washed) daily, except on 
Sunday when the museum was closed. 

Specimens were killed so as to insure uniform contraction and were then pre- 
served in formalin. 

The experiments were carried out mainly during a sabbatical leave in the States 
in 1926-27. Shortly before Japan entered the war, a summary of the results ob- 
tained was prepared and sent home from Burma. Original records, as well as 
specimens, were lost in the sack of Rangoon. 

The author's thanks are extended to Prof. G. H. Parker for provision of labora- 
tory facilities at Harvard University during the academic year 1926-27, to Prof. H. 
W. Rand for similar facilities in the U. S. Fish Commission building at Woods Hole 
during the summer of 1927, to Dr. Esther Carpenter for care of operated animals 
while the author was ill. 

NOMENCLATURE 

In one and the same article, an author once used "posterior end" to refer to : a 
posterior regenerate regardless of size; an anterior regenerate (heteromorphic) ; the 
anal region of an adult worm ; and long posterior portions of varying lengths up to 
a half or more of adult size. Similarly "anterior end" has had various meanings, in- 
cluding even that of tail (heteromorphic). Most confusing, however, has been a 
failure to distinguish adequately in discussions between the regeneration taking place 
at a single surface of amputation and that taking place at exactly the same level when 
there are two cut surfaces. In an attempt to avoid further complications, an effort 
has been made to restrict terms and phrases consistently to the meanings given 
herewith. 

(mouth present)" may refer to an anally sculptured cicatrix. Such sculpturing may be pre- 
liminary to growth of a tail regenerate. 

(g) Results of all operations behind 18/19 were lumped together. Mention was, however, 
made of three "B" heads at 19/20 which have also been listed above at that level. 

(h) No data as to number of segments in 1897 regenerates (Morgan, pp. 573-574) and no 
clues to warrant guesses as to nature of regenerates. 

(i) Results of 30 operations (Morgan, 1902, pp. 578-579) omitted because of uncertainty: 
(1) as to level of operation, said to have been "just behind the girdle (about the 25th segment)," 
i.e. either at 25/26 or 34/35; (2) as to nature of substrate, i.e. whether posterior or a two-surfaced 
fragment. Three months after operation, four specimens having died, the container had 36 speci- 
mens which were not examined for autotomy. Nevertheless, presence of one distinct new head and 
14 doubtful regenerates, of which "probably more were heads than tails" may be of considerable 
importance. 

Presence of extra worms in containers may have another explanation than autotomy. Just 
hatched juveniles are exceedingly difficult to find in either manure or soil. In absence of steriliza- 
tion of the manure used for culture medium, there was time, during the months allowed for 
regeneration, for young to attain adult size. In this connection a belief that regenerates became 
indistinguishable from substrates is perhaps important (Morgan, 1895, p. 424). 

# Regenerates referred to this class by Dimon were not characterized in any way. * Some 
"doubtful" regenerates of other authors are also included here. Others, that appear also to be 
doubtful, have been included in part. 



134 G. E. GATES 

In place of regenerant and regenerate, which are easily confused, substrate and regenerate 
are used respectively, to designate the portion of the original worm on which the new growth 
is formed and the new growth thus formed at the cut surface. This is in continuation of pre- 
vious practice (Gates, 1941). 

Posterior substrate refers to any posterior portion of the body, regardless of size, extending 
forward from the anal region to a single anterior cut surface. 

Healing after amputation may be cicatricial or enteroparietal, In the first, a cicatrix is 
formed across the cut surface, while in the second, cut edges of gut and body wall apparently 
heal together without definitely recognizable cicatricial tissue. 

A regenerate with no externally recognizable differentiation is a bud (indeterminate). As 
indications of buccal or anal sculpturing become recognizable, further characterization as cephalic 
or caudal is possible. With appearance of metameric differentiation the regenerate is a head 
or a tail. 

A considerable degree of deviation from normal structure may be possible in a regenerate 
without affecting its caudal or cephalic nature. Such variant regenerates are abnormal. A 
regenerate without cephalic or caudal characteristics, or with a mixture of cephalic and caudal 
characteristics, or with bifurcations, is a monstrosity. A growth without indication of caudal or 
cephalic nature is an indeterminate monstrosity. 

A metamerically normal regenerate may be cqiiimcric, hypcnncric, or hypnmcric, depending 
on whether it has the same number of segments, more than, or fewer than the excised portion. 

Heteromdrphosis indicates a more or less normal structure in a reversed or abnormal direc- 
tion. A head at a posterior amputation and a tail at an anterior amputation is heteromorphic. 
Homomorphic distinguishes the head or tail in normal position or direction. 

Levels are designated in two ways, by reference to the segments, as xxvi, and to the fur- 
rows bounding the segment as 25/26 and 26/27. The Roman numeral in lower case nleans the 
twenty-sixth segment beginning with the buccal as i; the prostomium of the Oligochaeta is not 
counted as a segment. The fractions refer to the intersegmental furrow at the anterior and 
posterior margin of segment xxvi, and make possible, with shorthand brevity, exact designation 
of level. Indication of level of amputation merely by reference to the segment, as "at the 
twenty-sixth segment," may be inadequate unless the context indicates which of the two possible 
levels, anterior or posterior, is involved. EL befcre the fraction means estimated level, the 
estimate usually that of the original author, otherwise made in accordance with his custom so 
far as is possible. 

The anal region of the body forward to the first complete intersegmental furrow is not 
regarded as an ordinary metamere but for purposes of segmental enumeration is taken as one 
segment (see Gates, 1948). Posterior substrates of unknown location with reference to the 
antero-posterior axis are characterized by a designation such as L14S, in that case meaning 
the last fourteen segments. 

Homoeosis, as ordinarily used in connection with earthworms, means : presence of an organ 
or pair of organs, or a series of organs, in a segment or series of segments, other than that, or 
those, in which usually or normally found. It refers primarily to individual variation within a 
species ; secondarily, to phylogenetic variation, for a species or a genus may be homoeotic with 
reference to other species in the genus, or other genera in the family. In case of individual 
homoeosis, the dislocation may involve one or both organs of a pair in a segment. The former 
is asymmetrical homoeosis, the latter symmetrical. 

SUMMARY OF RESULTS 

Healing at cuts in an anterior portion of the body was cicatricial, the cicatrix a 
low, flat-surfaced, circular disc without recognizable sculpturing. In some speci- 
mens no further development was recognizable. In others the cicatricial disc gradu- 
ally was protruded as a small, rather conical bud at first apparently unmarked by 
any sculpturing. In several cases the growth of the bud was inhibited at that stage. 
In the remainder the distal portion became sculptured to indicate a prostomium 
and mouth. Intersegmental furrows, setae, and finally pigment usually became 
recognizable in that order. 



REGENERATION IN AN EARTHWORM 135 

Regenerates always remained distinguishable from substrates by differences in 
pigmentation, segment size, setal intervals, etc. 

Several months' starvation resulted in reduction of size of substrates but no 
macroscopically recognizable reorganization was noted, either externally or inter- 
nally, behind the level of amputation. 

Reproductive organs were not found, in regenerate or (as result of reorganiza- 
tion) in substrate. 

A. After a single cut 

All substrates with cut surfaces at levels from 8/9 anteriorly, with one ex- 
ception, regenerated. At each level behind 8/9 one or more of the substrates did 
not survive operation long enough to regenerate or else failed to regenerate if 
surviving. Highest percentages of failure to regenerate were in the region around 
25/26. Further posteriorly, survival was better and percentages of successful re- 
generation higher. Results just mentioned were, however, minimal, as inhibited 
buds, rare monstrosities (indeterminate) and certain conditions to be considered 
later on were recorded as failures (to produce a more or less normal head or tail). 

Head regenerates were obtained at levels 1/2-23/24 only. Equimeric heads were 
obtained at levels 1/2-8/9 inclusive. Three regenerates at 4/5 were hypermeric 
(+1). 'All head regenerates at levels 9/10-23/24 were hypomeric, the maximum 
number of segments obtained being six. In a later series of operations, E43, of 
three head regenerates at 8/9, one had five, another had six, and a third had nine and 
a half segments, the half segment wedge-shaped and on the right side (+ I 1 /;)- 

Heteromorphic tail regenerates w r ere obtained, once each at levels 20/21 and 
23/24, and from 24/25 to 54/55. The largest number of setigerous segments 
differentiated in such heteromorphic tails was 25 at 40/41. the evidence available in- 
dicating increase in number of segments posteriorly to 40/41 and then a decrease. 

At levels behind 54/55 no regeneration whatever, including even buds and 
monstrosities, was obtained though numbers of substrates were under observation 
three to four months. 

B. After a previous regeneration 

In attempts to test for the effects of previous regeneration on anterior regenera- 
tion, several series of operations were made of which the following are mentioned. 

In series E41 posterior portions were removed at 34/35 and 35/36 and the 
substrates (anterior) were allowed to regenerate for twenty-three days. At that 
time the anterior eight segments were removed and discarded. Of the surviving 
substrates (8/9-34/35 or 35/36 + a tail regenerate), four regenerated heads 
anteriorly. Three were hypomeric with six segments each, and one was hyper- 
meric with nine segments ( -f 1 ) . 

In series E58 the last ten segments were removed from specimens having one 
hundred or more segments. At the end of twenty-two days' regeneration, an- 
terior portions were removed so as to leave ten or fifteen segments of the original 
substrates along with the tail regenerates. One of these small substrates had al- 
ready produced a bud at the anterior cut surface by the seventh day, at which time 
circumstances compelled termination of the experiment. Anterior regeneration in 



136 G. E. GATES 

this series, would, if completed, have taken place at levels behind 75/76, while normal 
posterior substrates, unconditioned by a previous regeneration, failed to regenerate 
at levels behind 54/55. 

In series E49 the posterior portion of the body was removed at 70/71. The 
anterior substrates were allowed to regenerate posteriorly for eighty days. At that 
time the tail regenerates were removed at the level of regeneration. One such tail 
regenerate, then acting as substrate, produced in twenty-seven days, at the an- 
terior cut surface (level 70/71 with reference to location on axis of original worm), a 
heteromorphic tail, unpigmented but with six setigerous segments and a small anal 
region without indication of production of further segments. Final substrates in 
this series were 7 to 10 mm. long and of 30-41 setigerous segments. 

C. After starvation 

To test for the effect of starvation, the following experiment was run (see also 
series E49 above for regeneration after 80 days' starvation). From worms that 
had been starved for seventy clays or longer, the anterior five or six segments were 
removed. Each surviving substrate regenerated a hypomeric head ( -- 1 to --3) 
with metameric differentiation complete and normal. 

DISCUSSION 

A first step towards obtaining a complete characterization of regenerative ca- 
pacity in E. foetida is determination of the morphological nature of the regenerate 
produced anteriorly, at each intersegmental level along the axis, by posterior sub- 
strates, as well as the number of segments in such regenerates. The latter, often 
neglected in the past apparently as of little importance, is of some interest with re- 
gard to morphogenesis in the Lumbricidae. 

Hypermery in head regenerates has now been recorded for the first time in E. 
foetida, and at two different levels, one of which, 8/9, is fairly well back. Hescheler 
(1896, p. 93) once secured a regenerate with more segments than had been removed 
but in a series of successive regenerations by a single individual, the worm even then 
still hypomeric by two segments (removal of 6% segments, regeneration of 5%; 
removal of 4, regeneration of 2 ; removal of 2, regeneration of 3). One hitherto un- 
noticed case of hypermery in the Lumbricidae has been found regeneration of four 
segments after removal of three by a specimen from which the nerve cord had 
been removed from the next two metameres behind the level of amputation (species 
unidentified, Goldfarb, 1909, p. 703, Table 4, No. 1.41). 

Hypermeric regenerates are of especial interest in connection with the problem 
of the origin of posterior homoeosis. In E. foetida posterior homoeosis of one 
segment only has been recorded and now in regenerates hypermery of one segment 
only. As all cases of symmetrical homoeosis in the species can now be considered 
to have resulted from hypomeric or hypermeric regeneration, postulation of some 
unknown embryonic cause is no longer necessary. 

The new data as to segment number in homomorphic anterior regenerates pro- 
vides confirmation of the cephalic nature of Michel's and Hescheler's regenerates of 
seven segments and of Morgan's regenerate of "7 or 8" segments, all of which seem 
to have been overlooked hitherto. 



REGENERATION- IX \\ T EARTHWORM 137 

Presence in a head regenerate of nine segments may indicate a possibility of 
equimeric regeneration back to 9/10 but is of especial interest in connection with 
the problem of the constitution of the "head." In the Oligochaeta homomorphic 
anterior regeneration is generally thought to be restricted to replacement of the 
"head." The latter, in the Lumbricidae. has been thought to comprise five seg- 
ments only. Six, seven, and eight (?) -segment head regenerates obtained by 
Michel and Morgan in E. joctida (Table I), and a six-segment regenerate at 9/10, 
as well as a seven-segment regenerate at 12 13 in AllolobopJwra tcrrestris 
(Hescheler, 1896). should have been taken into consideration in this connection. 
Carpenter's (1948) regenerates of six segments (Table I), and the author's of six 
to nine, show that regeneration of heads with more than five segments is not ex- 
ceptionally rare. Smaller numbers in previous work may have been due to less 
favorable conditions. 1 

The maximum number of segments now recorded for head regenerates in the 
family Lumbricidae is nine. With the exception of one pair of seminal vesicles and 
of spermathecae, both of which develop in connection with septum 9/10, reproduc- 
tive organs in the Lumbricidae are in the region from x posteriorly. All of the evi- 
dence available still indicates that Lumbricids regenerate anteriorly only a prego- 
nadal portion of the body. Regeneration, after amputation of the gonadal region, 
accordingly, is not sufficiently "complete" to enable an individual to reproduce. 
Although this has often been thought to be characteristic of earthworms generally, 
at present it appears to be applicable only to the Lumbricidae. In those representa- 
tives of other families that have been studied, regeneration of the gonadal region not 
only is possible but even usual (see Janda, 1926, for the Glossoscolecid Criodrilus 
lacuuin, and Gates. 1941. for the Megascolecid Periony.v excavatus) . The pattern of 
regenerative capacity, even with regard to this one matter, accordingly, is not uni- 
form throughout the earthworms. 

Such data as are now available with regard to segment number, and in particu- 
lar "7 or 8" segments in a head regenerate at EL 30/31 (Table I), do not appear 
to support current ideas as to decline in number of head segments regenerated as 
level of amputation recedes posteriorly (Hyman, 1940. p. 519) and gradient of head 
regeneration (Liebmann, 1943, p. 601, Fig. 12). 

New data given above as to the morphological nature of anterior regenerates 
agree with some hitherto overlooked in showing a region of definite bipotential re- 
generative capacity. On amputation within that region a worm may regenerate 
either a head or a tail. The individual variation in response to the same stimulus 
suggests a possibility of experimental modification of the nature of regenerates. 

The region of bipotential capacity, according to the author's results, is small 
and bounded by 20/21 and 23/24. Previous work indicates the possibility of con- 
siderable extension of those boundaries. Involved in estimation o-f the posterior 
limit of cephalic regeneration are two regenerates at EL 30/31 and EL 50/51 
(Morgan, 1899 and 1901). Both, it is important to note, were obtained after re- 
discovery of heteromorphosis. The cephalic nature of the first was proved from 
sections. The second, having five metamerically normal segments, presumably was 
large enough to be easily and correctly identified.. Level of the first amputation 
could have been from 31/32 to 38/39 (see note d, Table I), but was probably in re- 

1 Carpenter now reports obtaining in one series, seven six-segment, two seven-segment, 
and one eight-segment head regenerates at 10/11. 



138 G. E. GATES 

gion of 32 33-35/36. The other amputation, estimated to he at the middle of the 
hody, could have heen from 41/42-64/65 (see note d, Tahle I), but with probability 
of location at or even in front of 41/42. A level about midway between 30/31 and 
40/41, i.e., 35/36, appears at present to be as good an estimate as is possible in the 
circumstances. 

The anterior boundary for heteromorphic tails is extended to 18/19 by Dimon's 
results (Table I). However, some of her "uncertain" regenerates at 17/18-15/16 
presumably had, in absence of all reference to monstrosity, similarities to caudal re- 
generates. Morgan also had a regenerate at 15/16 thought to be "possibly a new 
tail" (1902, p. 579). All this seems to warrant placing the anterior boundary 
provisionally at 15/16. It is also noteworthy that at several still more anterior 
levels, to 10/11, some regenerates were "imperfect" or "very imperfect," character- 
izations apparently applied also to regenerates later found to be caudal. 

Gradient of segment number in heteromorphic tail regeneration appears, from 
the author's data, to be of an inverted V-shape rather than the even slope apparently 
anticipated by Morgan (1902, p. 577) from results obtained on small fragments. 

Failure, in the author's experiments, of normal worms to regenerate at levels 
behind 54/55 was unexpected in view of the results obtained from substrates as 
small as L14S in A. tcrrestrls (Korschelt, 1898, p. 80). Regeneration by tail re- 
generates from levels behind 54/55 (E49), and behind that level by substrates con- 
ditioned by a previous regeneration (E58), suggests a possibility that failures on nor- 
mal specimens were due to unfavorable conditions. 

Regeneration of heteromorphic tails anterior to 20/21 and of heads behind that 
level, and more important, of both heads and tails from the same levels, does not 
appear to be in accordance with Liebmann's hypothesis (1943) that specifically 
polarized, eleocytic aggregates in the coelomic cavities, a head aggregate in v-xx and 
tail aggregates behind 20/21, determine the nature of the regenerate. 

SUMMARY 

Posterior substrates of E. joetida, cut exactly at intersegmental furrows, re- 
generated homomorphic heads at levels 1/2-23/24, with equimery at 1/2-8/9 and 
hypermery (+ 1) at 4/5 and 8/9. Heteromorphic tails regenerated at 20/21 and 
from 23/24 to 54/55. Behind 54/55, regeneration of heteromorphic tails was ob- 
tained only from tail regenerates and substrates conditioned by a previous re- 
generation. Gradient of segment number in heteromorphic tails appears to be of an 
inverted V-shape. 

Starvation for 70 + days did not inhibit regeneration at 5/6 and 6/7 but all re- 
generates were hypomeric. 

Hypermery and hypomery provide an adequate explanation of the origin of 
symmetrical homoeosis. 

Regenerative capacity in a region from 20/21 to 23/24 is characterized as bi- 
potential since an anterior regenerate, in that region, may be a homomorphic head 
or a heteromorphic tail. 

Review of previous work on E. joetida provides indications that the region of 
bipotential regenerative capacity is even more extensive, with anterior limit of 
heteromorphosis possibly at or even in front of 15/16 and posterior limit of homo- 
morphosis in region of 35/36. 



REGENERATION IN AN EARTHWORM 



LITERATURE CITED 



139 



CARPENTER, E., 1948. Six-segment head regenerates in an earthworm, Eisenia foetida (Savigny) 

1826. Science, 108: 625-626. 
DIMON, A. C., 1904. The regeneration of a heteromorphic tail in Allolobophora foetida. Jour. 

E.vp. Zool, 1 : 349-351. 
GATES, G. E., 1941. Further notes on regeneration in a tropical earthworm, Perionyx exca- 

vatus, E. Perrier 1872. Jour. .r/>. Zool., 88: 161-185. 
GATES, G. E., 1948. On segment formation in normal and regenerative growth of earthworms. 

Growth, 12: 165-180. 
GOLDFARB, A. J., 1909. The influence of the nervous system in regeneration. Jour. Exp. Zool., 

7: 643-722. 
HESCHELER, K., 1896. Uber Regenerationsvorgange bei Lumbriciden. Jena. Zcit. Nativ., 30: 

177-290. 

HYMAN, L. H., 1940. Aspects of regeneration in Annelids. Amcr. Nat., 74: 513-527. 
JANDA, V., 1926. Die Veranderung des Geschlechtscharakters und die Neubildung des Gesch- 

lechtsapparats von Criodrilus lacuum Hoffm. unter kiinstlichen Bedingungen. Arch. 

Entii'ickmech. Org., 107: 423-455. 
KORSCHELT, E., 1898. Uber Regenerations- und Transplantationsversuche an Lumbriciden. 

Verb. Deutsch. Zool. Ges. 1898, 79-94. 
LIEBMAN, E., 1943. New light on regeneration in Eisenia foetida (Sav.). Jour. Morph., 73: 

583-610. 
MICHEL, A., 1898. Recherches sur la regeneration chez les annelides. Bull. Sci. France Bclg., 

31 : 245-420. 

MORGAN, T. H., 1895. A study of metamerism. Quart. Jour. Mic. Sci., 37 : 395-476. 
MORGAN, T. H., 1897. Regeneration in Allolobophora foetida. Arch. Entu'icknicch. Org., 5: 

570-586. 
MORGAN, T. H., 1899. A confirmation of Spallanzani's discovery of an earthworm regenerating 

a tail in place of a head. Anal. Anz., 15: 407-410. 
MORGAN, T. H., 1901. Regeneration. Columbia Univ. Biol. Series, 7. 
MORGAN, T. H., 1902. Experimental studies of the internal factors of regeneration in the 

earthworm. Arch. Entit'ickmech. Org.. 14: 562-591. 



FREE-ENERGY RELATIONS AND CONTRACTION OF 

ACTOMYOSIN J 

A. SZENT-GYORGYI 

Il.rficriinental Biology and Medicine Institute, Laboratory of I'liysieal Biology, National 

Institutes of Health, Bcthesda, Maryland and Institute jor Muscle Research, 

Marine Biological Laboratory, Woods Hole, Massachusetts 

There are two approaches to muscle. One is that of the physiologist, who 
studies function hoping to understand the nature and reactions of minute struc- 
tural elements. The other is that of the biochemist, who studies minute structural 
elements hoping to understand function. The physiologist carefully preserves 
structure and subtle qualities ; the biochemist wilfully destroys them. This de- 
struction may go as far as the dissolution of the system into single molecules. This 
approach was that of the author's laboratory, which has shown that the contractile 
matter of muscle is built of two proteins, actin (F. B. Straub 1942. 1943) and 
myosin. 

Destruction need not necessarily go that far. It may be limited to partial dis- 
solution, which leaves the contractile matter and its geometry untouched, or it may 
simply consist in the disturbance of certain equilibria by thermal or chemical means. 
In these cases, it is still convenient to call the system "muscle," but it should be 
clearly understood that by using this word no attempt is made to confuse such a par- 
tial system with the whole living and intact machinery. 

In themselves, neither myosin nor actin is contractile. If brought together in a 
suitable ionic milieu they unite to a complex : "actomyosin." According to the 
concentration and the nature of ions present, the actomyosin may be charged by 
the ATP and dissociate reversibly into its two components, or else it may be dis- 
charged and dehydrated excessively. If this reaction takes place in a hetero- 
geneous suspension, the actomyosin is precipitated. Owing to its violence, this 
precipitation was termed "superprecipitation" to distinguish it from the weaker 
dehydration and precipitation induced by salts alone in absence of ATP. If this 
reaction takes place in an actomyosin gel, it will lead to excessive shrinking, syn- 
eresis. If the elongated actomyosin particles are oriented, the shrinking will be 
anisodiametric and the gel shrinks in the direction of the axis of the particles and 
expands at right angles to this direction. Actomyosin threads or muscle fibres, 
under these conditions, may become shorter and wider without changing their vol- 
ume. If the reaction takes place in the muscle fibre where the elongated actomyosin 
filaments form a continuous system, the shortening will be able to do work by lift- 
ing weights, or develop tension under isometric conditions, and is usually called 
"contraction." 

The study of these phenomena suggested (see Szent-Gyorgyi, 1947) that the 

1 This research has been sponsored by a grant from the American Heart Association. 

140 



FREE ENERGY IN MUSCLE 



141 



contractile matter is built of functional units, "autones," and that contraction is an 
"all-or-none equilibrium reaction" of these autones, dependent on temperature. - 
Contraction, i.e. the dimensional change, in all probability, is secondary to another 
change in which charges are neutralized. The size of "autones" is independent of 
the colloidal particle size (1-1, 5 X 10 g) into which myosin breaks up on extrac- 
tion, and can be expected to be much smaller than this latter. Supposing that the 
actual shortening is proportional to the number of reacting units, the relative num- 
ber of charged and discharged units (that is the equilibrium-constant K) was cal- 
culated from the macroscopic length of the system. 



I200O 



10000 



8000 



6OOO 



4000 



2000 



1 1 000 



5500 







C e 



10 



20 



30 



40 



50 



FIGURE 1. The A F curve of the contraction of extracted frog and rabbit muscle and of 
frog and rabbit actomyosin threads. Ordinate : A F in calories. Abscissa : temperature in 
centigrades (quoted from L. Varga). 



From the temperature-dependence of K. the A F, i.e. the difference of the free- 
energy content of contracted and relaxed autones, was calculated. The final results 
of these calculations were summed up by L. Varga (1946) in a curve reproduced 
in Figure 1. The curve shows that the free energy of the system drops in con- 
traction and that the extent of this drop depends on temperature. In the rabbit it 
reaches 11,000 calories at 53 C, is 7-8000 calories at 37, and is at about C. 

- Buchtal and Knappeis pointed out in 1943 that certain mechanical features of muscular 
contraction are in accordance with the assumption that the fibre is built of smaller units con- 
tracting in an "all-or-none" fashion. 



142 A. SZENT-GYORGYI 

In the frog the same values were reached at 5 C. lower. The curve shows that the 
relaxed state is the high-energy metastahle state, the contracted state the low-energy 
stable state, contraction being a spontaneous process. 

In the first part of this paper material and methods will be discussed. In the sec- 
ond part, the theory will be tested along different lines, and in the third part, the ob- 
servations will be extended. 

PART I : MATERIAL AND METHODS 

Muscle is a very heterogeneous tissue. Not only are there different kinds of 
muscle (smooth, heart and cross-striated muscle), but there are considerable differ- 
ences between the different muscles of the same sort within the same animal. 

There is considerable difference in geometry between the various body muscles. 
In one muscle the fibres are parallel, while in others they follow a more complicated 
course, making evaluation of energy relations difficult. 

There is considerable difference, also, in the composition of various muscles. 
The contractile matter, actomyosin, is in its relaxed condition a soft gel which 
could easily be damaged by mechanical injury were it not protected by connective 
material, fasciae, collagen fibres and a sarcolem. Muscles lying closer to the surface 
will need more protection, and in these we will find strongly developed connective 
material and sarcolem. 3 An almost ideal material for the study of the contractile 
matter is the musculus psoas of the rabbit. This muscle lies sheltered in the body 
cavity, protected on one side by the vertebral column, and by the viscera of the belly 
on the other. Consequently, it contains very little connective tissue, and the sar- 
colem is poorly developed, which makes the elastic properties of the contractile 
matter come to the fore. It is built of very long, parallel fibres, stretching from one 
end of the muscle to the other fibres which, owing to the poverty of connective 
material, can easily be separated. It is easy to secure from a medium-sized rabbit 
very thin fibre bundles 8-10 cm. long which, if necessary, can be decomposed into 
single fibres of this length. Though occasionally frog sartorius and rabbit m. 
gracilis were also used, the major part of the experiments reported here were per- 
formed on the psoas. 

According to the theory outlined, contraction is a spontaneous process going 
hand-in-hand with a drop of free energy. Thus, contraction should occur spon- 
taneously wherever the ATP-actomyosin system is present in a suitable ionic milieu, 
and the system should persist in the low-energy stable contracted state. This is 
actually what happens any time we add ATP to an actomyosin gel or to muscle ex- 
tracted with water. In the intact resting muscle, however, we find ATP in an ac- 
tive form, linked to actomyosin (see below), but still the system does not con- 
tract contraction being inhibited by some unknown mechanism. If we want the 
muscle to go over into the contracted state, we have to abolish this inhibition. In 
the intact muscle this can be achieved by an electric shock or a "wave of excitation." 
These actions are fleeting and depend on subtle qualities of muscle, on "excitability," 
which makes them unfit for our present purpose. In order to study equilibria of 
energy relations, the inhibition had to be removed permanently and uniformly 

3 Ramsey and Street (1940), working with single fibres of the musculus semimembranosus 
of the frog, found the elastic properties of the contractile matter in resting muscle entirely 
covered up by the elastic properties of the sarcolem. 



FREE ENERGY IN MUSCLE 



143 



throughout the whole mass of the muscle, and the whole contractile matter made to 
go over into and remain in the contracted state. Poisons like caffeine, quinine, 
monojodo-acetic acid or chloroform, known to produce contracture, were found un- 
satisfactory because the tensions developed are very small, showing that only a 
small fraction of the contractile suhstance is at any time in the contracted state. 

A satisfactory method of abolishing inhibitions is freezing with subsequent thaw- 
ing, which method also has the advantage that the muscle can be kept in the frozen 
state, packed in dry ice, for days with undiminished contractility. 




MIN 



FIGURE 2. Isometric contraction of the frozen sartorius of the frog on thawing. The frozen 
muscle was immersed into Ringer of 20 C. at min. 

The experimental procedure was the following : The rabbit was killed by de- 
capitation, quickly skinned, eviscerated, and the front and the sidewalls of the belly 
cut off. This exposed the psoas which was liberated from its surroundings. The 
muscle was decomposed into smaller bundles by punching it through with a small 
forceps with closed tips and moving the forceps up and down while the index finger 
of the other hand kept the muscle somewhat lifted. If necessary, ligatures were 
put on the two ends of the muscle strip. Owing to the poverty of connective tis- 
sue, the muscle is rather soft and is easily cut through by ligatures. For this reason, 
relatively soft and thick threads were applied in dry condition (pearl cotton No. 5). 

If frozen strips were desired, fibre bundles of 2-3 mm. diameter were secured, 
placed on a celluloid ruler (to which the muscle does not stick), stretched to their 
rest length, the ligature being fixed by artery clamps. Then the strips were covered 
with freshly powdered dry ice. The strips used were mostly of the thickness of an 
average frog sartorius, weighing about 40 mg. per cm. Since at higher temperatures 
the muscle, after freezing, is rapidly damaged, it is important that it should reach 
temperature equilibrium quickly. So if experiments had to be 'performed above 30 
C., even thinner strips were used weighing 25 mg. per cm. 

On thawing, the frozen muscle, if containing the physiological amounts of ATP, 
contracts rapidly and develops maximal tension. 



144 A. SZENT-GYORGYI 

There are two phenomena which tend to disturb measurements. If the frozen muscle is 
suddenly placed into Ringer of room temperature, one side may thaw faster than the other and 
contract suddenly, which causes strong bending which damages the fibres. For this reason, the 
muscle was allowed to thaw first in Ringer before being transferred into a warmer Ringer 
of more than 15 C. The first movement of the lever indicates complete thawing which may 
take place in ten to sixty seconds. 

The contraction, elicited by the freezing and subsequent thawing, is developed relatively 
slowly while the sudden change in temperature may act, in itself, as an impulse and elicit a 
fast contraction. In this way, in frog muscle, a double peak is obtained, the second of which 
is mostly lower than the first (Fig. 2). In rabbit psoas this double contraction is less pro- 
nounced but still present. Below 10 C. "excitability" is low and the two waves fuse. At 
higher temperatures they can be made to fuse by making the temperature change less sudden 
by allowing the muscle to thaw in Ringer of C. before applying the higher temperature 
Tension given to the muscle also promotes fusion of the two peaks. The same is favored, also, 
by a thinner diameter. Correct values of maximal work or tension can be obtained only if the 
two contractions fuse into one. 

In order to show whether an observed effect was actually due to an interaction 
of ATP and actomyosin, the latter had to be prepared free of ATP. The effects 
observed on addition of ATP could then safely be ascribed to an interaction of the 
protein and the nucleotide.* 

Thus muscle fibres had to be prepared, free of ATP, and made permeable to this 
substance, without destroying the actomyosin structure. In earlier work this was 
done by extracting the muscle with water. In pure water, however, even at low 
temperature, muscle fibres preserve their full contractility only for a short time. 

Satisfactory results were obtained by employing a 50 per cent solution of glyc- 
erol. The fibre bundles, once extracted, can be preserved for weeks in this sol- 
vent at - 20 C. with undiminished contractility. The psoas was decomposed in 
, situ into fibre bundles of about one millimeter in diameter. A thin stick was laid 
alongside, and four or five such bundles were tied to it at both ends and cut out. In 
this way straight fibre bundles of rest length and attached to the stick were secured. 
If bundles of equilibrium length were desired, only one end of the bundle was fixed 
to the stick and the other end cut, whereupon the muscle contracted to its equilib- 
rium length. Then the other end of the bundle was fixed to the stick. In order 
to measure the difference between rest length and equilibrium length, a ligature 
was put on the free end of the bundle before cutting it, and the distance between the 
two ligatures was measured before and after cutting. 

The bundle, tied to the stick, was placed into 50 per cent glycerol of for 
twenty-four hours. Then the two ends of the muscle with the ligatures \vere cut 
off, whereby the muscle, detached from the stick, fell into the single bundles. The 
muscle was left in this condition for another day at in 50 per cent glycerol and 
then transferred in this solvent into the deep freezer kept at -- 20 C. 

4 Threads prepared from actomyosin are unfit as material for any experiment in which 
tensile strength is involved, since on extraction the continuous actomyosin filaments present in 
muscle are broken up, and actomyosin threads contain only their fragments. As will be shown 
later, one of the actions of ATP is to enable the actomyosin particles to slip alongside one 
another. Therefore, if an actomyosin thread is loaded or subjected to tension, and ATP is 
added, the actomyosin particles will contract, as they do in muscle, but they will also slip, and 
in spite of the contraction (observable in unloaded threads), the system will lengthen. This 
lengthening has led Buchtal, Deutsch, Knappeis and Petersen (1947) as well as Astbury, Perry 
and Reed (1948) to the erroneous conclusion that phenomena in actomyosin threads are funda- 
mentally different from those in muscle. 



FREE ENERGY IN MUSCLE 145 

The muscle in 50 per cent glycerol is too stiff to be decomposed into smaller 
bundles without straining, which causes the bundles to curl up. In water the 
muscle is too soft. For this reason, before the experiment, the bundles were trans- 
ferred from 50 per cent glycerol to 20 per cent glycerol for an hour or so and de- 
composed here to. the desired diameter, mostly into strips of 0.2 0.5 mm. diameter. 

The psoas is built of smaller fascicles, and it is well to follow the outlines of these 
preformed bundles in decomposing the muscle. The dissecting was clone by means 
of a pair of fine tweezers, used by watchmakers. The ends of the bundles are caught 
with these tweezers. By pulling them apart, the bundles can readily be separated. 
Before being subjected to experiment, the fibre bundles were examined under the 
microscope for continuity. 

The experiments were performed in Ringer containing 0.001 M MgCL. In 
all experiments glass-distilled water was used because of the deleterious action of 
copper usually present in common distilled water. 

Glycerol-treated fibre bundles of rest length and of 0.1 -- 0.2 mm. diameter, if 
placed into a 0.25 per cent ATP solution, contract rapidly. Diffusion being the 
limiting factor, the rate of contraction depends also on the diameter. Unloaded 
fibres contract at room temperature to one-fourth or one-fifth of their rest length. 
If connected to the isometric lever, on addition of ATP they develop tension' com- 
parable in intensity to that developed by intact muscle on maximal excitation. If 
loaded they will also lift weights isotonically, similarly to intact muscle fibre 
bundles of similar dimensions. 

This contraction of glycerol-treated muscle fibres under influence of ATP is one of the 
most striking biological phenomena and is very suitable for classroom experiments. Instead of 
ATP a freshly prepared boiled muscle juice may be used, or an ATP solution may be used 
prepared by elution of dried, alcohol-precipitated muscle. A smallish rabbit will provide mate- 
rial for a big class. Most of the experiments reported here were done by simple means and are 
suited for classroom experiments. Some of them have been repeated by the physiology class 
at the Marine Biological Laboratory at Woods Hole. 

In several experiments the maximal total amount of work had to be measured. 
Theoretically, this can be done in the following way: The muscle is connected to the 
isometric lever, made to contract, and the tension is measured. Then the muscle is 
allowed to shorten slightly and made to contract, and the tension measured, etc., till 
the muscle has contracted maximally and develops no more tension. If the length is 
plotted against tension, the area between length and tension represents the total 
amount of work. Such an experiment was performed on the frozen sartorius after 
thawing. Its result is schematically reproduced in Figure 3, where the hatched zone 
is the total amount of work. This experiment is a rather difficult one and can be 
performed only with limited materal and under specific conditions. In the rabbit 
psoas, contractility is lost after thawing even at C. in fifteen minutes, which makes 
the experiment impossible. In the frog the experiment can be done at low tem- 
peratures only, contractility being lost rapidly at higher temperatures. 

A simpler method had to be found which could be applied in any material in a 
wider range of temperatures. Two such methods are suggested by Figure 3. In 
this figure the total amount of work is equal to the area BDF, which is one-half of 
the area BDFH, and the double of the area CDEG. Accordingly, we could meas- 
ure the total amount of work in two different ways : (1) by measuring the maximum 
tension developed (DF) and multiplying it by the amount of maximum shortening 



146 



A. SZENT-GYORGYI 



B 




H 



30 



20 



10 



10 20 30 

6 TENSION 



40 



FIGURE 3. Schematic representation of muscular contraction. ( Sartorius of the frog. 
Temp. 2.5 C.) AD = initial rest-length, DF = maximum tension, AB = length of maximally 
contracted muscle, unloaded. Area BDF total amount of work. BF = length-tension dia- 
gram of the excited muscle. 

of the unloaded muscle (BD), then dividing the result by two. Psoas strips, after 
freezing and thawing, contract at room temperature by two-thirds of their rest length. 

The formula will thus be -~ = 1/3 It (1 == rest length, t == maximum tension). 

A. V. Hill (1913) developed a similar formula which in his case (frog muscle ex- 
cited electrically at 0) was 1/6 It. (2) Load the muscle with the weight corre- 
sponding to one-half of the maximum tension (DE), measure the distance by which 
the weight is lifted (EG), and take the double of the product of these two magni- 
tudes (the area CDEG). The product will be the biggest if the weight is just one- 
half of the maximum tension, but a small deviation from this value will not cause a 
considerable error making the area bigger in one dimension and smaller in the other. 
The first will be called the "isometric," the second, the "isotonic" method. Both 
methods may be criticized as to their exactness. The object of the present research 
is not to obtain exact numeric values, but to obtain information about the basic 
truth of the theory outlined. 



PART II 



Observation on heat contracture 



According to Figure 1, the A F, i.e., the free energy spent by the single units, 
rises with increasing temperature. The free energy of the phosphate bond in ATP 



FREE ENERGY IN MUSCLE 



147 



is 1 1 ,000 calories (Meyerhof, 1944) and according to the theory discussed, this energy 
is needed for relaxation. According to the curve in Figure 1, the expenditure of 
energy in contraction reaches 11,000 calories at 47 in the frog and at 53 in the 
rabbit, and exceeds 11,000 calories above these temperatures. If the theory is cor- 
rect, therefore, the muscle should be unable to relax at these temperatures and 
should persist in the full)' contracted state. 

In the frog, experiments were performed in the following way : the sartorius of 
Rana pipiens was provided with a ligature at both ends, was excised and loaded in 
one series with 2 g. (Fig. 4, circles) and in another series with 20 g. (triangles). 
The length between ligatures was measured and the muscle dipped into Ringer 
solution of varying temperature. Above 40 a rapid contraction ensued which was 
measured at the end of the second minute. The contraction reached its maximum 
at 47 C. The muscle remained in this maximally contracted state. The gradual 
lengthening at higher temperatures is due to the denaturation which takes place 
above 47 C. rather rapidly. 



12000 



10000 



8000 



6000 



4000 



20OO 




FIGURE 4. Heat contracture in the frog sartorius. The coordinate net corresponds to the 
right-hand side of Figure 1. The sloping straight line is the A F curve, and corresponds to the 
left-hand side ordinate. The points mark per cent of shortening and relate to the right-hand 
side ordinate of Figure 4. Abscissa : temperature in centrigrades. 

FIGURE 5. Same as Figure 2. Strips of the musculus gracilis of the rabbit. 



148 



A. SZENT-GYORGYI 



In the rabbit, experiments were performed in a similar way with the smaller 
weight (Fig. 5). Experiments were performed with strips of the musculus gracilis 
similar in dimensions to the frog sartorius. These strips were cut parallel to the 
fibres. The experiment was performed soon after the animal's death. The results 
were similar to those obtained in the frog. In both cases the muscle went into 
permanent maximal contraction at the temperature where the A F curve cuts the 
1 1,000 calorie level, as demanded by the theory. 

The fact that maximum and permanent contracture was reached only where the 
expenditure of F was 11,000 shows that the transference of energy from ATP to 
the contractile system goes without considerable loss. 

This heat contracture must not be confused with the shortening of muscle due to heat 
denaturation. If a muscle is immersed into Ringer of 70 C. an extensive shortening is pro- 
duced which is not due to the mobilization of the normal mechanism of contraction, but to 
denaturation. The difference between the two processes can easily be demonstrated. If the 
muscle is stored a few hours after death at room temperature or overnight in the ice box, the 
ATP disappears. No rapid contraction will be obtained at 53 in this muscle, but shortening 
will still be obtained at higher temperatures at which rapid denaturation is produced. This 
denaturation manifests itself, also, by a turbid appearance. The basic difference between the 
contraction obtained in the presence of ATP at 53 in the rabbit or 47 in the frog, due to the 
mobilization of the normal mechanism of contraction, and the shortening produced at higher 
temperatures and due to denaturation, can be demonstrated, also, by connecting the muscle to 



I2OOO 



10000 



8OOO 



6000 



4000 



2000 



UOOO 



5500 




O 



o 




O 




A * 



C 



10 



20 



30 



40 



50 



FIGURE 6. The work performed by strips of rabbit psoas, after freezing and thawing, at 
varied temperature, calculated for 35,000 gm. myosin present. The coordinates are identical 
with those of Figure 1. The sloping straight line is the theoretical AF curve. Triangles: 
isotonic measurement. Circles : isometric measurements. 



FREE ENERGY IN MUSCLE 149 

the isometric lever. While the theoretical maximal tension is produced in the first case, scarcely 
any tension is developed in the latter. The gradual lengthening of the loaded muscle above 
53 resp. 47 C. is evidently due to the denatttration. This denaturation in the rabbit sets in very 
rapidly above 53. 

Total u'ork in Ihc psoas 

Free energy being, by definition, tbat amount of energy which can do work, the 
most direct way of testing our A F curve is the measurement of the total work at 
different temperatures. 

The amount of work was measured by both the isotonic and the isometric 
method and the results are reproduced in Figure 6. The psoas strips were loaded or 
connected to the isometric lever in the frozen condition, a moderate tension being 
given to the lever. The work done was expressed in calories and calculated for 
35.000 g. of myosin. In these calculations the average myosin content of muscle 
(8 per cent) was taken into account, though it is probable that owing to poverty in 
connective matter, the psoas contains somewhat more myosin. In order -to find out 
the quantity of myosin present, the muscle was weighed immediately after the 
measurement was finished, its ends with the ligatures having been cut off. 5 

The results of the isotonic experiments are marked in the figure with triangles. 
No measurements could be taken above 45 C. owing to the great sensitivity of the 
muscle to high temperatures after freezing and thawing. As will be seen, the 
agreement with the A F curve is satisfactory and pleads for the basic truth of the 
theory. 

The values obtained in the isometric measurement are marked with circles. 
As the figure shows, at lower temperatures the agreement of the experimental 
values with the A F curve is satisfactory. This is true, however, only up to the 
5500 calorie level, at which it shows a break to become parallel to the abscissa. 
This means that the maximum of tension is reached at 28 C. and increases no 
more if the temperature is raised. 

This inability of the muscle to produce the expected tension at these higher tem- 
peratures is one instance of the so-called Fenn effect, named after its discoverer who 
found (1923) that the work clone by the muscle depended also on the sort of job 
the muscle had to do. 

The isometric method of calculating total work is based on the assumption that the length- 
tension diagram is a straight line. It is evident that in this region, where the isometric and 
isotonic curves differ, the 1-t diagram cannot be straight and the method cannot be used. If 
the results calculated by this method are reproduced, this is because they nevertheless show the 
fact that on raising the temperature the tension developed remains constant. 

The actual 1-t diagram of the psoas can be found in this region by loading the frozen 
muscle strips with different weights, bringing them to the temperature in question, and measur- 
ing the maximum distance to which the weights are lifted. The results of such an experiment, 
calculated for the same weight of muscle, are reproduced in Figure 7. 

The A F curve of jrog muscle 

Varga's A F curve admits but a very small expenditure of energy for the frog 
muscle at C. (600 cal. of the 35,000 gm. unit), which means that at this tem- 

5 After freezing and thawing, contracted muscle rapidly loses weight by pressing out water. 
This loss may exceed 30 per cent, and is in agreement with the assumption that contraction is 
connected with loss of charge and hydration. 



150 



A. SZENT-GYORGYI 



perature this muscle should IK- capable only of very feeble motion. It means, also, 
that the efficiency of the muscle would be exceedingly low because every autone 
would have to split one high-energy phosphate and pay 11,000 calories for the 600 
calories spent. 



N D 



B 






GLOAD 10 



20 



30 



40 



FIGURE 7. Length-tension diagram of strips of the psoas excited by freezing and subse- 
quent thawing at 40 C. AC = length of the muscle ; CH, CG, CF and CE are shortening 
with the weight CN, CM, CL and CK. The curved line connecting the corners of the squares 
(representing work done) is the 1-t diagram. 



As pointed out by A. V. Hill in the discussion following the author's lecture 
at the International Physiological Congress at Oxford (1947), frog muscle at C. 
is capable of rather strong motion if excited strongly by direct stimulation. Hill 
had shown previously (1913) that not only is the tension developed by frog muscle 
at C. rather high, but also the efficiency, which reaches 40 per cent (1939). The 
author was able to convince himself of the correctness of A. V. Hill's statements. 
Results of a few experiments on this line are reproduced in Figure 8. They show 
that the muscle actually spends much greater amounts of energy than allowed by 
the A F curve. The average expenditure around was found to be 4500 calories, 
which corresponds to a 40 per cent efficiency if the 4500 calories are paid for by 



FREE ENERGY IX MUSCLE 



151 



the ll,000. fi This agreement with Hill's results shows that freezing and thawing 
yields results similar to electric excitation. 

The reason for the discrepancy hetween Hill's and Varga's results obviously was 
to he sought in the different nature of the material. Hill worked with intact 
muscle, Varga with muscle extracted with distilled water and frozen, or with acto- 
myosin threads. It is easy to believe that in the animal the behavior of the acto- 
myosin system is adapted to life at low temperature by some sort of regulation no 
longer present in extracted muscle or actomyosin. 7 This assumption would become 
acceptable if it could be shown that the A F curve of the whole muscle can be 
tranformed into the type of Varga's curve by substances which are known to abolish 
physiological regulations. 



6000 



4000 



2000 



C" 



10 



20 



FIGURE 8. Work performed by the frozen sartorius after thawing, calculated for 35,000 
gm. of myosin present. Coordinates correspond to lower left corner of Figure 1. Sloping 
straight line : A F curve. 

Narcotics, at high concentration, inactivate many physiological mechanisms. 
Most of them also damage the contractile matter. Chlorated paraffins, however, 
like ethyl-chloride or chloroform, have no harmful action on actomyosin. 

The sartorii of the frog (Rana pipiens] were exposed, and provided at their 
ends with ligatures ; the distance between ligatures was noted. The muscle was ex- 
cised, fixed at its original length and placed for five minutes in Ringer of C.. 
saturated with chloroform. Then the muscle was covered with freshly powdered 
dry ice and frozen. Its working capacity was measured in the isotonic and isometric 
experiment at varied temperatures. Also, the Ringer in which the muscle w r as made 
to thaw and contract was saturated with chloroform. If the work was measured 
above 15 C., the muscle was connected to the lever or the weight and allowed to 
thaw first in Ringer of C., saturated with chloroform, and then transferred to the 
Ringer of higher temperature. 

6 When the work done by the right and left psoas was compared at slightly varied low 
temperatures (e.g. and 3 C.), the muscle was found to have a greater A F at higher tem- 
perature; the slope of the resulting A F curve cut the abscissa at about 30 C., and the 11,000 
cal. level slightly under 47. This suggests that the A F curve of intact frog muscle is similar 
to the curve of actomyosin, but has a different slope. 

7 The experiments were performed between November and January, and thus winter frogs 
were used. 



152 



A. SZENT-GYORGYI 



The work done, and thus the free energy spent, was calculated for 35,000 gm. 
of myosin. The results are reproduced in Figure 9. The isotonic measurements 
could not be extended further to the right, above 40 C., the frozen muscle being 
damaged rather readily by higher temperatures. The action of the chloroform is 
reversible, and if the chloroform is washed out the work done at lower temperatures 
increases. 



I2OOO 



IOOOO 



8000 




FIGURE 9. Work performed by the sartorius of Rana pipicns under influence of chloroform. 
The curve is analogous to Figure 5. Crosses : isotonic experiment with weights applied prior 
to freezing. 



As the figure shows, the results obtained are similar to those obtained in the 
rabbit; the curves have the same shape but lie 5 lower. Here, again, the isotonic 
curve becomes asymptotic on reaching the 5500 calorie level. 

In a series of experiments the muscle was loaded prior to freezing with the weight it was 
to lift later. The results are marked in Figure 9 by crosses connected by a dotted line. No 
correction was made for the elastic tension, but this correction would be smaller than the 
actual deviation from A F curve, which suggests that the expenditure of energy depends also on 
the tension which has to be overcome. It was observed repeatedly that the sartorius developed 
higher tension after freezing and thawing if stretched previously for a short while. 

To sum up the experience with frog muscle, we can state that the A F curves 
of whole frog muscle and extracted frog muscle are different, the latter being 
identical with the A F curve of actomyosin threads. By treatment with chloroform, 
the curve of the whole frog muscle can be transformed reversibly into a curve similar 
to that obtained by Varga in his extracted material. This brings out the point 



FRP:E ENERGY IN MUSCLE 



153 



that the A F curve of actomyosin can be greatly modified by accompanying sub- 
stances, and opens the possibility of adapting the contractile material to different 
physiological functions. Actomyosin is not a sharply defined substance and is ac- 
companied by other different substances, proteins and lipins which actually make 
part of the system, and it is not surprising to find that systems containing different 
substances may have different A F curves. Ionic equilibria, disturbed by ex- 
traction, may also contribute to shaping the A F curve. 

In the glycerol-treated psoas. immersed in 0.2 per cent ATP dissolved in 
Ringer, the A F curve, obtained by the isometric method, cuts the abscissa if ex- 
trapolated at - 10 C., while if extrapolated towards high temperatures, it cuts 
the 11.000 calorie level somewhat above 50 and has thus a different slope than un- 
treated muscle. 

Thermodynamic reversibility 

One of the most important implications of the A F curve (Fig. 1) is the 
thermodynamic reversibility of contraction, which means that the energy spent by 
the single units in contraction is a function of temperature on which it depends in 
a reversible way. It should thus be possible to increase or decrease the tension 
of the contracted muscle solely by variation of the temperature. 

Owing to secondary complications, not every material is suitable for this dem- 
onstration. The rapid loss of ATP and contractility in the rabbit muscle, after 
freezing and thawing, rules out this material. In the intact frog muscle, as shown, 
the effect of temperature is compensated. We can expect to be able to demonstrate 



50 




MIN I 



FIGURE 10. Tension developed by the frog muscle, treated with chloroform, at 13 and 
0-1 C. At the arrow the warmer Ringer was substituted by the cold one. Sloping straight 
line : tension demanded by the A F curve. 



154 



A. SZENT-GYORGYI 



thermodynamic reversibility in the frog muscle treated with chloroform, or in ex- 
tracted strips of the psoas immersed in a solution of ATP. 

Figure 10 illustrates a result obtained with frog muscle. The sartorius was 
treated with chloroform and frozen, connected to the isometric lever and dipped 
into chloroform Ringer at 13 C. After a short negative phase, usually seen in 
such conditions, the muscle rapidly contracted developing 48 gm. of tension. As 
the maximum was reached (26 sec.) the Ringer was exchanged for another chloro- 
form Ringer of C. The muscle suddenly relaxed. During the experiment the 
temperature of the Ringer rose 1 C. The sloping line in Figure 10 shows the 
theoretical tension of the muscle demanded by the A F curve. As can be seen, 
the tension of the muscle asymptotically approaches this line. The control ex- 
periment done with the other sartorius of the same frog showed that if the tem- 
perature is kept constant at 13 C., the tension remains high after a slight initial de- 
pression and does not fall more than a few per cent in five minutes. 



2000 



o 

CO 

jfjIOOO 

t- 

o 




T 



T 



I 



I 



T 



MIN 5 10 

FIGURE 11. Tension developed by extracted psoas fibres in ATP at 25 and 1 C. 

The following experiment, reproduced in Figure 11, illustrates thermodynamic 
reversibility in a fibre bundle of the psoas, treated with glycerol : The fibre bundle 
(42 mm. long and 0.5 mm. thick) was connected to the isometric lever, a tension 
of 200 ing. was given, and the muscle immersed in Ringer of 1 C. Then the 
Ringer was exchanged for another Ringer of the same temperature containing 0.2 
per cent ATP. The tension rose to 450 mg. At the arrow pointing upwards, the 



. FREE ENERGY IN MUSCLE 155 

fluid was exchanged for an identical solution of 25 C. The tension was noted 
at once and found to be 1800 mg. A reading was taken every thirty seconds and 
the warm ATP Ringer was exchanged for the cold one (arrow pointing down- 
wards). A reading was taken at once and subsequently every thirty seconds. At 
the arrow pointing upwards, again, the warmer Ringer was introduced, etc. As 
can be seen in the figure, the change in tension is immediate and reversible. As the 
experiment went on, the muscle gradually lost contractility. 

The measurement of the diameter has no pretense of accuracy. If the tension is 
calculated from the final measurement at 25 for one cm.-, a tension of 2^o kg. is ob- 
tained, which shows that the tension developed by a glycerol-extracted muscle under 
the influence of ATP is of the same dimension as the tension developed by an in- 
tact muscle under the influence of maximal stimulation. 

PART III 

Elasticity of the resting psoas 

The fresh, resting psoas shows a moderately high elasticity, as demonstrated by 
the following experiment (Fig. 12) : A strip of the psoas of the freshly killed 
rabbit was connected to the isometric lever, weight 56 mg., rest length 78 mm. 
(RL in fig.), equilibrium length (EL) immediately after excision, 62 mm. The 
muscle was slowly stretched, its length being increased by one millimeter in five 
seconds. After the rest length was reached, the muscle was released for a few 
seconds and then its equilibrium length measured. This was done by straightening 
the muscle out, measuring its length, and then applying a tension of 200 mg. and 
measuring the length again. The difference in length in both measurements was 
usually 2 mm. In the figure the average of these two measurements is given. 
After this measurement was completed, the muscle was stretched to the length from 
which it was released. This stretching was roughly twice as fast as the stretching 
before. Then the muscle was stretched further at the original lower rate and the 
procedure repeated after every 5 mm. of additional stretching till the muscle broke. 
The muscle was kept during the experiment in a wet chamber, immersed in a water- 
bath of C. In the figure the gradual stretching is symbolized by the upper 
straight line which refers to the ordinate (mm). The corresponding equilibrium 
length is reproduced in the middle curve. The single points of this curve lie under 
the point of the upper curve from which the muscle was released. The lowest curve 
shows the tension developed on stretching and refers to the ordinate. the numbers of 
which mean gram-tension in this case. The lowest straight line simply shows a 
slope of 45 and means that if the curve of tension is parallel to this line, the 
muscle obeys Hooke's law. 

The upper line shows that the muscle broke when extended to 173 per cent 
of its equilibrium length, and the middle curve shows that this extension was elastic 
in the whole range of measurements. The middle curve illustrates the well known 
fact of the poor reproducibility of the equilibrium length. The lowest curve shows 
that up to the rest length the contracted units can be stretched practically without 
resistance, but begin to develop resistance at this point. If the muscle were in vivo 
at its equilibrium length, it could develop no tension at the beginning of contraction ; 
if it were tensed any more, it would be spastic. 



156 



A. SZENT-GYORGYI 



0C 



110 



100 



90 



80 



70 



60 



50 



40 



30 



20 



10 



-Rtr 





FIGURE 12. Length-tension relations of the psoas at C. (see text). 



In Figure 13 an identical experiment is reproduced, performed at 23 C., 
weight of the muscle 130 mg. 

In this experiment, the muscle broke when extension reached 190 per cent of 
the equilibrium length. As the middle curve shows, this extension is, at its higher 
degrees, not completely reversible, and the elastic part of the extension is but 163 
per cent of the equilibrium length. The middle curve shows the rest length to be 
better reproducible at this temperature. The lowest curve again shows the con- 
tracted units to be extensible at the beginning with practically no resistance. The 
middle part of the curve obeys Hooke's law ; the upper part shows excessive tension. 
The transition from the region of low tension into the Hooke region is rather sharp 
and corresponds to the rest length. If the relaxed units contract, the tension de- 
veloped will be proportional to the contraction from the beginning which makes 
precise motion possible. On the other hand, having practically no tension, they 
will not impede the motion of their antagonist. 



FREE ENERGY IN MUSCLE 



157 



The muscle obeys Hooke's law up to one-half of the maximum of tension. At 
the point where it begins to develop excessive tension, stretching begins to be 
inelastic, causing slipping and permanent damage to the muscle. 

Elasticity, ATP, and flic slope oj the A F curve 

Freshly isolated strips of the psoas show high elasticity. After the death of the 
animal, its ATP gradually disappears in a few hours' time, as shown by M. Borbiro 
in a separate paper (pp. 162-7, this issue). Parallel to this disappearance of ATP, 
the elasticity of the muscle declines, and if the muscle is excised four hours after 
death, it will usually be found entirely inelastic. On stretching, the maximum 
tension is developed at once, and the muscle tears without considerably increasing 
its length. 

The question arises whether the high elasticity of fresh muscle is actually due to 
the ATP present, and whether the disappearance of this elasticity can actually be 
attributed to the decomposition of this nucleotide. It can be shown that this is 



23C 



110 
100 

90 
80 
70 
60 

50 
40 



RL 



z 



EL 



30 



20 



10 





FKU'KH 13. Same as Figure 12. at 23 C. 



158 



A. SZENT-GYORGYI 



actually the case : if strips of the psoas are extracted at equilibrium length with 50 
per cent glycerol, they are found to be entirely inelastic. At 0, in Ringer, they 
cannot be stretched at all without breaking, and even at 13 C. extensibility does not 
exceed two per cent. If, however, 0.2 ATP is added to the Ringer, the muscle 
again becomes extensible. Using fibre bundles of 0.3-0.4 mm. diameter, the 
muscle could readily be stretched at 0.5-1 C., on an average to 145 per cent of 
its rest length. 8 




10 



20 30 40 
TEMP C 



50 



FIGURE 14. Extensibility of extracted psoas fibres at varied temperature. 100 per cent of rest 
length (abscissa) means that the fibres are not extensible. 

The extensibility of the extracted muscle, in absence of ATP, is a function of 
temperature (Fig. 14). At the muscle is practically not extensible; extensi- 
bility rises slowly with increasing temperature, rising rapidly at body temperature. 
The extensibility at higher temeperatures, up to 53, is not due to denaturation, as 
shown by the relatively big force needed for extension. Denaturation rapidly sets 
in at a somewhat higher temperature, 5455, where the muscle soon becomes 
plastic, offering practically no resistance to stretching. 

This shows that, in absence of ATP, the actomyosin particles in muscle are 
surrounded by unbalanced forces which link neighboring particles together and 
make it impossible for them to move relative to one another, turning the system into 
a rigid, inelastic gel. They are counteracted by heat-agitation. 

The extensibility of muscle at low temperatures in presence of ATP shows that 
the elasticity of muscle actually depends on its ATP, which must be present in the 
resting state linked to the actomyosin. The interdependence of ATP and elasticity 
also shows that in the psoas the elastic properties observed were predominantly 

8 These results are in agreement with previous findings of Th. Erdos (1943) on the relation 
of ATP to rigor mortis. 



FREE ENERGY IN MUSCLE 159 

those of the contractile matter, actomyosin. and were not due to the sarcolem or the 
connective tissue present, no specific reaction being known to take place between 
these latter and ATP. 

It is evident that free energy is needed to abolish the cohesive forces linking 
the particles together, and if this is achieved by ATP, so it is also evident that part 
of the free energy of the ATP-actomyosin system will have to be spent in this reac- 
tion. The free energy spent in this reaction will manifest itself in the stability of 
the link between ATP and the actomyosin. The free energy needed to make acto- 
myosin elastic will decrease with increasing temperature, actomyosin becoming more 
elastic by itself on elevation of the temperature. This expectation is in agreement 
with the results of Mommaerts (1941-1942) who found that at low temperatures, 
the dissociation constant of the actomyosin- ATP complex was exceedingly low. 
F. B. Straub (1941-42) found that the binding of pyrophosphate to actomyosin 
greatly depends on temperature, being strongest at the lowest temperature. 

We can thus conclude that the free energy of the actomyosin-ATP system is 
spent in two successive steps. In the first step the ATP is linked to the myosin. 
cohesive forces are abolished, and a new system is formed in which particles are 
rendered capable of relative motion, contraction or extension. The free energy 
spent in this reaction, henceforth called "Reaction I," will decrease with increasing 
temperature. 

The second step, "Reaction II," entails the dimensional change called "con- 
traction." In the resting muscle, we find the actomyosin-ATP system in the state 
of Reaction I. but Reaction II is inhibited by some unknown mechanism. This 
inhibition is abolished by "excitation" which causes Reaction II to take place. In 
the glycerol or water-extracted muscle suspended in ATP this inhibitory mechanism 
is no longer present, and Reaction I is followed spontaneously by Reaction II. 

It is evident that Reaction II can only spend the free energy unspent by Reaction 
I, which may be involved in the slope of the A F curve of contraction (Fig. 1). 

Since actomyosin devoid of ATP-ase activity can still contract, as shown by 
Buchthal, Deutsch, Knappeis and Petersen (1947), we can conclude that no phos- 
phate is liberated in Reaction I or II, and the whole loss of free energy of the sys- 
tem takes place without splitting of high-energy phosphate links. 

The increase of extensibility of actomyosin under the influence of ATP was 
the first known specific effect of ATP on "myosin" discovered by Engelhardt, 
Ljubimova and Meitina (1941). 

If actomyosin is stored in dehydrated condition, links are developed which are 
not quantitatively split by ATP. Such links develop especially fast in contracted, 
thus discharged, actomyosin. Their development is favored by parallel setting. 

These observations on elasticity and its post mortem changes are in agreement 
with previous findings of Th. Erdos (1943), corroborated and extended by Bate- 
Smith and Bendall (1947). 

The weiglit of the ant ones 

The A F curve (Fig. 1) shows the free-energy change of the single autones 
at any given temperature. If the weight and myosin content (8 per cent) of the 
muscle are known and the A F curve and measurements of the total work are ac- 
cepted, the weight of the single autones can be found by simple numeric calculation. 



160 A. SZENT-GYORGYI 

If, for instance, at a given temperature the A F curve indicates an expenditure of 5500 cal- 
ories per unit, and our piece of muscle performed 0.0055 calories' worth of work and contained 
35 nig. of myosin, then the weight of the myosin-unit which has spent 5500 calories would 
have been 35,000 gm., and this would be the unit weight of myosin contained in one autonc. As 
has been shown (Figs. 6 and 9), isotonic measurements and isometric measurements up to the 
break indicated a unit weight of 35,000 gm. for myosin. Above the break, the isometric experi- 
ments do not yield correct values. At the temperature at which the A F curve cuts the 11,000 
calories level (53 C. in the rabbit and 47 C. in the frog), the unit weight calculated from 
the work done by the isometric method must be the double of 35,000 gm. In a series of experi- 
ments the unit weight of myosin was calculated from the work done by the sartorius as meas- 
ured by the isometric method at 47 C. The freshly isolated sartorius was in these experi- 
ments connected to the isometric lever and dipped into Ringer of 47 C. The results are given 
in Table 1. 

TABLE 1 

74,000 
66,000 
74,000 
74,000 
69,000 
70,000 
72,000 



Average 72,000 

This calculated unit weight of 35,000 gm. is based on the current myosin estima- 
tions. Should muscle be found to contain more myosin than 8 per cent, this would 
mean that the unit weight of myosin taking part in the building of one autone is 
correspondingly higher. There are indications suggesting that the psoas actually 
contains more myosin than 8 per cent. Moreover, if there is a loss of free energy, 
this also entails a bigger unit weight. So 35,000 gm. is rather an order of magni- 
tude and the lower limit than the absolute value, which might be equally well 70,000 
gm. H. B. Bull (1946) arrived along different lines at a unit weight of 40,000 gm. 

CONSIDERATIONS 

It may be asked how far the observations made on the psoas of the rabbit, 
a specific case, reflect a more general behavior. There are different kinds of muscle 
with widely different functions and structure. As reported before, the contrac- 
tile matter of all these different muscles seems to be similar, and actin and myosin 
prepared from cross-striated, smooth, or heart muscle, or even myomas, can be 
interchanged to form actomyosin which contracts on addition of ATP. Even clam 
muscle shows similar reactions (A. Lajta, 1947). 

There are indications suggesting that the regularities observed are not limited 
to the contractile matter. If the muscle is minced soon after death and suspended in 
an alkaline 0.6 M KC1 solution, a sticky extract is obtained 'which owes its high 
viscosity to the dissolved fibrous structural protein, actomyosin. The hydration and 
dissolution of this protein is not merely a result of its interaction with the salt- 
solution. The ATP present has a decisive influence, and if we store the minced 
muscle for a few hours prior to extraction, giving time for the decomposition of ATP, 
the subsequent extraction will yield an extract of low viscosity containing no 
actomvosin. Addition of ATP will restore conditions found in fresh muscle. 



FREE ENERGY IN MUSCLE 161 

As shown by Lajta (unpublished), kidney and other tissues behave in an analo- 
gous way. The fresh mince, if suspended in the alkaline salt solution, yields a sticky, 
highly viscous extract, and the strong double refraction of flow reveals the presence 
of dissolved fibrous structural proteins. If. however, the mince is incubated, the 
subsequent extraction yields a fluid of low viscosity containing no fibrous proteins. 
During the incubation the labile phosphate present disappears. Contrary to muscle, 
however, the original condition cannot be restored by the addition of ATP or a fresh 
boiled juice. The labile phosphate, the disappearance of which seems to be con- 
nected with this change, is found to be linked to nucleic acid present in the protein. 
The nucleic acid, prepared from fresh kidney, shows a high content of labile 
phosphate. 

This behavior is completely analogous to that found in muscle, with the dif- 
ference that instead of a single nucleotide, ATP, in kidney and other parenchyma- 
tous organs we find nucleotides united to long chains, to nucleic acid. In muscle, 
such long chains would interfere with motility. 

The close analogy with muscle suggests that in other organs, too, the pro- 
tein is built of small functional units, each correlated to a nucleotide which governs 
its physical state and enables the system to develop the two different states, the 
high-energy, charged, hydrated state corresponding to rest, and the low-energy 
level corresponding to activity. 

SUMMARY 

Material and methods of measurement of physical properties of muscle were 
discussed. 

Heat contracture, total work of muscle, and thermodynamic reversibility were 
studied and found to be in agreement with earlier assumptions. 

Elastic properties of muscle and their relation to ATP were studied. 

LITERATURE CITED 

ASTBURY, W. T., S. V. PERRY, AND R. REED, 1948. Conference at Kings College, London, 

April 7 and 8. 

BATE-SMITH, E. C., AND J. R. BENDALL, 1947. Jour. Physiol., 106: 177. 
BORBIRO, M., AND A. SzENT-GvoRGYi, 1949. Biol. Bull., 96 (2) : 162. 
BUCHTHAL, F., AND C. G. KNAPPEIS, 1943. Ada Physiol. Scand., 6: 123. 
BUCHTHAL, F., A. DEUTSCH, C. G. KNAPPEIS, AND A. PETERSEN, 1947. Acta Physiol. Scand., 

13: 167. 

BULL, H. B., 1946. Quart. Bull North-western Univ. Mcd. School, Chicago, 20: 175. 
ENGELHARDT, W. A., M. N. LJUBIMOVA, AND R. A. MEITINA. 1941. Sc. Acad. Sci. U.S.S.R. 

(N.S.), 30: 644. 

ERDOS, TH., 1943. Studies hist. Mcd. Cliem. Szeged, 3: 51. 
FENN, W. O., 1923. Jour. Physiol., 58: 175. 
HILL, A. V., 1913. Jour. Physiol., 46: 434. 
HILL, A. V., 1939. Proc. Roy. Soc. Scr. B, 127 : 434. 
LAJTA. A., 1947. Pnbl. Stat. Zoo!. Xafioli, 21 : 226. 
MEYERHOF, O., 1944. Ann. New York Acad. Sci., 54: 377. 
MOMMAERTS, W. F. H. M., 1941-2. Studies hist. Mcd. Chem. Sscged, 1 : 37. 
RAMSEY, R. W., AND S. F. STREET, 1940. Jour. Cell, and Camp. Physiol., 15: 11. 
STRAUS, F. B., 1941-2. Studies hist. Mcd. Chcm. Szeged, 1 : 43. 
STRAUB, F. B., 1942. Studies hist. Med. Chcm. Szeged, 2 : 3. 
STRAUB, F. B., 1943. Studies hist. Med. Chcm. Szeged, 3 : 23. 
SZENT-GYORGYI, A., 1947. Muscular Contraction. Acad. Press, New York. 
VARGA, L., 1946. Hungarica Acta Physiol., 1:1. 






ON THE RELATION BETWEEN TENSION AND ATP IN CROSS- 
STRIATED MUSCLE 

M. BORBIRO AND A. SZENT-GYORGYI ] 

Institute for Muscle Research, Marine Biological Laboratory, Woods Hole, Massachusetts - and 

Experimental Biology and Medicine Institute, Laboratory of Physical Biology, 

National Institutes of Health, Bcthcsda, Maryland 

According to the theory outlined in the preceding paper, the contractile matter of 
muscle is built of functional units containing myosin, actin, and ATP. Since 
muscle contains no free ATP, it can he expected that if the ATP concentration of 
muscle decreases, the number of contractile units decreases proportionately. The 
ATP concentration of muscle decreases after the death of the animal (Th. Erdos, 
1943). The object of the present research was to see whether the ATP content 
and the tension developed by muscle decrease proportionately. Such a parallelism 
would support the theory outlined, while a lack of parallelism would plead against it. 
For this reason, we measured, simultaneously, the tension developed by the muscle 
and the ATP concentration at various intervals after the death of the animal. 

The material used was the musculus psoas of the rabbit. At various intervals 
after the death of the animal, strips of this muscle were cut out and frozen at once. 
The tension developed on thawing was measured. 

The methods hitherto used for the estimation of ATP were found to be un- 
satisfactory for the following reason : we possess no direct method for the estimation 
of ATP. When this substance has to be estimated, extracts of the tissue are sub- 
jected to limited acid-hydrolysis, and the quantity of ATP is calculated from the 
quantity of labile phosphate liberated. Muscle contains in vivo a not inconsider- 
able amount of free phosphate. As the ATP is gradually decomposed post mortem, 
the amount of hydrolyzable phosphate decreases while the amount of free phosphate 
increases, and thus a slight error in the phosphate estimation makes the results of 
the ATP estimation doubtful. A new method of phosphate estimation had to be 
constructed in which the free phosphate did not interfere with the estimation of 
the ATP. 

In the first part of this paper this method will be described. In the second 
part, the results obtained by this method will be given. 

METHOD OF PHOSPHATE ESTIMATION 

The method is based on the ready solubility of phosphomolybdic acid in iso-butyl 
alcohol, described by Berenblum and Chain (1938), and on the yellow color with 
which the acid dissolves in this reagent. The muscle was extracted with tri- 
chloracetic acid. Ammonium molybdate was added to the solution. The free 
phosphate present combined with the molybdate and was shaken out with a mixture 

1 Special Fellow, U. S. Public Health Service. 
- Sponsored by the American Heart Association. 

162 



ATP AND CONTRACTILITY 163 

of iso-butyl alcohol and ethyl ether. Then the fluid \vas hydrolyzed and the free 
phosphate shaken out with iso-butyl alcohol and estimated colorimetrically. 

After the trichloracetic acid extract of the muscle is shaken out with butyl alcohol 
ether, it still contains a small quantity of phosphate. This quantity can be estimated 
and taken into account. If the extract is shaken out a second time with alcohol 
ether, no phosphate is left, and no correction has to be made on the final readings. 
In the present paper the former method was used. 3 

Muscle extract contains substances which, after boiling with HC1, yield products 
which interfere with the development of the yellow color. These substances are 
eliminated by the alcohol ether, since they are of lipoidic nature. 

The detailed description of the procedure is as follows: the rabbit (2-3 kg.) 
was decapitated, eviscerated, the side walls of the abdomen cut off and the psoas 
exposed. Two thin strips of the psoas were taken out, provided with ligatures, fixed 
and frozen with dry ice at their resting length, as described in the preceding paper. 
These strips were used for estimating the maximum tension developed by the muscle 
on thawing at 15 C. Simultaneously, a somewhat thicker strip of about one gram 
weight was cut out from the same region, weighed and frozen. This strip served as 
material for the ATP estimation. The remainder of the muscle, left in situ, was 
covered with cotton wool wetted with Ringer. The procedure was repeated once 
every hour. First the right and then the left psoas was used. Such samples were 
taken until the muscle showed no elasticity and no contractility after- thawing. One 
hour later a last sample was taken. 

Extraction : 25 ml. of 10 per cent trichloracetic acid was pipetted into a mortar 
which was pre-cooled to 20 C. The fluid solidified to a brei. The muscle, after 
having been weighed, was placed into the brei in frozen condition and ground to 
a fine suspension. On thawing, the suspension was transferred into a centrifuge 
tube and spun. The clear fluid was poured into a 50 ml. graduated measuring cyl- 
inder provided with a ground glass stopper. The volume was noted ; then for every 10 
ml., 1 ml. of 10 per cent ammonium molybdate solution was added and the fluid mixed. 
Then 1 ml. of iso-butyl alcohol was added for every 4 ml. of the fluid, and 4 ml. of 
ether added for every ml. of butyl alcohol used. The fluid was strongly shaken for 
twenty-five seconds and allowed to separate. If there was no ready separation of 
the two phases, the fluid was centrifuged. Then the ether butyl alcohol mixture was 
sucked off through a capillary glass tube. A few ml. of ether were added without 
shaking in order to wash off the remaining alcohol ether. The volume of the fluid 
was noted. If, after the shaking with alcohol ether, a heavy precipitate was formed, 
this was separated by centrifugation. The fluid was divided into samples, each of 
which corresponded to 100 mg. of muscle, and pipetted into test tubes. Out of nine 
samples four were put aside. To five samples, 1/10 parts of concentrated HC1 
(approximately 10 N) was added and the tubes placed into the boiling water-bath 
for seven minutes and then rapidly cooled. To the unboiled samples, the same 
amount of HC1 was added. To all tubes one drop of 0.1 per cent potassium per- 
manganate was added which stained the fluid a rose color. This color persisted 
for about half a minute. This was done in order to oxidize any reducing agent 
present which would reduce the phosphomolybdate. Then 10 per cent ammonium 

3 If for any reason the quantity of free phosphate present in the muscle extract had to be 
known, this could be estimated colorimetrically in the combined alcohol ether extracts. 



164 



M. BORBIRO AND A. SZENT-GYORGYI 



molylxlate was added to the- unboiled lubes, and 5 nil. iso-lmtyl alcohol to all 
samples. The butyl alcohol used here was shaken out previously with water. 
(This is necessary in order to prevent the butyl alcohol from taking up waler 
later.) The fluid was shaken strongly for five seconds, the opening of the tube 
being closed by the thumb covered by a rubber glove. After the two phases sepa- 
rated, the watery phase at the bottom was sucked off by means of a thin glass tube, 
connected to the vacuum by a thin rubber tube which was pinched tight while the 
tip of the tube was passing the alcohol. Then the alcohol was poured over into the 
colorimeter tubes which were marked at their 5 ml. volume. Usually the volume of 
butyl alcohol is less than 5 ml. It was filled up to 5 ml. with butyl alcohol which 
was used to rinse the tubes that contained the extract previously. Then to every 
tube 1 ml. of ethyl alcohol was added and the color estimated in the Klett- Summer- 
son colorimeter with the S 42 blue light filter (400-460 m/x). 

As a standard, a solution of KH 2 PO 4 was used, containing 0.01 mg. per ml. 
Samples of 1, 2 and 3 ml. of this fluid were filled up with water to 4 ml., 0.5 ml. cc. 
HC1 and 0.5 ml. of 10 per cent molybdate were added ; then the fluid was shaken 
out with 5 ml. butyl alcohol which was treated as described above. 

EXPERIMENTAL RESULTS 

Before embarking on the problem proper, a few minor points had to be cleared 
up. First, is the method of P estimation reliable, and is the distribution of ATP in 
the psoas homogeneous ? 

A rabbit was killed and six samples of 1 g. were taken from different parts 
of the two psoas muscles. In Table I the actual colorimeter readings are repro- 
duced. The six upper columns related to the unhydrolyzed extract are thus the zero 
values. The corresponding readings of the hydrolyzed samples are reproduced 
in the lower columns. 

As can be seen, the readings are very uniform. The one value in the fourth 
column, marked with an asterisk, is evidently due to some rough mistake and has 
to be discounted. The other single values do not differ from the average by more 
than five per cent. The average of the value was substracted from the average of 
the hydrolyzed product. From this the ATP was calculated. The standard with 



TABLE I 





40 


38 


30 


29 


24 


29 




39 


35 


30 


69* 


24 


28 




39 


37 


30 


29 


24 


28 




202 


194 


190 


204 


198 


204 




200 


189 


200 


200 


194 


202 




204 


194 


198 


206 


196 


202 




190 


196 


189 


200 


200 


198 




196 


199 


187 


204 


208 


194 




195 


186 


198 


214 


195 


195 


Average 


198 


194 


194 


205 


198 


199 


ATP 


3.55 


3.50 


3.47 


3.70 


3.68 


3.60 mg. per gm. 



ATP AND CONTRACTILITY 165 

0.02 mg. phosphate gave a reading of 80. The quantity of P found was multiplied 
by 8.4 to give the ATP, which is noted in the last horizontal line. This shows 
the ATP content of the psoas to be very uniform, 3.6 mg. ATP per gm. 

According to the literature, muscle contains 2-2.5 mg. ATP per gm., thus con- 
siderably less than psoas. This difference is probably due to the shielded position 
of the psoas and the consequent poverty of connective material. In order to eluci- 
date this point, samples of different muscles of a freshly killed rabbit were taken 
and subjected to analysis. Results are reproduced in Table II. 

TABLE II 

Psoas 3.55 (mg. ATP per gm.) 

Deep muscles of the back 3.04 

Big adductor muscle 2.56 

Musculus gracilis 2.10 

Superficial muscle of the back 2.10 

Smaller muscles from the gluteal region 1.96 

These values show that the more superficial the position and the richer the con- 
nective tissue, the lower the ATP content. The muscles of the whole animal would 
give an average of about 2.5 mg. ATP per gm. 

The third question which had to be cleared up was whether the ATP content 
of the psoas decreases uniformly in all its parts after the death of the animal. 
Preliminary experiments have shown that the rate of disappearance of ATP post 
mortem depends on the temperature and the oxygen supply. If the muscle is cut 
into thin strips which are exposed to air, the disappearance becomes much slower. 
While the ATP in the muscle left in situ may disappear within three to four hours,' 
muscle strips exposed to air may contain ATP and thus remain contractile at room 
temperature even twenty-four hours after the death of the animal. Experiment 
also showed that in the muscle left in situ, the ATP disappeared faster in the deeper- 
lying dorsal than in the superficial ventral part. 

Experiment : The rabbit was killed, the psoas exposed as usual and covered 
with wet cotton wool. Three hours later, five strips, weighing approximately 1 gm., 
were cut out and analyzed for ATP. One of the strips was taken from the lateral edge 
of the muscle, two from the ventral surface, two from the deeper-lying dorsal surface. 
ATP (mg.) per gm : 

edge: 2.53 

ventral: 2.77 2.77 

dorsal: 1.80 1.95 

If the ATP content of muscle and its tension are to be measured simultaneously, 
it is essential that strips from the same region be used for both measurements. 
Even with this precaution, considerable scattering of results can be expected. 

The relation between ATP content and tension was studied in eight experi- 
ments. The following example may be cited (Fig. 1) : Samples of muscle were 
taken every hour after the death of the animal. The ATP content in milligrams per 
gram of muscle is marked in the curve by points. They relate to the left ordinate. 



166 



M. BORBIRO AND A. SZENT-GYORGYI 



The tension developed is marked with circles and refers to the right-hand side 
ordinate. (The scale of this ordinate is arbitrary and is chosen in such a way that 
the numbers, if multiplied by 100, give the total working capacity in calories calcu- 
lated for 35,000 gm. myosin by the formula: % tension X length X 0.000023.) 

As the curve shows, tension and ATP content run parallel. At the end of the 
third hour the muscle develops no more tension and does not contract on thawing, 
and is found to be completely inelastic. At this point, the ATP curve shows a 
break and becomes roughly parallel to the abscissa. 



HOURS 



40 



o 
en 

z. 

UJ 



20 



As the curve shows, the muscle at this point still contains a not inconsiderable 
amount of labile phosphate. Whether this hydrolyzable ATP is derived from ATP 
or some other source (ADP?) cannot be stated at present. If this hydrolyzable P 
is derived from ATP, this ATP must be in some way different from the rest, be- 
cause it is no longer split by the muscle (or is split only exceedingly slowly) and 
has no influence on contractility and elasticity. This "residual" hydrolyzable 
phosphate was found in approximately the same proportion in all experiments. 

The second point, equally borne out by the other experiments, is that the de- 
crease of ATP concentration is linear : the rate of its disappearance is independent of 
its concentration. The most likely interpretation of this rather unexpected fact is 
that the splitting of ATP depends on some change in the contractile matter. As 
has been shown by A. Biro and A. E. Szent-Gyorgyi (unpublished), myosin is enzy- 
matically active in its contracted condition only. 

In two out of the eight experiments, the ATP concentration did not fall at all 
during the first hour after death. This can be explained by the presence of creatine- 
phosphate which rephosphorylates the ADP formed. 

All experiments gave similar results. In most of them the scattering was 
stronger than in the quoted example. Nevertheless, all experiments bore out the 
close parallelism between tension developed and the quantity of ATP present. 



ATP AND CONTRACTILITY 167 

SUMMARY 

A new colorimetric method of ATP estimation is described. In the psoas of 
the rabbit the post morlem decomposition of ATP and the loss of contractility are 
parallel. 

LITERATURE CITED 

BERENBLUM, J., AND E. CHAIN, 1938. Biocliciu. Jour., 32 : 295. 
ERDOS, TH., 1943. Studies Inst. Med. Chem. Ssegcd, 3: 51. 



EARLY STAGES IN THE LIFE HISTORY OF THE COMMON 
MARINE SHRIMP, PENAEUS SETIFERUS (LINNAEUS) 

WILLIAM W. ANDERSON, 1 JOSEPH E. KING, 12 AND MILTON J. LINDNER" 

The purpose of this report is to assemble information concerning the early 
stages in the life history of the shrimp, Pcnacus sctifcnts (Linnaeus), which sup- 
ports the most valuable commercial fishery of the South Atlantic and Gulf of Mexico 
regions of the United States. 

P. setijerus is an organism of high reproductive potential. A count made by 
the authors on the ripe ovaries of a female, 172 mm. total length with spermatophore 
attached, revealed a total of approximately 860,000 eggs. Burkenroad (1934) 
states that the ovary of a large shrimp may contain 500,000 eggs. Heldt (1938) 
counted about 800,000 eggs in the ovaries of P. trisulcatns, a European species of 
similar size and closely related to P. setijcnis. It may be expected, therefore, that 
a female will produce from 500,000 to 1 ,000,000 eggs in a single spawning. 

DESCRIPTION OF THE EGG 

The size of the ripe ovarian egg has been given by Weymouth, Lindner and 
Anderson (1933) as ranging from 0.25 to 0.33 mm. in diameter with an average of 
0.277 mm. Burkenroad (1934) believed the egg to be about two-thirds this size, 
or about 0.185 mm. Later he changed this estimate to " about 0.25 mm. or less," 
(Burkenroad, 1939). Pearson (1935), through the use of a plankton net, secured 
nauplius-bearing eggs varying in diameter from 0.38 to 0.42 mm. Later he re- 
ports (Pearson, 1939) that the diameter of twenty-five live eggs, also secured with 
a plankton net, uniformly measured 0.28 mm. Gutsell (1936) obtained measure- 
ments ranging from 0.192 to 0.300 mm. on ripe eggs from a female with spermato- 
phore attached. He found that fresh oocytes dissected out in sea water were about 
0.30 by 0.36 mm. Much of the variation in the data cited may be due to varying age 
of the eggs and varying manner in which they were handled (some were measured 
fresh, others after fixation). 

According to Pearson (1939) "the egg of P. setiferus is demersal and sinks 
promptly in still sea water." It is "non-adhesive and spherical" and "possesses a 
thin transparent membrane, or chorion, that in living and preserved eggs shows a 
characteristic purplish-blue color in reflected light under the microscope." 

LARVAL DEVELOPMENT 

Our knowledge of the larval development of P. setiferus depends largely on the 
work of Pearson (1939). To quote from the summary of his paper: "The larval 
development of Penaeus setiferus, the common commercial southern shrimp, con- 
sists of ten distinct stages excluding the demersal spherical egg. These stages 

1 Chief, Gulf Investigations, U. S. Fish and Wildlife Service. 

- Aquatic Biologist, Gulf Investigations, U. S. Fish and Wildlife Service. 

3 Aquatic Biologist, Office of Foreign Activities, U. S. Fish & Wildlife Service. 

168 



EARLY LIFE HISTORY OF THE COMMON SHRIMP 169 

are made up of five forms generally included under the name of nauplius, three 
forms included under the name of protozoea, and two forms included under the 
name of mysis." In addition to these ten larval forms, Pearson descrihes two post- 
larval stages which precede the true adult form. 

In brief, the larval development of P. sctifcnis requires from two to three weeks. 
Some twenty to twenty-four hours after the egg is spawned the nauplius hreaks the 
chorionic membrane and emerges. Its ovoid body, of 0.30 to 0.34 mm. in length, 
bears a single, simple eye and three pairs of oar-like appendages which are to be- 
come eventually the first and second antennae and the mandibles. Although this 
minute organism is to a great extent at the mercy of the prevailing currents, it is 
capable of some movement. In the next twenty-four to thirty-six hours the nau- 
plius undergoes five successive molts to become a protozoea of approximately 1 mm. 
in length. It now has seven pairs of appendages, a pair of sessile compound eyes 
in addition to the ocellus, and a complete alimentary tract consisting of mouth, 
esophagus, stomach, intestine, and anus. Prior to this stage the food of the nau- 
plius has been the yolk material carried over from the egg. This food supply is 
now exhausted and henceforth the protozoea must capture its own food if it is to 
survive. This transitional period is without doubt a critical one in the animal's life 
history. 

The third protozoea stage is followed by the first mysis, which is about 3.5 mm. 
in length, possesses fourteen pairs of functional appendages, and on the abdomen 
five pairs of buds which will soon become the pleopods. In the second mysis the 
pleopods are well developed, and rudimentary gills have made their appearance on the 
thoracic somites. With the succeeding molt the organism ends its larval phase and 
assumes the general proportions of a miniature adult. At the end of two post-larval 
stages and fifteen to twenty days after hatching, the young shrimp is only 5 to 6 mm. 
in length and is still planktonic. During this period of early development, the 
young shrimp have moved from the saline offshore spawning area to the brackish 
inside marshes, bays, and estuaries (Weymouth, Lindner and Anderson, 1933). 
Upon reaching these "nursery grounds" they adopt for the first time, it is believed, 
a benthic existence. 

The factors responsible for this inshore movement of larval and post-larval P. 
setifcrus have not been determined. We believe, however, that for the young to 
reach the nursery grounds they must encounter a favorable incoming current. 
While capable of some movement, and perhaps responsive to a salinity gradient, 
they would certainly be quite helpless against out-going currents. P. setiferus has a 
long spawning season, which in Louisiana extends from March to September ; conse- 
quently, at intervals during this period the young are bound to encounter favorable 
conditions for their inshore migration. 

Although spawning usually takes place offshore, schools of adult shrimp have 
been known to approach the coast and spawn close to inlets. When such a spawn- 
ing occurs, the eggs may be swept through the passes on incoming currents, and 
the larvae may reach the nursery grounds within a few hours. 

YOUNG SHRIMP 

As stated above and previously reported by Weymouth, Lindner, and Anderson 
(1933), young shrimp approximately 7 mm. in length are found during the early 



170 



W. W. ANDERSON, J. E. KING, AND M. J. LINDNER 



spring months in the brackish inside areas which serve as their nursery grounds for 
the next four to eight weeks of their existence. This hahitat is a rich feeding ground 
characterized hy shallow water, muddy bottoms, rather widely fluctuating seasonal 
temperatures, and moderate to low salinity. Numerous seine and frame-net col- 
lections in these areas have yielded quantities of small shrimp 7 to 10 mm. in length ; 
whereas frequent hauls with the same gear during the same period along the ocean 
and Gulf heaches have failed to yield any P. sctijcrus of this size, although other 
species of shrimp were found. 

As the young grow, they move from the shallow waters of marsh, bayou, and 
lagoon into the deeper creeks, rivers, and bays, making their first appearance on the 
inside fishing grounds when about 50 mm. long. In Louisiana the lower limit of the 
size distributions obtained from operations with commercial gear in the fishery was 
43 mm., in Georgia 58 mm., and in Texas 63 mm. 



LAMPADOZIER 
20 40 60 



EASTCIELD RIVER 
zo 40 go 









20 40 eO 

LCNCTM IN MILLIMCTCR5 






?0 40 80 



FIGURE 1. Size distribution of young shrimp (Pcnacus sctiferns) seined from the nursery 
ground areas of Lampadozier and Eastfield River, Georgia. Males and females combined. For 
the Lampadozier series the curves are based on 200 shrimp in June, 200 in July, 100 in August, 
200 in September, 100 in October, and 92 in November. The Eastfield River series are based 
on 100 shrimp in June, 400 in July, and 100 in August. 



KARLY LIFE HISTORY OF THE COMMON SHRIMP 171 

To illustrate the population on a typical nursery ground area, the length fre- 
quency distributions of small shrimp from seining and frame-net operations in two 
localities in Georgia are shown in Figure 1. The Lampaclozier series, which covers 
a period from June to November, was obtained entirely by seining in one particular 
locality in a section representing the inner reaches of the nursery grounds. The 
young shrimp of the Eastfield River series were somewhat larger in size ; they repre- 
sent collections (covering a period from June to August) taken with both seines and 
frame-nets in an area midway between the upper nursery grounds (represented by 
the Lampaclozier series) and the lower bays or sounds. The apparent reverse or- 
der in the sizes of shrimp in the Eastfield River series is due, it is believed, to the 
exodus late in June of the larger shrimp, a product of an early spawning, and the 
entrance in great abundance into the River in July and August of the young from 
the peak spawning period of May and June. 

In the Lampaclozier section during June and July the average length of the shrimp 
was 18 mm. with a range from 8 to 48 mm., although in July a few scattered longer 
individuals were obtained. In August the average length had increased to 23 mm. 
with a range from 8 to 53 mm. During September the average length was main- 
tained at 23 mm. with the bulk of the population ranging between 8 and 38 mm., 
although scattered individuals up to 78 mm. in length were secured. By October 
the average length had increased to 38 mm. with a range from 18 to 53 mm. During 
November the average length rose to 48 mm. with a range of 28 to 63 mm. 

From the Lampadozier data, the increase in the lower limits of the length fre- 
quency distribution from 8 mm. in September to 18 mm. in October and to 28 mm. 
by November, indicates that after September no new recruits were appearing on the 
nursery grounds. September marks the end of the spawning season in Georgia 
(Anderson, Lindner and King, 1948). 

RELATIONSHIP OF NURSERY GROUNDS TO COMMERCIAL CATCH 

The distribution of the shrimp fishery in itself obviously indicates that passes 
and the adjacent inland waters are of prime importance to the species. Louisiana, 
which has a combination of more passes and a vastly larger inland water area 
landward of these passes than any other state, produces about two-thirds of the 
shrimp caught each year throughout the entire South Atlantic and Gulf region. 
Likewise, Georgia and. South Carolina, whose shorelines have the most numerous 
passes and favorable inside waters on the South Atlantic Coast, develop the greatest 
numbers of shrimp in that section. As a consequence, we conclude that the num- 
ber of openings to the outside waters and the extent of favorable nursery grounds 
are two of the major physical factors influencing the production of shrimp in the 
various sections of the fishery. 

In addition to the number of passes and the area of nursery grounds, a coastal 
or nearby offshore area of relatively shallow water, high salinity, and mud or clay 
bottom also seems to be a requisite. The Florida peninsula between Fort Pierce 
on the east coast around almost to St. Marks on the west coast lacks this and like- 
wise lacks shrimp. It is not yet known whether this factor is a requirement of 
adults or larvae or of both. 



u 

^ 





172 W. W. ANDERSON, J. E. KING, AND M. J. LINDNER 

LITERATURE CITED 

ANDERSON, WILLIAM W., MILTON J. LINDNER, AND JOSEPH E. KING, 194<S. Observations on 
certain phases of the reproductive cycle in the common shrimp, Penaeus setiferus 
(Linn.). (Manuscript submitted for publication.) 

BURKENROAD, MARTIN D., 1934. The Penaeidae of Louisiana with a discussion of their world 
relationships. Bull. Amcr. Mus. Nat. Hist., 68 : 61-143. 

BURKENROAD, MARTIN D., 1939. Further observations on Penaeidae of the Northern Gulf of 
Mexico. Bull. Bini/hani Occanographic Collection, Peabody Museum of Natural His- 
tory, 6: 1-62. 

OUTSELL, JAMES S., 1936. A study of the ovaries of the common shrimp, Penaeus setiferus 
(Linn.), with reference to the life history. Unpublished manuscript in the files of the 
Gulf Investigations, U. S. Fish and Wildlife Service, Sarasota, Florida. 

HELDT, JEANNE HENRI, 1938. La reproduction chez les crustaces decapodes de la famille des 
Peneides. Annals dc I' Institute Oceanographique, 18: 31-206, Paris. 

PEARSON, JOHN C., 1935. Eggs of a Peneid shrimp. Science, 82: 172. 

PEARSON, JOHN C., 1939. The early life histories of some American Penaeidae, chiefly the 
commercial shrimp Penaeus setiferus (Linn.). Bull, of the U. S. Bureau of Fisheries, 
49: 1-73. 

WEYMOUTH, F. W., MILTON J. LINDNER, AND W. W. ANDERSON, 1933. Preliminary report on 
the life history of the common shrimp, Penaeus setiferus (Linn.). Bull, of the U. S. 
Bureau of Fisheries, 48 : 1-26. 



MITOCHONDRIA!. ARRANGEMENT IN ALVEOLAR EPICYTES 

AND FOAM CELLS OF MOUSE LUNGS. PARTICULARLY 

AS INDUCED BY THE VACUOLOIDS 1 

CHARLES C. MACKLIX 

Detriment of Histolof/ical Research of the Faculty of Medicine of the University of Western 

Ontario, London, Ontario, Canada 

INTRODUCTION 

The problem of the causation of the design taken collectively by the mitochon- 
dria in a living or a fixed and stained cell has interested cytologists for many years. 
For instance. Lewis and Lewis (1915, p. 352), in studying the living cells of tissue 
cultures, ask: "What is it that governs the arrangement of the mitochondria? 
Is it the shape of the cell, the influence of the central body or of the nucleus, the 
internal structure of the cytoplasm, or do the metabolic activities of the cell govern 
the size, shape and arrangement of the mitochondria?" Full answers to these 
important questions have not yet been forthcoming, and the problem is a compli- 
cated one. No doubt each of the factors mentioned plays a part. 

It seems clear, too, that the physical influence of mechanically inert bodies in the 
cytoplasm is a formative factor which may be predominant, for mitochondria occupy 
the general cytoplasm and not the special masses, living or dead, which may find a 
place in it ; and hence anything which molds this general cytoplasm will incidentally 
establish the morphological pattern of the mitochondria in it. Thus Cowdry (1914) 
found that the mitochondria of large spinal ganglion cells occurred between the 
flakes of Nissl substance, and Thurlow (1917) observed that in nerve cells of the 
cranial nuclei the mitochondria avoided the canalicular apparatus. Similarly the 
Lewises (1915) showed, in their Figure 26, the mitochondria arranged in a net- 
work around the fat droplets of a tissue culture cell. Foreign body inclusions act 
likewise. This simple mechanical influence on mitochondrial arrangement is herein 
shown to be exerted by vacuoloids in the pulmonic alveolar epicytes and foam cells 
to such a degree that the ensuing picture is outstanding and characteristic. 

Alveolar epicytes are the residual epithelial cells in the pulmonary alveolar walls 
(Macklin, 1946) . They are also called "septal cells," "niche cells" and other names. 
Although in the marginal alveolar bases (Macklin, 1945) and other places they 
have but one air face and rest upon connective tissue, in the interalveolar partitions 
they frequently have two air faces. These are often of unequal size, the larger over- 
lying the head of the cell and the smaller the foot. In silverwashed material the 
head and foot are each encircled by a line of silverized material which is part of the 
silver lineation of the alveolar walls and bronchioles (Macklin, 1938). At alveolar 
wall intersections the epicytes, as herein discussed, not seldom have three air faces 
(Fig. 5). This trifaciality is like that found in certain dust cells of mouse lungs 

1 A grant in aid of this investigation by the National Research Council of Canada is grate- 
fully acknowledged. 

173 



174 CHARLES C. MACKLIN 

treated with ammoniacal silver solution (Macklin, 1948). Epicytes may assume a 
phagocytic role (Macklin, 1946). Other functions have been ascribed to them 
(Hayek, 1942; Sjostrand and Sjostrand, 1938). That they may, on occasion, be- 
come malignant, so initiating a primary cancer of the_ lung, has been admitted as a 
possibility (Macklin, 1938). Their most prominent and characteristic feature is an 
array of vacuoloids which occupies much if not most of the cytoplasm (Macklin, 
1947a). These are clear, round, discrete, non-lipoidal bodies averaging 0.5 /x to 
0.75 p. in diameter, which do not take stains (Brodersen, 1933). They are relatively 
stable, indenting the nucleus in fixed and stained sections. 

Alveolar foam cells of mammalian lungs have been described with special 
reference to their vacuoloids (Macklin, 1947b). They are found in ordinary his- 
tological sections, and some of them may be recovered by what has been termed the 
"gash-irrigation" technic, in which the fresh, collapsed lung is incised through a 
drop of physiological saline solution and the preparation inverted over a glass slide 
on which is received the emerging fluid carrying loose cells from the peripheral 
alveoli. This fluid is then spread and stained as for blood. Foam cells are re- 
garded as originating from epicytes and possibly also from the diversifying epi- 
thelium of the bronchioles at the marginal zone adjoining the alveolar ducts, and as 
being developmental brothers of the dust cells. Difficulty may be encountered in 
distinguishing the smaller foam cells from well developed epicytes, in sections. The 
alveolar foam cells are thus of entodermal origin, and are not to be confused with the 
mesodermal "foam cells" of the pathological literature. As the name implies, they 
have a foamy appearance, the numerous vacuoloids accounting for the clear 
spots, which in sections often misleading seem to be merged. In dry smears the 
vacuoloid diameter may reach 1.5 p.. 

There is no reason to suspect that the mitochondrial arrangements hereinafter 
described are peculiar to the alveolar epicytes and foam cells of the mouse. They 
are probably to be found in these cells throughout the mammalian class at least. 

MATERIAL AND METHOD 

This short study is upon albino mouse lungs freed from as much blood as pos- 
sible by hemorrhage and moderately distended by the prompt intratracheal injec- 
tion of Regaud's fixing fluid. Paraffin sections, stained by Bensley's adaptation of 
the Altmann technic (Cowdry, 1943), reveal the mitochondria in brilliant ruby-red. 
Most of the mitochondria in epicytes and foam cells are of round or oval form. 
These are seen in all parts of the general cytoplasm. The ovals grade into short 
thick rods with rounded ends. Filaments, often beaded, occur. Mitochondria 
differ in size, the largest being conspicuous while the smallest are seen with difficulty. 
It is possible that the degree of differentiation with picric acid has something to do 
with the optical impression of size, which seems less in over- than in under-differ- 
entiated cases ; but this factor can hardly be in operation when mitochondria in 
the same cell are being considered, for these have presumably been subjected to 
uniform technical action. It -is the impression that the mitochondrial content of the 
well developed epicytes is more conspicuous than that of the simpler epicytes on the 
one hand and the larger foam cells on the other. These mature foam cells are 
lighter in color, lacking the marked rosy hue typical of the mature epicytes, and in 
them the mitochondria appear generally smaller and more weakly staining than in 
the smaller foam cells and epicytes, and are predominantly of coccoid form. Thus 



MITOCHONDRIA!. ARRANGEMENT 175 

there is considerable variation in the relative prominence <>l the mitochondria! pic- 
ture in the various cells examined. 

PF.RIVACUOLOIDAL GROUPING OF M FTIH IIOXDRIA 

The design of groups of mitochondria in typical epicytes and foam cells, as seen 
in well stained thin sections, is dominated by the presence and spacial disposition 
of the vacuoloids and appears characteristically as a round-meshed sieve with circles 
of mitochondria, the perivacuoloidal clusters, hounding these clear spheroidal bodies. 
Mitochondria are never admitted to the interiors of the vacuoloids. 

In Figure 2 is shown one of the smaller bifacial epicytes in which the structure is 
relatively simple, the vacuoloids being in only one layer. Perivacuoloidal groups 
[* 1- (/ surround these bodies. Those mitochondria which lie just beneath the cell 
membrane may take part as well in the formation of the inframembranal group 
/ /;; //, and similarly those which are immediately around the nucleus participate as 
well in the makeup of the perinucleal group [> n </. This is a good example of an 
epicyte which goes right through the alveolar wall, and it shows a larger end. the 
head, well rilled with vacuoloids, and a comparatively narrow shank or trunk with 
a small foot. 

"Where greater numbers of vacuoloids occur, the appearance is more complex. 
In the epicyte seen in Figure 3. there are many perivacuoloidal groups, mainly of 
ovals and spheroids. This cell appears lodged in a crotch ot alveolar walls. It has 
a free air-face above, another to the left and a third to the right, represented in the 
small foot at the end of the narrow shank which is within a space of the alveolar wall 
like a pore. Above are rods just beneath and parallel with the free face / ;;/ ;/. 

In the larger epicyte^ and foam cells the vacuoloids are in several layers, and 
hence the perivacuoloidal mitochondria comprise relatively much more of the total 
content. Figure 7 gives a good impression of this sievelike pattern. It is from a 
very thin slice of a foam cell at the side of the nucleus. The inframembranal layer 
is incomplete. In thick sections one can focus up and down through the numerous 
vacuoloids and Find impressive numbers of mitochondria in the cytoplasm around 
them. 

Figures 8, 9 and 10 >how this perivacuoloidal arrangement in other foam cells. 
Under the oil immersion lens the overall picture is uniquely beautiful, and once seen 
is not forgotten. It is like chains of brilliant rubies festooned about large luminous 
pearls. Photographs at best give an inadequate representation. The numerous 
and often large mitochondria are mainly round or oval, and most of them are about 
the vacuoloids, with an incomplete layer under the cell membrane and another over 
the nuclear membrane. 

Sometimes epicytes which appear to be underdeveloped are found, showing 
relatively few vacuoloids or mitochondria. In Figure 1, for instance, there is a 
single row of vacuoloids present only on the air surface. Hut one mitochondrion ap- 
pears on the side next to the air. and it is between two vaculoids. This is an ex- 
ample of a very simple distribution of mitochondria. They are massed above and to 
the right, in the cytoplasm of the surface which rests on connective tissue. 

It does not appear that the mitochondria are attracted by the vacuoloids, 
but rather that they occupy inertly the available space around them. By no 
means all of the vacuoloidal surface is contiguous to mitochondria. Most of the 
mitochondria around the vacuoloids are of the spheroid or ovoid type; but there 



176 



CHARLES C. MACK1.1X 

PLATE I 

_ f w 

a/ 






a/ 




"P w 



al 



al 




^ ?V S 








-png 



The ten figures are photomicrographs at 1900 diameters made with a Bausch and Lomb 
1.9 mm. 1.32 N.A. oil immersion fluorite system objective and 10 X ocular from 3M Altmann 
sections of mouse lungs prepared as described. On the prints the mitochondria were intensified 
v/ith India ink applied with a fine pen on consultation of the original cell under the oil immer- 
sion lens. The first six figures are regarded as epicytes and the remainder as foam cells. 
Detain of mitochondrial arrangement are given in the -text. 



MITOCHOXDRIAL ARRANGEMENT 177 

are many short rods and these are found with the long axis lying tangentially to 
the vacuoloidal surface. There is no reason to suggest that mitochondria are in 
any way concerned in the formation of the vacuoloids or that they are influenced in 
form, size or any other way 1>y contiguity with the vacuoloids. Experimental swell- 
ing and distortion of the vacuoloids is reflected in spreading and attenuation of 
mitochondrial arrangements around them. 

ARRANGEMENTS NOT DETERMINED BY THE VACUOLOIDS 

When epicytes are so cut as to show the long axis of the cell approximately 
parallel with the optical plane, we may see mitochondrial rods of the intratruncal 
group i t g lying more or less parallel with one another in the trunk or shank and 
reaching to the foot (Figs. 2, 3 ). This region of the epicyte lies within the alveolar 
wall close to the capillaries. In cross sections of such shanks the now dotlike 
mitochondria are disposed in a circle. This arrangement suggests the shrunken 
staves of an empty barrel. When cut at a slant such a group appears as in Figure 4. 
No reason for this peculiar pattern is apparent. 

Mitochondria have been noted in epicytes and foam cells lying close to the cell 
membrane / m g. As rods and filaments they often lie parallel with this membrane, 
and sometimes occur in a double row (Fig. 9). Another layer, which may be in- 
definite and typically composed of shorter forms, has been noted in the perinucleal 
cytoplasm p u g (Figs. 3, 8. 10). In the edges of the heads of epicytes, where the 
inframembranal cytoplasm underlying the air surface merges with that adjoining the 
connective tissue surface, the mitochondria of the peripheral group /> p g (Fig. 5) in 
lateral view may have a curious pointed appearance like a pile of sticks, as in the 
profile of the supports of a North American Indian wigwam, which is difficult to 
photograph ; while other groups simulate the downdrooping branches of the tops of 
balsam trees. Such clusters contain rounded and oval forms as well as rods. 
Sometimes mitochondria in these edges are packed in a dense triangular mass. 
Again, no explanation for these curious formations has been found. 

Epicytes on marginal alveolar walls (those which rest on connective tissue) are 
well endowed with mitochondria. One of these is represented in Figure 6 in the 
angle between adjoining alveoli. To the left, a group of mitochondria juts into 
the partition p TV separating the upper from the lower alveolar space. Perivacuo- 
loidal formations are seen here as in other epicytes. 

Substantially the same representation of mitochondrial arrangement in these cells 
was obtained after the use of Bensley's acid violet-safranin O (Bensley, 1911; 
Lillie, 1948, p. 98), and Regaud's modification of the iron hematoxylin method 
of Heidenhain (Cowdry, 1918), though hitherto, in the author's hands, less bril- 
liantly. 

Abbreviations : 

a I alveolus, 

i m g inframembranal group of mitochondria, 

it g intratruncal group, 

m w marginal alveolar wall, 

p n g perinucleal group, 

p /> g peripheral group, 

p : </ perivacuoloidal group, 

p w partitional alveolar wall. 



178 CHARLKS C. MAC KLIN 

SUMMARY 

In epicytes and foam cells the combined perivacuoloidal groups of mitochondria 
present, in thin sections, an outstanding and characteristic lacelike picture based 
on the disposition and condition of the vacuoloids. Its determination is probably 
mechanical. Mitochondria are never found within the vacuoloids. An incom- 
plete layer immediately underneath the cell membrane, and another around the nu- 
cleus, are found. In the former the rods often lie parallel with the membrane and 
sometimes in double rows. 

In epicytes there is often a distinctive group, mostly ol rods, which suggest the 
outer layer of the fasces, and lie in the long axis of the shank, arranged in a circle 
around a central area devoid of them. Bizarre angulated and branched arrange- 
ments are noted in the peripheries of the heads of epicytes. 

In foam cells the mitochondria! content varies, often being abundant and con- 
spicuous, and again, perhaps in older cells, relatively inconspicuous. 

ACKNOWLEDGMENT 

I wish to thank Mr. Charles K. Jarvis for his excellent work in the preparation of the 
sections and photomicrographs. 

LITERATURE CITED 

BENSLEY, R. R., 1911. Studies on the pancreas of the guinea pig. .liner. Jour. of Anat.. 12: 
297-388. 

BRODERSEN, J., 1933. Uber die Staub-, Korner- und Schaumzellen der Lunge und ihre Funk- 
tion. Zeits. f. niikros.-anat. ForscJiunij, 32: 73-83. 

COWDRY, E. V., 1914. The comparative distribution of mitochondria in spinal ganglion cells of 
vertebrates, .liner. Jour, of Anat., 17: 1-30. 

COWDRY, E. V., 1918. The mitochondrial constituents of protoplasm. C out rib. to Einbryol., 
No. 25, The Carnegie Institution of Washington, 8: 39-160. 

COWDRY, E. V., 1943. Microscopic technique in biology and medicine. Baltimore, The Wil- 
liams & Wilkins Co. 

HAYEK, H. VON, 1942. Uber Bau und Funktion der Alveolarepithelzellen. Anai. Auz.. 93: 
149-155. (See Abst. 846 by W. W. Ballard in Biol. Absts., 1946, 20: 103.) 

LEWIS, MARGARET REED, AND WARREN HARMON LEWIS, 1915. Mitochondria (and other 
cytoplasmic structures) in tissue cultures. The Aincr. Jour, of Anat., 17: 339-401. 

LILLIE. R. D., 1948. Histopathologic technic. The Blakiston Co., Philadelphia. 

MACKLIN, CHARLES C., 1938. The silver lineation on the surface of the pulmonic alveolar 
walls of the mature cat, produced by applying weak silver nitrate solution and exposing 
to sunrays or photographic developer. Jour, of Thor. Sure/., 7: 536-551. 

MACKLIN, CHARLES C., 1945. The marginal and partitional types of base in mammalian pul- 
monic alveoli. Anat. Rcc., 91 : 288. 

MACKLIN, CHARLES C., 1946. Residual epithelial cells on the pulmonary alveolar walls of 
mammals. Trans. Roy. Soc. of Canada, Sect. V, 3rd Series, 40: 93-111. 

MACKLIN, CHARLES C., 1947a. The epicytes of mammalian lungs. Anat. Rcc., 97: 397. 

MACKLIN, CHARLES C., 1947b. The foam cells of mammalian lungs with special reference to 
the vacuoloids. Paper given at the Congressus VI Internationalis Cytologicus, Stock- 
holm, Sweden, July 10th to 17th, 1947. (Publication pending in Acta Physiologica 
Ccllularis.) 

MACKLIN, CHARLES C., 1948. Silverized dust cells inserted into alveolar pores. Anat. Rcc., 
100: 693. 

SJOSTRAND, F., AND T. SjosTRAND, 1938. Uber die granulierte Alveolarzelle und ihre Funk- 
tion. Zcitsch. f. inik.-Anat. Forsdi.. 44: 370-411. 

THURLOW, MADGE DfcG., 1917. Quantitative studies on mitochondria in nerve-cells. Contribu- 
tions to Embryology Xo. 16, The Carnegie Institution of Washington, 6 : 37-44. 



THE CHARACEAE OF THE WOODS HOLE REGION, 

MASSACHUSETTS 

R. D. WOOD 
Botany Department, Rhode Island State College, Kingston, Rhode Island 

INTRODUCTION 

It is extremely doubtful that any other comparable area in North America has 
been subjected to such constant investigation for fresh-water algae over such a long 
period of time as has the region in the vicinity of Woods Hole, Massachusetts. 
Specimens which have become widely distributed in herbaria in this country and 
abroad show that an almost continuous series of collections has been made since 
the days of Agassiz and the establishment of the original marine biological labora- 
tory on Penikese Island. Among these collections, many specimens of Characeae 
have been accumulated by generation after generation of students and investigators, 
until today one finds an abundance of herbarium material available for study. The 
objectives of the present paper are primarily to present a workable local flora for the 
Characeae for use of students of phycology and the independent investigator who 
employs these plants in his research; and secondarily to provide a systematic treat- 
ment of the local species based upon a study of available specimens. 

During the summer of 1947, the writer undertook a survey of all accessible 
bodies of fresh and brackish water in the Woods Hole region. Specimens of 
Characeae were collected from all places where found, and herbarium mounts and 
preserved material were prepared. Duplicates of representative forms have been 
distributed to the New York Botanical Garden, to the herbarium of the Marine 
Biological Laboratory at Woods Hole, and to certain private herbaria. 

The Woods Hole region as delimited for the present paper is approximately the 
same as that covered by Croasdale (1935) with the addition of certain islands. 
Roughly, the area includes the islands of Nantucket and Martha's Vineyard, the 
Elizabeth Islands, and the southern tip of Cape Cod. Names of localities and 
ponds (cf. Table I) follow those used by Croasdale. Ponds in areas not included 
by that writer and for which the local names are not known to the present writer 
are assigned local names as indicated on herbarium labels. 

The ponds of the region vary greatly as ecological habitats. In general, the 
isolated inland ponds result from the glacial knob-and-kettle topography, and 
tend to be slightly to strongly acid. The coastal ponds are largely lagoons which 
have become partly or totally isolated from the ocean by barrier beaches or bars. 
These tend to be markedly alkaline, and salinity varies from strictly marine to weakly 
brackish. These differences are reflected in the quite distinct characean flora of 
the two types of ponds. Correlations of distribution and ecological variables will 
be reported elsewhere. 

Despite the wide range of aquatic habitats, the characean flora of the Woods 
Hole region is not great. With the possible exception of Nitclla Morongii Allen, 
all the species reported also occur on the mainland. On the contrary, a number of 

179 



180 



R. D. WOOD 



PLATE I 




I -A 





I-B 





2-B 



I-C 



I-D 





2-C 







3-A 



3-B 



3-C 



3-D 




4-A 



4-B 



4-C 



4-D 



4-E 



4-F 



WOODS HOLE REGION CHARACEAE 181 

species are lacking which could well be expected in the region studied, because of 
their frequent occurrence on the adjacent mainland. Among those noticeable by their 
absence are Chara contrarla Kiitz., C. zntlgaris L., C. sejnncta Braun, C. gym- 
nopitys Braun (--C. fihrosa Ag. ex Bruz.), 1 C. fragilis Desv. ap. Lois. (--C. 
(/lobiilaris Thuillier), N. opaca (C. Agardh ex Bruz.) C. Ag., N. acuminate/ Braun 
ex Wallm., N. capillaris (Krocker) Groves & B.-W., N. tenuiss'una (Desv.) Kiitz., 
and Tolypella spp. 

In addition to the specimens obtained during this survey, the writer has had the 
opportunity to inspect material from the region in various herbaria, including the 
following: Brown University (BRU) courtesy of Dr. W. H. Snell ; Chicago 
Natural History Museum (F) courtesy of Dr. F. Drouet ; Farlow Reference 
Library and Herbarium ( FH) courtesy of Dr. W. L. White; Marine Biological 
Laboratory (MBL) ; Maria Mitchell Association of Nantucket (MMA) courtesy 
of Grace Wyatt ; New York Botanical Garden (NY) courtesy of Dr. D. P. 
Rogers, Dr. F. J. Seaver. and Rosalie Weikert ; University of California (UC)- 
courtesy of Dr. H. L. Mason and Dr. G. F. Papenfuss ; Yale University (Y) ; and 
the private herbaria of E. T. Moul (ETM), Dr. M. S. Doty (MSD), and Dr. W. R. 
Taylor (WRT). The abbreviations indicated for these herbaria and employed 
throughout the text follow Lanjouw (1939) with the exception of the writer's her- 
barium (ROW), other private herbaria (ETM, MSD, WRT), and those for which 
no standard abbreviation is listed (MBL, MMA). 

The literature of the Woods Hole Characeae consists of a few scattered papers. 
Halsted (1879) reported TV. gracilis (Sin.) Ag. from Nobska Pond and C. coronata 
var. Schweinitzii Braun from shallow ponds at Woods Hole. T. F. Allen (1880; 

iZaneveld (1940: 153). 

PLATE I 

Diagrams illustrating terminology and certain morphological details of the Characeae. 

FIGURE 1. Nitella. Diagnostic features: 1-A, branchlets in the whorls (once) furcate; 
two true branches shown. 1-B, coronula of oogonium with ten cells (6 visible) in two tiers. 
1-C, axial node, without stipulodes ; axis ecorticate. 1-D, one fertile branchlet twice furcate 
(divided) into, four secondary rays at the first furcation and 3-4 tertiary rays (dactyls) at tine 
second furcations; three antheridia shown terminally on rays in whorl of rays of the next order; 
one oogonium shown lateral at the first branchlet node. 

FIGURE 2. Chara. Diagnostic features : 2-A, branchlets in whorls, not furcate : no true 
branches. 2-B, coronula of oogonium with five (3 visible) cells in one tier. 2-C, axial node 
with two rows of stipulodes (diplostephanous) ; axis corticated; branchlet not furcate; antheridia 
below oogonia at branchlet nodes ; two elongated bracteoles shown subtending each oogonium ; 
reduced bracts at sterile branchlet node and at fertile nodes abaxial to bracteoles. 

FIGURE 3. Chara. Terminology of axial nodes: 3-A, ecorticate, haplostephanous (stipu- 
lodes in one row). 3-B, haplostichous, haplostephanous. 3-C, diplostichous. diplostephanous 
(stipulodes in two rows). 3-D, triplostichous, diplostephanous. 

FIGURE 4. Chara. Terminology of axial cortication. A few cortical filaments shown on 
a segment of internode. Primary cortical cells drawn solid, secondary cortical cells dotted. 
4-A, ecorticate (without cortication). 4-B, haplostichous, secondary cortical cells of each corti- 
cal node not elongated. 4-C, diplostichous. secondary cortical cells about half the length of 
primary cortical internodal cells, resulting in one row of secondary cells between each row of 
primary cells. 4-D, triplostichous, secondary cortical cells about equal in length to the primary 
cells, resulting in two rows of secondary cortical cells between each row of primary cells. 4-E, 
triplostichous, aulacanthous (secondary cortical cells greater in diameter than primary cortical 
cells'). 4-F, triplostichous, tylacanthous (primary cortical cells greater in diameter than sec- 
ondary cortical cells). 



182 R. 1). WOOD 

1882a) reported C. crinita f. leptosperma; and later (1896) a form near Nitclla 
in hmta or N. batrachospenna, possibly a new species N. maxceana Allen, from Nan- 
tucket ; and the description of N. Morongii Allen. Maria Owen (1888) listed the 
following species from Nantucket : Nitclla hatrachosperma, N. fle.vilis, N. flcxilis var. 
subcapitata, N. Morongii, N. inucronata, Chara coronata, C. coronata var. 
Schweinitzii, C. crinita, C. crinita f. leptosperma, C. fray His var. dclicatnla. The 
most recent work is by Croasdale (1935) who reported Nitella yracilis from Wood 
and Golf Ponds, N. fle.rilis from Cuttyhunk, N. inucronata var. gracilliina from 
Wood Pond, Chora cancsccns and C. jragilis from Chara Pond, and C. delicatula 
from Cuttyhunk. This work included a diagnostic key to the species. 

The terminology employed in the present paper follows the majority of recent 
workers in the field. Because of variations in usage of certain terms in the litera- 
ture, the writer's usage of each term is defined in the discussions of the class, family 
and genera. Further, the structures are labeled in Plate I. 

The nomenclature is that which, in the writer's opinion, represents the valid 
name ; and where marked deviations from recent monographic treatment exist, 
reasons for such changes are given. Synonyms listed include only those which 
occur in recent literature. For more complete synonymies, the reader is referred 
to Migula (1897), Groves and Bullock- Webster (1920; 1924), Zaneveld (1940). 
and Wood (1948b) for the genus Nitella in North America. 

The descriptions and figures, with exception of illustrative diagrams, were taken 
from specimens collected in the region, and thus reflect only the characteristics of 
the local forms. All such specimens are in the writer's herbarium (RDW). It 
should be noted that the description of each species is based entirely on a single 
specimen. Variations from this plant are to be found in the discussion of varia- 
tions following each description. 

In addition to the persons and institutions indicated above to whom the writer 
is deeply indebted for the opportunity to study herbarium material, he is especially 
indebted to Dr. Hannah Croasdale who guided the writer to many collecting sites, and 
to Dr. M. S. Doty for his encouragement and aid in the completion of the present 
work. Most sincere gratitude is due G. O. Allen, Esq., of England, Dr. W. R. 
Taylor, and Dr. M. S. Doty for timely criticisms of the original manuscript. 

THE KEY 

The following key is intended to be strictly analytical in nature, and employs 
vegetative characteristics as far as possible. The most convenient characteristics 
are listed first in each entry. The key disregards the distinctions between genera. 
A differential synopsis of tribes and genera may be found under "Family Characeae." 

la. Main axes uncorticated 2 

Ib. Main axes corticated 6 

2a. Branchlet whorls not subtended by stipulodes ; branchlets one or 
more times furcate (forked): antheridia born terminally on rays; 
oogonia borne laterally at furcations of branchlets, thus situated 

below antheridia 3 

2b. Branchlet whorls subtended by one series of stipulodes ; branchlets 
not furcate ; antheridia borne laterally at branchlet nodes ; oogonia 
above antheridia 5. Chara Braunii, p. 193 



WOODS HOLE REGION CHARACEAE 183 

3a. Ultimate rays (dactyls) of branchlets not terminated by a small pointed 

cell (mucro) 1. Nitella flc. \-ilis, p. 187 

3b. Ultimate rays of branchlets terminated by one or more such small cells 4 

4a. At least some of the dactyls more than two-celled, the tips varying 
from a single-celled mucro to a tip with two or more such mucros ; 
oogonia geminate (two at a node) to aggregate (more than two at 
a node), but occasionally solitary; plants large and robust, main 
axes in mature plants generally exceeding 400 p. in diameter. 

2. Nitella mcyacarpa, p. 188 

4b. Dactyls strictly two-celled including the small terminal mucronate 
cell; oogonia solitary (one oogonium at a fertile node) ; plant dis- 
tinctly small and delicate, main axes generally less than 350 p, in di- 
ameter 5 

5a. Branchlets once or twice furcate ; fertile whorls reduced to small heads ; 
oospore membrane appearing roughened, but not reticulate. 

3. Nitella Morongii, p. 189 
5b. Branchlets 3 or more times furcate ; fertile whorls not reduced to heads ; 

oospore membrane reticulate 4. Nitella transilis, p. 191 

6a. Spines (at least some) on main axes generally in groups of 2-5 
(fascicled) ; cortication haplostichous (number of corticating cells 
equal to the number of branchlets in an adjacent whorl). 

6. Chara eaneseens, p. 196 
6b. Spines (if present) on main axes not fascicled (very rarely paired). 

solitary ; cortication of the main axis' appearing diplostichous to 
triplostichous (corticating cells 2-3 times as many as branchlets in 

adjacent whorl) 7 

7a. Plants monoecious, the oogonia and antheridia on same plant, though by 
loss of one or other of the sex organs they may appear dioecious. 

7. Chara delieatnla. p. 198 
7b. Plants dioecious, the oogonia and the antheridia borne on separate plants 

(thorough inspection of a number of plants must be made before the dioecious 
condition can be considered proved) 8. Clwm aspcra, p. 199 

CLASS CHAROPHYCEAE 

More or less bushy, green, submerged, attached, aquatic plants which vary in 
size from 0.5 cm. to nearly 2 meters in height. Rhizoidal portions hyaline, and 
ramifying in the soil. Laterals borne in whorls along an erect axis. Certain forms 
deposit an external incrustation of lime ; some become dark brown to black. Both 
conditions may be uniform or may develop as bands encircling the cells. Habitat 
strictly fresh water in most species, some species more tolerant of low salinity, and 
some restricted to brackish water. No marine forms are known. 

Vegetative portions fundamentally consisting of a uniaxial filament, the fila- 
ment exhibiting alternating elongate internodal cells and short nodal cells. The 
internodal cells merely elongate, but the nodal cells divide three or more times in 
such a way as to form central nodal cells and a series of peripheral nodal cells. One 



184 R. D. WOOD 

or more central nodal cells at each node cut off apical cells, which continue develop- 
ment of filaments (main axis). A dominant filament so formed is considered the 
main axis, others are considered as branches (laterals of unlimited growth). The 
peripheral nodal cells each cut off meristematic initials which form laterals of limited 
growth a whorl of branchlets at each node. Other peripheral nodal cells (e.g., 
Chara) cut off meristematic initials which form corticating filaments lying along the 
outside of the internodal cell. This layer is the cortication. From other peripheral 
nodal cells may arise elongated cells, the stipulodes, which spread laterally sub- 
tending the hranchlets (PI. I. Fig. 3). The hranchlets may divide (furcate) into 
two or more segments or rays (PI. I. Fig. 1-D) at each node to the fourth or fifth 
order (Nitella), or the hranchlets may remain strictly undivided (Chara), and 
consist of alternating nodes and internodes (except for terminal ecorticated cells). 
Undivided hranchlets produce only one central nodal cell at each node with ac- 
companying peripheral nodal cells. Spine-like processes from these peripheral 
branchlet tiodal cells form bracts (PI. IV, Fig. 4-C) at each branchlet node. The 
hranchlets may he completely or partly corticated or totally ecorticate. 

The oogonium or egg-producing cell is fundamentally an apical cell terminal 
borne on a modified lateral and ensheathed in an encasement of five spirally twisted 
laterals of limited growth, the enveloping cells. At first these are straight, hut be- 
come tightly spiralled about the oogonium in maturity. The writer employs the 
term com'olutions to indicate the resulting spirals apparent on the female gametan- 
gium. Each enveloping cell is terminated by one small cell in the tribe Chareae 
and by two small cells in the tribe Nitelleae, which results in a coronula of five cells 
or ten cells in two. superimposed tiers, respectively. The entire female gamentan- 
giuni is known taxonomically as the oogonium. 

The oogonia generally arise as laterals of the antheridial stalk (see below) in 
Chara, or from a peripheral nodal cell of a branchlet in Nitella and species of 
Chara where gametangia are not conjoined. In the genus Chara, elongated cells, the 
Irracfcolcs which often closely resemble bracts, also develop from the antheridial 
stalk. The oospore develops within the enveloping cells of the oogonium. The im- 
pression of the enveloping cells on the oospore results in spiralled ridges, the 
striae or striations. The outer membrane of the oospores frequently develops char- 
acteristic oospore membrane patterns. 

The biflagellate, spiral sperms are produced singly in cells which occur in fila- 
ments (capitular filaments) borne terminally on laterals (manubria) which project 
inwardly from the centers of four or eight shield plates. The shield plates coalesce 
to form a spheroid structure, and radiating lines (partial septa [modified cells?]) 
project from the periphery toward the center of each shield plate. The entire 
spherical male gametangium is known taxonomically as the antheridiuin (PI. I, 
Fig. 1-D). The antheridia arise from peripheral nodal cells of branchlets (Chara) 
or from the apical cell of a ray (Nitella). 

The vegetative plants are haploid ; meiosis, as far as is definitely known, oc- 
curs in the germination of the zygote. Both monoecious and dioecious species occur. 

For a more detailed discussion of the morphology, the reader is referred to 
Fritsch (1935:447-465). 



WOODS HOLE REGIOX CHARACEAE 185 

FAMILY CHARACEAE 

The only existing family with characteristics of the class. The family has been 
divided into tribes and genera based upon both sexual and vegetative characters. A 
synopsis to this classification is given below. 

Coronula of the oogonium consisting of ten cells in two tiers Tribe NITELLEAE 

Ganterer (1847: 8). pro parte, emend. Leonhardi (1863: 69). 
Anthericlia apical on a ray in the furcation of the branchlets Genus NITELLA 
C. A. Agardh (1824: xxvii). pro parte. emend. Braun (1849a, b: 195. 292). 
Antheridia lateral at the furcation of the branchlets Genus TOLYPELLA 

(Braun. 1849a: 199) Leonhardi (1863: 72). 
Coronula of the oogonium consisting of five cells in one tier Tribe CHAREAE 

Leonhardi (1863: 72). 
Stipulodes at the base of the branchlets lacking. 

Bracts present, one to two at a node Genus NITELLOPSIS Hy (1889: 

397). 

Bracts absent Genus PROTOCHARA Womersley and Ophel (1947: 311). 
Stipulodes at the base of the branchlets present, although sometimes rudi- 
mentary. 

Oogonia normally situated below antheridia ; axes ecorticate Genus 
LAMPROTHAMNIUM Groves (1916: 336). emend. Ophel (1947: 322). 
Oogonia normally lateral with respect to antheridia. an oogonium situ- 
ated between two antheridia : axes corticate Genus LYCHNOTHAMNUS 
(Ruprecht 1845: 11) pro partc, Leonhardi (1863: 72). 
Oogonia situated above the antheridia ; axes corticate or ecorticate Genus 
CHARA Vaillant ex Linnaeus (1754: 491). 

Womersley and Ophel (1947) recently described a new genus Protochara. This 
genus is founded upon P. australis Worn. & Oph., a new species described in the 
same article. Nitellopsis inftata Filarszky & Allen was transferred to the new genus 
in the combination P. in flat a (Fil. & Allen) Worn. & Oph. Ophel (1947), in an- 
other article, emended Lamprothamnium Groves, and included Chara macropogon 
Braun in the combination Laniprothamnimn inacropogon (Braun) Ophel. He also 
suggested removing L. Hanscnii Sender to Chara Hansenii (Sender) Ophel. The 
material and evidence to support these changes have not as yet been examined by 
the present writer. 

Of the six or seven known genera, three occur in North America ; namely. 
Chara, Nitella, and Tolypella. Of these, Chara and Nitella are represented in our 
region ; although Tolypella is known to occur as near as the Finger Lakes of New 
York, and has been recorded from Vermont. 

Genus Nitella 

Branches commonly two or more at a node. Branchlets one or more times fur- 
cate into two or more rays (PI. I. Fig. 1-D) at each furcation. The ultimate rays 
of a branchlet beyond the last furcation are known as dactyls (PI. I, Fig. 1-D). In 
many species a small terminal cell occurs on the dactyl, and is known as a mucro 
(PI. Ill, Fig. 2-A). In some species the fertile branchlets are greatly reduced and 



186 



R. D. WOOD 



PLATE II 




WOODS HOLE REGION CHARACEAE 187 

modified; in others the fertile hrunchlet whorls and the branch upon which they are 
borne are reduced and form more or less dense heads (PI. II, Fig. 2). Fertile 
heads may be enveloped in mucus a hyaline, gelatinous material. An oogonium 
arises from peripheral nodal cells of a branchlet. thus replacing a ray, and projects 
laterally below the whorl of rays. An antheridium occurs terminally on a ray amid 
the whorl of rays of the next order, replacing the apical cell of the ray. Cells of the 
coronula of the oogonium ten, in two superimposed tiers of five cells each. Oospores 
are laterally compressed. 

In our region, specimens of Nitella can be immediately distinguished from 
Chara in the field by the obvious character that the branchlets are divided, whereas 
they are strictly undivided in Chara. 

1. Nitella flexilis (Linn., pro partc) C. A. Agardh, Syst. Alg., p. 124. 1824. 

Chara fle.vilis Linn., Spec. Plant., p. 1157. 1753. 
References for the region : A r . fle.vilis: Owen ( 1888 : 74) ; Croasdale ( 1935 : 94) ; var. 

snbcapitata: Owen I.e. 

(Plate II. Fig. 1 ; Plate III, Fig. 1 ) 

Plant monoecious. 20 cm. high, diffuse but robust. BrancJilcts about eight in 
a whorl, once furcate into two long dactyls (one occasionally lacking). Dactyls 
( 1-) 2 L> , equal to or exceeding the primary ray in length, terminated by a sharp- 
pointed mucronate tip (not a distinct cell). Gametangia generally aggregate, (1-) 
2 (-3) oogonia and an antheridium at each fertile node. Oogonia 850-875 p. long 
by 690-710 /x broad; coronula about 75 ^ broad by 36 /x high, ultimately deciduous; 
convolutions 7. Oospores 530-550 /A long by 472-500^ broad, black; striae ap- 
parently 5; membrane smooth. Antheridia 340-415 /x (immature; 515-555 /x in 
R. D. Wood 2014} in diameter. (Descr. from R. D. Wood 2013 (ROW).) 

Variations among the local forms result from differences in development of 
vegetative structures. In size, plants vary up to 35 cm.- high. The dactyls vary 
from short (% length of primary ray) in Maria Owen 3, to greatly elongated 
(longer than primary ray) in R. D. Wood 2014. The rays vary from those which 
are all equal in length and give a distinctly regular appearance to the whorls as in 
R. D. Wood 2030, to those which are very unequal in length and give a ragged ap- 
pearance to the whorls as in T. Morong 3. The branchlets vary from elongate in 
R. D. Wood 2014 giving loose whorls, to very short in T. Morong 2b giving ap- 

- G. O. Allen (corresp., 1948) reports that three dactyls occasionally occur in British 
specimens. 

PLATE II 

Drawn from the indicated herbarium specimens which are extant in the writer's her- 
barium. X 0.5. 

FIGURE 1. Xitclla fle.vilis ( L. pro parte) C. Agardh. (2030.) 

FIGURE 2. Nitella Morongii Allen emend. Wood. (E. T. Moul 3173.} 

FIGURE 3. Nitella mcgacarpa Allen. (2060.) 

FIGURE 4. Nitella transilis Allen. (2021.) 

FIGURE 5. Chara dclicatula C. Agardh. (2058.) 

FIGURE 6. Chara cancscens Lois. (2027.) 

FIGURE 7. Chara Braiinii Gmelin. (2004.) 



188 R. D. WOOD 

pearance of reduced clumps isolated on elongated internodes. The fertile whorls 
are generally similar to the sterile whorls, but in W. R. Taylor (17085 WRT) the 
heavily fertile whorls are somewhat reduced into loose heads. The color of dried 
specimens is generally translucent greenish brown, but certain specimens become 
opaque dark brown as in T, Morong 1 and thus suggest N. opaca (but our speci- 
mens are monoecious!). These extreme characteristics appear to occur in random 
combination, and could permit designation of easily ten or more different forms; 
but none of the combinations seem sufficiently constant to merit assignment to 
varietal status. Allen (1871: 9; 1880: 11) and Owen (1888: 74) have reported 
those forms in which fertile whorls are somewhat reduced as var. subcapitata 
(Hartm.) Groves (== var. nidifica Groves, H. & J.). and Allen (1880: pi. 5) as- 
signed forms with extremely shortened branchlets and stout internodes to var. 
crassa Braun and to form superne brachyphylla (in herb. NY). 

N. fiexilis is easily distinguished in the field, as it is the sole species in our local 
flora in which the branchlets are only once divided. However, occasional sterile 
branchlets may not be furcate, and inspection of the fertile whorls may be necessary 
to demonstrate this feature. 

Illustrations: Allen (1892: pi. 6, 6a, 6b) : Groves & Bullock-Webster (1920: pi. 8); 
Migula (1897: 133; 1925: 214, fig. 4, 5); Woods (1894: pi. 26). 

Exsiccatae: Phyc. Boreali-Amer., No. 1435, 1691; Char. Amer. Exsicc., No. 28, 29, 30. 

Localities: CAPE COD: Weeks Pond R. D. Wood 2016, July 26, 1947 (RDW) ; CUTTY- 
HUNK: Clubhouse Pond R. D. Wood 2030, July 31, 1947 (RDW, NY) ; Sheep Pond IV. R. 
Taylor, June 28, 1932 (17085 WRT, NY) ; Sheep Spring jr. R. Taylor, Tune 27, 1933 
(16981. 17010 WRT) ; July 3, 1934 (16769 WRT) ; MARTHA'S VINEYARD: Chilmark Pond- 
R. D. Wood 2014, July 20, 1947 (RDW, NY) ; Tiasquam Dam R. D. Wood 2013, July 20, 
1947 (RDW); NANTUCKET: Cato's Pond T. Morong 3, July 22, 1887 (NY); New Lane 
Pond, pond west of New Lane and S. of Grove Lane Grace Wyatt, Sept. 4, 1947 (RDW) ; 
Polpis Mary Owen 3, July, 1879 (NY), as var. snhcapitata ; R. R. Track Pond, pool E. of 
town [Nantucket] near R. R. tracks T. Morong 1, July 16, 1887 (NY) ; Sesachacha, drainage 
ditch S.W. of pond Grace Wyatt, Aug. 12, 1947 (RDW) ; Weweeder Pond T. Morong 2b, 
July 16, 1887 (NY) ; July 15, 1887 (NY) ; NONAMESSET: South Pond Hannah Croasdale 55, 
June 15, 1935 (MBL, preserved; 2012 RDW); PASQUE: West End Pond//'. R. Taylor, 
July 2, 1941 (20840 WRT, EH); ? Pond, west end of island//'. R. Taylor. Tuly 3, 1940 
(19691 WRT). 

2. Nitella megacarpa Allen, Characeae Americanae Exsiccatae, No. 3. 1880. 

Nitella microcarpa subsp. incyacarpa (Allen) Nordstedt apud Braun. Frag- 

mente einer Monographic der Characeen, p. 73. 1882. 

References for the region: N. inucronata: Owen (1888: 74 [pi. not seen]); var. 
(jracilllma Groves B.-W. : Croasdale (1935: 94). 

(Plate II, Fig. 3; Plate III, Fig. 4) 

Plant monoecious, 25 cm. high, robust, bright green. Branchlets (4-) 5-6 (-7) 
in a whorl, up to 3.5 cm. long, spreading widely ; whorls at apex of main axes partly 
convergent and forming a rather broad, terminal clump; branchlets bear gametangia 
throughout the season, 3-4 times furcate into 5 secondary rays, 2-3 tertiary rays, 
2-3 quaternary rays, and occasionally 2-4 quinary rays. Dactyls 2-3, 2-3 (oc- 
casionally 4- more) -celled; ultimate cell a conical mucro 37-74/1 long; basal cell 
100-500 /JL long; intermediate cells (when present) generally one or two subcylindri- 



WOODS HOLE REGION CHARACEAE 189 

cal cells a little longer than broad. Gametang'ui borne on branchlets of all whorls, 
present throughout the season, solitary to aggregated, not enveloped in mucus. 
Oogonia about 710 ju. long by 510 /x broad, 1-2 (-3 or more) at a branchlet node; 
coronula about 25 //, long by 36 ^ broad ; convolutions 9. Oos pores about 420 ^ long 
by 370 fj. broad, yellow when immature, darkening to deep brown on maturity, often 
nearly spherical; striae 5-6; membrane strongly reticulate; reticulae 2. 2-3.6 /t in 
diameter, subquadradic, 9-11 across a fossa. Antheridia 265-445 /A in diameter. 
(Descr. from R. D. Wood 2060 (RDW).) 

This large, bright Nitella is by far the most beautiful species in the region, and 
one which cannot be confused once it has been seen in the field. In size, the plants 
vary up to nearly 40 cm. high. 

Nordstedt (1882) and Zaneveld (1940) included A', mcgacarpa within the 
limits of A 7 ", microcarpa Braun. Our form certainly is very similar in most funda- 
mental respects to this species, and is doubtlessly very close phylogenetically. The 
local form differs in being a much larger plant with much larger gametangia. The 
greater general size suggests polyploidy from N. microcarpa. If this proves to 
IDC the case, the erection of a distinct species is almost required. Furthermore, the 
writer has seen no specimens which exhibit complete serial intergradation between 
the two forms. Therefore, until intergrades are seen, or until cytological investiga- 
tions are completed, the writer prefers to consider N. megacarpa Allen a distinct 
species in agreement with G. O. Allen (corresp., 1948). 

A characteristic feature of this plant is the manner in which the main axis is easily 
separated at the nodes. As a result, specimens are commonly collected, especially 
with plant hooks, which consist of only the terminal clump of whorls of branchlets. 
These fragments do not give the impression of massiveness characteristic of the 
plant. 

Illustrations : No habit sketches of this species have been published previously. 

Exsiccatae: Phyc. Boreali-Amer., Fasc. 32, No. 1588, Fasc. E, No. CII ; Char. Amcr. 
Exsicc., No. 3. 

Localities: CAPE COD: Ashumet Pond /. F. Leivis, Sept. 4, 1926 (WRT) ; Leech Pond 
C. W. Palmer, July 27, 1936 (17199 WRT) ; Summerfield Pond, South C. C. Jao, Sept. 4, 
1933 (16771 WRT) ; Weeks Pond Hannah Croasdale, July 28, 1947 (2019 RDW, MBL, 
MSD, ETM) ; R. D. Wood 2031, Aug. 1, 1947 (RDW), 2060, Sept. 6, 1947 (RDW) ; Wood 
Pond W. R. Taylor, July 2, 1921 (3668 WRT). 

3. Nitella Morongii Allen, Bull. Torrey Bot. Club 14: 214. 1887; emend. 
Wood, Rhodora 51 (602) : 16. 1949. 

Nitella maxceana Allen, spec, dub., Char. Amer. 2(3) : 27. 1896. 
References for the region: N. batrachosperma: Owen (1888: 74) ; N. gracilis: Hal- 

sted (1879: 176), Croasdale (1935: 95); f. brachyphylla: Collins in Phyc. 

Boreali-Amer., No. 1195; N. maxceana: Allen, I.e.; N. Morongii: Owen, I.e., 

Allen, l.c., Wood, I.e. 

(Plate II. Fig. 2; Plate III, Fig. 2) 

Plant monoecious, 14 cm. high, delicate, and with characteristic heads. Branch- 
lets of two types, including: (1) the normally expanded sterile or lightly fertile 
branchlets, and (2) the greatly reduced fertile branchlets. Sterile branchlets 2-5 
in a whorl, 15-26 mm. long. 1-2 times furcate into 3-5 secondary rays, 2-3 terti- 
ary rays. Dactyls of sterile branchlets 2-3, 2-celled, the ultimate cell a conical mucro 



190 



K. I). WOOD 
PLATE III 




WOODS HOLE REGION CHARACEAE 191 

which is early deciduous. Fertile braiiehlets 6-7 in a whorl, twice furcate, greatly 
reduced, 1-3 mm. long ; 3-5 such reduced whorls borne on a reduced branch, the en- 
tire complex resembling a dense head ; heads apparently axillary in sterile whorls or 
terminal, enveloped in mucus. Dactyls of fertile braiiehlets 2 (-3), 2-celled, one 
commonly shorter than the other, terminated by an elongated mucro. Gainetangia 
solitary, an oogonium and an antheridium at all fertile branchlet nodes, enveloped in 
a weak mucus. 3 Ooyonia 290-386 /A long by 210-288 p. broad; coronula 35 X 35 p. 
Oosporcs 238-268 |U long by 1 80-210 /x broad; striae of 5 prominent ridges; mem- 
brane roughened with anastomosing lines, almost appearing very finely reticulate. 
Anthcr'uiia somewhat flattened longitudinally. 134-148 ,w long by 174-179 ^ broad, 
short stipitate. (Descr. from E. Moid 3173 (RDW).) 

Specimens which have been collected since 1888 are quite consistently of a dif- 
fuse form as in E. Moid 3173, described above. A compact form as in the TYPE 
COLLECTION T. Morong, July 21, 1887, has very short branchlet rays, more fertile 
whorls at a fertile branch, and early deciduous sterile branchlets. This modifica- 
tion is thought to be the result of extreme ecological conditions exerted by constant 
trampling by the feet of animals and the muddy water (cf. Wood, 1949). In size, 
some plants reach 20 cm. high. 

Illustrations: Allen (189-1: pi. 16), excellent for the original material, but not as emended. 

Exsiccatae : Phyc. Boreali-Amer., No. 1195 as N. gracilis, 1382. 

Localities: CAPE COD: Golf Pond G. M. Gray, July. 1931 (17006 WRT) ; IV. R. Taylor, 
July 6, 1917 (2293 WRT): Hannah Croasdale. June 24, 1935 (17078 WRT, NY, 950899 F, 
RDW) : Harper Pond E. T. Afoul 3173, July 7, 1947 (ETM, RDW, NY) ; Urda K. Wood, 
July 11, 1947 (2011 RDW, NY) ; Nobska Pond W. A. Setchcll and W. J. V. Osterhout 644, 
Tuly 15, 1894 (MBL, FH, 315652 UC) ; W. A. Setchcll, July 15, 1893 (P. B.-A. No. 1195, as 
N. gracilis, NY); ? Pond, Woods Hole [Coll.? }, 1883 (NY); NANTUCKET : Maxey's Pond 
T. Morong, July 7, 1887 (NY, Type of N. ma.rceana Allen) ; Siasconset, small pond south 
side of old Sconset Road, opposite "Bloomingdale" L. L. Dainc, July, 1886 (NY, in one P. B.-A. 
packet No. 1382) ; in a very muddy pool on the roadside near Siasconset T. Morong 4, July 
21, 1887 (FH, NY, TYPE COLLECTION) ; in a small pool near Siasconset F. S. Collins, Aug. 
23, 1896 (P. B.-A. No. 1382, NY, BRU) ; NAUSHON : Petchett (or Peckett [?]) Pond- 
Hannah Croasdale, July 5, 1946 (MBL, preserved). 

4. Nitella transilis Allen, Char. Amer. 2(3) : 24. 1896. 
References for the region : none known. 

3 Allen (1888) in his synopsis enters .V. Morongii Allen under "heads not enveloped in 
mucus," but this point is reported with some question later (Allen, 1894: 15). 

PLATE III 

All figures have been drawn with the aid of a camera lucida from specimens preserved in 
2-3 per cent formalin. The indicated specimens are extant in the writer's herbarium. 

FIGURE 1. Nitella fle.rilis (L. pro partc) C. Agardh. 1-A, tip of terminal branchlet cell, 
X 35. 1-B, dactyls, X 14. 1-C, fertile branchlet node, one antheridium and two oogonia, X 14. 
1-D, oogonium, X 35. (2013.) 

FIGURE 2. Nitella Moronyii Allen emend. Wood. 2-A, tip of dactyl with terminal mucro, 
X 35. 2-B, dactyls, < 14, 2-C, fertile branchlet, X 14. 2-D, oogonium, K 35. (Urda K. 
Wood 2009.) 

FIGURE 3. Nitella transilis Allen. 3-A, tip of dactyl with terminal mucro, X 35. 3-B, 
dactyls, X 14. 3-C, axial node and part of one branchlet, X 14. 3-D, oogonium, X 35. (A and 
B from 2015; C and D from 2059.) 

FIGURE 4. Nitella meyacarpa Allen. 4-A, tips of dactyls showing variations in form of 
mucros, X 35. 4-B, dactyls, < 14. 4-C. fertile branchlet (a very small specimen), < 14. 
4-D, oogonium, X 35. (2019.) 



192 K. 1). WOOD 

(Plate II, Fig. 4; Plate III, Fig. 3) 

Plant monoecious, 12 cm. high, very fine and delicate. Branchlets 6-7 in a 
whorl (branches occasionally two at a node), 3-4 times furcate into 6 secondary rays, 
5 tertiary rays, and 4 (-5) quaternary rays; quinary rays when present 2-3; 
branchlets usually exceeding the axial internodes in length, primary rays about % 
the total length of the branchlet. Dactyls 2-3 (-5), 2-celled. Gametangia solitary, 
an antheridium and an oogonium borne on the second and third, rarely at the first, 
branchlet nodes, enveloped in mucus. Oogonia about 309 //, long by 240 ^ broad ; 
convolutions 7-8. Oospores about 276 p. long by 170^, broad; striae of 6 ridges; 
membrane strongly reticulate, reticulae 10-12 across the fossae. Anihcridia about 
178 p. in diameter, stipitate on stalks about 37-59 /x long; markings on shield ex- 
tending about half wav to base of manubrium, irregular. (Descr. from R. P. Jl'ood 
2015 (RDW).) 

The local form of this species is very constant, and apparently does not ex- 
hibit modifications found in other parts of eastern North America. In size it reaches 
16 cm. in height. Some specimens might be confused with N. Morongii because of 
the size and occurrence of mucus. 

Whether N. transilis Allen is sufficiently distinct to be separated from N. 
tenuissima (Desv.) Kiitz. has been discussed (corresp., 1946-1948) with G. O. 
Allen. The European N. tenuissima f. gracilior (L. Chcvallier, Aug. Sept. 1893. 
Gallia accident alls: In stagnis circa "Bazouges" (7185 WRT)) approaches N. 
transilis rather closely. Other intergrades of European N. tenuissima exhibit 
diffuse branching resembling N. transilis. Further significant information is ac- 
cumulating in the writer's herbarium in the form of a series of intergrades from 
various stations in New England. It seems probable that our specimens may well 
be included under N. tenuissima. At present, the characters which seem sufficient to 
retain it as a separate species from A r . tenuissima include: (1) the very regular dif- 
fuse whorls, (2) the branchlets exceed the internodes in length, (3) the rather 
frequent occurrence of gametangia at the first branchlet node, and (4) the stipitate 
antheridia. 

Illustrations: Allen (1896: pi. 23). 

Exsiccatae : Char. Amer. Exsicc., No. 31, sub. now. N. tenuissima f. longifolia clongata. 

Localities: CAPE COD: Ashumet Pond M. S. Doty & L. Spiegel 7470, Aug. 17, 1948 
(MSB, RDW) ; John Pond, Mashpee G. IV. Prcsco'tt 5, Aug. 30, 1937 (17973 WRT) ; 
Weeks Pond Hannah Croasdalc, July 14, 1946 (6615 MSD), July 28, 1947 (2021 RDW, 
ETM, MBL, MSD, NY) ; R. D. Wood 2015, July 26, 1947 (RDW, NY) ; R. D. Wood 2059, 
Sept. 6, 1947 (RDW, mature). 

Genus Chara 

Branches formed occasionally at nodes. Branchlets not furcate, divided by 
nodes and internodes into a continuous series of articulations ; the terminal two or 
more articulations may lack nodes, and be merely separated by cell walls. Spine- 
like processes (bracts, PL IV, Fig. 4-C) arise from peripheral cells of the branch- 
let nodes, and form a more or less distinct whorl at sterile and fertile nodes. 
Branchlet articulations corticated, partly corticated, or uncorticated. Main axes 
corticated or uncorticated (ecorticatc, PL I, Fig. 3-A, 4-A). Cortication varies 
from triplostichons (PL I, Fig. 3-D, 4-D) in which the secondary cortical cells 



WOODS HOLE REGION CHARACEAE 193 

are nearly equal in length to the primary cortical cells, thus forming two series of 
cells between each primary cortical series (and three times as many cortical filaments 
as branchlets at an adjacent node) ; to diplosticlious (PI. 1, Fig. 3-C, 4-C) in which 
the secondary cortical cells are about half the length of the primary cortical cells 
so that the secondary cells from adjacent primary cortical cells series lie end to 
end forming one continuous series of cells between each primary cortical series (and 
thus twice as many cortical filaments as branchlets at an adjacent node) ; to 
haplostichous (PI. I, Fig. 3-B, 4-B) in which the secondary cortical cells do not 
become enlarged, thus only the primary cortical cells are apparent in the cortication 
(and thus the same number of cortical filaments as branchlets at an adjacent node). 
The details of cortication are best seen by inspecting the cortication of very young 
axial internodes (fide G. O. Allen, corresp., 1948) before they become unduly elon- 
gated. One series of stipulodes (haplostephanous, PI. I, Fig. 3- A) or two series 
(diplostephanous, PI. I, Fig. 3-C) may develop from axial nodal peripheral cells and 
subtend the branchlets. Cortical nodal cells divide longitudinally into an inner and 
outer cell. The outer cell may merely swell into a papillns or spine, or may cut off 
several elongated processes (spines) in groups of 2-5 (fascicled, PI. IV, Fig. 2-B). 
Gametangia borne primarily at the branchlet nodes ; in monoecious species the an- 
theridium is generally directly below the oogonium (conjoined), or isolated at a 
separate node (sejoined) ; in dioecious species borne on separate plants. An- 
theridia arise from peripheral nodal cells of branchlet nodes. Oogonia (in con- 
joined monoecious species) arise from laterals of antheridial stalk, on the abaxial 
side; spine-like processes (bracteoles, PI. I, Fig. 2-C) which develop from cells 
of the antheridial lateral appear to subtend the oogonium. These may closely re- 
semble the bracts. Starch bulbils regularly formed on rhizoids of certain species. 
Oospores terete, not laterally compressed. 

5. Chara Braunii Gmelin, Fl. Badensis Alsatica 4: 646. 1826. 

Chara coronata Ziz. (ined., c. annum 1814) ; Braun, Ann. Sci. Nat., ser. II, 1: 

353. 1834. 
References for the region: C. coronata: Owen (1888: 75) ; var. Schweinitzii: Hal- 

sted (1879: 181); Owen, I.e. 

(Plate II, Fig. 7; Plate IV, Fig. 1) 

Plant monoecious, totally ecorticate, 20 cm. high. Branchlets 8-9 in a whorl, 
widely spreading to closely convergent; articulations of branchlets (3-) 4 (-5), 
first and second nodes generally fertile; bracts 300-455 fj. long to greatly reduced; 
terminal internodal cell occasionally somewhat inflated, terminated by a crown of 
3 mucro-like cells, each 150-274 /* long. Stipnlodcs in one series (haplosteph- 
anous), 730-1035 p. long, alternating with the branchlets, spreading. Gametangia 
at first and second branchlet nodes, fundamentally conjoined, but antheridia fre- 
quently early deciduous ; occasionally the sex organs geminate (2 oogonia and an- 
theridia at a node). Oogonia 660-960 ^ long by 384-490 /JL broad; coronula about 
150 /JL long by 90 p. broad, the cells with short, divergent tips; convolutions 10; 
bracts about equal in length to the oogonia. Oospores 470-590 //. long by 259-340 u 
broad, black; striae 8-10; membrane smooth and nearly opaque. Antlieridia 268- 
282^ in diameter. (Descr. from Hannah Croasdale (2008 RDW) ; details of the 
gametangia from Urda K. Wood, July 16, 1947.) 



194 





2-A 



3-A 



R. D. WOOD 
PLATE IV 



4 -A 




WOODS HOLE REGION CHARACEAE 195 

Variations among the local forms result from differences in development of 
vegetative structures. In size, some plants reach 35 cm. in height. The general 
plant structure varies from diffuse, attenuate forms, as in Hannah Croasdale (2020 
RDW). typical of relatively deep, quiet water to short, compact forms as in R. D. 
Wood 2033, characteristic of shallow littoral hahitats. The hracteoles vary from 
shorter than mature oogonia in R. D. Wood 2005 to li/o to 2 times as long in 
Hannah Croasdale (2020 RDW). The crown of end cells terminal on branchlets 
varies in length from equal to the diameter of the branchlet in R. D. Wood 2005 to 
less than half the diameter in R. D. Wood 2029. The relative lengths of the 
branchlet articulations vary from those in which the basal internode is about half 
the length of the others in R. D. Wood 2005, to those in which this articulation is 
extremely short in (Coll.?, Aug.). to those in which the terminal articulation is 
shortened and sometimes swollen in B. F. D. Rnnk 457 . The elongation of the 
bracts at the primary branchlet nodes varies from about equal to other bracts in 
R. D. Wood 2033 to those in which these bracts are twice the length of the bracts 
of other nodes in B. F. D. Rnnk 457. The stipulodes vary greatly in length on the 
same specimen, but are characteristically short in Hannah Croasdale (2008 RDW) 
and long in B. F. D. Rnnk 457 . The apparent general trend is for plants growing 
in shallow littoral zones to be tufted, form tightly connivent whorls, have shortened 
branchlet articulations and elongated bracts and stipulodes. Plants of deep, quiet 
water tend to be diffuse, have elongated branchlet articulations and reduced bracts, 
bracteoles, and stipulodes. These appear to be ecological variants, and range from 
what has been considered var. Schweinitzii Braun, in which bracteoles exceed ma- 
ture oogonia in length, to var. Braunii (Braun) Zanev.. in which bracteoles are 
equal to or shorter than mature oogonia. Oospores vary in length from 450-600 /* 
and thus are intermediate between the limits of var. Schweinitsii (550-650 /A) and 
var. Braunii (420-550 /JL) as delimited by Zaneveld (1940: 139). It is thus con- 
cluded again (cf. Wood, 1947: 250; Allen, 1882b) that attempts to differentiate 
varieties of this species collected from various localities of North America are im- 
possible in view of the facts. The writer has made a practice of annotating ex- 
treme specimens as approaching either the one variety or the other, but emphasizes 
that this is but a convenient manner of indicating differences in expression of the 

PLATE IV 

All figures have been drawn with the aid of a camera lucida from specimens preserved in 
2-3 per cent formalin. The indicated specimens are extant in the writer's herbarium. < 14. 

FIGURE 1. Chara Braunii Gm. 1-A, terminal cell? of branchlet. 1-B, axial node showing 
stipulodes at base of branchlets and lack of cortication. 1-C, oogonium. 1-D. oogonium sub- 
tended by antheridium. 1-E, bracts at sterile branchlet node. (Hannah Croasdale (2008 
RDW).) 

FIGURE 2. Chara cancsccns Lois. 2-A, terminal cells of branchlet. 2-B, axial node show- 
ing two rows of stipulodes, spines in fascicles, and haplostichous cortication. 2-C. bracts of 
sterile branchlet node. 2-D, oogonium at fertile branchlet node. 2-E, axial cortication. (2004.) 

FIGURE 3. Chara dclicatula C. Agardh. 3-A. terminal cells of branchlet. 3-B, axial 
node showing one row of stipulodes and diplostichous tylacanthous cortication. 3-C, fertile 
branchlet node with oogonium and antheridium. 3-D, fertile branchlet node with an oogonium. 
3-E, axial cortication. (2002.) 

FIGURE 4. Chara dclicatula C. Agardh var. barbata Groves & B.-W., pro*. 4-A, ter- 
minal cells of branchlet showing elongated penultimate cell. 4-B, axial node showing well- 
developed two rows of stipulodes, and nearly isodiametric primary and secondary cortical cells. 
4-C, bracts at sterile branchlet nodes. 4-D, axial cortication. (2024.) 



106 R. I). WOOD 

vegetative characteristics. One peculiar specimen was found {Coll.?, Aug.), which 
had a definite glomerate appearance. This resulted from the development <f a 
somewhat reduced hranch at each node, combined with elongation of hracts and 
stipulodes, and typical compactness of shallow-water forms. No further collections 
of this form have been seen; and, until other specimens have been reported, it will 
be considered an anomaly. 

This species is the favorite local Charad for physiological investigation, and in- 
vestigators commonly mistake the plants for a Nitella. There should be no con- 
fusion once it is recognized that this species is an ecorticate Chara, and that it is 
the only local species of Characeae which is ecorticate, has stipulodes at the base 
of the branchlets, and in which the hranchlets are not divided. The latter two 
characters easily distinguish the species in the field. 

Illustrations: Allen (1882b); Groves & Bullock-Webster (1924: pi. 26); Woods (1894: 
pi. 30); Migula (1897: 324; 1925: 226, fig. 1, 2). 

Exsiccatae : Phyc. Boreali-Amer., No. 822; 1383 as C. Sckweinitsii; Amer. Alg., No. 256, 
529; Char. Amer. Exsicc., No. 8, 12, 13, 14. 

Localities: CAPE COD: Chara Haven R. D. Wood 2005, July 7, 1947 (RDW, ETM) ; 
Chara Pond B. F. D. Runk 406, June 20, 1941 (1099774 F, RDW) ; Desmid Haven E. T. 
Moid, July 11, 1945 (ETM) ; Harper Pond/. /. Copeland, July, 1928 (13800 WRT) ; Ice 
House Pond M. S. Doty, July 10, 1946 (6624 MSD) ; Little Pond/?. D. Wood 2033, Aug. 
1, 1947 (RDW, NY) ; Nobska Pond W. G. Farloiv, Sept. 15, 1876 (FH) ; Oyster Pond- 
Urda K. Wood, July 16, 1947 (RDW, preserved) ; Salt Pond/?. D. Wood 2034, July 30, 
1947 (RDW) ; "Sandwich Pond," pond on W. side of road, route No. 6, E. of Sandwich 
R. D. Wood & Urda K. Wood (2007 RDW) ; Weeks Pond Hannah Croasdalc, July 14, 1946 
(6613 MSD), July 28, 1947 (2020 RDW) ; R. D. Wood 2017, July 26, 1947 (RDW) ; CUTTY- 
HUNK: Gosnold Pond W. R. Taylor, July 6, 1921 (3670 WRT) ; Sheep Pond/. F. Leivis 
(MBL); W. R. Taylor, July 9, 1925 (10173 WRT, MICH) ; /?. D. Wood 2027 (RDW) and 
2029 (RDW, NY), July 31, 1947; NANTUCKET: Long Pond W 7 . PI. Sheldon, Aug. 7, 1934 
(MMA) ; Sesachacha Pond [coll.? ], Aug. (NY) ; Weweeder Pond [T. Morong] 2a, July 
15, 1887 (NY); NAUSHON : French Watering Place W. R. Tavlor, July 12, 1921 (3669 
WRT) ; July 13, 1922 (7585 WRT) ; PASQUE: West End Pond B. 1'. D. 'Runk 457, July 1, 
1941 (1099770 F, RDW) ; ? Pond, small pool 2nd or 3d pool south of West End Pond 
W. R. Taylor, -]u\y 2, 1941 (FH, 20841 WRT); ? Pond, smaller pond near the southwest 
end of island Hannah Croasdalc, July 6, 1933 (16982 WRT); PENIKESE : Typha Pond 
Hannah Croasdale, July 6, 1947 (2008 RDW, MBL, MSD). 

6. Chara canescens Desvaux ct Lois, a pud Loiseleur-Deslongchamps, 4 Not. Fl. 
France, p. 139. 1910. 

Chara crinita Wallr., Annus Bot., p. 190. 1815. 

Chara crinita var. ainericana Allen, Char. Amer., Part 2, plate 2. 1879. 
References for the region: Chara crinita: Owen (1888: 75) ; f. leptospenna: Allen 

(1880: 5 ; 1882a : 41 ) ; Owen, I.e. ; C. canescens: Croasdale (1935 : 95). 

(Plate II, Fig. 6; Plate IV, Fig. 2) 

Plant dioecious, 12 cm. high, densely hirsute. Main a.vcs haplostichous, cortical 
nodes bearing spines solitary to fascicles of 2-5, one secondary cortical cell commonly 
apparent at cortical nodes and extending Vs to l / the way to the next node (some- 

4 Loiseleur-Deslongchamps has consistently been given the credit for this species. In a 
footnote (p. 135), he stated that Desvaux had recognized several new species in the genus 
Chara and desired that they be published for him pending a more extensive comparative ex- 
amination of the species. The present species, however, was entitled "Chara canescens. N." ; 
and since "N." indicates nobis (by us), both men are necessarily recognized in the author 
citation. 



WOODS HOLE REGION CHARACEAE 197 

times greatly developed so as to give a nearly diplostichous appearance). Branch- 
lets 6-8 in a whorl, articulations 4-6, corticated except for terminal 1-2 cells, ter- 
minal cell rounded at tip; bracts varying widely in length, blunt. Stipulodes, 
diplostephanous (in 2 series), 2 pairs at each branchlet, tipper series 297-447 //. 
long, generally exceeding the lower series in length, blunt. Gametangia borne on 
separate plants, i.e., dioecious. Oogonia about 890 /A long by 442 ^ broad, borne 
regularly. at first branchlet node, occasionally at second and third; convolutions 10; 
coronula short and broad, truncate, 86 ^, tall by 147 /A broad ; bracteoles blunt to 
acute, about equal to oogonia in length. Oosporcs about 590 /x long by 318 /x 
broad, black; striae about 10; membrane opaque. Anthcridia, none seen on local 
specimens. (Descr. from R. D. Wood 2004 (RDW).) 

Little variation is exhibited among the local specimens collected. In size, some 
plants reach 20 cm. in height. The spines on the axes vary from very abundant and 
nearly twice as long as the diameter of the axis in T. F. Allen, Aug. 30, 1895, to 
rather sparse and shorter than the diameter of the axis in R. D. Wood 2004. Brac- 
teoles vary from about equal in length to the mature oogonia in R. D. Wood 2004, 
to one to two times the length in F. S. Collins, July 25, 1882. The differences in 
expression of length of stipulodes, bracts, and spines appear to be more directly cor- 
related with local physiochemical factors, particularly salinity, rather than depth as 
in C. Brannii. This follows logically since most local specimens of C. canescens 
have been found growing in water less than one foot deep. All the local specimens 
seen exhibited the angular oospores which characterize the form leptosperma 
Braun (1834: 356) as described by Allen (1882a: 41, pi. XVIII), but the present 
writer prefers to temporarily ignore forms as nomenclatural entities. 

This species is highly characteristic in its very spinous appearance of the main 
axes. It is restricted to brackish-water ponds. Salinity determinations by means 
of silver nitrate titrations on fresh samples of water from various habitats during 
July and August, 1947. showed extremes of 4 to 22 o oo NaCl, with the optimum 
vegetative development at about 10 o oo NaCl. 

Although no antheridia were discovered on local specimens, a very similar 
monoecious species, apparently C. cvoluta Allen, was found in 1948 by Dr. V. I. 
Cheadle and Dr. E. A. Palmatier in Little Compton, Rhode Island, just across the 
state line of Massachusetts. 

Illustrations: Allen (1879: pi. 2; 1880: pi. 2) ; Migula (1897: 352, 353: 1925: 226, fig. 6) ; 
Groves & Bullock-Webster (1924: pi. 27). 

Exsiccatae : Phyc. Boreali-Amer., No. 823 as C. crinita. 

Localities: CAPE COD: Chara Pond J. M. Fogg, Jr., June 29, 1925 (MBL, 10240 WRT) ; 
W. R. Taylor, July 30, 1937 (17937 WRT, NY, FH) ; H. T. Croasdalc, July 14, 1946 (6612 
MSD) ; R. D. Wood 2004, June 28, 1947 (RDW, MBL, MSB. ETM); Little Pond F. S. 
Collins, July 25, 1882 (19776 WRT, RDW, NY, 132461 UC ;' dupl. herb. T. F. Allen, as C. 
crinita) ; W. A. Setchell and W. /. V. Osterhout, Aug. 17, 1895 (MBL) ; F. S. Collins, July, 
1886 (Y) : July 25, 1882 (NY, 132461 UC, RDW) ; Salt Pond Urda K. Wood, June 28, 
1947 (2003 RDW) ; R. D. Wood 2023, July 30, 1947 (RDW, NY) ; ? Pond, Woods Hole 
/. /. Copeland (16772 WRT) ; NANTUCKET: Coskata Pond T. Morong 5, July 15, 1887 (NY) ; 
II'. H. Sheldon, Aug. 6, 1931 (MMA) ; [coll.?], Aug. 21, 1896 (MMA) ; Polpis Maria Oivcn, 
July, 1879 (NY) ; Sesachacha Pond Dame, Jenks and Swin, July 14, 1880 (NY) ; F. S. 
Collins, July, 1886 (Y, NY, 7929 WRT, 823 P. B.-A. as C. crinita f. leptosperma) ; L. L. 
Dame, Aug. 1886 (NY) ; E. P. Bicknell 11633, Sept. 16, 1899 (NY) ; Wawinet T. F. Allen, 
Aug. 30, 1895 (132435 UC, NY, 19782 WRT, RDW; dupl. herb. T. F. Allen, as C. crinita 
Wallr., det. T. F. Allen) ; ? Pond L. L. Dame, Aug., 1880 (NY) ; July 11, 1886 (NY). 



K. D. WOOD 

7. Chara delicatula C. A. Agardh, Syst. Alg., p. 130. 1824. (iwm. illeg.) "' 

(Non) Cham delicatula Desvaux c.r Lois., Not. Fl. France, p. 137. 1810 

(== C. asficra (Dethard.) Willdenow, fide Migula (1897: 654)). 
Chara fragilis var. delicatula von Leonhardi, Verhandl. Naturf. Vereins Briinn 

2:209. 1864. 

Chara vcrrucosa Itzigsohn, Bot. Zeit. 8: 338. 1850, fide Robinson (1906: 280). 
References for the region : Chara fragilis: Croasdale (1935 : 96 [pi. not seen] ) ; var. 
delicatula: Owen (1888: 75) ; C. delicatula: Croasdale (1935: 96). 

(Plate II, Fig. 5; Plate IV, Fig. 3, 4) 

Plant monoecious, 10 cm. high; slightly incrusted with lime; starch bulbils abun- 
dant on rhizoids. Main axes regularly triplostichous, secondary cortical cells gen- 
erally somewhat smaller in diameter than the primary cells ; spines at cortical nodes 
reduced to mere papillae. Branchlets 8 in a whorl, articulations 7-10, corticated 
except for terminal 1-2 cells ; bracts at sterile nodes inconspicuous. Stipulodes 
apparently haplostephanous, reduced to mere papillae at some nodes. Gametangia 
conjoined at branchlet nodes, but frequently one or other sex appears to be lacking. 
Oogonia 850-1000 /x long by 450-500 /x broad; convolutions 12, very nearly per- 
pendicular to long axis of oogonium ; coronula elongate at maturity, 250-270 p. tall, 
cells generally connivent. Oosporcs 460-500 p. in diameter, black; striae 12-13; 
membrane opaque. Anthcridia 410-455 p. in diameter. (Descr. from R. D. Wood 
2032 (ROW).) 

Variations among the local forms are numerous, and result from differences in 
expression of the vegetative characteristics. In size, some plants reach 25 cm. in 
height. Other variations appear to be of two distinct orders : ( 1 ) a remarkably 
constant differentiation into forms with two well-developed series of stipulodes 
(diplostephanous) as in R. D. Wood & Urda K. Wood (2058 RDW) and forms 
with one well-developed series (haplostephanous) as in R. D. H'ood 2032; and 
(2) less constant differences which appear in random combination. The haplo- 
stephanous form is the typical form as described by Groves and Bullock- Webster 
(1924: 65) ; whereas the diplostephanous form (PL IV, Fig. 4) agrees closely with 
the description of var. barbata (Ganterer) Groves and Bullock-Webster (1924: 68, 
pi. 44, fig. 9). The features subject to less constant variation are several. In gen- 
eral habit, the plants vary from very attenuate form in W. R. Taylor (10171 WRT) 
to densely compact form in R. D. U'ood 2000. The number of ecorticated terminal 
branchlet articulations varies from 3-6 in R. D. U'ood 2002 to 1-2 in [/'. R. Taylor 
(10171 WRT). Generally, the penultimate cell of branchlet is not swollen, but it 
may be rather inflated as in R. D. U'ood 2022 or very elongate as in R. D. Wood 
2002. The general trend in expression, as in C ' . Braunii, is toward more attenuate 

5 The problem of just what is the valid name for this species is one of long standing. The 
plant has become known as C. delicatula C. Ag., but this name is a later homonym of C. 
delicatitla Desv. (= C. aspera, fide Braun ; cf. Groves & Bullock-Webster, 1924: 51, 67). 
Robinson (1906: 280) decided upon C. vcrrucosa Itzigsohn (1850: 338), but it is not known 
if he saw the type specimen of that plant. There are at least three synonyms in the literature 
prior to 1850, including C. pilifera C. Ag. (1824: xxviii), C. rirgata Kiitzing (1834: 56), and 
C. joliolata Hartman (1820: 378). The problem can be solved only by inspection of the type 
specimens, a task most easily accomplished by students in the countries in which the critical 
specimens are to be found. 



WOODS HOLE REGION CHARACEAE 199 

plants with longer branchlet articulations, shorter bracts, bracteoles, and stipulodes 
in quiet, deeper water; and compact, tufted, frequently bulbiliferous (with abundant 
starch bulbils on rhizoids) plants with longer bracts, bracteoles, and stipulodes in 
shallow littoral. 

This species varies widely, and approaches C. (jlobularis Thuill.(== C. fragilis 
Desv.) very closely. At one extreme, it is almost identical in vegetative characters 
to C. aspera. Croasdale's (1935) record of C. fragilis for Falmouth Heights was 
probably C. delicatula. 

Illustrations: Typical form Groves, H. & J. (1880: pi. 207, fig. la); Migula (1897: 
753; 1925: 242, fig. 7) ; Groves & Bullock-Webster (1924: pi. 44, figs. 1-8). Var. barbata- 
Groves & Bullock-Webster (1924: pi. 44, fig. 9). 

Exsiccatae: Phyc. Boreali-Amer., No. 1199 as C. frac/ilis subsp. dclicatula; Char. Amer. 
Exsicc No 22 

Localities f CAPE COD: Chara Haven R. D. Wood 2010, July 7, 1947 (ROW, NY); 
R D Wood & Urda K. Wood, Sept. 6, 1947 (2058 RDW) ; Chara Pond M. S. Doty, June 
28, 1947 (2001 RDW, MBL, MSD, ETM) ; R. D. Wood 2002, June 28, 1947 (RDW, NY) ; 
Little Pond, Falmouth Heights/?. D. Wood 2006, July 7, 1947 (RDW, NY), 2032, Aug. 1, 
1947 (RDW); Oyster Pond Annex [?]. S.E. of Oyster Pond W. R. Taylor, July 4, 1925 
(10172 WRT, MICH) ; Salt Pond W. R. Taylor, July 15, 1917 (2294 WRT) ; Hannah 
Croasdale, July 14, 1946 (6614 MSD) ; R. D. Wnvd 2000, June 28, 1947 (RDW, NY), 2018, 
July 26, 1947 (RDW), 2024. 2025 (NY, RDW), 2022, 2026, July 30, 1947 (RDW); CUTTY- 
HUNK: Gosnold Pond W. R. Tavlor, July 15, 1919 (2931 WRT) : Sheep Pond W. R. Taylor, 
July 9, 1925 (10171 WRT), June 28, 1932 (17012, 17083 WRT, NY), July 3, 1934 (16767, 
16766 WRT) ; R. D. Wood 2028, July 31, 1947 (RDW, NY), collected on inland edge of pond, 
2029, July 31, 1947 (RDW, NY) collected on seaward edge of pond; NANTUCKET: Sesachacha 
Pond L. L. Dame, July, 1886 (NY); F. S. Collins. July, 1886 (NY); NAUSHON : French 
Watering Place W. R. Taylor. July 6, 1920 (3105 WRT). 

8. Chara aspera Willcl.. Ges. Naturf. Fr. Berlin 3: 298. 1809. 

The writer has seen no herbarium specimens which are undisputably of this 
species for the region, and as such regards the local records as highly questionable. 
Of the specimens seen, the sterile condition rendered differentiation from C. delica- 
tula almost impossible, and recently collected fertile specimens have all proved to 
be C. delicatula Ag. However, during the summer of 1948, Dr. E. A. Palmatier 
and Dr. V. I. Cheadle collected excellent male and female specimens of C. aspera in 
Rhode Island just across the Massachusetts state line in Ashawonk's Swamp, 
Little Compton. The chances are good that C. aspera should be found in our re- 
gion in brackish-water ponds. 

The best practical distinguishing feature between the American forms of these 
two species seems to be the fact that C. aspera is dioecious, whereas C. delicatula is 
monoecious. A generalized description of C. aspera is almost identical to C. deli- 
catula in vegetative parts. In C. aspera, the female plant has whorls of branchlets 
which are more spreading than the closely convergent branchlets of the male plants. 
Antheridia are borne at most of the nodes of fertile branchlets of male plants. 
Oogonia are borne at the first three (occasionally fourth) fertile nodes of female 
plants. G. O. Allen (corresp., 1948) stated that normally C. aspera has long spine- 
cells [spines] which are short in C. delicatula. The writer has studied the New 
England specimens, particularly those for Massachusetts and Rhode Island, and 
concludes that this is helpful but finds that in this region C. aspera may be practically 
devoid of spines ; hence, this character of European plants does not appear to hold 
for our specimens. 



200 



R. D. WOOD 



Illustrations: Allen (1882b: pi. 21, fig. A, 1-3, 7); Migula (1897: 656, 657); Groves 
& Bullock-Webster (1924: pi. 39). 

Exsiccatae : Phyc. Boreali-Amcr., No. ]]V6; Char. Amer. Exsicc., No. 26. 27. 
Localities : none known. 

SPECIES EXCLUDED FROM LOCAL FLORA 

Cliara fragilis Desv. (== C. globularis Thuill.) ; from Chara Pond, Cape Cod, fide 
Croasdale (1935) - ? Chara dclicatula Ag. (specimen not seen by author). 

Nitella batrachosperma (Reich.) Braun (~N. Nordstedtiana Groves, H. & J.) ; 
from Nantucket, fidf Owen (1888, determined by T. F. Allen) == N. Moronyii 
Allen emend. Wood. 

Nitella gracilis (Sm.) C. Ag. ; from Wood Pond, Golf Pond, Cape Cod, fide Croas- 
dale (1935), Nobska Pond, fide F. S. Collins (P. B.-A., No. 1195} -- N. 
Morongii Allen emend. Wood. 

Nitella nia.vceana Allen, spec, dub.; from [Maxey's Pond] Nantucket, fide T. F. 
Allen (1896) = N. Morongii Allen emend. Wood. 

Nitella mucronata (Braun) Miquel var. gracillinia Groves & Bullock- Webster ; from 
Wood Pond, Cape Cod, fide Croasdale (1935) " N. megacarpa Allen. 

TABLE I 





Nitella 
flexilis 


Nitella 
mega- 
carpa 


Nitella 
Morongii 


Nitella 
transilis 


Chara 
Braunii 


Chara 
canescens 


Chara 
deli- 
catula 


Cape Cod 
















Ashumet Pond 





P 





X 











Chara Haven 














X 





X 


Chara Pond 














X 


PX 


X 


Desmid Haven 














X 








Golf Pond 








p 














Harper Pond 








X 





p 








Ice House Pond 














X 








John Pond 











p 











Leech Pond 





p 

















Little Pond 














X 


p 


X 


Nobska Pond 








p 





p 








Oyster Pond 














X 








Oyster Pond Annex 




















p 


Salt Pond 














X 


X 


PX 


Sandwich Pond 














X 








Summerneld, South 





p 

















Weeks Pond 


X 


X 





X 


X 








Wood Pond (Gansett Pond) 





p 

















Cuttyhunk 
















Clubhouse Pond 


X 




















Gosnold Pond 














p 





p 


Sheep Pond 


p 











PX 





PX 


Sheep Spring 


p 




















Martha's Vineyard 
















Chilmark Pond 


X 




















Tiasquam Dam 


X 





















WOODS HOLE REGION CHARACEAE 



201 



TABLE I Continued 





Nitella 
flexilis 


Nitella 
mega- 
carpa 


Nitella 
Morongii 


Nitella 
transilis 


Chara 
Braunii 


Chara 

canescens 


Chara 
deli- 
catula 


Nantucket 
















Cato's Pond 


P 




















Coskata Pond 

















P 





Long Pond 














P 








New Lane Pond 


X 




















Maxey's Pond 








P 














Polpis. 


P 














P 





R. R. Track Pond 


P 




















Sesachacha Ditch 


X 




















Sesachacha Pond 














P 


P 


P 


Siasconset Pond 








P 














Wawinet Pond 

















P 





Weweeder Pond 


P 











P 








Naushon 
















French Watering Place 














P 





P 


Petchett (Peckett) Pond 








X 














Nonamesset 
















South Pond 


P 




















Pasque 
















West End Pond (Nitella Pond) 


X 











X 








? Pond, near West End Pond 














X 








? Pond, smaller pond near S. W. 
















end of island 














P 








? Pond, near west end of island 


X 




















Penikese 
















Typha Pond 














X 









LOCALITIES FOR CHARACEAE IN WOODS HOLE REGION 

In the above table, all localities are listed from which specimens of Characeae 
have been seen by the writer. The substantiating specimens are listed in the text 
under "localities" for each species. Because of the great effect of the hurricane of 
1938 on the coastal ponds of the region, the writer has chosen this date as the 
critical year. Specimens collected prior to 1938 are recorded as "P." Specimens 
collected since 1938 are recorded as "X." If no collections have been seen by the 
writer, this is recorded as "0." 



EXSICCATAE CITED 

ALLEN, T. F., 1880-1893. Characeae Americanae Exsiccatae, Fasc. I-V. (For dates of issue, 

cf. Wood (1948).) 
COLLINS, F. S., I. HOLDEN, AND W. A. SETCHELL, 1895-1919. Phycotheca Boreali- Americana, 

Fasc. I-XLV, A-E. 
TILDEN, JOSEPHINE E., 1894-1909. American Algae, Fasc. I, Cent. 1-7. 



202 R. I). WOOD 

LITERATURE CITED 

AGARDH, C. A., 1824. Systema Algarum. 1. Lund. 

ALLEN, T. F., 1871. Characeae. But!. Torrcy Rot. Club, 2 (3) : 9-10. 

ALLEN, T. F., 1879. Characeae Americanae. 1: pi. 1; 2: pi. 2. New York. 

ALLEN, T. F., 1880. The Characeae of America. 1 : 1-8, pi. 1-3; 2: 9-14, pi. 4-6. Boston. 

ALLEN, T. F., 1882a. Development of the cortex in Chara. Hull. Torrcv Hot. Club, 9: 37-47, 

pi. 15-22. 
ALLEN, T. F., 1882b. Observations on some American forms of Chara coronata. Aincr. Nat., 

16: 358-369, 1 pi. 

ALLEN, T. F., 1887. Some notes on Characeae. Bull. Torrcv Bot. Club, 14: 211-215, pi. 71-75. 
ALLEN, T. F., 1892. The Characeae of America. 2 (1): 1-8. pi. 1-8. New York. (For 

system used for citation and numbering of plates, cf. Wood, 1948b: 334.) 
ALLEN, T. F., 1894. Ibid., 2 (2) : 9-17, pi. 9-17. 
ALLEN, T. F., 1896. Ibid., 2 (3) : 19-28, pi. 18-27. 
BRAUN, A., 1834. Esquisse monographique de genre Chara. Ann. Sci. Nat., ser. II, 1: 349- 

357. 
BRAUN, A., 1847. Uebersicht der schweizerischen Characeen. Ncitc Dcnkschr. Schwcis. 

Gesell. Natunc., 10 (3) : 1-23. 
BRAUN, A., 1849a. Charae australes et antarcticae. Hooker's Journ. Bot. and Kat> Card. 

Misc., 1 : 193-203. 

BRAUN, A., 1849b. Characeae Indiae orientalis et insularum maris pacificis. Ibid., 1 : 292-301. 
BRAUN, A., 1882. Fragmente einer Monographic der Characeen. Nach den hinterlassenen 

Manuscripten A. Braun's herausgegeben von Dr. Otto Nordstedt. pp. 1-211, pi. 1-7. 

Berlin (also in Abli. K. Akad. ll'iss. Berlin (1882): 1-211, pi. 1-7. 1883). 
CROASDALE, HANNAH T., 1935. The Fresh Water Algae of Woods Hole, Massachusetts. 134 

pp., 8 maps. Philadelphia. 
DESVAUX, A. N., 1810. /;; Loiseleur-Deslongchamps. (Text for new species accredited to 

Desvaux in footnote to p. 135.) 

FRITSCH, F., 1935. The Structure and Reproduction of the Algae. 1. 791 pp. Cambridge. 
GANTERER, U., 1847. Die bisher bekannten osterreichischen Charen. (Dissert, pp. 1-21, 2 pi.) 

Vienna. 

GMELIN, C. C., 1826. Flora Badensis Alsatica., 4: (cf. pp. 643-647). Carlsruhe. 
GROVES, H. AND J., 1880. A review of the British Characeae. Jour. Bot., 18: 97-103, 129- 

135, 161-167, pi. 207-210. 

GROVES, J., 1916. On the name Lamprothamnus, Braun. Jour. Bot., 54 : 336-337. 
GROVES, J., AND G. R. BULLOCK-WEBSTER, 1920. British Charophyta. 1. London. 
GROVES, J., AND G. R. BULLOCK-WEBSTER, 1924. Ibid., 2. 
HALSTED, B. D., 1879. Classification and description of the American species of Characeae. 

Proc. Boston Soc. Nat. Hist., 20: 169-190. 

HARTMAN, C. J., 1820. Handbok i Skandinaviens Flora. Stockholm. 
HY, F., 1889. Sur les modes de ramification et de cortication dans la famille des Characees, 

et les caracteres qu'ils peuvent fournir a la classification. Bull. Soc. Bot. France, 36: 

393-398. 

ITZIGSOHN, H., 1850. Charologisches. Bot. Zcit., 8: 337-340. 
KUTZING, F. T., 1834. Beschreibung einiger neuen Arten der Gattung Chara. Flora, 17 : 

705-707. 
LANJOUW, J., 1939. On the standardization of herbarium abbreviations. Cliron. Bot., 5: 142- 

150. 

LEONHARDI, VON, H., 1863. Die bohmischen Characeen. Lotos, 13: 55-80, 110-111. 
LEONHARDI, VON, H., 1864. Die bisher bekannten osterreichischen Armleuchter-Gewachse. 

Vcrhandl. Naturf. Vercins Briinn, 2: 122-224. 
LINNAEUS, C., 1753. Species Plantarum. 2. Stockholm. 
LINNAEUS, C., 1754. Genera Plantarum, ed. 5. Stockholm. 
LOISELEUR-DESLONGCHAMPS, J. L. A., 1810. Notice sur les Plantes a aj outer a la Flore de 

France. Paris, 1810. 
MIGULA, W., 1890-1897. Die Characeen Deutschlands, Osterreichs und der Schweiz. In 

L. Rabenhorst, Kryptogamen-Flora von Deutschland, Oesterreich und der Schweiz. 

Ed. 2. 5 : 1-765, fig. 1-149. 



WOODS HOLE REGION CHARACEAE 203 

MIGULA, W.. 1925. Charophyta (Charales). In A. Pascher, Die Siisswasserflora Deutsch- 

lands, Osterreichs und der Schvveiz. 11: 207-243, fig. I-XIV. 

NORDSTEDT, C. F. O., 1882. In A. Braun, Fragmente einer Monographic der Characeen. 
OPHEL, I. L., 1947. Notes on the genera Lychnothamnus and Lamprothamnium (Characeae) 

Trans. Roy. Soc. S. Australia 71 (2) : 318-322, fig. 1-2. 

OWEN, MARIA L., 1888. Plants of Nantucket. A catalogue of plants growing without cultiva- 
tion in the county of Nantucket, Mass. Northampton. 

ROBINSON, C. B, 1906. The Chareae of North America. Bull. -V. V. Dot. Card., 4: 244-308 
RUPRECHT, F. J., 1845. Distributio Cryptogamarum vascularium in Imperio Rossico. Bcitrage 

zur Pflanzcnkitndc dcs Rnssischcn Rcichcs.. 3: 7-18. Petrograd. 
WALLROTH, C. F. W., 1815. Annus Botanicus. Halle. 

WILLDENOW, C. L., 1809. Fiinf neue Pflanzen Deutschlands. Gcscll. Xat. Frcundc, 3 : 298. 
WOMERSLEY, H. B. S., AND I. L. OPHEL, 1947. Protochara, a new genus of Characeae from 

Western Australia. Trans. Roy. Soc. Australia, 71 (2) : 311-317, 2 figs. 
WOOD, R. D., 1947. Characeae of the Put-in-Bay region of Lake Erie (Ohio). Ohio Jour. 

Sci, 47 : 240-258, pi. 1-4. 
WOOD, R. D., 1948a. Proposed dates for T. F. Allen's exsiccatae (Characeae). Far! anna, 3 

(3) : 327-329. 
WOOD, R. D., 19485. A review of the genus Nitella (Characeae) of North America. Far- 

hw'm, 3 (3) : 331-398, pi. 1, 2. 
WOOD, R. D., 1949. Monographic studies of the Characeae. I. Emendation of Nitella 

Morongii Allen. Rhodora, 51 (602) : 13-18. 1 pi. 
\VOODS, A. F., 1894. Characeae. In Flora of Nebraska. 2: 122-128, pi. 25-36. Published 

by the Botanical Seminar. Lincoln. 
ZANEVELD, J. S., 1940. The Charophyta of Malaysia and adjacent countries. Bluinca, 4: 1- 

224, fig. 1-20. 



Vol. 96, No. 3 June, 1949 

THE 

BIOLOGICAL BULLETIN 

PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY 



RADIOACTIVE SODIUM PERMEABILITY AND EXCHANGE 

IN FROG EGGS 

PHILIP H. ABELSON 
Department of Terrestrial Magnetism. Carnegie Institution of Washington 

AND 

WILLIAM R. DURYEE 
National Cancer Institute 

STATEMENT OF THE PROBLEM 

The transfer of ions in both directions across the cell membrane is a matter 
of major biological importance. While problems involving permeability have led 
to a vast and prolix literature, only meager attention has centered on the specific 
distribution of electrolytes within single cells. The limitations of earlier techniques 
were emphasized by Hastings (1941) in his Harvey lecture on the tissues and body 
fluids, in which he early recognized the value of radioactive isotopes. The subject 
of permeability has been recently reviewed by S. C. Brooks (1945). Our interest 
has centered in the dynamic equilibria of a protoplasmic system, their control by 
diffusion and the blocking of internal exchange. Use of radioactive tracer tech- 
niques has given new precision to the qualitative and quantitative study of small 
ionic transfers within cellular dimensions. Accordingly, in this paper are given 
results of an investigation undertaken to determine how much and how fast traced 
sodium (containing Na 24 ) exchanges with normal sodium (Na'- 3 ) in a verte- 
brate egg. 

MATERIALS AND METHODS 

Ovarian "winter" eggs of the frog, Raua plpiens, were chosen because they are 
single cells, spherical and of large size. Throughout the fall and winter quarters 
of the year, they are readily available in quantity (without hormone stimulation). 
In addition to the vitelline membrane, ovarian eggs possess two thin epithelial 
layers which could be removed successively by fine-pointed forceps. However, in 
most of the experiments, these membranes were left intact, because of the possi- 
bility of injury to the cell surface. Amphibian eggs are ideal for volume measure- 
ments. One unique advantage is that they possess an extremely large nucleus, or 
germinal vesicle, approximately 0.5 mm. in diameter, which may be isolated easily 
under a dissecting microscope in a Ca-free medium (Duryee, 1937). 

205 



206 



PHILIP H. ABELSON AND WILLIAM R. DURYEE 



Eggs were isolated individually in standard Ringer solution : NaCl, 0.66 g. ; 
KC1, 0.014 g. ; CaClo, 0.012 g. ; distilled H 2 O, 100 ml. All solutions were buffered 
to pH 7.6 with NaHCO;.. As described below, Na L>! was incorporated in experi- 
mental solutions as NaCl. 

Active healthy frogs were selected. After ovaries of pithed animals were 
removed as bloodlessly as possible, fresh Ringer solution was poured over them 
before transfer to small beakers. A small lobe of an ovary was next cut off with 
iridectomy scissors taking care not to injure the cells. Groups of 20-30 eggs were 
next transferred to Syracuse dishes where individual eggs were cut apart. All 
small yellow or transparent eggs were removed and the ovarian wall was trimmed 
off flush with the theca membranes of each full-size egg. In this way, using a 
dissecting microscope to insure precise excision, it was possible to obtain from 
40 to 60 separate eggs per hour. Average volume was 2.86 mm 3 , based on an 
average diameter of 1.76 mm. An important step was the reexamination of indi- 
vidual eggs to reject those which might have been nicked or crushed. Aspherical 
eggs were also discarded along with any less than 1.65 mm. in diameter. Follow- 
ing these procedures, a final screening for uniformity of diameter was made. This 
consisted of shaking approximately 100 eggs in a flat bottom shallow dish to obtain 
a single layer with close packing. Any cell not obviously tangent to its six neigh- 
bors was discarded. 

Eggs were conveniently handled individually with a pipette having an internal 
diameter just larger than that of the cell itself. In a few experiments where short 
exposures involving minimum transfer of external radioactive material was essen- 
tial, each egg was advantageously picked up by the short flap of theca membrane 
with sharp forceps. 




FIGURE 1. Sectional view of plexiglas cup for measuring radioactivity in frog egg. Actual size. 

For experiments on dead cells, a method of killing involving minimal change 
was sought. After rejecting reagents such as alcohol, acetic acid and KCN, we 
standardized on heating to 65 C. for ten minutes in normal Ringer. 

Groups of 10-20 eggs were exposed in small beakers containing 20 ml. of Ringer. 
Temperature was maintained in water baths controlled to 0.5 C. The presence 
of water vapor surrounding the beakers kept down evaporation, which by weighing 
was found to be negligible over periods up to 24 hours. 

Upon removing a cell from the radioactive Ringer, it was put through two 
washes of normal Ringer before transfer to the measuring cup. One or two eggs 
were first transferred in a vertically held pipette allowing them to sink to the 
meniscus. By merely touching the surface of the washing fluid, the eggs dropped 



PERMEABILITY TO RADIOACTIVE SODIUM 207 

from the pipette, thus avoiding transfer of more than one or two cubic millimeters 
of previous fluid. 'Washing vials each held 7 ml. It was considered advisable to 
use a fresh pipette for each successive transfer. Since it was found that Na 24 
could be washed from the cell interiors, the process was reduced to a standard 
thirty seconds. 

For measurement in the Geiger counter, eggs were transferred from the second 
washing vial to special plexiglas cups (see Fig. 1) again in a vertically held 
pipette. Not more than 5 mm 3 of the second wash fluid 'was thus transferred 
with the egg. Tests of this fluid showed negligible radioactivity. 

Radioactive solutions 

The radioactive Na 24 was prepared in the cyclotron by bombardment of metallic 
sodium with 16 m.e.v. deuterons. The metal was dissolved in ethyl alcohol and 
converted into NaCl with 12 N hydrochloric acid. The mixture was then dried 
and ignited at red heat to produce a neutral salt free of organic material. Weighed 
amounts of the radioactive salt were converted into amphibian Ringer solution. 
To obtain a relation between quantity and radioactivity of Na 24 , a known aliquot 
of the Ringer was evaporated to dryness and measured with a Geiger counter. A 
typical result was as follows : 1 ml. of amphibian Ringer was diluted to 500 ml. 
A volume of 0.039 ml. of this solution was dried and measured on the counter. 
Since 1 ml. of the Ringer contained 0.026 g. Na, the aliquot possessed 0.026 X 1/500 
X 0.039 == 2.03 X 10- 7 g. Na. This sample gave 1600 counts/min. Thus 1000 
counts/min. represented 1.27 X 10~ 7 g. Na. 

For each solution made up with radioactive sodium, controls were made up 
with identical amounts of normal NaCl. The radioactivity of the solutions was 
such that the radiation level was less than an amount which would produce 1 
r.e.p./min. No visible cytological changes were produced by this level of radiation. 
In view of the well-known tolerance of frog eggs to much larger dosage of X-rays, 
it is fair to assume that radiological effects were negligible in our results. 

Radio-autographs 

A new variant of conventional methods of making radio-autographs has been 
devised to fit the purpose of recording radioactivity distribution within a single cell. 
Our technique consisted essentially of quick-freezing in liquid air, followed by 
sectioning the cell to a known thickness, and then exposing the section in a light- 
proof cold box to a photographic plate. 

Eggs were rapidly rinsed (5 sec.) in two washes of normal Ringer (7 ml. 
each), oriented in a 1.8 mm. hole in thin calibrated bronze strips (1.5 X 3.5 cm. 
and either 80 or 200^. thick) and plunged into liquid air. The orientation was 
such that the axis of the egg was parallel to the surface of the strip. It w r as calcu- 
lated that freezing of a 1.7 mm. egg at - - 180 C. would be completed at the center 
in about 0.2 sec. This time should be compared with the ten minutes necessary 
for partial equilibrium of ionic diffusion in the egg at normal temperatures. The 
bronze strip containing the frozen cell was then placed on a previously cooled 
brass block (see Fig. 2) and both were transferred to the stage of a dissecting 
microscope. Using a cooled razor blade mounted in a special non-conducting 
handle, the frozen egg was sliced down to expose the nucleus. Orientation was 



208 



PHILIP H. ABELSON AND WILLIAM R. DURYEE 



thus re-checked to insure that the plane of section was being made parallel to the 
egg axis. Improperly oriented eggs were discarded. Subsequent slices through 
the lateral third of the nucleus were made, making the surface even and parallel 
with the bronze strip. The nucleus always stood out sharply as a white circle in 
a grey background of frozen cytoplasm. Finally the strip was inverted and the 
cell was sectioned similarly from the other side. Frozen sections were conveniently 
temporarily stored in a cold slotted brass holder in an insulated box, cooled with 
solid CO 2 . 




FIGURE 2. Brass cooling block for holding frozen egg and supporting strip during section- 
ing. Note that both holes corresponding to eggs in holder are displaced laterally from the 
main axis of the block, so that the strip, when inverted after first sectioning, brings cut surface 
against flat portion of the block. Screw at each end makes convenient attachment for handle. 

Actual exposure of the section to film took place in a well-insulated chest fitted 
with a copper bottom and copper slide holder. One hundred pounds of dry ice 
kept the temperature in the slide holder at approximately - 40 C. for a week. 
Medium-contrast lantern slide plates 1 were laid in the slide holder, emulsion side 
up, and the cold bronze strips with the frozen sections were laid on the plates, 
being weighted to insure even contact. With the activity of our solutions, it was 
found necessary to expose from two to twelve hours to provide the desired darken- 
ing. Plates were developed in Eastman D-19 developer. 

A typical calculation indicates the amounts of radioactivity involved. A section 
of a frog egg 80 ^ thick contained 2.0 X 10~ s g. of exchanged sodium. This sample 

1 We have learned from Dr. Kenneth Endicott, of the National Institutes of Health, that 
Ansco non-screen X-ray films are particularly useful where only low intensities of radiation 
are present. 



PERMEABILITY TO RADIOACTIVE SODIUM 209 



gave 320 counts/min. on the counter. The actual number of disintegrations oc- 
curring in the egg section was approximately 20/sec. An exposure of four hours 
sufficed to give a good darkening showing a clear differential distribution between 
nucleus and cytoplasm. This amounts to 288,000 electrons, about half of which 
went into 0.026 cm 2 of the plate emulsion. 

RESULTS 
Sodium content of ovarian eggs 

The sodium content of frog eggs is not well known. Bialaszewicz (1929) has 
given an approximate figure of 42 mg. Na/100 g. wet weight of eggs of Rana 
temporana. It seemed desirable, therefore, to determine sodium on our experi- 
mental animals. For this purpose, both ovaries were removed from six animals. 
The ovaries were rinsed quickly in distilled water, blotted, weighed, and cautiously 
ashed over low heat by concentrated HNO :( , followed by concentrated H 2 SO 4 . 

After removal of a small amount of insoluble material, probably CaSO 4 , the 
sodium was determined as (UO,) :! ZnNa(CH s COO) 9 -6H,O according to the 
method of Barber and Kolthoff (as given in Hillebrand and Lundell, 1929). Re- 
sults obtained from six pairs of ovaries gave a preliminary value of 137 mg. Na 
per 100 g. wet weight of eggs (Duryee and Abelson, 1947). Further study has 
shown these results to be too high and that PO 4 interferes with the accuracy of 
the method. This is at least in part due to the formation of an insoluble uranium- 
phosphorus compound which tends toward giving high results for the sodium value. 
Accordingly the procedure was modified. Following wet ashing, excess acid was 
driven off, the residue dissolved in distilled water, and the solution made alkaline 
with NH 4 OH. A 25 per cent barium acetate solution was added until no further 
precipitate formed, followed by a few drops excess. The mixture was allowed to 
stand for half an hour and then centrifuged. The precipitate was washed twice 
with distilled water and the washings added to the first supernatant. The solution 
was evaporated to dryness, the residue dissolved in 1 ml. of distilled water, and 
the sodium precipitated with the uranyl zinc acetate reagent. The following results 
were obtained from six pairs of ovaries : 

Wt. of ovaries 

(grams) mg. Na mg. Na/g. ovary 

8.1 6.8 0.84 

9.3 7.40 0.80 
9.0 7.4 0.82 
8.5 7.0 0.82 
7.0 5.9 0.80 

8.4 7.1 0.84 



Average 0.82 mg. Na 

There remained the question of how well the sodium content of the whole ovary 
approximates that of the eggs. Since the connective tissue, blood vessels and 
peritoneum formed only a minor fraction of the ovary, it seemed that the error 
introduced by neglecting this factor would be small. A sodium determination on 
separated eggs gave 0.80 mg. Na/g. wet weight. Eggs averaged 1.76 mm. in 
diameter with a volume of 2.86 mm 3 , a density of 1.10, and a calculated sodium 
content of 25.8 X 10~ 7 g. Na per egg. 



210 



PHILIP H. ABELSON AND WILLIAM R. DURYEE 



Water content of eggs 

In analyzing the results, it was found desirable to measure the water content 
of the eggs. Accordingly four determinations were made two with separated 
eggs and two with ovaries. Excess moisture was blotted up with filter paper. The 
eggs were weighed and then dried. to constant weight at 120 C. for one hour. A 
water content of 52 per cent was found. This corresponded to 1.63 mg. of water 
for our standard 1.76 mm. diameter egg. 

An interesting observation gave evidence on the water content of the nucleus. 
In six sets of eggs which were frozen whole, the half sections, when thawed and 
dried, showed a negative mold of the nucleus with only a flake remaining. This 
indicates the relatively higher water content of the nucleus above that of the yolk- 
cytoplasm. 



40 - 



30 - 



Z 

CO 

2 
< 

cr 
o 



i 
o 



SODIUM CONTENT OF NORMAL EGG 



20 - 



10 - 




DEAD EGG 



LIVE EGG 



A.R. - 



01234 

HOURS 

FIGURE 3. Comparison of uptake of Na 24 by live and dead eggs in amphibian Ringer. 
Average value of sodium content of single egg, as determined by chemical analysis, shown by 
dotted line. Each point represents average measurements on ten separate cells. 



PERMEABILITY TO RADIOACTIVE SODIUM 211 

Sodium exchange 

In the course of the research a total of twenty experiments were performed 
involving 400 individual eggs. The experiments gave a reproducible set of values 
with a probable variation of plus or minus ten per cent. A comparison of the 
exchange of sodium in live and dead eggs is shown in Figure 3. Each point on 
the curve represents an average of ten measurements. In the case of live eggs, 
the following features are to be noted : Initially there was a rapid penetration of 
the traced sodium into the egg. Within an hour, ho\vever, the amount of the 
traced substance within the cell ceased to increase. The level reached amounts to 
12 per cent of the total sodium content of the egg. By testing samples of cyto- 
plasm and later by making radio-autographs, it was shown that the traced sodium 
was principally inside the egg. 

With dead eggs (Fig. 3) the exchange of sodium was extremely rapid. The 
amount of traced sodium which could be found in the egg rose above the total 
sodium content of the live cell as determined chemically. This entrance of extra 
sodium can be related to at least two facts : We have observed a loss of potassium 
from the cell on death. The second fact is that the sodium content of amphibian 
Ringer for a volume equal to that of the frog egg is 78.4 X 10~ 7 g., or more sig- 
nificantly, the sodium present in a volume of Ringer equal to that of the cell's 
water content is 42.4 X 10 7 g. Na. 

To study further the nature of the sodium uptake process, eggs were first 
exposed to radioactive Ringer, washed and then immersed in an inactive Ringer 
solution. The results are shown in Figure 4. During the first hour, a typical 
curve was obtained for penetration of the traced sodium. The downward curve 
was obtained during exposure to inactive Ringer. The time required for the 
traced sodium to leave the cell was about the same as the time involved in the 
initial penetration. Chemical analysis showed that the total sodium content of the 
egg did not change during these experiments. When dead eggs containing tracer 
sodium are exposed to inactive Ringer, the traced sodium likewise leaves the egg 
very quickly. 

Since calcium is thought to be important in permeability relationships, experi- 
ments were designed to investigate the effect of this ion on sodium exchange. 
Three solutions were made up with tracer sodium : The first, an ordinary Ringer 
solution ; the second, with calcium not present ; and a third, with double the usual 
calcium content. When eggs were exposed to these solutions and measured as 
before, the curves shown in Figure 5 were obtained. These curves show that 
calcium only slightly influenced that fraction of the sodium (12 per cent) which is 
exchangeable. After several hours, however, lack of calcium resulted in an 
increased exchange of the sodium. 

A series of experiments was made to determine the effect of temperature on 
exchange of sodium. Experiments were conducted at 9 C., 20 C., and 30 C. 
While the speed of penetration of sodium was somewhat faster at the higher tem- 
perature, the level reached after an hour was almost identical in all cases. After 
three hours approximately 6 X 10~ T g. Na was found per cell, while the low tem- 
perature values were essentially those of the controls at 20 C. It is apparent, 
therefore, that sodium exchange is not highly dependent on cellular metabolism. 



212 



PHILIP H. ABELSON AND WILLIAM R. DURYEE 



.^ 
$ 

O 




Q 
O 



/ 
/ 



/ 



3 
o 

i fi 



c/1 
u 



P 5 
kl .> 



" r7 

03 PH 

03 g 
C u 



P< 

3 



SWVtiO - 



O 



rt 

a. 

o 
U 



o 
i 

fe 



PERMK AIMLITV TO RADIOACTIVE SODUM 



213 





T 1 ' T ' ' 


1 


M 




i 




| 




1 


1 










i 




\ fY 




CJ I 




t 





% ' 




u \ < 


(J 


5 -~- 




' \ 


i + 


|JCJ 




\ 




^ _|_ 




\ 




r 


. 


k 




"~* S 

* i 4-' 

ro 




\ 




j/j | 









~ '5 




\ 





1> O 




1 




be u 




< \ 




'T, n 




1 \ 


I 


.S "5 






; 


CO II 




z 




o: s ^ 




Li. J 


; - 


CVJ D x Si 




o 


i 


O ^-S 






; 


1^"* CH 
.3 












~7 


i 


'O 1U 




LJ 




in .S 




1- 


i 


M-H 

O t3 




o 


; 


^ "5 




(J 


1 


re 








1; ^. 




D 


i 


~^ - ' 

r- 





* 


4- - 


'j 




O 




r ^- 






I 


1^ 




4 


it 


M-l J2 




\ \* 


s S 




1 " 


w re 




\\\ 


|-a 




^^^^^1 

A\ 


i 5 




1 1 1 1 1 1 ^ 




? iX 


:> O C 


C\J 



214 



PHILIP H. ABELSON AND WILLIAM R. DURYKK 




FIGURE 6. Radio-autograph of ovarian egg exposed to Na" 4 for thirty minutes. Cell \\as 
frozen in liquid air and sectioned parallel to main axis. Dark portion in upper third corre- 
sponds to size and position of the cell nucleus. 




-CUE 7. Radio-autograph of dead ovarian egg exposed to Na" 4 for five minutes, 
"ring" is proportional to diffusion rate of sodium inside the cell. 



Width of 



PERMEABILITY TO RADIOACTIVE SODIUM 215 

The question of where the sodium goes inside a cell seemed of fundamental 
importance. Many attempts were made to answer this question with but little 
success. It was of course possible to isolate single nuclei from exposed eggs. 
Such nuclei were washed twice in calcium-free nuclear medium to remove outside 
"cytoplasmic" sodium and were then placed individually on plastic cups for meas- 
urement with the Geiger counter. The counts per nucleus were always signifi- 
cantly higher than background, but showed disappointing variation. .It w r as 
concluded that the washing process introduced an uncontrollable variable. Con- 
sequently, we turned to the radio-autograph technique. In Figure 6 is shown an 
enlargement of a typical radio-autograph of a frog egg frozen in liquid air and 
sectioned meridianally. Of 47 autographs of single cells exposed to Na 24 for 
more than 30 minutes, all showed denser silver particles in the nuclear area than 
in the cytoplasm, when the plane of section was through the germinal vesicle. In 
those autographs of eggs sectioned to one side of the nucleus, a uniform density 
was found throughout the endoplasmic area. 

In the analysis of sodium diffusion rates inside the cell, advantage was taken 
of the radio-autographic records of penetration. In Figure 7 is shown an enlarge- 
ment of a radio-autograph obtained by exposure of a dead egg to radioactive Ringer 
for five minutes followed by washing, freezing, and sectioning. When live eggs 
were exposed to the active Ringer, for five minutes, followed by standard technique, 
a similar ring-shaped autograph was also obtained with the density of the darkening 
much less. 

DISCUSSION 

To serve as a basis of comparison, a calculation ~ has been made of the time 
required for diffusion processes to exchange sodium in an element of fluid the size 

2 An exact calculation of diffusion in an object similar to the frog egg would be practically 
impossible. First, there is the inhomogeneity of nucleus and cytoplasm. Second, is the fact 
that only a part of the sodium in the egg exchanges. One important factor in the calculation is 
that these experiments trace the behavior of Na 24 in a medium under conditions where there is 
no net transport of sodium or chloride ions. Therefore, in calculating the diffusion of Na 24 in 
Ringer solution, one should use the self-diffusion coefficient of Na + rather than that of NaCl. 
Fortunately, the self-diffusion of Na has been measured in sodium chloride solutions by Jehle 
(1938) and in sodium iodide solutions by Adamson (1947). In the experiments the concentra- 
tion of salt is the same on both sides of a diaphragm, and a very small amount of radioactive 
sodium is added to one of the solutions. From the rate of appearance of tracer sodium in the 
second solution the self-diffusion coefficient can be measured. The value observed was 1.2 X 10~ 
cnr./sec. in 0.113 M NaCl solution, and 1.23 X 10"'"' cnr./sec. in 0.113 M sodium iodide solutions. 

A calculation giving useful information regarding diffusion into a sphere can be made by 
employing formulas presented by Barrer (1941). The formulas have been applied to "Ionic 
Exchange Absorption Processes" by G. E. Boyd et al. (1947). In the case of a completely 
permeable sphere initially free of the diffusion ion, the time required for the solute to reach half 
of its equilibrium value is given by 

00 

<2/<2, = 1/2 = 1- 6/7T 2 2 1 /n z exp. ( -D^n^t/r 2 ) 

n=l 

where Q^ is the equilibrium value of the quantity of traced sodium, Q is the value at any time 
/, D is the diffusion coefficient of Na taken here as 1.23 X 10~ 5 cm 2 ./sec., and r the radius is 0.088 
cm. By a method of successive approximations the above equation is satisfied when Dw 2 //r 2 = 
0.3 and t = 19 seconds. 



216 PHILIP H. ABELSON AND WILLIAM R. DURYEE 

of a frog egg. Assuming no retardation at the surface of the object and a diffusion 
coefficient of 1.23 X 10 5 cnv/sec. internally, the time required for half of the 
sodium to exchange is 19 seconds. In the case of the actual egg, the time required 
for half of the exchangeable sodium to exchange is 15 minutes. The exchange of 
sodium in the egg may be slower for at least two reasons : delay in passing through 
the cell membrane, and the existence of a low diffusion coefficient within the egg. 
The fact that we have obtained "ring" radio-autographs after exposure of five 
minutes is explainable only on the basis that the movement of sodium in the egg 
is much slower than in Ringer solution, and that the membrane has little limiting 
effect on the exchange. 

On the assumption that the membrane presents no barrier, one can calculate a 
value for the diffusion coefficient within the egg. Thus, for Q/Q : : 1/2, Dirt/r' 
- 0.3, and D " 2.6 X IQ- 7 cnr./sec. 

From the curves given in Figures 3 and 5, it is clear that only 12 per cent of 
the sodium in the ovarian frog egg is readily available for exchange. Part of the 
remaining sodium can be exchanged over a period of many hours. Since the 
factors which govern this slow exchange are wholly within the egg itself, some 
form of internal blocking must occur. Speculation concerning the mechanism is 
still unwarranted. We believe that the finding that 12 per cent of the sodium 
behaves differently from the remainder is of considerable importance to others 
who are performing tracer experiments. In this experiment, the behavior of the 
traced substance was not characteristic of the behavior of all the sodium of the cell. 
Upon the other hand, it would appear that the difficulty raised might be turned 
into an advantage. One has a technique for studying the various degrees of 
binding and the chemical activity of cellular components. 

It is of special interest that sodium could be traced into the germinal vesicles. 
Our experiments thus show that the nuclear membrane is permeable to sodium. 
They also show that sodium is normally present in a cell nucleus. It is especially 
significant that the concentration of this cation in the nucleus is at least double 
that of the cytoplasm. The relatively large amount of active sodium found in the 
nucleus may be connected with the fact that the nucleus has a much higher per- 
centage of water than does the yolk cytoplasm. 

Our data on the increased permeability of dead cells to Na 24 offer a basis for 
explaining some of the empirical observations of Quimby (1947). Working with 
second and third degree burns she was able to show that the more severely dam- 
aged tissues took up much greater amounts of tagged sodium. These results must 
be due to the presence of dead cells. Furthermore, she found that addition of 
hypertonic saline materially aided recovery. It is clear, therefore, that in both 
sets of observations a fundamental factor is operating. This epitomizes, in the 
case of sodium, what many physiologists have long believed to be a basic diagnostic 
character of life namely, that a living cell can discriminate between ions both 
quantitatively and qualitatively in its extracellular environment and can build 
specific internal gradients and unequal distributions, which a dead cell certainly 
cannot do. As we have shown in the case of the amphibian egg, permeability to 
radioactive sodium can be used as an indication of life or death in a cell. 



PERMEABILITY TO RADIOACTIVE SODIUM 217 

SUMMARY 

1. The ovarian egg of the frog Rana pipicns is freely permeable to Na'-' 4 . 

2. At least two different types of binding limit the internal diffusibility of 
sodium within the egg. Only 12 per cent of the normal sodium is readily exchange- 
able. The remainder exchanges very slowly. 

3. The implications of finding non-equilibration of such a simple ion as sodium 
are presented. 

4. A new technique for making radio-autographs of single cells shows that after 
half an hour sodium is distributed almost uniformly throughout the cytoplasm. 

5. Calculations based on rate of exchange of sodium into the egg plus radio- 
autograph evidence give a value of 2.6 >: 10~ 7 cm-. /sec. for the diffusion coefficient 
of sodium within the egg. 

6. At equilibrium the nucleus possesses approximately twice as much traced 
sodium per unit volume as the cytoplasm. 

7 . By direct chemical analysis the sodium content of frog eggs was found to 
be 0.082 per cent of the wet weight.- 

LITERATURE CITED 

ADAMSON, A. W., 1947. Measurement of Na* ion diffusion by means of radiosodium. Jour. 

Chem. Phys., 15: 762. 

BARKER, R. At., 1941. Diffusion in and through solids, p. 29. Cambridge Press. 
BIALASZEWICZ, K., 1929. Recherches sur la repartition des electrolytes dans le protoplasme 

des cellules ovulaires. Protoplasuia, 6: 1-50. 
BOYD, G. E., A. W. ADAMSON, AND L. S. MYERS, JR., 1947. The exchange adsorption of ions 

from aqueous solutions by organic zeolites. Jour. Aincr. Chan. Soc., 69: 2838. 
BROOKS, S. C, 1945. Permeability. Annual Rciicw of Physiology, Annual Reviews, Inc., 

Stanford University. 
DURYEE, W. R., 1937. Isolation of nuclei and non-mitotic chromosome pairs from frog eggs. 

Arch. f. c.vp. Zcllf., XIX: 171. 
DURYEE, W. R. AND P. H. ABELSON, 1947. Permeability to radioactive sodium in frog eggs. 

Blol. Bull, 93 : 225. 
HASTINGS, A. B., 1941. The tissues and body fluids. The Harrcy Lectures, Science Press, 

Lancaster, Pa. 
HILLEBRAND, W. F. AND G. E. F. LUNDELL, 1929. Applied inorganic analysis, p. 522. John 

Wiley and Sons. 

JEHLE, L. P., 1938. Ph.D. Thesis, University of California at Berkeley. 
QUIMBY, E. H., 1947. Radioactive sodium as a tool in medical research. Aincr. Jour. 

Rocntgcnol. and Rod. Therapy, 58: 741. 



STUDIES IN THE REGULATION OF BLOOD-SUGAR CONCENTRA- 
TION IN CRUSTACEANS. I. NORMAL VALUES AND 
EXPERIMENTAL HYPERGLYCEMIA IN 
LIBINIA EMARGINATA 

L. H. KLEINHOLZ,i WITH THE ASSISTANCE OF 
BARBARA CHASE LITTLE 

Marine Biological Laboratory, Woods Hole, Mass., and Reed College, Portland, Oregon 

INTRODUCTION 

Studies of blood-sugar concentrations of < invertebrates were, until relatively 
recently, confined for the most part to defining the range of glycemic values in 
different species. Stimulated by investigations of the effects of insulin on blood- 
sugar concentrations in mammals, these early studies among invertebrates demon- 
strated that considerable variations existed in the amount of glucose in the blood, 
even within a single species. The reviews of Beutler (1939) and of Benazzi- 
Lentati (1941) summarize many of these observations. 

The wide range of glycemic concentrations in crustaceans reported by different 
investigators, who at times studied the same species, was soon recognized to be 
due, in part, to the different analytical methods employed and, perhaps even to a 
greater extent, to the varied physiological states of the animals at the time blood 
samples were taken for analysis. This latter possibility led to observations on 
animals maintained under more critical laboratory conditions and to studies of 
factors that influenced the amount of sugar in the blood. 

Thus, where Hemmingsen (1924a) had found an increase in blood sugar in 
Astacus after feeding, Kisch (1929) reported a decrease in Carcinus inaenas during 
starvation. These results were confirmed both by Stott (1932) and by Florkin 
(1936) for the same species of Carcinus. On the other hand, Roche and Dumazert 
(1935) found that the blood glucose of Cancer pagunis starved for one month did 
not differ significantly in concentration from that of freshly captured individuals. 
Asphyxiation was reported by Stott (1932) to cause marked hyperglycemia in 
C. pagurus, Portunus puber, and Carcinus niocnas; this observation was confirmed 
by Roche and Dumazert (1935) on Cancer pagunis. Stott (1932) also observed 
a high concentration of glucose in the blood of newly-molted crustaceans (within a 
few hours of ecdysis) compared with animals before the molt. 

Hemmingsen's studies (1924b) on the crayfish Astacus led him to believe a 
regulatory mechanism was present for maintaining a constant level of blood sugar. 
The basis for this view was his observation that samples of concentrated glucose 
solution when injected into Astacus disappeared from the blood stream too rapidly 
to have been oxidized to CO 2 during the experimental period, and yet no glucose 
was excreted in detectable amount into the water in which the injected animals 

1 Fellow of the John Simon Guggenheim Memorial Foundation. 

218 



BLOOD-SUGAR CONCENTRATION IN CRABS 219 

were maintained. These results Hemmingsen regarded as evidence for a hypo- 
glycemic regulatory mechanism. 

In addition, a number of pharmacological substances had been reported as 
being effective in inducing hyperglycemia in crustaceans. The controversy in this 
branch of the general problem lay not so much in the interpretation of such hyper- 
glycemias when they occurred, but whether these substances really induced hyper- 
glycemia. Thus, Medvedeva (1936) reported that injection of adrenalin in 
Potamobius (Astacus) caused hyperglycemia, but that injection of insulin was 
without any definite effect. Roche and Dumazert (1935), on the other hand, 
reported that neither adrenalin nor insulin had any appreciable effect on the blood- 
sugar concentration of Cancer pagurus. Kalmus and Waldes (1936) stated that 
not only adrenalin and insulin, but also such non-specific substances as hydro- 
quinone and sodium chloride solution, effected marked hyperglycemias when in- 
jected into crayfish. Florkin and Duchateau (1939), using more carefully con- 
trolled procedures, reported that insulin had no effect while adrenalin produced a 
hyperglycemia in the crayfish, thus confirming the observations of Medvedeva. 

These scattered observations, controversial though they may have been, indi- 
cated the possible existence of a hyperglycemic mechanism, just as Hemmingsen's 
studies had indicated the possibility of a hypoglycemic mechanism. The first 
indication of a definite anatomical structure which might be involved in regulating 
sugar metabolism in crustaceans was made by Abramowitz, Hisaw and Papandrea 
(1944). These authors found that injection into Callincctcs sapidus of aqueous 
extracts of crustacean eyestalks increased the concentration of blood sugar within 
an hour. More specific localization of the source of this diabetogenic factor was 
demonstrated by the preparation and injection of extracts prepared from the sinus 
glands of Hanstrom that had been removed from eyestalks. The injection of such 
extracts resulted in a marked hyperglycemia amounting to nearly four times the 
normal basal concentration of blood glucose. When extracts, prepared from the 
remainder of the eyestalks from which the sinus glands of Hanstrom had been 
previously removed, \vere injected, they were practically without hyperglycemic 
effect. These investigators' complementary experiments, which consisted of re- 
moving the sinus glands by ablation of both eyestalks, to determine whether hypo- 
glycemia would ensue, gave negative results ; in fact, over a period of seven days 
after eyestalk removal, there was an anomalous, slight increase in concentration 
of blood sugar. 

Our own studies were undertaken to define in greater detail the nature of such 
glycemic changes under experimental conditions and to investigate the physiology 
of the regulatory processes. This is the first report in detail of our investigations, 
some of which have appeared in abstract form (Kleinholz, 1948; Kleinholz and 
Little, 1948; Kleinholz and Havel, 1948). 

MATERIALS AND METHODS 

The animals used in this study were the marine spider crab, Libinia emarginata. 
A large stock of animals was maintained by the laboratory collectors in a live-car. 
Other than the occasional placing of a freshly killed fish into the live-car, no 
regular feeding of these stock animals was undertaken. When groups of animals 
were removed for use in the laboratory, they were starved for three days before 



220 L. H. KLEINHOLZ AND BARBARA CHASE LITTLE 

blood samples were taken, to insure a basal level of blood-sugar concentration, and 
were not otherwise fed except where indicated. Such experimental animals were 
marked for identification by painting serial numbers with lacquer on the dorsal 
surfaces of the carapace of each ; the crabs were then placed in individual containers, 
similarly numbered, through which a stream of sea water circulated. It was hoped 
that the hyperglycemic effect of crowding reported by Abramowitz et al (1944) 
would be reduced or obviated by such isolation. Only male individuals were used 
in this study. 

Tuberculin hypodermic syringes of 1 ml. capacity, graduated in hundredths of 
a milliliter, were used for taking blood samples. Each syringe was calibrated 
to deliver 0.5 ml. by weighing the volume of water delivered between the 0.60 ml. 
and 0.10 ml. marks on the barrel of the syringe. Blood for analysis was taken 
from the sinuses of the walking legs, the arthroidal membrane between the base of 
the leg and the body being first wiped dry with filter paper or absorbent cotton. 
The syringe was filled slightly beyond the 0.60 ml. mark and, after withdrawing 
the needle from the sinus, was emptied to this mark, the excess droplet of blood 
being removed by touching the tip of the needle to filter paper. A 0.5 ml. sample 
of blood could thus be delivered into tubes containing the deproteinizing mixture. 
To avoid injury to the arthroidal membrane that would ensue from the repeated 
bleeding of the same individual, samples were taken from different legs on both 
sides of the animal. In all of the experiments reported here, blood samples from 
control and from experimental animals were taken in the daytime, in most cases 
in the forenoon. The possibility of a diurnal variation in concentration of blood 
sugar was thus avoided. 

It was found most convenient to work with groups of six Libinia at a time. 
In most instances blood samples were taken from the individuals of a group before 
a particular treatment, and then again after the experimental treatment, each crab 
thus serving as its own control. The control blood samples and the experimental 
blood samples were carried through the analytical procedure simultaneously and 
received comparable handling. The method for determining the amount of blood 
glucose was that described by Miller and Van Slyke (1936). It is reported that 
this method, when used with mammalian blood, gives "true" blood-sugar values, 
which do not include non-fermentable reducing substances. We have found a 
significant amount of non-fermentable reducing substance present in Libinia blood, 
so that in our hands the method must be considered as expressing total reducing 
substances as glucose equivalents. The procedure was essentially as described by 
Miller and Van Slyke. The dilute eerie sulfate for the titration was prepared 
fresh daily from the stock solution. 

Two blanks, consisting of the reagents used in the glucose determination, were 
used with each set of blood samples. These blanks required about 0.15-0.25 ml. 
of the dilute eerie sulfate to reach the same end-point obtained in the titration of 
the blood samples. This wide range was due to the preparation of a second lot 
of stock reagent solutions during the course of the work. For the first set of stock 
solutions the blanks varied from 0.18-0.25 ml., the average for 38 blanks being 
0.22 ml. ; with the second set of stock solutions the blanks varied from 0.13-0.18 ml., 
the average for 20 blanks being 0.15 ml. The average for the total 58 blanks was 
0.195 ml. of the dilute eerie sulfate. 



BLOOD-SUGAR CONCENTRATION IN CRABS 221 

The accuracy of the Miller and Van Slyke method in our hands was tested by 
determining the amount of glucose in prepared solutions of known concentration. 
These concentrations ranged from 16200 mg. of glucose per 100 ml. of solution. 
The average percentage of error for 13 such determinations was 3.9 per cent. 

Where Libinia without eyestalks were used to determine the effect of absence 
of the sinus glands on the blood-sugar level, bilateral eyestalk ablation was done 
with the aid of fine dissecting scissors. Bleeding from the cut surface was very 
slight and ceased upon the formation of a blood clot in the orbit. 

The distinction between total reducing substances and non-fermentable reducing 
substances in the blood was made by fermenting one of two blood samples with a 
10 per cent suspension of Fleischmann's yeast. The yeast was prepared by sus- 
pension in distilled water, centrifuging, and pouring off the supernatant. After 
three such washings, the final 10 per cent suspension was kept in the refrigerator 
until used. In the fermentation, 3.5 ml. of the yeast suspension and the 0.5 ml. 
blood sample were mixed and allowed to remain at 22 C. for one hour, after which 
the mixture was centrifuged and the supernatant poured off into a second tube 
containing acid cadmium sulfate. The yeast and blood residue was similarly 
washed and centrifuged three times with 1 ml. portions of distilled water, the 
supernatants each time being added to the first one. A second 0.5 ml. blood 
sample taken from the same animal had been prepared for the routine analysis. 
Both blood samples were carried through the analytical procedure simultaneously, 
using adequate blanks (washings of a 3.5 ml. aliquot of yeast suspension) for the 
fermented samples. 

The reliability of this method of fermenting glucose in blood was tested with 
samples from six Libinia. To a 0.5 ml. portion of blood from each animal was 
added 0.5 ml. of solution containing 5.06 mg. of glucose (thus equivalent to adding 
1012 mg. per cent of glucose to the blood sample) and 3.5 ml. of the 10 per cent 
yeast suspension. The blood samples were fermented^ and treated as described 
above. The glucose-equivalent in reducing substances present in these samples 
ranged from 6.3-11.1 mg. per cent, with an average of 8.9 mg. per cent, showing 
practically complete fermentation of the added glucose. 

OBSERVATIONS 

A. Normal and eycstalklcss animals 

The studies of Abramowitz, Hisaw and Papandrea (1944) pointed to the sinus 
glands as being mediators in the hyperglycemic response following injection of 
prepared extracts. Their attempts to observe whether removal of this gland (by 
ablation of both eyestalks) resulted in hypoglycemia yielded paradoxical results, 
a gradual hyperglycemia being observed in such animals over a period of seven days. 

Our own observations made over a longer period in a comparable series of 
experimental animals, do not confirm the latter results of these investigators. Of 
eighteen animals brought into the laboratory at the same time, the eyestalks of 
twelve were ablated, while the remaining six served as normal control animals. 
The animals of the control group were isolated in individual containers ; six of the 
operated Libinia were designated as Group A and were similarly isolated, while 
the remaining six operated animals, constituting Group B, were placed in a com- 



222 



L. H. KLEINHOL2 AND BARBARA CHASE LITTLE 



mon tank. These animals were not fed during the time the experiment was in 
progress. Beginning on the morning of the third day after eyestalk removal, blood 
samples were taken from the individuals of Group A and of the control group ; 
on the following morning, the fourth day after ES removal, samples were taken 
for analysis from individuals of Group B. By alternating in this fashion and 
taking blood samples every third day. the observations were extended over a 
period of twenty-six days. The results which are shown in Table I are the 
averages and the standard deviations for the six animals constituting each group. 

TABLE I 

Comparison of the blood-sugar concentrations in two groups of Libinia without eyestalks, with 
that of a normal control group. The figures are the averages for the 6 crabs of each group and their 
standard deviations. 



Days after eyestalk 
removal 


Blood-sugar concentration in mg.-per cent 




Group A 


Group B 


Control 


3 


10.1 2.5 





6.9 1. 


2 


4 





11.1 2.1 







6 


12.0 3.1 





9.9 1. 


5 


7 


- 


9.0 2.5 







10 


10.2 3.4 





7.4 1. 


3 


11 





9.3 3.6 







17 


10.2 4.3 


. 


7.3 1. 


8 


18 





9.6 3.7 







24 


10.1 3.2 





8.7 1. 


7 


26 





7.9 2.6 








As might be expected, there were variations in the concentration of blood sugar 
not only among the individuals of a group, but also in the same individual at the 
different intervals when blood was taken for analysis. These variations are prob- 
ably due to a combination of actual fluctuations in glucose concentration and of 
slight artifacts in the analytical procedure. The relatively low average of the 
blood-sugar concentration in the control group on the third day, compared with 
later averages, is probably to be explained on this basis, for the average glycemic 
values determined for three different normal groups, which had been starved three, 
six, and ten days, were, respectively, 7.9, 8.3 and 9.1 mg. per cent. We conclude 
from the data of Table I that removal of the sinus glands has no marked effect on 
the basal level of the blood-sugar concentration. The hyperglycemia reported by 
Abramowitz et al. after eyestalk removal in Callinectes could not be confirmed 
with Libinia. 



B. Total reducing substances and true blood sugar 

It has been known that the blood of mammals contains, in addition to glucose, 
other reducing substances which, in the analytical methods currently employed, 
may contribute significantly to the total value obtained as "apparent" glucose. 
For an accurate measure of the amount of glucose in a blood sample the supple- 



BLOOD-SUGAR CONCENTRATION IN CRABS 



223 



mentary use of a yeast fermentation method along with the conventional determina- 
tion permits the distinction to be made between total reducing substances or ap- 
parent blood glucose, and non-fermentable reducing substances; the difference 
between the two such determinations is then considered to represent the ferment- 
able glucose. 

The application of methods devised for the analysis of mammalian blood to that 
of invertebrates would require similar supplemental yeast fermentation methods, 
since little is known about the presence or the nature of non-fermentable reducing 
substances in blood of the latter group. Such yeast fermentation analyses of the 
blood of Libinia, using the procedure described under "Methods," \vere conducted 
in parallel with samples taken at the same time for the determination of total reduc- 
ing substances. The results for experimental and control animals are arranged 
in Table II. 

TABLE II 

True blood-sugar concentrations in groups of eyestalkless and of normal Libinia. Each group 
consisted of 6 animals. TRS, total reducing substances; NFRS, non-fermentable reducing substances; 
TBS, true blood sugar. Figures are averages for the animals of a group and the standard deviations 
from the mean. 



Animals and condition 


Concentration in mg. per 100 ml. blood 


TRS 


NFRS 


TBS 


Group A, starved 24 days 
Group B, starved 26 days 
Controls, starved 24 days 
Normal, starved 6 days 


10.1 3.2 

7.9 2.6 

8.7 1.7 
8.3 1.3 


7.4 0.8 
5.0 1.3 
6.8 1.7 
6.3 0.7 


2.7 
2.9 
1.9 
2.0 



The data shown in this table represent the averages for four groups of Libinia, 
each group consisting of six animals. Three of these groups consisted of individ- 
uals whose blood analyses for total reducing substances had been made at intervals 
over nearly four weeks, as shown in Table I. The animals of Group A, Group B, 
and the control group are described in the text above and in the preceding table. 
On the twenty-fourth and twenty-sixth days when final analyses were being made 
on these three groups, additional samples were taken at the same time for deter- 
mination of the non-fermentable reducing substances after the blood sample had 
been mixed with yeast suspension and fermented. The fourth group of Table II 
consisted of normal animals which had been starved six days, in comparison with 
the twenty-four days of starvation undergone by the control group to the eyestalk- 
less condition. The figures which are given for concentration of true blood sugar 
are averages for the six individuals of a group, representing the differences be- 
tween the average concentrations of total reducing substances present in one set 
of samples and the average amounts of non-fermentable reducing substances found 
in similar samples after they had been fermented by the yeast suspension. 

In all groups the amount of true blood sugar is quite low. There appears to 
be no difference in giycemic level between normal animals starved for a short 
period (6 days) and 'hose starved for an appreciably longer time (24 days). At 



224 L. H. KLEINHOLZ AND BARBARA CHASE LITTLE 

first glance the slightly higher level of true blood sugar in the groups of eyestalkless 
individuals as compared with that in the normal control animals might seem to 
indicate an alteration in glucose metabolism as a result of eyestalk removal, but in 
view of the small number of animals involved in the experimental groups and the 
comparatively high standard deviations of the averages for each group, it is doubted 
that these differences from the controls can be regarded as significant. 

C. Hyperglycemia as a result of injection of eyestalk extract 

The hyperglycemic effects obtained by Abramowitz, Hisaw and Papandrea 
(1944) in Callinectes upon injection of eyestalk extracts showed a rough agree- 
ment between dosage and the increment of the resulting hyperglycemia. But since 
their determinations were in terms of total reducing substances, with no distinction 
being made between fermentable and non-fermentable components, closer examina- 
tion was made of these components of the total reducing substances at the same 
time that we tried to confirm their observations. 

Blood samples from a group of six Libinia from which both eyestalks had been 
ablated were analyzed on the first and fifteenth days after eyestalk removal. At 
these times the average concentrations of total reducing substances were respectively 
7.1 1.5 mg. per cent and 6.3 1.6 mg. per cent, confirming previous observa- 
tions made in Table I that no significant change follows in the glycemic level of 
animals from which both eyestalks have been removed. On the morning of the 
seventeenth day after eyestalk ablation, each of the crabs was injected with 0.1 ml. 
of extract prepared from the eyestalks of Libinia, so as to receive the equivalent of 
one eyestalk. One hour after the injection, blood samples were taken from each 
animal for determination of both the total reducing substances and the non- 
fermentable reducing substances. Five of the six injected Libinia showed striking 
increments in total reducing substance in the blood, the concentrations after injec- 
tion being from twice to nearly five times those obtained before injection ; in the 
sixth animal the amount of total reducing substance after injection was about 60 
per cent greater than before the injection. The average value^ for total reducing 
substance for all six animals was 17.3 6.4 mg. per cent. The average for non- 
fermentable reducing substances of the post-injection samples after yeast treatment 
was 4.5 0.4 mg. per cent; average fermentable blood sugar after injection was 
therefore 12.8 mg. per cent. Yeast fermentations were not made on blood samples 
taken on the first and fifteenth days after eyestalk removal, but if the average value 
for true blood sugar is assumed to be comparable to those shown in Table II, then 
the average increase in fermentable blood sugar after injection of eyestalk extract 
is well over 400 per cent. [We are thus able to confirm the observation of Abramo- 
witz et al. that injection of crustacean eyestalk extract induces a marked hyper- 
glycemia in crustaceans, and to show, furthermore, that this increase is apparently 
a fermentable sugar. 

D. Hyperglycemia as a result of asphyxia 

Stott (1932) had reported a large increase in blood sugar of crustaceans which 
had been kept for ten hours in containers of sea water that had been tightly covered. 
This change he attributed to asphyxia due to the decrease in oxygen content of the 



BLOOD-SUGAR CONCENTRATION IN CRABS 



225 



water during the period of the experiment, because when an adequate air supply 
was again made available to the animals by removing the cover, the blood-sugar 
concentration returned to the normal level. Roche and Dumazert (1935) con- 
firmed this observation by reporting that removing animals from sea water and 
keeping them in air for 30-60 minutes resulted in a marked hyperglycemia. Both 
studies reported this hyperglycemia as a direct observation, with no attempt to 
investigate in further detail the mechanism of this response. 

Similar results were obtained by us with Libinia, and a possible mechanism 
for what we shall call the hyperglycemia of asphyxia was indicated by further study. 
In these experiments, groups of six normal and six eyestalkless animals were em- 
ployed. To obtain partial asphyxia during which the animals could be kept under 
observation, the method of Roche and Dumazert was used : removing the animals 
from sea water and keeping them in air for 60 minutes, and then removing a blood 
sample from each for analysis and for comparison with samples before asphyxia. 
The results of these experiments are shown in Table III. 

TABLE III 

Effect of asphyxia on the blood-sugar concentration of normal animals and animals without sinus gland 



Animal group 


Eyestalk 
condition 


Days 
starved 


Concentration in mg.-per cent 


Before asphyxia 


After asphyxia 


Group B 
Nos. 13-18 


ESoff 
31 days 


31 


(7.9 2.6)* 


7.0 2.8 


Controls 


Normal 


31 


(8.7 1.7)* 


16.1 8.3 


Nos. 19-24 










Nos. 31-36 


ESoff 


18 


7.1 1.5 


6.0 1.1 




1 day 








Nos. 25-30 


Normal 


19 


7.3 1.0 


22.0 12.2 



* See Table I . 

The first two groups of crabs tested consisted of eyestalkless individuals which 
had been under observation for several weeks (the animals constituting Group B 
of Table I), and a similar number of normal Libinia which had been their controls. 
Both groups were removed from sea water and placed in individual finger-bowls in 
air. After one hour of such exposure, blood samples were taken for analysis (at 
this time the animals were limp and showed a marked loss of muscular tone ; fol- 
lowing their return to sea water recovery was rapid). No blood samples were 
taken in this experiment directly before the asphyxiating experience, the glycemic 
values which had been determined at regular intervals for the preceding twenty-six 
days being considered sufficient to serve as a standard. As can be seen from 
Table III, the effect of this asphyxia was different in the two groups, the crabs 
without eyestalks showing no appreciable change in their average concentration of 
blood sugar, while the group of normal animals showed a marked increase in the 
glycemic average for the group. 



226 L. H. KLEINHOLZ AND BARBARA CHASE LITTLE 

The experiment was then repeated with two additional groups of similar ani- 
mals. This time a blood sample was removed before subjecting the animals to 
asphyxia, and the second sample was taken immediately after the 60 minutes of 
asphyxia. The results were similar to those obtained previously; the normal 
animals (with eyestalks) showed a marked hyperglycemia, the average concen- 
tration being three times the pre-asphyxia level, while the animals without eye- 
stalks showed no significant change from the glycemic level before asphyxia. 
These results show that the observed hyperglycemia is dependent upon the intact 
eyestalk and indicate the possibility that the response may be mediated by the sinus 
gland. The results of more exact studies, in which sinus glands were removed 
from otherwise intact eyestalks, to define the mechanism of the hyperglycemic 
response, will be reported later. 

E. Alimentary hyperglycemia 

A number of investigators have reported the effects of feeding and inanition 
upon blood-sugar levels in crustaceans. The studies of Hemmingsen (1924a) and 
Stott (1932) showed that feeding resulted in a rise in blood-sugar concentration. 
Stott had found that in a group of starved Carcinus macnas, the glycemic level 
ranged between 5-8 mg. per cent. When such animals were fed mussels, the 
blood sugar rose to values of 20 mg. per cent or more over a period of several 
hours ; about fourteen hours after such feeding, the level of blood sugar returned 
to a concentration of approximately 5 mg. per cent. 

In view of the part played by the eyestalk and sinus gland in mediating the 
hyperglycemia resulting from asphyxia, as described in the preceding section, it 
was thought advisable to determine whether alimentary hyperglycemia was simi- 
larly regulated. Seven Libinia from which both eyestalks had been removed three 
days previously, and which had been starved for three days, were isolated in 
individual containers. The average glycemic value immediately before feeding was 
9.1 2.8 mg. per cent. Each animal was then supplied with 5-10 grains of the 
visceral mass of Venus mcrcenaria, which was devoured within fifteen minutes. 
Blood samples taken three hours after this feeding showed a marked rise in sugar 
content in each of the seven animals, the average for the group after feeding being 
18.3=t4.7 mg. per cent. The results therefore indicate that alimentary hyper- 
glycemia is not mediated by the sinus glands in the eyestalks. 

SUMMARY 

1. Removal of the sinus glands by eyestalk ablation in unfed Libinia emarginata 
has no significant effect on the blood-sugar concentration when compared with 
similarly unfed controls. 

2. Values for true blood sugar, as distinguished from total reducing substances, 
were determined after yeast fermentation of blood samples. In starved animals 
the concentration of total reducing substances is between 8-9 mg. per cent ; that of 
non-fermentable reducing substances, 6-7 mg. per cent ; that for true blood sugar 
is therefore about 2 mg. per cent. 

3. Injection of eyestalk extract increases the concentration of total reducing 
substances in the blood. This increase is in the fermentable component, amounting 



BLOOD-SUGAR CONCENTRATION IN CRABS 227 

to over 400 per cent of that in the uninjected animal, and therefore probably repre- 
sents a true hyperglycemia. 

4. Asphyxia also causes hyperglycemia, the total reducing substances in blood 
samples being two to three times the concentration preceding asphyxia. 

5. Removal of the sinus gland by eyestalk ablation prevents the appearance of 
the hyperglycemia of asphyxia. The sinus gland may be a mediator in certain 
hyperglycemic responses of crustaceans, but does not seem to be concerned in 
alimentary hyperglycemia. 

LITERATURE CITED 

ABRAMOWITZ, A. A., F. L. HISAW AND D. N. PAPANDREA, 1944. The occurrence of a diabeto- 

genic factor in the eyestalks of crustaceans. Biol. Bull., 86 : 1-5. 
BENAZZI-LENTATI, G., 1941. Sulla distribuzione del glicogeno e sulla glicemia vera degli 

Jnvertebrati. Arch. Zool. Italiano, suppl. 29 : 35-70. 
BEUTLER, RUTH, 1939. Vergleichende Betrachtungen iiber dem Zuckergehalt des menschlichen 

und tierischen Blutes. Ergcb. d. Biol., 17 : 1-104. 
FLORKIN, M., 1936. Sur le taux de la glycemie plasmatique vraie chez les crustaces decapodes. 

Bull. Acad. Roy. Belgique, 22: 1359-1367. 
FLORKIN, M. AND G. DUCHATEAU, 1939. La glycemie de 1'ecrivisse apres 1'inj action d'adrenaline 

ou d'insuline. Compt. Rend. Soc. Biol. Paris, 132: 484-486. 
HEMMINGSEN, A. M., 1924a. The blood sugar of some invertebrates. Skand. Arch. PhysioL, 

45 : 204-210. 
HEMMINGSEN, A. M., 1924b. Blood sugar regulation in the crayfish. Skand. Arch. PhysioL, 

46: 51-55. 
KALMUS, H. AND V. WALDES, 1936. 1st die durch Adrenalin bewirkte Glycolyse beim Fluss- 

krebs spezifisch? Zcit. vergl. PhysioL. 23: 712-714. 
KISCH, B., 1929. Der Gehalt des Blutes einiger Wirbelloser an reduzierenden Substanzen. 

Biochcm. Zeit., 211 : 292-294. 
KLEINHOLZ, L. H., 1948. Experimental hyperglycemia in the marine crustacean, Libinia emar- 

ginata. Anat. Rec., No. 4, 101 : 84. 
KLEINHOLZ, L. H. AND VIRGINIA JOHNSON HAVEL, 1948. The hyperglycemic effect of adrenalin 

injection in the crayfish, Astacus trowbridgei. Anat. Rec., No. 4, 101 : 85. 
KLEINHOLZ, L. H. AND BARBARA CHASE LITTLE, 1948. Blood-sugar values in the marine crus- 
tacean, Libinia emarginata. Anat. Rec., No. 4, 101 : 84. 
MEDVEDEVA, NAT., 1936. Le probleme de la reactivite specifique des invertebres aux increts 

des vertebres. IV. Action de 1'adrenaline et de 1'insuline sur certains invertebres. 

Mcdichnii Zhurnal vscukrain. Acad. U.S.S.R., Kiev, Nauk 6: 385-387. 
MEDVEDEVA, NAT., 1936. Le probleme de la reactivite specifique des invertebres aux increts 

des vertebres. Du mecanisme de la regulation du sucre de 1'hemolymphe chez dif- 

ferents invertebres sous Faction de 1'adrenaline et de la surcharge glucosee. Alediclmii 

Zhurnal vseiikrain. Acad. U.S.S.R.. Kiev, Nauk 6: 805-807. 
MILLER, B. F. AND D. D. VAN SLYKE, 1936. A direct microtitration for blood sugar. Jour. 

Biol. Chew., 114: 583-595. 
ROCHE, J. AND C. DUMAZERT, 1935. Sur la glycemie de Cancer pagurus. Compt. Rend. Soc. 

Biol. Paris, 120: 1225-1227. 
STOTT, F. C., 1932. Einige vorlaiifige Versuche iiber Veranderungen des Blutzuckers bei 

Dekapoden. Biochem. Zeitschr., 248 : 55-64. 



OVARIAN INHIBITION BY A SINUS-GLAND PRINCIPLE IN THE 

FIDDLER CRAB 

FRANK A. BROWN, JR. AND GWEN M. JONES 

Department of Zoology, Northwestern University, and the Marine Biological Laboratory, 

Woods Hole, Mass. 1 

The action of the sinus gland in inhibiting ovarian development was first demon- 
strated by Panouse (1943, 1944, 1946) working with females of the shrimp, 
Leander serratus. In these animals, amputation of both eyestalks or bilateral 
removal of the sinus glands resulted in a great acceleration of ovarian growth, 
maturation of the oocytes, and even laying of mature eggs, during a period when 
these structures are normally quiescent or just beginning the normal growth phase. 
Implantation of sinus glands into abdomens of destalked animals resulted in an 
inhibition of ovarian development. Similar results following eyestalk removal 
were obtained with the crayfish Cambarus immunis by Brown and Jones (1947). 

The following experiments were performed upon the fiddler crab, Uca pugi- 
lator, to ascertain whether this phenomenon of ovarian inhibition by a blood-borne 
principle from the sinus glands also obtained in the division, Brachyura, of the 
Crustacea. 

MATERIALS AND METHODS 

The animals used in the experiments were females of Uca pugilator collected 
near Woods Hole, Massachusetts, on July 10, 1948. The carapace widths ranged 
from 15 to 20 mm. at the widest point. They were kept in the laboratory at room 
temperature (about 25 C.) in individual containers each holding sea water to a 
depth of a quarter of an inch. The water was changed daily. The animals were 
not fed during the course of the investigation. 

Removal of eyestalks was accomplished by amputation at their bases and the 
wounds were allowed to close spontaneously by clotting of the blood which welled 
slowly from them. 

Sinus glands were obtained from donor animals for the purpose of implanting 
according to the following procedure. Eyestalks were removed as above, placed 
in sea water, and the contents of the eyestalks exposed by a dorsal splitting of the 
chitinous sheath. The sinus gland, a discrete bluish organ, was dissected free of 
surrounding tissue and drawn into the lumen of a 25 gauge needle by means of a 
tuberculin syringe. The gland, plus a minute quantity of sea water, was injected 
into the ventral hemocoele of the recipient animal's abdomen. The chitinous mem- 
brane of this region of the body is transparent and thus it is possible to see the 
actual extrusion of the contents of the needle. The dissections and implantations 
were accomplished with the aid of a dissecting microscope. 

1 This investigation was supported by a research grant from the graduate school of North- 
western University. 

228 



OVARIAN INHIBITION BY SINUS GLAND 



229 



In order to observe the influences of the above procedures, experimental and 
normal control animals were autopsied as they died, or were sacrificed for dissec- 
tion at six-day intervals. The carapace and hypodermis were removed, and the 
ovary, an H-shaped organ lying over the hepatopancreas and just below the hypo- 
dermis, was dissected out in sea water and placed in a tared \vatchglass for weigh- 
ing. Excess moisture was removed with filter paper, and fresh weights were 
taken. 

RESULTS 

On July 11, 1948, ten normal animals were sacrificed and the ovaries removed. 
Eight of these ovaries were found to be in an immature state ; the oocytes were 
very small and the organs as a whole were slender and of a light yellow-pink color. 
The other two ovaries contained somewhat larger oocytes and the color of the 
organ was a deep shade of pink. The average of the ten ovarian weights was 
12.6 mg.. the extremes being 6.8 nig. and 18.8 nig. respectively 

On Julv 12, 120 animals of nearly uniform size were selected and divided into 
three lots. Eyestalks were removed from two lots of forty of them. Two days 
later, one sinus gland was implanted into each of one lot of forty of the eyestalkless 
animals using the technique described earlier. The implants were then repeated 
every fifth day for the duration of the experiment. 

TABLE I 

Number of specimens, and ranges and averages of ovarian fresh weights in milligrams 



Days 


Destalked 


Destalked. receiving sinus 
gland implants 


Controls 


No. spec. 


Range 


Av. 


No. spec. 


Range 


Av. 


No. spec. 


Range 


Av. 


1-6 


6 


11.8-46.2 


24.0 


14 


7.2-45.2 


20.6 


6 


9.2 16.2 


13.5 


7-12 


4 


17.9-61.2 


32.6 


10 


6.4-67.6 


24.7 


4 


6.6-13.1 


10.1 


13-18 


5 


32.6-54.9 


42.2 


4 


12.5-35.8 


21.0 


4 


4.2-34.2 


18.4 


19-24 


11 


23.0-165.6 


54.7 


5 


8.8-32.3 


19.5 


1 1 


5.8-23.6 


13.9 


25-30 


6 


33.8-160.4 


66.4 


4 


15.8-22.3 


17.9 


6 


4.4-19.4 


11.0 



In Table I are summarized the ovarian weights of the three groups of animals 
(destalked. destalked and receiving sinus gland implants, and normal controls) 
which died or were sacrificed during five succeeding six-day periods. Each value 
obtained represents the average of data from 4 to 14 animals. It will be observed 
from Table I that over the thirty-day period the average ovarian fresh weights of 
the destalked animals increased approximately linearly with time from an original 
12.6 mg. to 66.4 mg., a more than five-fold increase. The ovarian weights of 
destalked animals receiving sinus-gland implants showed an initial rise, with an 
approximate level being maintained at values somewhat higher (a total average of 
7 mg.) than those of the control animals. 



230 I- RANK A. BROWN, JR. AN* I) (i\YKX M. JOXES 

( Hhcr changes observed in the ovaries of the destalked animals, in addition to 
the gro>s si/.e alteration, \vere gradual increases in oocyte diameter and a shift of 
their color from the previously mentioned pinkish-yellow to a deep purple-red. 
The color change became most striking as the ovary attained a weight of approxi- 
mately 15-18 mg. 

In general, the color and size of oocytes of the destalked animals receiving sinus- 
gland implants were found to lie somewhere between the extremes offered by the 
destalked and control animals. 

During the course of the experiment, only 7 of the 31 control animals were 
found at autopsy to have ovaries in the apparently mature condition typical of the 
destalked ones. On the other hand, after the first six-day period, in no case did 
any of the latter group contain oocytes presenting an immature appearance, either 
in size or in color. 





A B 

K 1. Ovaries of two fiddler crabs removed September 1, 1948: A, from an animal 
destalked one month earlier: B, from a normal animal. 

Figure 1 is a photograph of two ovaries removed from animals of the same 
carapace width 17 mm. The animal from which ovary A was removed was de- 
stalked on July 30, 1948, and sacrificed for dissection on September 1, 1948. The 
animal from which ovary B was removed was a normal control maintained under 
identical laboratory conditions during the same period and sacrificed on the same 
day. These two organs are typical of those removed from destalked and normal 
animals, respectively, during the course of the experiment. The approximate wet 
weights are : ovary A, 80 mg. ; ovary B, 10 mg. 

It is also of interest to note that during the time that the investigation was in 

>gress, five females which had been deprived of eyestalks and received no sinus- 

I tissue laid mature eggs, and one female, also eyestalkless, which had re- 

ceivi one sinus gland implant, did likewise. In none of these cases w r ere the 



OVARIAN INHIBITION BY SINUS GLAND 231 

eggs fastened to the pleopods of the animal as normally occurs. No eggs were 
laid by any of the control animals. 

DISCUSSION 

It seems apparent from the foregoing results that the sinus gland in Uca, as in 
Leander and Cambarus, is the source of an ovary-inhibiting principle which, when 
absent, allows for a period of ovarian growth and development even at a time when 
no such gonadal activity would otherwise be manifested. 

Implantation of the quantity of sinus-gland tissue utilized in this work into 
the abdomens of destalked animals tends to suppress the gonadal growth, but 
allows the ovary to be maintained at a stage somewhat more mature than that 
characteristic of the normal animals in possession of both sinus glands. 

There is no indication from these experiments whether the principle from the 
sinus glands inhibits the ovary directly or serves to inhibit the production of a 
gonad-stimulating principle normally produced elsewhere in the body. On the 
latter hypothesis one possible explanation of the ovarian growth during the first 
six-day period in the gland-implanted animals is that during the two days elapsine 
between eyestalk (sinus gland) removal and the first implant, the blood titer of 
the inhibitor dropped to such a point that a gonad-promoting principle was per- 
mitted to be liberated into the blood. The first implant might be presumed to 
inhibit further production of the stimulating principle but not counteract the action 
of this factor already present. 

An explanation based upon an hypothesis of a direct inhibition of ovarian 
growth is as follows : During the two days which elapsed between amputation of 
the eyestalks and the initial implantation of the sinus-gland tissue, there was a 
drop in titer of the inhibitory substance to an ineffectual level which permitted 
nearly as rapid growth in these ovaries as occurred in the destalked animals which 
received no implanted sinus glands. There may also be a delay in the production 
of an inhibiting concentration by the implants. 

There is a suggestion in Table I that sinus-gland implants in the eyestalkless 
animals not only are able to inhibit growth in the partially developed ovaries, but 
may even effect a reduction in their size. 

SUMMARY 

1. Removal of the eyestalks of adult females of Uca pugilator results in a period 
of rapid ovarian growth in which the increase in fresh weight of the gonad is 
approximately five-fold in a thirty-day period. 

2. The period of ovarian growth is characterized by increase in oocyte diameter 
and a color change from light pink to a deep purple-red. 

3. Implantation of sinus-gland tissue into the abdomens of destalked females 
serves to inhibit to a large degree this rapid growth. 

4. Six of the animals which had been deprived of their eyestalks laid mature 
eggs during the course of the experiment ; none of the controls did so. Eggs pro- 
duced by the experimental animals failed to become attached to the pleopods. 



FRANK A. BROWN, JR. AND GWEN M. JONES 

LITERATURE CITED 

BROWN, F. A., JR. AND GWEN M. JONES, 1947. Hormonal inhibition of ovarian growth in the 

crayfish, Cambarus. Anat. Rcc., 99 : 657. 
PANOUSE, J. B., 1943. Influence de 1'ablation clu pecloncle oculaire sur la croissance de 1'ovaire 

chez la Crevette Leander serratus. C. R. Acad. ScL, Paris, 217: 553-555. 
PANOUSE, J. B., 1944. L'action de la glande du sinus sur 1'ovaire chez la Crevette Leander. 

C. R. Acad, ScL, Paris, 218: 293-294. 
PANOUSE, J. B., 1946. Recherches sur les phenomenes humoraux chez les Crustaces. Annalcs 

dc L'lnstitut Occanographique, 23 : 65-147. 



PIPERAZINE DIHYDROCHLORIDE AND GLYCYLGLYCINE AS 

NON-TOXIC BUFFERS IN DISTILLED WATER 

AND IN SEA WATER 1 - 2 

MARSHALL E. SMITH AND LYNWOOD B. SMITH a 
The Marine Biological Laboratory, Woods Hole, Mass. 

A wide selection of buffers is necessary in biological work, since it is often 
desirable to repeat a particular experiment with a different buffer. Piperazine 
dibydrochloride and glycylglycine are crystalline, non-volatile, very soluble solids 
readily obtainable in pure form. Piperazine is relatively non-toxic to man (Hanz- 
lik, 1917) and to rats (Dieke, Allen, and Richter, 1947) and has been used as an 
apparently non-toxic buffer by certain biologists at our suggestion (Cornman, 
1940, 1941 ; Evans, Beams, and Smith, 1941). The buffer merits of glycylglycine 
in sea water have been previously pointed out by Tyler and Horowitz (1937). 
This relatively non-toxic material is a normal constituent of many proteins. A 
wide-range buffer is simply and accurately prepared from only these two substances 
and sodium hydroxide. Used in sea water there is no observed precipitation of 
salts until a pH of 9.9 is reached. The commonly used phosphate buffer precipi- 
tates calcium and magnesium phosphate from sea water at a much lower pH, thus 
disturbing the salt balance and adding uncertainty to conclusions from experi- 
ments. The shortcomings of many of the buffers in common use have recently 
been mentioned by Gomori (1946). We have not used the new buffers suggested 
by him and cannot compare his buffers with ours, except to point out that our 
buffers have a wider range. 

For special cases where it is desired to have no inorganic ions in a buffer, it is 
possible to obtain buffers from a pH of 7.0 to 1 1 .0 by titrating glycylglycine with 
the free base of piperazine. However, we are presenting no data on this subject. 

In this paper we present a table indicating the preparation of several buffers, 
using piperazine dihydrochloride, glycylglycine and equimolecular mixtures of the 
two substances in distilled water and in sea water. We also present pK x and pK 2 
values of piperazine dihydrochloride. 

EXPERIMENTAL 

The piperazine was purchased from the Eastman Kodak Co. in the form of 
the hexahydrate. Because the free base of piperazine absorbs carbon dioxide and 
moisture from the air, it was converted into the stable dihydrochloride (Sieber, 
1890) before use. 

1 A brief report of this work was presented at the Florida Academy of Science Meeting, 
Tampa, Fla., December 1946. 

2 We are indebted to Dr. W. Mansfield Clark for certain suggestions relative to the manu- 
script, and to H. G. Smith for help with the experiments. 

3 Present address : 418 West Platt St., Tampa 6, Fla. 

233 



234 MARSHALL E. SMITH AND LYNWOOD B. SMITH 

Piperazine dihydrochloride is prepared by dissolving 50 g. of piperazine 
hexahydrate in 100 ml. of 95 per cent ethanol, and adding slowly 100 nil. of con- 
centrated hydrochloric acid. Heat is evolved. As the mixture cools, crystals of 
the dihydrochloride hydrate are formed. The mixture is cooled in an ice bath 
and is filtered. The crystals are washed several times with cold ethanol, and are 
air-dried. The material is ready for use after it has been dried at 100 C. for 
eight hours. The yield is 33 g. Anhydrous piperazine dihydrochloride is slightly 
hygroscopic. 

Analytically pure glycylglycine was purchased from the Amino Acid Manu- 
factures of the University of California at Los Angeles and was used without 
further purification. The material was dried at 100 C. for six hours just prior 
to use. Glycylglycine is not appreciably hygroscopic. 

The pH measurements were made -at 25 C. ( 0.2) with a Leeds and 
Northrup potentiometer-electrometer No. 7660, equipped with Leeds and Northrup 
glass dip electrode Std. 1199-12 made of Corning 015 glass, and a reference 
saturated calomel half-cell electrode Std. 119913 with a potassium chloride capil- 
lary salt bridge. Before and after each titration the electrode was checked against 
"standard acetate," for which the pH value of 4.64 was taken (Maclnnes, Belcher, 
and Shedlovsky, 1938). As is the practice in standardizing buffers, the liquid 
junction potentials were neglected. 

The sodium ion error for the higher pH values was corrected by using the 
following equation adapted from Powney and Jordan (1937) to fit the sodium 
ion errors found experimentally when our glass electrode was calibrated with the 
hydrogen electrode. 

Log ApH == 0.50 pH -- 5.86 + 0.46 log [Na + ] 

The possible error in these readings increases with increasing alkalinity, but below 
a pH of 9.0, the accuracy was within the limits of 0.02. 

Although stock solutions of piperazine dihydrochloride and glycylglycine may 
be prepared, it is preferable to prepare the solutions fresh, since on long standing 
glycylglycine may undergo hydrolysis and piperazine dihydrochloride might form 
toxic products (Greenbaum, 1937). 

Solutions of piperazine and glycylglycine and equimolecular mixtures of the 
two were titrated with standardized sodium hydroxide and numerous readings 
were taken. From these readings a table for the preparation of buffers was made 
(Table I). 

Several pH determinations were made with the glass electrode and the hydro- 
gen electrode on solutions which were equimolecular with respect to piperazine 
dihydrochloride and the monohydrochloride (pK/) and on solutions which were 
equimolecular with respect to the monohydrochloride and free piperazine (pK ') 
(Table II). 

DISCUSSION 

We have used the available data in Table II in making approximate calculations 
of the ionization exponents of piperazine at infinite dilution, since this has not been 
previously reported. Using the standard Debye-Hiickel equation for moderately 
dilute solutions, we have found values for piperazine dihydrochloride for pK^ of 



PIPERAZINE AND GLYCYLGLYCINE BUFFERS 



235 



TABLE I 

Table for Preparation of Buffers at 25 C. 

(1) 0.1591 g. piperazine dihydrochloride diluted to 100 ml. with distilled water to which is added 
0.1000 N sodium hydroxide as indicated below in column (1). 

(2) 1.591 g. piperazine dihydrochloride diluted to 100 ml. with distilled water to which is added 
1.000 N sodium hydroxide. 

(3) 15.91 g. piperazine dihydrochloride diluted to 100 ml. with distilled water to which is added 
1.000 N sodium hydroxide. 

(4) 0.1321 g. glycylglycine diluted to 100 ml. with distilled water to which is added 0.1000 N 
sodium hydroxide. 

(5) 0.1591 g. piperazine dihydrochloride plus 0.1321 g. glycylglycine diluted to 100 ml. with dis- 
tilled water to which is added 1.000 N sodium hydroxide. 

(6) 0.1591 g. piperazine dihydrochloride diluted to 100 ml. with filtered sea water (pH 8.0) to 
which is added 1.000 N sodium hydroxide. 

(7) 0.1591 g. piperazine dihydrochloride plus 0.1321 g. glycylglycine diluted to 100 ml. with filtered 
sea water (pH 8.0) to which is added 1.000 N sodium hydroxide. 



Buffer 
pH 


ml. of NaOH to be added to above solutions 


(1) 


(2) 


(3) 


(4) 


(5) 


(6) 


(7) 


4.4 


0.66 








0.007 






4.6 


1.11 


0.78 






0.054 






4.8 


1.71 


1.18 


7.0 




0.120 






5.0 


2.47 


1.72 


11.1 




0.202 






5.2 


3.38 


2.48 


17.4 




0.302 






5.4 


4.44 


3.46 


25.8 




0.419 


0.088 


0.025 


5.6 


5.65 


4.55 


35.5 




0.538 


0.200 


0.140 


5.8 


6.74 


5.73 


46.6 




0.651 


0.353 


0.262 


6.0 


7.62 


6.80 


58.1 




0.745 


0.493 


0.399 


6.2 


8.35 


7.67 


69.0 




0.827 


0.615 


0.524 


6.4 


8.89 


8.35 


78.0 




0.899 


0.718 


0.655 


6.6 


9.27 


8.89 


85.0 




0.953 


0.797 


0.765 


6.8 




9.23 


90.0 




0.999 


0.854 


0.852 


7.0 






93.6 


0.57 


1.045 


0.895 


0.933 


7.2 








0.88 


1.091 


0.923 


1.017 


7.4 








1.37 


1.152 


0.947 


1.105 


7.6 








2.07 


1.231 


0.965 


1.195 


7.8 








2.94 


1.329 


0.983 


1.305 


8.0 








3.93 


1.445 


1.001 


1.435 


8.2 








5.03 


1.571 


1.027 


1.573 


8.4 








6.14 


1.697 


1.065 


1.695 


8.6 


10.66 






7.17 


1.825 


1.113 


1.821 


8.8 


11.00 


10.83 


107.9 


8.01 


1.950 


1.176 


1.949 


9.0 


11.51 


11.23 


112.1 


8.65 


2.064 


1.262 


2.070 


9.2 


12.19 


11.85 


117.9 


9.07 


2.185 


1.362 


2.194 


9.4 


13.09 


12.70 


125.2 


9.40 


2.313 


1.488 


2.338 


9.6 


14.18 


13.75 


134.6 




2.451 


1.642 


2.520 


9.8 


15.39 


14.90 


145.6 




2.587 


1.865 


2.750 


10.0 


16.60 


16.05 


156.6 




2.717 






10.2 


17.68 


17.09 


166.6 




2.832 






10.4 


18.60 


17.98 


176.2 




2.937 






10.6 


19.35 


18.74 


184.4 




3.042 






10.8 




19.39 


190.5 




3.153 






11.0 




19.94 


195.2 











236 



MARSHALL E. SMITH AND LYNWOOD B. SMITH 



TABLE II 

Relations Between pK' and Ionic Strength for Piperazine 
Dihydrochloride at 25 C. 



Ionic strength 


pKi' 


Ionic strength 


pKY 


0.0100 


5.44 


0.0174 


9.74 


0.0238 


5.49 


0.0519 


9.78 


0.100 


5.56 


0.174 


9.82 


0.238 


5.68 


0.571 


9.87 


1.00 


5.79 


0.800 


9.88 


1.67 


5.86 







5.32 and pK 2 of 9.70. Bredig (1894) found a second ionization constant of 6.4 
X 1O 5 at 25 C., but failed to report a first ionization constant. Kolthoff (1925, 
1925) reported a pK/ of 4.05 and a pK 2 ' of 8.34 for piperazine at 15 C. Since 
the temperature he used is different from that used in the present experiment, the 
results are not comparable. A search of the literature reveals no other reports of 
the ionization exponents of piperazine. 

On the other hand, because of interest in amphoteric electrolytes and in dipep- 
tides, numerous studies of the K a , K b , pK/, pK 2 ', pK 1? and pK 2 values of glycyl- 
glycine have been made (Euler, 1907; Dernby, 1916, 1917; Eckweiler et al.. 1921 ; 
Harris, 1923; Levene et al., 1924; Taufel and Wagner, 1927; Branch and Miya- 
moto, 1930; Mitchell and Greenstein, 1930;. Fromageot and Watremez, 1930; 
Stiasny and Scotti, 1930; Greenstein, 1933; Johnson and Peterson, 1935; Neu- 
berger, 1937; Konikov, 1938; Carr and Shutt, 1939; Glasstone and Hammel, 1941 ; 
Smith and Smith, 1942). Included above are numerous titration curves for 
glycylgylcine in different media, but no actual tables for the preparation of buffers 
have been previously reported. 

SUMMARY 

The advantages of piperazine dihydrochloride and glycylglycine as buffers 
include the low toxicity, the lack of volatility, the solubility, the availability of the 
pure products, the convenience and accuracy of buffer preparation, and the lack of 
precipitation of calcium and magnesium salts from sea water below a pH of 9.9. 

Table I indicates the preparation of solutions of known pH involving piperazine 
dihydrochloride, glycylglycine, and mixtures of these two in distilled water and in 
sea water at 25 C. Because the determinations were not made with the hydrogen 
electrode this table must be considered as being susceptible to small error, especially 
on the alkaline side of pH 9. 

Table II shows the pK/ and pK 2 ' values for piperazine dihydrochloride for 
several ionic strengths. Using the Debye-Hiickel equation, the extrapolated pK 
values of piperazine at infinite dilution were found to be 5.32 for pK t and 9.70 
for pK 2 . 

LITERATURE CITED 

BRANCH, G. E. K., AND S. MIYAMOTO, 1930. Dissociation constants and heats of ionization of 
some simple amino acids and peptides. Jour. Amcr. Chem. Soc., 52 : 863-868. 

BREDIG, G., 1894. On the great affinity of bases. Z. physlk. Client., 13: 313. 

CARR, W. AND W. J. SHUTT, 1939. Dielectric properties and ionization constants of amino 
acids. Trans. I'araday Soc., 35 : 579-587. 



PIPERAZINE AND GLYCYLGLYCINE BUFFERS 237 

CORNMAN, I., 1940. Echinochrome as the sperm activating agent in egg water. Biol. Bull., 

79: 365. 

CORNMAN, I., 1941. Sperm activation by Arbacia (punctulata) egg extracts, with special ref- 
erence to echinochrome. Biol. Bull., 80: 202-207. 
DERNBY, K. G., 1916. Kinetics of an enzymic hydrolysis of glycylglycine. Compt. rend. trav. 

lab. Carlsbcrg. Ser. chim., 11 : 263-295. 
DERNBY, K. G., 1917. Studies on the proteoclastic enzymes of yeast and their relationship to 

autolysis. Biochcin. Z., 81 : 165. 

DIEKE, S. H., G. S. ALLEN, AND C. P. RICHTER, 1947. The acute toxicity of thioureas and 
related compounds to wild and domestic Norway rats. Jour. Pharmacol. Exp. Thcrap., 

90: 260-270. 
ECKWEILER, H., H. M. NOYES, AND K. G. FALK, 1921. Amphoteric properties of some amino 

acids and peptides. Jour. Gen. Physio!., 3 : 291-308. 

EULER, H., 1907. Fermentative cleavage of dipeptides. Z. physiol. Chem., 51 : 213-225. 
EVANS, T. C., H. W. BEAMS, AND M. E. SMITH, 1941. Effects of Roentgen radiation on the 

jelly of the Arbacia egg. Biol. Bull. 80: 363-370. 
FROMAGEOT, C. AND M. WATREMEZ, 1930. Comparison between the buffer power of glycocol 

and glycylglycine. Compt. rend., 190: 1459-1462. 
GLASSTONE, S. AND E. F. HAMMEL, JR., 1941. Physicochemical studies of the simpler poly- 

peptides. Jour. Anier. Chcm. Soc., 63: 243-248. 
GOMORI, G., 1946. Buffers in the range of pH 6.5 to 9.6. Proc. Soc. Expil. Biol. Mcd., 62 : 

33-34. 
GREENBAUM, F. R., 1937. New water-soluble theophylline compound. Amer. Jour. Pharm., 

109 : 550-554. 
GREENSTEIN, J. P., 1933. Studies of the peptides of trivalent amino acids. Jour. Biol. Chcm., 

101 : 603-621. 
HANZLIK, P. J., 1917. Piperazine and other organic urate solvents. Jour. Lab. Clin. Mcd., 

2 : 308-327. 
HARRIS, L. J., 1923. The titration of amino and carboxyl groups in amino acids, polypeptides, 

etc. Proc. Roy. Soc. (London), 95B : 440-484. 
JOHNSON, M. J. AND W. H. PETERSON, 1935. The peptidase system of Aspergillus parasiticus. 

Jour. Biol. Chcm., 112: 25-34. 
KOLTHOFF, I. M., 1925. The dissociation constant, solubility product, and titration of alkaloids. 

Biochcm. Z., 162 : 289-353. 
KOLTHOFF, I. M., 1925. The electrometric titration of alkaloids. Pharm. Wcekblad, 62: 1287- 

1293. 
KONIKOV, A. P., 1938. The effect of a neutral salt on the dissociation of ampholytes. Jour. 

Gen. Chcm. (U. S. S. R.), 8 : 1194-1203. 
LEVENE, P. A., H. S. SIMMS, AND M. H. PFALTZ, 1924. The relation of chemical structure to 

the rate of hydrolysis of peptides. Jour. Biol. Chem., 61 : 445-464. 
MACINNES, D. A., D. BELCHER, AND T. SHEDLOVSKY, 1938. Meaning and standardization of 

the pH scale. Jour. Amer. Chcm. Soc., 60: 1094-1099. 
MITCHELL, P. H. AND J. P. GREENSTEIN, 1930. Electrometric determination of the dissociation 

of glycocoll and simple peptides. Jour. Gen. Physiol., 14: 255-275. 
NEUBERGER, A., 1937. Dissociation constants and structures of amphoteric ions. Proc. Roy. 

Soc. (London), 158A : 68-96. 

POWNEY, J. AND D. O. JORDAN, 1937. The application of the glass electrode to the measure- 
ment of hydrogen-ion concentration in alkaline solution. Jour. Soc. Chem. Ind. (Lon- 
don), 56T: 133-137. 

SIEBER, J., 1890. On diethylenediamine. Bcr., 23: 326-327. 
SMITH, E. R. B. AND P. K. SMITH, 1942. Thermodynamic properties of solutions of amino 

acids and related substances. Jour. Biol. Chem., 146: 187-195. 
STIASNY, E. AND H. Scorn, 1930. Acid- and alkali-binding power of peptides. Ber., 63B : 

2977-2983. 
TAUFEL, VON, K. AND C. WAGNER, 1927. On the significance and determination of potential 

acidity. Z. angcu'. Chem., 40: 133-141. 
TYLER, A. AND N. H. HOROWITZ, 1937. Glycylglycine as a sea-water buffer. Science, 86 : 

85-86. 



CHROMATOPHOROTROPINS IN THE CENTRAL NERVOUS 
ORGANS OF THE CRAB, HEMIGRAPSUS OREGONENSIS 

THOMAS E. BOWMAN 1 
Department of Zoology, University of California, Berkeley 4 

INTRODUCTION 

Early work on the humoral control of crustacean chromatophores has demon- 
strated that the sinus gland is the most important source of chromatophorotropic 
suhstances. Investigations leading to this conclusion are discussed in Brown's 
review (1944, pp. 130-134). Later work hy Brown (1946) and Brown and 
Saigh (1946) has shown that most crustacean central nervous systems also possess 
at least two chromatophorotropic principles, one causing all portions of the body 
of Crago except the telson and uropods to become pale (Crago body-lightening 
hormone, CBLH), and a second (Crago-darkening hormone, CDH) darkening 
the telson and uropods, and in the absence of CBLH, the body also. CDH, how- 
ever, was absent in the Brachyura studied. 

This paper reports experiments undertaken for the purpose of determining 
whether or not chromatophorotropins are present in the central nervous system 
of the Pacific coast shore crab, Hemigrapsus oregonensis. Most attention was 
given to the optic ganglia, but some experiments were performed to test the brain 
and thoracic ganglia. I wish to thank Dr. R. I. Smith for his many helpful 
suggestions and criticisms. 

MATERIALS AND METHODS 

Only male crabs were used. Their eyestalks were ligated with number 80 
cotton thread on successive days before injections were made. After the melano- 
phores were completely punctate, the crabs were injected with Carcinides perfusion 
fluid (Pantin, 1934), and those whose melanophores responded at all were not 
used in subsequent experiments. 

Organs from which extracts were made w T ere rinsed in several changes of 
Carcinides perfusion fluid to remove any blood adhering to them and transferred 
to a roughened depression slide containing a drop or two taken from a measured 
quantity of perfusion fluid. Here they were torn apart, crushed, and triturated 
with fine forceps under a dissecting microscope, care being taken to ensure as com- 
plete extraction as possible. The extract was then transferred with an eyedropper 
to the measured quantity of perfusion fluid, and the depression slide was rinsed 
with this perfusion fluid several times. The extract was boiled for a few seconds, 
allowed to settle, and the supernatant fluid was used for injections. Sterile needles 
and syringes were used for all injections. 

1 Present address : Scripps Institution of Oceanography, La Jolla, California. The material 
reported herein is from a thesis submitted to the Faculty of the University of California in 
partial fulfillment of the requirements for the degree of Master of Arts. 

238 



CHROMATOPHOROTROPINS IN HEMIGRAPSUS 239 

To record the responses of the melanophores, an arbitrary index of four stages 
was used, from complete concentration of the pigment (stage 1) to complete dis- 
persion (stage 4). The somewhat opaque and pigmented cuticle of Hemlgrapsus 
oregonensis, especially in the larger crabs, obscures the chromatophores over much 
of the body, and observations were therefore made on the arthrodial membranes 
at the bases of the legs. 

In preliminary experiments it was found that the melanophores of H. orego- 
nensis responded to injections of sufficiently strong extracts of muscle and gill, as 
well as to weak egg albumin solutions (Fig. 2). Such preparations, particularly 
the latter, certainly do not contain chromatophorotropins, and the melanophore 
response in such cases probably is part of a rather generalized stimulation resulting 
from the introduction of foreign substances into the hemolymph. Since it was 
essential to eliminate such responses when testing extracts of central nervous or- 
gans for chromatophorotropins, this was done by making up the extracts from 
comparable volumes of tissue, small enough so that extracts of them would not 
contain sufficient protein or other unknown non-humoral material to affect the 
chromatophores. In this way it was intended to distinguish specific chromato- 
phorotropic effects from the non-specific effects resulting from injection of large 
tissue masses. In order to make up such extracts it was necessary to know the 
amounts of tissue in the different organs extracted. Since the organs, especially 
the sinus glands, were too small to weigh on an analytical balance, measurements 
were made of their volumes using a procedure suggested by the method of Weil 
and Pantin (1931) for measuring volume changes in the turbellarian, Gunda ulvac. 
The organ was carefully dissected out, placed in the ruled area of a hemacytometer 
counting chamber and flattened under the cover glass. An enlarged (1 mm. = 
2 in.) outline of the organ was drawn on a piece of paper containing a copy of the 
ruled area of the hemacytometer ; this was traced onto a piece of medium weight 
drawing paper, cut out, and weighed. By comparing this weight with that of a 
similarly enlarged square millimeter (0.1 cu. mm.) the volume of the organ could 
be roughly determined. Table I gives the results of these measurements. All 
crabs used had a carapace width of 1.7 cm. The numbers are the weights of the 
paper cutouts in milligrams. 

These measurements are admittedly crude, most of the error being due to 
dissection. They do, however, give some idea of the relative size of the organs 
involved. The dilution factors are selected values, based on the relative volumes 
by which extracts of the different organs were diluted to give approximately equal 
volumes of tissue in the same amounts of extract. For organs other than those 
listed in Table I (brain, leg nerve, etc.) the volume was measured by the pre- 
ceding method, and the extract was diluted accordingly. 

In preparing extracts, the size of the crab from which the organ was extracted 
was considered. It was assumed that the size of the sinus gland and other organs 
varies directly with the weight of the crab ; thus, for example, the sinus gland from 
a crab 1.9 cm. wide (3.0 g.) would be twice the volume of that from a crab 1.5 
cm. wide (1.5 g.). 

The amount of extract injected was always 5 per cent of the body weight, 
assuming a specific gravity of 1.00 for the extract. To obviate weighing each 
crab, a large number of crabs were weighed and the weight plotted against the 



240 



THOMAS E. BOWMAN 



TABLE I 

Comparative size of sinus gland and optic ganglia 





Sinus 
gland 


Medulla 
terminalis 


Medulla 
interna 


Medulla 
externa 


Lamina 
ganglionaris 


1 mm.- 
paper 




19 


607 


308 


311 


207 


377 




14 


767 


284 


293 


282 


364 




16 


501 


272 


341 


250 


355 




18 


579 


249 


250 


198 


365 




12 


550 


236 


259 


157 


376 




14 


350 


210 


279 




374 


av. 


15.5 


559 


260 


289 


219 


369 


av. 


0.004 


0.151 


0.070 


0.078 


0.059 


0.100 


3690 


Relative volume 


1 


36.1 


16.8 


18.7 


14.17 




Dilution factor 


1 


30 


15 


15 


12 





J I 



SINUS GLAND 0.5 so 

av 28 crabs 



J I 



MEDULLA TERMINALIS 



ov 14 crabs 50 SO 
-o 

,,-' ' o 




av 9 crabs 0-5 SO 



I I I I I 



15 30 45 60 75 90 105 120 



15 30 45 60 75 90 105 120 



SINUS GLAND 3.0SG 

cw 6 c r abs 



J I 



J I 



J I 



15 30 45 60 75 90 105 120 



MEDULLA INTERNA 



av. 8 crobs 5.0 SG _ _-<>- 

,O'" 




jf 



I I I I I 



15 30 45 60 75 90 105 120 



SINUS GLAND 5.0 SG 
a 3 c'abs 



J I 



I I I 1 



J I 



15 30 45 60 75 90 105 120 



MEDULLA EXTERN A 0.5 SG 

8 crabs 




r4*-- o.-. o 

LAMINA GANGLIONARIS 0.5 SG 

Ov 7crob5 



15 30 45 60 75 90 105 120 



FIGURE 1. Responses of Hemigrapsus melanophores to injections of extracts of various 
organs. Abscissae: time (minutes) after injection. Ordinates : degree of dispersion of melanin 
(1 = complete concentration; 4 = complete dispersion). 



CHROMATOPHOROTROPINS IN HEMIGRAPSUS 



241 



4 







3 

2 


THORACIC GANGLIA 0.5 SG 

ov. 4 crabs 




1 


-"BRAIN 0.5 SG 

av. 4 crobs 
1 1 1 1 1 1 


1 } 



15 30 45 60 75 90 105 120 



EGG ALBUMEN 



~-av. 2 crobs 0.5 SG 




I I I I I I I I 

15 30 45 60 75 90 105 120 



4 i 
3 

2 
I 



LEG NERVE 5.0 SG 
ov. (0 crobs 



^lYESTALK MUSCLE 0.5 SG 

ov 6 crabs 
I I I I I I I 



15 30 45 60 75 90 105 120 



ACETYL CHOLINE CHLORIDE 
av. 9-crobs 9-5" IO' 8 




15 30 45 60 75 90 105 120 



FARADIC STIMULATION OF EYESTALK STUBS 
av. 9 crobs 



I 



I 



I 



I 



I 



15 30 45 60 75 90 105 120 

FIGURE 2. Responses of Hemigrapsus melanophores. Explanation same as for Figure 1. 

carapace width. This made it possible simply to measure the carapace width and 
to inject the proper amount. Injections were made at the base of a walking leg. 
The strength of each extract is expressed in terms of the volume of a sinus 
gland of the crab injected. Thus "0.5 SG" (cf. Fig. 1) indicates that a crab 
injected with 5 per cent of its body weight of this extract received a volume of 
tissue approximately equal to 0.5 times the volume of one of its own sinus glands. 

EXPERIMENTS AND RESULTS 

The results of the injections are shown in Table II and in the graphs of Figures 
1 and 2. Responses are classified as "weak" w r hen the melanin was not dispersed 
beyond stage 2, and "good" when stage 3 was reached. The average responses 
do not include, those animals which failed to respond. The validity of averaging 
the arbitrary figures of the melanophore index is subject to criticism (Parker, 
1948, pp. 1415), and the variability of response, shown in Table II, must be con- 
sidered when evaluating a response. 

By far the most potent extracts were those of sinus glands. Other extracts 
from comparable volumes of tissue, while in some cases acting as rapidly as sinus 
gland extracts, did not produce the maximum and sustained responses which always 
followed injections of the latter. Moreover, the only extracts which always pro- 
duced 100 per cent "good" responses were those of sinus glands. The more rapid 



242 



THOMAS E. BOWMAN 



TABLE 1 1 

Summary of experiments 



Organ extracted 


Strength of extract 
(vol. of one of own 
sinus glands = 1 ) 


No. of crabs 
injected 


Responses 


None 


Weak 


Good 


Sinus gland 


0.5 SG 


28 








28 


Sinus gland 


3.0 SG 


6 








6 


Sinus gland 


5.0 SG 


3 








3 


Medulla terminalis 


0.5 SG 


11 


2 


4 


5 


Medulla terminalis 


5.0 SG 


15 


1 


5 


9 


Medulla interna 


0.5 SG 


13 


4 


5 


4 


Medulla interna 


5.0 SG 


8 





1 


7 


Medulla externa 


0.5 SG 


8 





5 


3 


Lamina ganglionaris 


0.5 SG 


17 


10 


5 


2 


Brain 


0.5 SG 


8 


4 


4 





Thoracic ganglia 


0.5 SG 


12 


8 


3 


1 


Eyestalk muscle 


0.5 SG 


13 


7 


5 


1 


Leg nerve 


5.0 SG 


21 


11 


9 


1 


Egg albumin 


0.5 SG 


14 


12 


2 





Egg albumin 


2% sol'n 


11 





2 


9 


Acetylcholine 


9.5 X 10~ 8 


26 


17 


5 


4 


Acetylcholine 


10~ 4 


5 


5 








Faradic stimulation of 




10 


1 


1 


8 


eyestalk stubs 













responses to the weaker (0.5 SG) extract are difficult to understand, but because 
of the small number of crabs injected with the stronger sinus gland extracts and 
the extent of individual variability in responsiveness, it is not possible to compare 
the responses adequately. 

The responses to optic ganglia extracts with a concentration of 0.5 SG were 
in most cases weak, but these extracts probably did contain specific chromatophoro- 
tropins, since the responses to 0.5 SG egg albumin solutions were so slight, and 
the responses to eyestalk muscle extracts of the same concentration were also 
insignificant. However, the amounts of hormone in these optic ganglia extracts, 
especially those of lamina ganglionaris, appear to have been close to the threshold 
for the melanophores of Hemigrapsus oregonensis. 

The responses to optic ganglia extracts of the concentration 5.0 SG were more 
definite, although they were much weaker than the responses to sinus gland ex- 
tracts. It can be safely said, therefore, that the medulla terminalis and medulla 
interna (and probably the other optic ganglia) contain material that causes dis- 
persion of the melanophores in Hemigrapsus oregonensis. The importance of this 
to the normal crab might be determined by removing the sinus glands without 
destroying the optic ganglia. 

The possibility remained that the melanophores were responding to the acetyl- 
choline present in the extracts. Welsh (1939) found large amounts of acetyl- 
choline in leg nerves and ventral ganglia of Carcinides ( Carcinus), there being 
about five times as much in ganglia (about 10y/g.) as in fibers (about 2y/g.), 
while Smith (1939) found up to 20y/g. in nerve fibers and up to 66y/g. in 



CHROMATOPHOROTROPINS IN HEMIGRAPSUS 243 

the ganglia of Cainbanis liuiosus. It is improbable, however, that the chromato- 
phorotropic effects of Hemigrapsus ganglia extracts are due to their acetylcholine 
content, since cholinesterase is probably also present in these extracts (Marnay 
and Nachmansohn, 1937). Moreover, Abramowitz and Abramowitz (1938) ob- 
tained slight responses of the chromatophores in only 20 per cent of the Uca they 
injected with acetylcholine. When injected with 5 per cent of their body weight 
of 9.5 X 10~ s acetylcholine chloride, each Hemigrapsus received the amount of 
acetylcholine that would have been present in tissue equal in volume to five times 
one of its own sinus glands, assuming 50y/g. as the concentration of acetyl- 
choline in this tissue. Seventeen out of twenty-six crabs thus injected failed to 
respond, and five crabs injected with the much stronger 10 4 acetylcholine also 
showed no response. This makes it fairly certain that the responses to optic 
ganglia extracts were not caused by the acetylcholine contained in them. 

Responses to extracts of brain and thoracic ganglia of the concentration 0.5 SG 
w 7 ere in most cases weak or absent, and they give little information as to whether 
or not chromatophorotropins are contained in these organs. A few injections of 
much stronger extracts of brain resulted in good responses, but these experiments 
were nor well controlled. The good response to about ten seconds' faradic stimu- 
lation of one of the eyestalk stubs with a Harvard inductorium shows, however, 
that substances affecting the melanophores can be released in the eyestalkless 
Hemigrapsus, most probably from some part of the central nervous system. Deep 
probing of eyestalkless Hemigrapsus with a hypodermic needle at the base of the 
third or fourth leg also caused melanin dispersion in some cases. It is possible 
that the melanin dispersion following injections of muscle extracts and egg albumin 
solutions is an indirect response, caused by the release of chromatophorotropins 
from central nervous sources. 

It is interesting to note that responses to leg nerve extracts were weak or 
absent, indicating that if a chromatophore hormone is present in nervous tissue it 
may be produced by or concentrated in the central nervous system rather than the 
peripheral nerves. 

DISCUSSION 

It must be emphasized that these experiments do not compare the total amount 
of chromatophorotropic hormone available to the animal from one organ with that 
available from another organ, but indicate that while this hormone is most con- 
centrated in the sinus gland it is not absent from certain parts of the central nervous 
system. It is entirely possible that in Hemigrapsus orcgonensis as much or more 
hormone is present in central nervous system sources as in the sinus glands, al- 
though the present work does not provide quantitative information concerning this 
point. Although Brown (1940) found that 80 per cent of the chromatophorotropic 
material in the eyestalks of several species of shrimps and crabs was referable to 
the sinus gland. Smith (1948) has recently presented evidence that only about 
one-third of the retinal pigment activator in the eyestalks of Hemigrapsus orcgo- 
nensis and two other species of grapsoid crabs resides in the sinus glands. It 
seems not unlikely that the distribution of the melanophore activator in the eye- 
stalks of Hemigrapsus oregonensis is comparable. 

The concentration of chromatophorotropins in the histologically specialized and 



244 THOMAS E. BOWMAN 

well innervated sinus gland may represent an adaptation for the storage and more 
especially the release of active substances in effective amounts and within short 
periods of time. Production of the active principles themselves might be by nervous 
tissues in general, or, as seems more likely, might be limited to more or less 
restricted regions of specialized cells within the central nervous system, including 
the sinus gland itself. Thus we could imagine that the chromatophorotropins in 
any given mass of nervous tissue are derived from a relatively few cells, each as 
specialized as sinus gland cells. These cells could be evenly distributed, resulting 
in a uniform distribution of hormone throughout the central nervous system, as 
Brown and Saigh (1946) found for CDH in the isopod. Idothca baltica. On the 
other hand, as in the case of CDH and CBLH in Crago, they could be restricted 
to a single organ (the tritocerebral commissure. Brown, 1946). The sinus gland, 
as Turner (1948, p. 561) points out, probably represents the highest evolutionary 
stage in the differentiation of endocrine tissue from the central nervous system in 
the Crustacea, and would therefore be expected to contain the highest concentra- 
tions of active materials. The experiments reported herein show this to be the 
case for chromatophorotropins in the sinus gland of Hcmigrapsus oregonensis. 

SUMMARY 

1. The melanophores of Hemigrapsus oregonensis become punctate after eye- 
stalk removal. 

2. Chromatophorotropins, which cause dispersion of the melanin when injected, 
are present in greatest concentration in the sinus gland, and are also present in the 
optic ganglia and possibly in the brain and thoracic mass of ganglia. 

3. The melanin dispersion in response to electrical stimulation of the eyestalk 
stubs and to deep probing with a hypodermic needle indicates that some source 
of releasable chromatophorotropins exists other than the eyestalks. 

4. The total amount of chromatophorotropins in the sinus gland is not neces- 
sarily greater than in any of the central nervous organs. The specialized structure 
and the innervation of the sinus gland suggests that its importance lies in its ability 
to store and rapidly release effective amounts of chromatophorotropins. 

5. Injection of sufficient amounts of certain substances, including muscle and 
gill extracts and egg albumin solution, also induces melanin dispersion in eyestalk- 
less Hetnigrapsus oregonensis. It is suggested that these substances do not con- 
tain chromatophorotropic hormones, but the response to them is the result of a 
more general stimulation causing the release of chromatophorotropins from central 
nervous sources. 

LITERATURE CITED 

ABRAMOWITZ, A. A. AND R. K. ABRAMOWITZ, 1938. On the specificity and related properties 

of the crustacean chromatophorotropic hormone. Biol. Bull., 74 : 278-296. 
BROWN, F. A., JR., 1940. The crustacean sinus gland and chromatophore activation. Physiol. 

Zool., 13 : 343-355. 
BROWN, F. A., JR., 1944. Hormones in the Crustacea : their sources and activities. Quart. 

Rev. Biol., 19: 32-46, 118-143. 
BROWN, F. A., JR., 1946. The source and activity of Crago-darkening hormone (CDH). 

Physiol. Zool, 19 : 215-223. 






CHROMATOPHOROTROPINS IN HEMIGRAPSUS 245 

BROWN, F. A., JR. AND L. M. SAIGH, 1946. The comparative distribution of two chromato- 

phorotropic hormones (CDH and CBLH) in crustacean nervous systems. Biol. Bull., 

91 : 170-180. 
MARNAY, A. AND D. NACHMANSOHN, 1937. Cholinesterase dans le nerf de Homard. C. R. 

Soc. Bio}., Paris, 125 : 1005. 
PANTIN, C. F. A., 1934. On the excitation of crustacean muscle. I. Jour. E.rp. Biol.. 11 : 

11-27. 
PARKER, G. H., 1948. Animal colour changes and their neurohumours. Cambridge University 

Press, Cambridge, England. 
SMITH, R. I., 1939. Acetylcholine in the nervous tissues and blood of crayfish. Jour. Cell. 

Comp. Physio!., 13 : 335-344. 
SMITH, R. I., 1948. The role of the sinus gland in retinal pigment migration in grapsoid crabs. 

Biol. Bull., 95: 169-185. 

TURNER, C. D., 1948. General endocrinology. W. B. Saunders Co., Philadelphia. 
WEIL, E. AND C. F. A. PANTIN, 1931. The adaptation of Gunda ulvae to salinity. II. The 

water exchange. Jour. E.rp. Biol.. 8: 73-81. 
WELSH, J. H., 1939. Chemical mediation in crustaceans. I. The occurrence of acetylcholine 

in nervous tissues and its action on the decapod heart. Jour. E.rp. Biol., 16: 198-219. 



SOME EFFECTS OF CENTRIFUGING UPON PROTOPLASMIC 

STREAMING IN ELODEA 1 

H. W. BEAMS 
Zoological Laboratories, State University of loiva, Iowa City 

As pointed out by Ewart (1903), protoplasmic streaming was probably ob- 
served before the existence of protoplasm as such was recognized. Notwithstand- 
ing the many studies that have been made upon this interesting and complicated 
biological phenomenon, certain of the basic problems such as motive force, function 
and mechanism of flow remain largely unsolved. This is not surprising, because 
to understand protoplasmic streaming requires a rather complete knowledge not 
only of protoplasm itself, but also of the physics and chemistry of streaming as 
well. In fact, certain theories of protoplasmic structure are inadequate because 
they fail to account for a suitable structural mechanism to allow for protoplasmic 
streaming. 

Of the extensive literature dealing with protoplasmic streaming, few papers 
have been published which are concerned directly with the effects of centrifugal 
force on this process as such, although numerous studies have been made upon 
protoplasmic viscosity by aid of the centrifuge. Accordingly, it seems desirable 
to record here the results of some studies made upon protoplasmic streaming in 
the leaf cells of Elodea by use of the ultracentrifuge. 

MATERIAL AND METHODS 

Leaves of Elodea canadensis were removed from a region one to two inches 
back of the tip of an actively growing stem. They were placed in a dish where 
about one-fourth inch of the tip was cut off in order that the piece be of suitable 
size to fit in the cell of the air-driven rotor. The tips were then placed directly 
in a water mount where they were observed under the high dry and oil immersion 
lenses. Here observations were made upon the frequency and direction of stream- 
ing in the cells of a selected area near the tip of the leaf. The piece was then 
removed to the rotor of the ultracentrifuge with the long axis parallel to the 
centrifugal force. It was then centrifuged at forces varying from 135,000 to 
350,000 times gravity for intervals varying from five minutes to four hours. After 
centrifuging, the piece was again removed to a slide and the region formerly 
studied selected for observation. 

Some of the pieces, after having the cells stratified in one direction, were 
reversed in the centrifuge so that stratification in the opposite direction occurred. 
Certain of the pieces were killed immediately upon removal from the ultracentrifuge 
by immersion for a short interval in boiling water. 

The minimum time required to stop the centrifuge, remove the piece to a slide 
and find the area formerly studied was about three to four minutes. 

1 Aided by grant from the National Institutes of Health, administered by J. H. Bodine. 

246 



CENTRIFUGE EFFECTS ON ELODEA 247 

OBSERVATIONS 

As is well known, the long axis of the cells of Eloclea is arranged parallel to 
the long axis of the leaf. Near the mid-rib of the leaf the cells are usually longer 
and narrower and are more likely to be found undergoing streaming. Usually, 
if the leaf has been taken from near the tip of an actively growing stem, a few 
minutes' stand on the stage of the microscope, exposed to light and a slightly 
elevated temperature, is sufficient to initiate active rotational streaming in a high 
percentage of the cells. The streaming in Elodea has been referred to as rota- 
tional ( Seifriz, 1943) because the protoplasm is chiefly confined to a peripheral 
layer between the sides of the cell and the central vacuole. In Plate I, Figure 1, 
is illustrated a control cell in active streaming. The chloroplasts at the periphery 
are blurred because ol their movement within the interval required to take the 
photograph. 

Under normal conditions, according to Pfeffer (1906), the flow is in one 
direction only within the cells, clockwise or counter clockwise. In addition, both 
Pfeffer (1906) and Ewart (1903) maintain that the flow is in opposite directions 
on the two sides of the dividing walls between each pair of contiguous cells. \Ve 
have not been able to confirm this statement in the Elodea studied here. Counts 
of a hundred pair of actively streaming adjacent cells revealed that in only about 
70 per cent of them was the direction of flow opposite on the two sides of adjacent 
cell walls. The reason for this discrepancy is not clear, but it should be recalled 
that Berthold (1866) reported inconsistency in the direction of streaming between 
adjacent cells of Elodea. However, why, in the majority of adjacent cells, the 
direction of streaming on the two sides of the adjacent walls should be opposite, 
is unknown. 

Figure 2 shows a low power view of a number of cells that were centrifuged 
at approximately 135,000 times gravity for five minutes. It will be observed that 
the chloroplasts are packed at the centrifugal pole. If the rotor is allowed to 
accelerate rapidly, the chloroplasts, but not the cytoplasm, are probably displaced 
within ten seconds. In other words, the movement of the chloroplasts takes place 
very rapidly through the protoplasm of the cell. It is of interest that the proto- 
plasm is not killed by this rapid displacement of the chloroplasts through it. In 
fact, stratified cells (Fig. 4) may be reversed in the centrifuge and the chloro- 
plasts thrown to the opposite end (Fig. 5). This process may be repeated several 
times without killing the cell. However, Northen and Xorthen ( 1938) have 
found that repeated centrifugation and displacement of the chloroplasts through 
the cells of Spirogyra produces a marked lowering of the viscosity. 

High centrifugal force, 350,000 times gravity (Fig. 3), causes rapid displace- 
ment of the cellular materials. From the centrifugal to the centripetal pole the 
elements are stratified as follows: (1) chloroplasts, cytoplasm and nucleus; (2) 
vacuole. It will be noted that a sharp boundary exists between the centrifugal end 
of the vacuole and the cytoplasm, although such is not always the case for the 
chloroplasts, cytoplasm and nucleus. Apparently the vacuole is displaced cen- 
tripetally and is forced in contact with the cell wall. Xo cytoplasm can be ob- 
served remaining along the sides of the cell. What happens to the plasma mem- 
brane if such be a permanent structure in Elodea. is not known. Xo evidence of 



248 



H. W. BEAMS 

I 'I \TE I 




CENTRIFUGE EFFECTS ON ELODEA 249 

organized streaming of the protoplasm is apparent in such cells, although Brownian 
movement may be evident. 

Figures 6, 7, 8 and 9 are a series of photographs showing stratification and 
partial recovery of three cells centrifuged for thirty minutes at 350,000 times 
gravity. A complete stratification of the cellular contents occurs here (Fig. 6) 
as was also noted in Figure 3. Immediate examination upon removal from the 
ultracentrifuge revealed the cytoplasm in Brownian movement. All indications of 
anv type of organized streaming or even churning movements are absent. How- 
ever, within thirty minutes after centrifuging, the Brownian movement of the 
cytoplasm pushes the chloroplasts back beyond the edge of the vacuole so that it 
is no longer obvious (Fig. 7). A gradual migration of the cytoplasm toward the 
centripetal end, followed by a movement of some of the chloroplasts along both 
sides of the cells, occurs. There is in these cells 110 organized streaming of cyto- 
plasm at this time. Within sixty minutes after centrifuging (Fig. 8), the cytoplasm 
has become continuous around the sides of the cells and has started to flow in an 
organized fashion. However, the rate of flow is very slow when compared to that 
of the normal cells. Ninety minutes after centrifuging (Fig. 9), the chloroplasts 
in the upper two cells are still largely bunched at the centrifugal end although some 
isolated chloroplasts are observed being carried around the cell in the stream. In 
the lower right hand cell the bunched chloroplasts are rotating around the cell as 
a mass. However, the movement is not continuous but by jerks, due to the forces 
encountered in trying to move a large mass through a relatively small stream. In 
some cells treated in this manner, complete recovery with normal streaming and 
normal distribution of chloroplasts occurs. In others, the cells are killed. In still 
others, as in Figures 6, 7, 8 and 9, injury occurs from which the cells partially, but 
not completely, recover within a six-hour interval. Less than one per cent of the 
cells have their walls ruptured and their contents missing (Fig. 15). 

A cell centrifuged at 135,000 times gravity for five minutes is illustrated in 
Figures 10, 11, 12 and 13. Here the chloroplasts are not as tightly packed as they 
are in the cells centrifuged at higher forces. Figure 10 shows the cell about four 
minutes after removal from the centrifuge. In addition to the chloroplasts the 
nucleus may be clearly observed. Rapid Brownian and churning movements of 
the protoplasm are evident at the centrifugal end of the cell, and the massed chloro- 

PLATE I 

All figures in Plates I and II (except Fig. 15) are of living unstained cells <>f Elodea. In 
all centrifuged cells the force was directed at various angles toward the bottom of the plate 
except when otherwise indicated. 

FIGURE 1. Control cell actively streaming. 

FIGURE 2. Low power view of the general effects of centrifugal force on the position of 
the chloroplasts. Centrifuged at approximately 135.0(10 times gravity for five minutes. 

FIGURE 3. High power view of cells showing stratification effects. Centrifuged at 350,000 
times gravity for thirty minutes. 

FIGURES 4 AND 5. Same cell centrifuged first in one direction and then in the opposite 
direction (at 135,000 times gravity tor five minutes in each case). Centrifugal lorce in Figure 
5 is toward the top of the plate. 

FIGURE 6. Group of cells photographed about six minutes after being centrifuged at 350,000 
times gravity for thirty minutes. 

FIGURE 7. Same cells thirty minutes later. 



250 



H. W. BEAMS 



PLATE II 




CENTRIFUGE EFFECTS ON ELODEA 251 

plasts are being pushed centripetally. Here the reestablishment of flow is up the 
right side of the cell. The chloroplasts gradually migrate in the stream along the 
right side of the cell (Figs. 11, 12) and eventually a complete separation of them 
occurs and recovery is effected (Fig. 13). In this cell the nucleus was not ob- 
served to be actively carried about in the streaming cytoplasm. However, they 
often do rotate in cells so treated. Figure 14 is a cell treated as in Figure 10. 
Here the redistribution of the chloroplasts is along both sides of the cell instead of 
one side only. In other words, organized streaming was delayed in this cell for 
a longer time than it was in the cell in Figure 10, and recovery was eventually 
established. 

In addition to the methods of recovery already described for centrifuged cells, 
other conditions may occur. In some cells the packed chloroplasts at the cen- 
trifugal end may be forced back as a mass to a position near the middle or even 
to the centripetal end (Figs. 16, 17). This seems to occur as a result of the 
clumped chloroplasts partially blocking the movements of the redistributing cyto- 
plasm. Consequently, they are simply forced ahead of it. The nucleus, too, may 
be carried along with the massed chloroplasts. When the cytoplasm breaks around 
the chloroplasts or develops new streams across the cell, the clumped chloroplasts 
break away into the stream, usually in small groups at first, but eventually they 
become isolated. It may take thirty minutes or more for normal distribution of 
the chloroplasts to become established. 

Observations on the direction of streaming in one hundred cells that were 
subsequently centrifuged at 135,000 times gravity for five minutes, showed that 
approximately 7 per cent of them had undergone a reversal in direction of flow. 

Clumped chloroplasts such as those shown in Figures 16 and 17 sometimes, 
but not always, cause formation of protoplasmic strands which cross between the 
wall and vacuole in almost any direction, resulting in many diverse and irregular 
patterns of flow. Two or more protoplasmic strands may join on the upper 
surface of the vacuole, giving rise to a churning motion of the protoplasm at the 
point of juncture. Flow in a single strand may be in opposite directions. When 
this condition occurs, the velocity of flow is much slower at the point of contact of 
the two opposite flowing protoplasmic streams. The small granules moving in 

PLATE II 

FIGURE 8. Same cells sixty minutes later. 

FIGURE 9. Same cells ninety minutes later. 

FIGURES 10, 11, 12 AND 13. Series of photographs showing recovery from centrifuging at 
135,000 times gravity for five minutes. 

FIGURE 14. Cell in process of recovery from centrifuging at 135,000 times gravity for 
five minutes. 

FIGURE 15. Cell showing broken wall and chloroplasts missing. Centrifuged at 350,000 
times gravity for thirty minutes. Such conditions are rare. 

FIGURES 16 AND 17. Cells centrifuged at 135,000 times gravity for five minutes. Photo- 
graph taken ten minutes later. The chloroplasts are being forced back from the centrifugal end 
by Brownian movement of the cytoplasm. (Centrifugal force directed to left in figures.) 

FIGURES 18, 19 AND 20. Starch grains in various stages of displacement within chloroplasts. 
The starch grains are on end of chloroplasts originally directed centrifugally. Brownian move- 
ment has caused the chloroplasts to lose the position taken up in the centrifugal field. 

FIGURE 21. Starch grain that has been freed from the chloroplast presumably by the cen- 
trifugal force. 



252 



H. W. BEAMS 



opposite directions at the point of contact of the two streams may be observed to 
collide and bounce around each other. Streaming in opposite directions within a 
single strand is usually a temporary condition. ' Direction of flow in the strands has 
also been observed to be in one direction for a time and subsequently reversed. 
Reversal of flow, however, is usually of short duration and appears to be caused by 
the difference in flow pressure produced by the main channels of the cytoplasm 
along the two opposite sides of the cells in which the strands are usually directly 
or indirectly connected. The difference in flow pressure of the two opposite sides 
of the cells is often due to the massed chloroplasts blocking the normal channels of 
flow. Hence, strands are developed at the points of least resistance to the proto- 
plasmic flow. 

In some of the thin protoplasmic strands which extend across the cell, a chloro- 
plast may become included within its streaming cytoplasm (Text Fig. 1, A). 
The chloroplast, being greater in diameter than the stream, moves relatively slowly 
along its channel (Text Fig. 1, B). In a few such cases we have observed that 






TEXT FIGURE 1 

FIGS. A, B AND C. Centrifuged cell partially recovered showing movement of chloroplast 
by means of contraction within small strand. At A the chloroplast is flowing slowly toward 
the lower right side of cell. At B it has almost reached the opposite end of strand. However, 
suddenly a rapid backward movement of the chloroplast occurs as shown at C. 

FIGS. D, E AND F. These figures illustrate contraction of a cytoplasmic strand. Figure D 
shows position of strands. At E strand has moved with current down along side of cell. 
Figure F shows return of strand to near former position. 

a quick return of the chloroplast from a position indicated in Text Figure 1, B to 
that of Text Figure 1, C occurs. Here the reversed movement of the chloroplast 
seems to be due to a contraction of the strand, because its movement is much too 
fast to be accounted for as a simple reversal of flow of the cytoplasm within the 
strand. 

Other protoplasmic strands extending across the cell from the upward channel 
of flow of one side of the cell to the downward channel of flow of the other side 
sometimes change position rapidly (Text Fig. 1, D, E and F). For example, the 
end of the strand in connection with the downward channel of flow may move 
with the current to a position indicated in Text Figure 1, E. When this occurs 



CENTRIFUGE EFFECTS ON ELODEA 253 

the strand appears to be stretched and its diameter diminished. However, sud- 
denly such a strand may "snap back" very quickly to a point near its former 
position (Text Fig. 1, F). This change seems to be due to a property of the 
protoplasm of the strand as a whole. The contraction described above may be 
repeated several times. We have also observed that "balls" of protoplasm con- 
siderably larger than the diameter of the strand may flow along its channel. Such 
occurrences are not rare and have been described by others in normal protoplasmic 
streaming. 

Figures 18, 19, 20 and 21 are centrifuged cells with chloroplasts showing starch 
grains. The starch inclusions appear heavier than the chloroplasts and are dis- 
placed centrifugally. Figure 18 illustrates a chloroplast with the starch grain 
displaced so that it appears to be partially extruded. The positions taken up by 
smaller starch grains are indicated in Figure 19. The upper chloroplast in Figure 
20 shows not only a partially displaced starch inclusion, but evidence of a partial 
stratification of the other materials as well. However, this is rare, for in the 
majority of the cells little, if any, stratification can be detected within the un- 
stained chloroplast. This indicates either that the chloroplast as a whole is very 
dense, or that its contents vary only slightly in relative specific gravity. 

In Figure 21 the lower inclusion is an isolated starch grain which has probably 
been pulled away from the chloroplast by the centrifugal force. It is difficult to 
say anything definite concerning the nature of the surface "membrane" of the 
chloroplast other than that considerable resistance is met at the surface in the 
displacement of the starch grains through it. In other words, the presence of some 
form of limiting "membrane" is indicated, although such a structure could not be 
seen in the centrifuged unstained chloroplast. 

DISCUSSION 

It is unnecessary here to review the extensive literature dealing with proto- 
plasmic streaming, since excellent reviews on this subject have been published by 
Ewart (1903) and more recently by Seifriz (1943). 

Protoplasmic streaming is known to be affected by many agents such as tem- 
perature, visible light, ultraviolet light, salts, acids, alkalies, oxygen, organic sub- 
stances, anesthetic agents, x-rays, radium, electricity, hydrostatic pressure, me- 
chanical manipulation and supersonic waves (see Seifriz, 1943, for references). 
In this paper it has been demonstrated that it is also affected to varying degrees 
by ultracentrifugal force. However, both Andrews (1915) and Vexler (1935) 
report that centrifuging with the usual laboratory type centrifuge produces little 
effect upon protoplasmic streaming. In fact Vexler (1935) found that centrifug- 
ing stimulated streaming in Myxomycetes. 

Marsland (1939) has suggested that in Elodea the streaming is motivated by 
sol-gel reactions and consequently is a phenomenon fundamentally related to ame- 
boid movement. The evidence for this is that with increasing hydrostatic pressure, 
the rate of protoplasmic streaming in Elodea is diminished. Complete obliteration 
of streaming with decreasing viscosity of the cytoplasm occurs at between 400 to 
500 atmospheres. This reaction is reversible. To apply the theory of sol-gel 
reversibility of protoplasm as the chief mechanism of streaming in a cell like Elodea 
is not easy, for according to Pfeffer (1906) only a very thin ectoplasmic membrane 



254 H. W. BEAMS 

exists in contact with the cell wall in actively streaming cells. Furthermore, ac- 
cording to Ewart (1903), the adjacent surfaces of both the protoplasm and vacuole 
flow. In addition, to account for rotational streaming by a sol-gel mechanism 
requires several assumptions. Because of this, Seifriz (1943) suggests an alter- 
nate view, namely, that "streaming is occasioned by a contractile force which need 
not involve a viscosity change or a sol-gel transformation." 

If the sol-gel mechanism is the main source of the motive force in protoplasmic 
streaming in Elodea, as there is certainly good evidence that it is for protoplasmic 
streaming in certain other cells (Mast, 1931; Lewis, 1942), the inhibition of 
streaming produced by centrifuging may be due to a lowering of the viscosity 
resulting in a disruption of the sol-gel mechanism. 

Moore (1935) found that centrifuging at 75,000 times gravity for five min- 
utes deforms and retards proliferation (and I presume streaming) for fifteen 
hours in plasmodium. He interprets this result as due to a separation of a heavy 
and light component of the cytoplasm. When allowed to stand for sufficient time, 
these heavy and light components of the cytoplasm return to their normal spatial 
relationships, and proliferation and streaming are reestablished. 

High centrifugal force has been demonstrated to displace most of the visible 
cellular materials (Beams and King, 1939; Beams, 1943) and certain ultramicro- 
scopic structures in the liver cell (Claude, 1943), as well as various types of protein 
molecules in non-living colloidal solutions (Svedberg, 1934). Yet, convincing 
evidence has not been obtained that a disruption of the vital ultramicroscopic 
organization of the protoplasm of Ascaris eggs occurs even at forces on the order 
of 900,000 times gravity for thirty minutes (Beams and King, 1937; Beams, 1943). 
This indicates that the intermolecular forces contributing to the vital structural 
protoplasmic framework are sufficiently great to resist disruption by high cen- 
trifugal force. 

However, whether or not the inhibition of protoplasmic streaming in Elodea 
reported here may be explained on the same basis as that given by Moore (1935) 
for plasmodium is unknown. 

In spite of the fact that all the visible cellular materials, including the cytoplasm, 
may be displaced to one end of the cell, recovery in the majority of cells does not 
result in a reversal of the direction of streaming. No experimental procedure has 
as yet been developed to produce consistent reversal in streaming of Elodea cells. 
However, Ewart (1903) states that change in direction of streaming can sometimes 
be observed in cells of Elodea after the application of stimuli sufficiently great to 
produce death in some cells and temporary stoppage of streaming in others. Seifriz 
(1943) has observed in treated Elodea that the chloroplasts flow as a belt around 
the "w r aist" of the cell. It is difficult to understand where the factors for polarity 
of streaming reside within the cell. All of the cytoplasm, including the peripheral 
membrane, seems to be displaced, at least insofar as could be detected with the 
microscope. 

Both elasticity and contractility have been observed in streaming cytoplasmic 
strands. In thin strands the thickness of the cortical layers must be very small. 
Hence, the contractile properties of the strand probably include the whole of its 
protoplasmic structure. 

The chloroplasts seem to be of a highly viscid consistency. The starch granules 
were the only elements within them that could be consistently displaced by high 



CENTRIFUGE EFFECTS ON ELODEA 255 

centrifugal force. This is in agreement with the observations made with the 
micro-dissection apparatus, to the effect that chloroplasts appear to be composed 
of an "elastic jelly of doughy consistency" (Scarth, 1927). Large starch grains 
often appear in normal storage tissue attached to the surface of a chloroplast 
(Zirkle, 1926). However, there seems little doubt that the displacement of the 
starch grains reported here is due to centrifugal force. 

CONCLUSIONS 

High centrifugal force produces a rapid stratification of the visible cellular 
materials in Elodea. From the centrifugal to the centripetal end, the materials 
are stratified as follows: (1) chloroplasts, cytoplasm and nucleus; (2) vacuole. 
In cells examined immediately after centrifuging at high forces, evidence of organ- 
ized streaming is usually absent, but Brownian movement is often apparent. Most 
of the cells survive exposure to 350,000 times gravity for thirty minutes. 

The inhibition of streaming may be due to a lowering of the viscosity as a 
result of the rapid displacement of the chloroplasts through it. 

Recovery from centrifuging usually occurs in the following order : (1) Brownian 
movement at the centrifugal end aids in a redistribution of the granular cytoplasm 
along one or both sides of the cells ; (2) initial streaming movements are usually of 
an unorganized type; and (3) organized streaming is slowly established which 
results in a slow irregular rotation of the large massed chloroplasts. Eventually 
the bunched chloroplasts separate, and the normal velocity of streaming, as well as 
the normal distribution of chloroplasts, is established. 

In only about 7 per cent of the cells could the direction of streaming be reversed 
by centrifuging. 

Evidence of elasticity and contractility within thin strands of protoplasm has 
been observed. 

Displacement of starch grains within chloroplasts has been observed. 

LITERATURE CITED 

ANDREWS, F. M., 1915. Die Wirkung der Zentrifugalkraft auf Pflanzen. Jahrb. IViss. Bot.. 
56: 221-253. 

BEAMS, H. W., 1943. Ultracentrifugal studies on cytoplasmic components and inclusions. 
Biol. Symf>., 10: 71-90. 

BEAMS, H. W. AND R. L. KING, 1937. The suppression of cleavage in ascaris eggs by ultra- 
centrifuging. Biol. Bull., 73: 99-111. 

BEAMS, H. W. AND R. L. KING, 1939. The effect of centrifugation on plant cells. Bot. Rev., 
5: 132-154. 

BERTHOLD, G., 1866. Studien iiber Protoplasmamechanik. Leipzig. 

CLAUDE, A., 1943. Distribution of nucleic acids in the cell and the morphological constitution 
of the cytoplasm. Biol. Symp., 10: 111-129. 

EWART, A. J., 1903. On the physics and physiology of protoplasmic streaming in plants. 
Clarendon Press, Oxford. 

LEWIS, W. H., 1942. The relation of the viscosity changes of protoplasm to amoeboid locomo- 
tion and cell division. Structure of Protoplasm, 163-197. Ames, Iowa. 

MARSLAND, D. A., 1939. The mechanism of protoplasmic streaming. The Affects of high hydro- 
static pressure upon cyclosis in Elodea canadensis. Jour. Cell, and Comf. Physiol., 13 : 
23-30. 

MAST, S. O., 1931. Locomotion in Amoeba proteus (Leidy). Protoplasma, 14: 321-330. 



256 H. W. BEAMS 

MOORE, A. R., 1935. On the significance of cytoplasmic structure in plasmodium. Jour. Cell. 

and Comp. Physlol, 7: 113-129. 
NORTHEN, H. T. AND R. T. NORTHEN, 1938. Studies on protoplasmic structure in Spirogyra. 

II. Alterations on protoplasmic elasticity. Protoplasina, 31 : 9-19. 
PFEFFER, W., 1906. The physiology of plants. Oxford. 
SCARTH, G. W., 1927. The structural organization of plant protoplasm in the light of micrurgy. 

Protoplasma, 2 : 189-205. 

SEIFRIZ, W., 1943. Protoplasmic streaming. Bot. Rev., 9 : 49-123. 

SVEDBERG, THE, 1934. Molecular weight analysis in centrifugal fields. Science, 79: 327. 
VEXLER, 'D., 1935. A value for the tension at the surface of a Myxotnycete. Proc. Soc. Exp. 

Biol. Mcd., 32: 1539-1541. 
ZIRKLE, C, 1926. The structure of the chloroplast in certain higher plants. Amcr. Jour. Bot., 

13: 301-341. 



THE PRESENCE OF THE TRICARBOXYLIC ACID CYCLE 
IN THE CILIATE COLPIDIUM CAMPYLUM * 

GERALD R. SEAMAN 2 - 3 

The Marine Biological Laboratory, Woods Hole, Mass, and the Biological Laboratory, 

Fordham University, Nciv York 

Colpidium cainpylwn is a ciliate which can be easily cultured bacteria-free in 
a liquid medium. It has been demonstrated that when cultured in proteose-peptone 
from which the lipids have been extracted, the organism is capable of synthesizing 
large amounts of fatty acids (Wilber and Seaman, 1948). 

Since the tricarboxylic acid cycle is a link between protein and carbohydrate 
metabolism, it seemed desirable to make a study of this cycle as the first step 
toward the elucidation of the pathway for fatty acid synthesis from protein in this 



organism. 



While there have been many investigations of this cycle in vertebrate tissue 
and in bacteria, there has been little done with protozoa. Van Niel, Thomas, 
Ruben and Kamen (1942) found that the ciliate Tetrahymena gcleii assimilates 
carbon dioxide in the anaerobic formation of succinate during the fermentation of 
glucose. Baker and Baumburger (1941) found cytochrome c, b, and a t to be 
present in this same organism with indications of the presence of cytochrome a 2 . 
Hutchens, Jandorf and Hastings (1941) ascertained the DPN content of the 
flagellate Chilonwnas paramecium. Hutchens (1940) also identified the presence 
of cytochrome c in Chilomonas. Laurie (1935) demonstrated the presence of 
succinic dehydrogenase in the ciliate Glaucoma pyrifonnis. 

MATERIALS AND METHODS 

Colpidia were grown in sterile, pure cultures in 150 cc. Erlenmeyer flasks con- 
taining 50 cc. of 3 per cent Difco proteose-peptone solution from which the carbo- 
hydrate had been precipitated with copper sulfate (Peters and Van Slyke, 1931) 
and the lipids extracted with hot alcohol (Bloor, 1943). The organisms used were 
obtained from cultures maintained in the Biological Laboratory, Fordham LTniver- 
sity and are the same strain as was used in a previous investigation (Wilber and 
Seaman, 1948). For use in this investigation, new cultures were inoculated with 
1 cc. of organisms from a three-day culture and allowed to grow for two days at 
a temperature of 22 2 C. At this time the cultures were at the mid-point of 
the logarithmic phase of growth (population about 40,000 colpidia per cc.). 

The organisms for use were concentrated by centrifugation and aliquots with- 

1 Portion of a dissertation submitted in partial fulfilment of the requirements for the degree 
of Doctor of Philosophy at Fordham University. 

2 The author wishes to express his sincere gratitude to Dr. Charles G. Wilber for invaluable 
aid and encouragement. 

3 U. S. Public Health Service Fellow. 

257 



258 



GERALD R. SEAMAN 



drawn for ascertaining the dry weights of the cells (Ormsbee, 1942). The re- 
maining cells were washed three times with Hahnert's solution (Hahnert, 1932), 
to which was added magnesium sulfate to make a final concentration of 0.02 M 
(final pH adjusted to 5.6). The cells were then starved for twelve hours before 
use. At the end of this period the organisms were again concentrated, resuspended 
in the modified Hahnert's solution and 2 cc. portions (containing approximately 
10 mg. dry weight of cells) transferred into standard Warburg vessels. 

Oxygen uptake was measured by the conventional Warburg direct method. 
In all cases the total volume of each vessel was 3.5 cc. Vessels were shaken at a 
rate of 120 cycles per minute through an arc of 5 cm. 

Sodium pyruvate was prepared by the method of Robertson (1942) ; oxalo- 
acetic acid by the method of Krampitz and Werkman (1941). All other sub- 
strates were obtained commercially. Concentrations of substrates are given as 
final concentration. 

Pyruvic and a-ketoglutaric acids were estimated according to the method of 
Friedmann and Haugen (1943) ; succinic acid according to Krebs (1937) ; oxalo- 
acetate according to Edson (1935) ; fumaric acid according to Krebs, Smyth and 
Evans (1940). 

RESULTS 

Pyruvate is rapidly metabolized by Colpidium. When 0.02 M pyruvate is 
added to cells respiring in modified Hahnert's solution there is an immediate 



a 



1 




40 80 

time in minutes 



120 



FIGURE 1. Effect of pyruvate on oxygen uptake in Colpidium. Modified Hahnert's solution, 
pH 5.6. Gas phase, CK. Temperature, 25.5 C. At arrow, 0.02 M pyruvate added. 



METABOLISM IN COLPIDIUM 



259 



30 



25 



20 



0) 

o 



_o 
o_ 

3 



1 




10 20 30 

time in minutes 



40 



50 



60 



FIGURE 2. Effect of malonate and fumarate on oxygen uptake in Colpidium. Modified 
Hahnert's solution, pH 5.6. Gas phase, O 2 . Temperature, 25.5 C. Curve 1, no added sub- 
strate ; curve 2, 0.02 M pyruvate ; curve 3, 0.02 M pyruvate + 0.02 M malonate ; curve 4, 0.02 M 
pyruvate + 0.02 M malonate + 0.001 M fumarate. 

increase in the rate of oxygen uptake (Fig. 1). There is a utilization of 0.081 
mg. of pyruvate per mg. dry weight of cells per hour (Table II). 

If the tricarboxylic acid cycle plays a role in the metabolism of Colpidium, the 
oxygen uptake in the presence of pyruvate should be inhibited by malonate. This 
inhibition should be released upon the addition of fumarate. Figure 2 shows that 
0.02 M pyruvate increases the Q 02 from the endogenous value of 13.2 to 26.3, 
an increase of 99 per cent. In the presence of pyruvate and 0.02 M malonate 
the Qo 2 is 15.4, 83 per cent inhibition of the pyruvate effect. The Qo 2 is restored 
to a value of 23.6 by the addition of 0.001 M fumarate, an 89 per cent recovery 
of the malonate inhibition. 

The effect of other acids of the tricarboxylic acid cycle on oxygen uptake is 
shown in Table I. Succinate results in an increased Q 02 of 105 per cent; a- 
ketoglutarate 102 per cent; fumarate 90 per cent; malate 97 per cent; and oxalo- 
acetate, an increase of 85 per cent. 

The quantities of metabolites recovered from various substrates are shown in 
Table II and III. Fumarate and a-ketoglutarate are recovered in approximately 
equal amounts when pyruvate is the substrate. The addition of fumarate to 
pyruvate increases the recovery of a-ketoglutarate by 142 per cent. Fumarate 
and pyruvate are recovered in a ratio of approximately 1 to 4 when oxaloacetate 
is utilized as a substrate. 

Table III shows that as a result of the fumarate release of malonate inhibition, 
there is an added utilization of 0.051 mg. pyruvate per mg. dry weight of cells 
per hour, and an added recovery of succinate amounting to 0.013 mg. per mg. dry 
weight of cells per hour. 



260 



GERALD R. SEAMAN 



TABLE I 

Effect of acids of the tricarboxylic acid cycle on oxygen uptake in Colpidium. Modified Hahnert's 
solution, pH 5.6. Gas phase, 02. Temperature, 25.5 C. Concentration of all substrates except 
fumarate, 0.02 M; fumarate, 0.001 M. 

Substrate 



Qo 2 

13.2 
27.1 
26.8 
25.1 
26.1 
24.5 



succmate 

a-ketoglutarate 

fumarate 

malate 

oxaloacetate 



TABLE II 

Utilization of substrates and recovery of intermediate metabolites in Colpidium. Modified 
Hahnert's solution, pH 5.6. Gas phase, 2 . Temperature, 25.5 C. Qsubstrate is mg. substrate 
utilized ( ) or mg. metabolite formed (recovered) ( + ) per mg. dry weight of cells per hour. Pyruvate, 
0.02 M; oxaloacetate, 0.002 M; fumarate, 0.001 M. 





Substrate added 


Usubstrate 


pyruvate 


pyruvate + fumarate 


oxaloacetate 


>"' /pyruvate 








0.044 


\ /pyruvate 


0.081 


0.065 





\\) ketoglutarate 


0.012 


0.029 





( /oxaloacetate 








0.121 


(< /fumarate 


0.016 




0.014 



TABLE III 

Formation of succinate in Colpidium. Modified Hahnert's solution, pH 5.6. 
Temperature, 25.5 C. Malonate, 0.02 M; pyruvate, 0.02 M; fumarate, 0.001 M. 



Gas phase, 02. 



Qsubstrate 

' ^pyruvate 
( i )succinate 



Substrate added (in addition to malonate) 
pyruvate pyruvate + fumarate 



0.016 
0.003 



0.067 
0.016 



TABLE IV 

Effect of succinate, a-ketoglutarate, and citrate in releasing malonate inhibition in Colpidium. 
Modified Hahnert's solution, pH 5.6. Gas phase, 2 . Temperature, 25.5 C. Malonate, pyruvate, 
a-ketogluterate, succinate, 0.02 M; citrate, 0.008 M; fumarate, 0.001 M. 

Substrate (in addition to pyruvate which was 
present in all vessels) 



malonate 

citrate 

citrate + malonate 

citrate + malonate + fumarate 

a-ketoglutarate + malonate 

succinate + malonate 



26.3 

15.4 
26.1 
13.7 
22.8 
28.4 
27.6 



METABOLISM IN COLPIDIUM 261 

Added citrate in final concentrations ranging from 0.002 M to 0.01 M has no 
effect on the oxygen uptake. Table IV shows the ability of citrate in releasing 
malonate inhibition as compared to the ability of succinate and a-ketoglutarate 
to release the inhibition. Succinate and a-ketoglutarate release the malonate inhi- 
bition to approximately the same extent as does fumarate (compare Fig. 2), 
whereas citrate does not release the inhibition. 

DISCUSSION 

It would be desirable to compare the Q 02 values obtained for Colpidium in this 
investigation with values obtained for other protozoa. However, it is impossible 
to make such a comparison, since it was found (Ormsbee, 1942) that the Qo- 2 
of the same species of Tetrahymena varies from 6.2 to 77.7 depending upon the 
age of the culture, the length of the starvation period before oxygen uptake is 
measured, and the composition of the suspending medium. Other factors affect- 
ing Qo 2 values in protozoa are the rate of shaking of the manometer vessels (Hall, 
1938) and the concentration of cells used (Pace and Lyman, 1947). Hutchens 
(1941) found that in Chilomonas paramecium the oxygen uptake per hour per 
10,000 cells varies with different strains, even though both strains are studied 
under identical conditions. 

Since added citrate does not increase oxygen uptake or release malonate inhi- 
bition, and since fumarate, succinate, and a-ketoglutarate do cause increased oxygen 
uptake and do release malonate inhibition, it must be concluded (Stare, Lipton, 
and Goldinger, 1941) that citrate does not occupy a major position in the tri- 
carboxylic acid cycle as it occurs in Colpidium. 

It appears from the data of Von Dach (1942) that the tricarboxylic acid cycle 
is not present in the colorless flagellate, Astasia. In this organism, succinate, 
fumarate and malonate have no significant effect on the oxygen uptake. In Para- 
mecium caudatimi, succinate increases oxygen uptake by only 8 per cent (Leich- 
senring, 1925). 

Elliott (1935) found that pyruvic acid (0.5%) inhibits growth in Colpidium 
campylum and in C. striatwn. On the other hand, Bond (1933) found that 
pyruvic acid stimulated growth in C. campylum. However, he found that suc- 
cinate (1.0%) and malate (1.0%) inhibit growth. These findings are unusual 
if, as has been demonstrated in this paper, these compounds are metabolites. It 
must be noted that the concentrations used by these authors were very much higher 
than those used in the present investigation. It is well known that normally 
occurring metabolites in high concentrations may cause inhibition of metabolic 
functions, as measured by oxygen uptake. It would be desirable to ascertain the 
effects of acids of the tricarboxylic acid cycle on the growth of Colpidium when 
used in concentrations which are known to be physiologically active (0.001-0.02 M). 
Such an investigation is now in progress. 

SUMMARY 

1. Evidence is presented for the presence of the tricarboxylic acid cycle in the 
metabolisms of the ciliate Colpidium campylum. 

2. Apparently citrate does not occupy a major position in the tricarboxylic acid 
cycle as it occurs in Colpidium. 



262 GERALD R. SEAMAN 



J 



LITERATURE CITED 



BA^ER, E. S. G. AND J. B. BAUMBURGER, 1941. The respiratory rate and cytochrome content 

of a ciliate protozoan (Tetrahymena geleii). Jour. Cell. Comp. Physiol., 17: 285-303. 
BLOOR, W. R., 1943. Biochemistry of the fatty acids. Reinhold, N. Y., 387 pp. 
BOND, R. M., 1933. A contribution to the study of the natural food cycle in aquatic environ- 
ments. Bull. Bing. Ocean. Coll., 4: 1-89. 
EDSON, N. L., 1935. Ketogenesis-antiketogenesis. I. The influence of ammonium chloride on 

ketone-body formation in liver. Biochcm. Jour. 29: 2082-2094. 
ELLIOTT, A. M., 1935. Effects of certain organic acids and protein derivatives on the growth 

of Colpidium. Arch. f. Protis., 84: 472-494. 
FRIEDMANN, T. E. AND G. E. HAUGEN, 1943. Pyruvic acid. II. The determination of keto- 

acids in blood and urine. Jour. Biol. Chcm., 147 : 415-442. 
HAHNERT, W. F., 1932. A quantitative study of reactions to electricity in Amoeba proteus. 

Biol. Bull., 80 : 265-274. 

HALL, R. H., 1938. The oxygen consumption of Colpidium campylum. Biol. Bull., 75: 395-408. 
HUTCHENS, J. O., 1940. The need of Chilomonas paramecium for iron. Jour. Cell. Comp. 

Physiol., 16: 265-267. 
HUTCHENS, J. O., 1941. The effect of the age of the culture on the rate of oxygen consumption 

and the respiratory quotient of Chilomonas paramecium. Jour. Cell. Comp. Physiol., 

17 : 321-332. 
HUTCHENS, J. O., B. J. JANDORF, AND A. B. HASTINGS, 1941. Synthesis of diphosphopyridine 

nucleotide by Chilomonas paramecium. Jour. Biol. Chcm., 138 : 321-325. 
KRAMPITZ, L. O. AND C. H. WERKMAN, 1941. The enzymatic decarboxylation of oxaloacetate. 

Biochcm. Jour., 35: 595-602. 
KREBS, H. A., 1937. The role of fumarate in the respiration of Bacterium coli commune. 

Biochcm. Jour., 31 : 2095-2124. 
KREBS, H. A., D. H. SMYTH, AND E. A. EVANS, 1940. Determination of fumarate and malate 

in animal tissues. Biochcm. Jour., 34 : 1041-1045. 
LAURIE, N. R., 1935. Studies on the metabolism of protozoa. II. Some biochemical reactions 

occurring in the presence of the washed cells of Glaucoma pyriformis. Biochcm. Jour., 

29 : 2297-2302. 

LEICHSENRING, J. M., 1925. Factors influencing the rate of oxygen consumption in unicellular 
, organisms. Am. Jour. Physiol., 75 : 84-92. 

v ORMSBEE, R. H., 1942. The normal growth and respiration of Tetrahymena geleii. Biol. Bull., 

82 : 423-437. 
PACE, D. M. AND E. D. LYMAN, 1947. Oxygen consumption and carbon dioxide elimination 

in Tetrahymena geleii. Biol. Bull., 92 : 210-216. 
PETERS, J. P. AND D. D. VAN SLYKE, 1931. Quantitative clinical chemistry. Vol. 2. 

Methods. Williams and Wilkins, Baltimore. 

ROBERTSON, W. B., 1942. The preparation of sodium pyruvate. Science, 96 : 93-94. 
STARE, F. J., M. A. LIPTON, AND J. M. GOLDINGER, 1941. Studies on biological oxidation. 

XVIII. The citric acid cycle in pigeon muscle respiration. Jour. Biol. Chcm., 141 : 

981-987. 
VAN NIEL, C. B., J. O. THOMAS, S. RUBEN, AND M. D. KAMEN, 1942. Radioactive carbon as 

an indicator of carbon dioxide utilization. IX. The assimilation of carbon dioxide by 

protozoa. Proc. Nat. Acad. Sci., 28: 157-161. 
VON DACH, H., 1942. Respiration of a colorless flagellate, Astasia klebsii. Biol. Bull., 82: 

356-371. 
WILBER, C. G. AND G. R. SEAMAN, 1948. The lipids in Colpidium campylum. Biol. Bull., 94 : 

29-32. 



GAMETOGENESIS IN THE OYSTER UNDER CONDITIONS OF 

DEPRESSED SALINITY 

PHILIP A. BUTLER 

Chesapeake Shellfish Investigations, Fish and Wildlife Service, U. S. Department of the 

Interior, Annapolis, Maryland 

The American oyster, Ostrea virg'mica Gmelin, flourishes naturally in brackish 
waters ranging in salinity from 16 to 27 parts per thousand. But the salt tolera- 
tion of the animal is such that it can survive in waters having a much broader range 
of salt content. In many localities, commercial production of oysters is maintained 
where seasonal floods may expose the bars to entirely fresh water for short periods 
of time. Some of the more important seed-producing areas on the Atlantic Coast 
consistently have a salt content of less than 15 /oo- Consequently the effects of 
lowered salinity on oyster physiology and reproductive ability have long been of 
interest. The opportunity presented itself in 1946 to examine the gonads of oysters 
living under unusually great variations in salt content. Extensive flood waters 
from the Susquehanna River watershed into the upper reaches of Chesapeake Bay 
during the summer of 1945 and spring of 1946 caused salinity depressions from a 
normal range of 10 to 15 /oo to zero for protracted periods. Oyster beds located 
twenty miles south of the entrance of the river into the bay were frequently exposed 
to fresh water. In the period following these extremes, mortalities up to 70 per cent 
of the population were recorded on the bars in this area (Engle, 1946). The 
oysters remaining viable were of unusually poor quality. The body tissues were 
edematous and nearly transparent. The adductor muscle lacked tonus so that the 
valves could be separated easily and frequently were gaping. 

Samples of ten or more oysters from this low salinity area, designated here 
as the LS group, were collected weekly in the summer and at longer intervals dur- 
ing the fall and winter of 1946. Transverse sections of the gonad were prepared 
for histological examination. For comparative purposes, a similar series of oysters, 
designated as the HS group, was collected in another part of the bay where the 
salinity was higher and remained relatively unaffected by the flood conditions. 
These oysters were of good market quality and during the summer produced a 
set of young oysters of commercial proportions indicating normal gonad develop- 
ment and spawning reactions. Routine hydrographical observations were made 
at the time of each sampling, as well as plankton tows and notes on the feeding 
activity and general condition of the oysters. 

Of the 185 oysters in the LS group over three years old examined, 40 per cent 
were females, 33 per cent were undifferentiated, 26 per cent were males and 1 per 
cent were sex reversals. Of the 221 specimens in the HS group, 70 per cent were 
females, 29.5 per cent were males and 0.5 per cent were hermaphroditic. The ab- 
sence of undifferentiated gonads in the HS group was striking in comparison with 
the LS group. 

263 



264 



PHILIP A. BUTLER 



The orderly sequence of events in the development of functional gametes in the 
American oyster has been described (Coe, 1932; Loosanoff, 1942), and the resume 
of the stages given here for the HS oysters growing in Chesapeake Bay differs in 
no important respect from conditions found elsewhere except with regard to 
timing (Loosanoff and Engle, 1940). Spawning is initiated when water tempera- 
tures rise to levels approximating 18 to 20 C, and consequently its occurrence 
varies from year to year at any particular geographical location. Typically, after 
the final spawning of the population in late summer, there is a short period of rest 
in which the gonadal tissue is made up of undifferentiated gonial cells. These 
soon proliferate and early maturation takes place. By this time, usually late Decem- 
ber in Chesapeake Bay, water temperatures have decreased to the extent that the 
oyster becomes inactive and the gonad remains quiescent until the following March. 
Thus in early spring, gonad sections from the HS oysters are characterized by fairly 
large numbers of auxocytes. As water temperatures increase, differentiation and 
growth proceed at a rapid pace, and mature gametes first appear in May when 
spawning may begin. In June most of the gonads are filled with ripe sexual 
products, and from that time until early September, successive waves of spawning 
may continue. By the end of September the majority of gonads are in the resting 
condition. 

In contrast to this typical picture, section of the gonads of the LS group revealed 
that 5 to 40 per cent of each sample contained gonads which were in the resting 



Early Maturation 



Goniol Proliferation 



Resting Gonlols_ 



Spawned Out 



Partially Spawned. 



Mature Gametes. 



Late Maturation 



Early Maturation 



Gonlat Proliferation 



Retting Gonioli 




* 1 1 I 1 . L L_ ' 

May June July Auguit September October November December Jonuo ry 

FIGURE 1. Seasonal progression of stages in the development of gametes in oysters from 
a low salinity area (LS Group) and from a higher salinity area (HS Group). Each point 
represents the predominant activity in a sample of 10-40 oysters collected during a two-week 
or longer period. Initial and final phases in the growth of auxocytes, termed Early and Late 
Maturation, are normally separated by the winter hibernating period in this area. 



EFFECT OF LOW SALINITY ON OYSTERS 265 

gonial or undifferentiated stage until the middle of August. This condition must 
have persisted from the close of the spawning period of the previous year, the time 
of its normal occurrence. By the end of August there was a marked improvement 
in the appearance of the oysters, arid the gonads reached stages of activity which 
had characterized the HS oysters examined two months earlier. In early Novem- 
ber, a majority of the LS oysters were spawned out, and from this time until 
January, early maturation continued at a high level. When the oysters finally en- 
tered the hibernating stage at temperatures of less than 5 C., the majority of LS 
oysters were indistinguishable, with respect to the histology of the gonad, from the 
oysters living in the higher salinity area. 

In about 90 per cent of the specimens of LS oysters examined, the gametogenic 
cycle lagged approximately two months behind that of the high salinity group, 
but in the remaining 10 per cent of the specimens, the gametogenic cycle showed the 
same timing pattern as in the HS group. In order to portray graphically the dif- 
ferences between the two populations, the successive stages in normal gonad activity 
were assigned arithmetic values from one to ten, depending on the preponderant 
condition or cell type present. The average arithmetic value was then obtained 
for each sample of gonads collected over a two-week or longer period and has been 
plotted against time for the two areas studied (Fig. 1). 

It was observed that during the summer, developmental stages in different 
gonads of a sample overlapped or were concurrent because of the relatively long 
period of four months in which eggs are produced. The average values shown in 
Figure 1 demonstrate the seasonal trend of the gametogenic cycle, but they do not 
show the wide variations found within each of the samples of oysters. Earlier 
investigators (Nelson, 1928; Loosanoff, 1942) have noted the variations in gonad 
response found in some individuals of a sample where, for unknown reasons, 
maturation may be delayed or physiologically mature gametes may be retained long 
after the general population has spawned. This condition is especially prominent 
in the LS oysters examined. In the first week of August, individuals from one 
sample demonstrated all stages in gonad development from the undifferentiated 
gonial cells to the spawned-out stage. The degree of variation among individuals 
was far less extensive in the HS group, in which for the same period the gonads 
were fairly equally divided between the partially spawned and the spawned-out 
stages. The percentage distribution of each stage within the samples collected is 
tabulated to illustrate this disparity in the two populations (Fig. 2). 

During the first two weeks of August there was a significant change in the 
appearance of the gonad sections from the oysters of the LS group. Wide varia- 
tions in the stages of activity attained by the individual oysters continued, but all 
of the gonads suddenly advanced beyond the indifferent and early maturation stages, 
and 50 per cent of them were partially or almost completely spawned. In this period 
there were only minor fluctuations in the temperature but the salinity rose abruptly 
from less than 3 /oo to more than 8 %>o- No other environmental changes of 
importance were noted during this period. 

The recovery of oyster larvae from the plankton tows made at the two stations 
corresponds, in general, with the histological picture. 1 In the high salinity area, 

1 The writer is indebted to Mr. James B. Engle of the U. S. Fish and Wildlife Service, 
who provided the data on plankton. 



266 



PHILIP A. BUTLER 



larvae were found two weeks after mature gametes were observed in the sections. 
The last plankton sample taken, October 8, still contained numerous larvae, al- 
though it was from two to three weeks after the apparent absence of gametes from 
the gonad sections. There were two seasonal peaks in larval production : the first 
week of July and the last week of August. In the low salinity area there was but 
one seasonal peak toward the end of August. No larvae at all were collected here 
until seven weeks after their initial appearance in the HS area, although through- 
out this period 10 per cent of the gonad sections had contained apparently mature 
gametes. The failure to find larvae in the water at that time may be attributed to 
inadequate sampling methods or, more probably, to the inhibition of spawning. 
The observed tissue edema may have interfered with the activity of the adductor 
muscle in the spawning reaction (Galtsoff, 1938) or have partially closed the gill 
ostia, thus preventing the passage of ova to the exterior (Hopkins, 1936). 



EARLY 


L 


H 


L 


H 


L 


H 


L 


H 


L 


H 


L 


H 


L 


H 


L 


H 


L 


H 


L 


H 


L 


H 


L H 
















































MATURATION 
































25 


9 


74 


II 


75 


10 100 


55 100 


GONIAL 


PROLIFERATION 
































5 




21 


II 


20 


30 




45 


RESTING 


GONIALS 
















10 




20 


16 


63 


30 


4 
55 


9 
9 


10 
50 


9 
9 


5 


II 


5 


20 






SPAWNED OUT 


PARTIALLY 


SPAWNED 




II 


7 


75 


16 


84 


6 

12 


37 
53 


24 


60 


47 


37 


20 
50 


41 


82 


10 


55 
16 




56 
II 




40 




- 


MATURE GAMETE 


LATE 


MATURATION 




39 


7 


25 




16 


































- 


EARLY 


MATURATION 


32 


39 


36 


4 


32 




35 




21 




5 
























- 


GONIAL 


PROLIFERATION 


21 


1 1 


29 




26 




41 




21 




5 
























- 


RESTING 


GONIALS 


47 




21 




26 




6 




34 




27 




























MAY 
16- 31 


JUNE 
1-15 16-30 


JULY 
1-15 16- 


31 


AUGUST 
1- 15 16-31 


SEPTEMBER 
1- 15 16- 


10 


OCT 


NOV 
DE C 


a 


JAN 



FIGURE 2. The percentage distribution of different stages of gonad activity in each sample 
of oysters collected from the low (L) and high (H) salinity areas. See legend under Figure 1 
for description of samples. 



EFFECT OF LOW SALINITY ON OYSTERS 



267 



Water temperatures throughout the period of observations were normal for the 
region (Fig. 3). In the low salinity area, the bottom temperature was 16.8 C. 
in the middle of May, approached 20 C. in the first week of June, and reached the 
summer maximum of 26 C. on the first day of August. It then decreased gradually 
to 15.8 C. at the end of October, and to less than 5 C. in the period December 
through January. Bottom water temperatures in the high salinity area regularly 
followed the same levels within one or two degrees. 




May 



June 



July 



August September Octobf' November December Jnnnnry 



FIGURE 3. Seasonal fluctuations in salt content of bottom waters in the high (HS) and 
low (LS) salinity areas. The bottom temperature curve (T) is shown for the LS area. Tem- 
peratures in the HS area did not vary more than one degree plus or minus from these data. 

The salt content of the water was more variable (Fig. 3). In the LS area, 
bottom salinities fluctuated from zero (fresh) to 6 / o in the period from the 
middle of May until the first of August. One-third of the records for this time 
showed fresh water. In August the salt level increased steadily to 13 /oo and 
then gradually dropped to 11 /oo by the end of the year. In the HS area, the 
lowest salinity of 6 /oo was recorded toward the end of June. Before and after 
that time, the salinity increased steadily to 15 /oo- 

Only four specimens, one per cent of the total examined, gave evidence of the 
instability of the sex mechanism in this species of oyster. Three of the individuals 
were clearly defined protandric reversals in which the gonaducts contained residua 
of spermatozoa, and the walls of the follicles were lined almost exclusively with 
oocytes in early stages of maturation. These specimens were obtained early in 
September, which indicates that reversal of sex had taken place when the majority 
of the population were spawning. Loosanoff (1942) has suggested that sex 



268 PHILIP A. BUTLER 

reversal takes place when the gonads are made up of undifferentiated gonial cells, 
usually in late October in Long Island Sound. It would appear most reasonable 
that sex reversal should take place during this stage, but the specimens found here 
suggest that if it does, the indifferent stage may occur much earlier in the summer, 
i.e., July and August, in at least part of the population. In one of the four speci- 
mens mentioned above, the gonadal tissue was made up of fairly equal numbers of 
developing oocytes and spermatocytes within each follicle. The developmental 
stages attained were the same as in other unisexual specimens collected at the same 
time, indicating that this oyster would have been a functional hermaphrodite when 
the general population spawned. The percentage of intersexes found in this rather 
small sampling agrees with observations by other workers. In the 221 oysters 
from the HS area there was a ratio of females to males of 2.41. This figure is 
comparable with observations in Galveston Bay, Texas, but contrasts with the ap- 
proximately 50-50 sex ratio found along the Atlantic Coast (Hopkins, 1931). 

The deleterious effect of the environment on the physiology of the oyster, as 
evidenced by the delayed production of gametes until such time that the water 
temperatures made their survival improbable, would appear to be due to the low 
salinity of the water. That this effect was not a direct inhibition of gametogenesis 
is indicated by the fact that 10 per cent of the LS group elaborated mature gametes 
at the usual time in the early summer. The factors directly affected by lowered 
salinities which may be operating here to prevent gametogenesis include several 
possibilities. It has been shown (Hopkins, 1936) that during exposure to fresh 
water the oyster's valves may be closed most of the time and also that even when 
open, the passage of water through the gills may decrease or stop entirely. Either 
one or both of these factors would seriously curtail the feeding of the animal. It 
is also possible that during this time necessary food elements were absent from 
the plankton, or that tissue edema prevented the normal assimilation of food. In 
.any event the end result appears to have been, fundamentally, a tissue starva- 
tion. Hopkins (I.e.) theorized such an end result after studying the feeding 
mechanism in O. gigas in the presence of artificially lowered salinities. It was 
noted that in the small group of LS oysters which produced gametes at the usual 
time in late spring, there was a moderate reserve of stored food which gave the 
tissues a typical opaque appearance. These oysters, as well as the ones having no 
visible food storage, had empty digestive tracts at the time of examination. This 
would indicate that the reserve food had been held over from the previous fall rather 
than that this small group had been able to continue feeding during the period of 
lowered salinities. The evidence is clear, moveover, that soon after the salinity 
level rose above 6 / 00 in the first week of August, the animals commenced feed- 
ing, there was an obvious improvement in the appearance of the tissues, and gonad 
activity started to approach the normal picture. 

SUMMARY 

Histological examination of oyster gonads from an area naturally exposed to 
prolonged periods of fresh water, when compared to oyster gonads from an adjacent, 
unexposed area, showed : 

1. Gametogenesis was inhibited in 90 per cent of the surviving population until 
salinity levels rose above 6 parts per thousand. 



EFFECT OF LOW SALINITY ON OYSTERS 269 

2. Following the salinity increase, oysters rapidly improved in condition but 
required from three to four months to attain the same final level of gonad activity 
as the unaffected group. 

3. Marked variation and suppression of gonad activity in the exposed oysters 
is attributed to variations in food availability, rather than to direct inhibition of 
sexual activity by less saline water. 

4. Sex ratios and extent of intersexuality in the population sampled, as well as 
details of the gametogenic cycle, agree for the most part with published observa- 
tions on Ostrea virginica in other parts of its geographical range. 

LITERATURE CITED 

COE, W. R., 1932. Sexual phases in the American oyster (Ostrea virginica). Biol. Bull., 63: 

419-441. 
ENGLE, J. B., 1946. Commercial aspects of the Upper Chesapeake Bay oyster bars in the light 

of recent oyster mortalities. Mar\land Brd. of Nat. Res., Third Annual Report, pp. 

134-140. 

GALTSOFF, P. S., 1938. Physiology of reproduction of Ostrea virginica. I. Spawning reac- 
tions of the female and male. Biol. Bull., 74 : 461-486. 
HOPKINS, A. E., 1931. Factors influencing the spawning and setting of oysters in Galveston 

Bay, Texas. Bull. U. S. Bur. Fish., 74: 57-83. 
HOPKINS, A. E., 1936. Adaptation of the feeding mechanism of the oyster (Ostrea gigas) to 

changes in salinity. Bull. U. S. Bur. Fish., 48: 345-364. 
LOOSANOFF, V. L. AND J. B. ENGLE, 1940. Spawning and setting of oysters in Long Island 

Sound in 1937, and discussion of the method for predicting the intensity and time of 

oyster setting. Bull. U. S. Bur. Fish., 74: 217-255. 
LOOSANOFF, V. L., 1942. Seasonal gonadal changes in the adult oysters, Ostrea virginica, of 

Long Island Sound. Biol. Bull., 83: 195-206. 
NELSON, T. C., 1928. Relation of the spawning of the oyster to temperature. Ecology, 9 : 

145-154. 



NUCLEAR AND CYTOPLASMIC INTERRELATIONS IN THE 
FERTILIZATION OF THE ASTERIAS EGG 

ROBERT CHAMBERS AND EDWARD L. CHAMBERS 
Lilly Research Laboratories, The Marine Biological Laboratory, Woods Hole, Mass. 

The existence of a functional relation between nucleus and cytoplasm is gen- 
erally accepted, but there are relatively few instances in which the relationship can 
be demonstrated experimentally. Among ova an extreme case is that exhibited 
by the maturing ovum. Fol (1877, 1879), in his classic work ! on the maturation 
and fertilization of the egg of Asterias glacialis, was probably the first to associate 
the maturation of the ovum with the breakdown of the germinal vesicle. In gen- 
eral, the significance of maturation of the ovum has been too closely limited to the 
elimination of the polar bodies. More attention should be given to what is probably 
the basic feature of the phenomenon, namely, changes incurred in the cytoplasm 
through the admixture of nuclear material from the enlarged germinal vesicle of 
the ovarian egg (cf. maturation cytoplasmique of Delage, 1901 ; and R. Chambers, 
1921). It has been recently proposed (R. Chambers, 1949) that the cytoplasm of 
the maturing and mature egg be termed karyocy to plasm. 

There is also to be considered a relationship between the male and female nu- 
clear elements of the fertilized egg and of both elements with the maturing karyo- 
cytoplasm. Many observers have ascribed the movements of the male and female 
pronuclei to their mutual attraction across the intervening cytoplasm of the egg. 
An early attempt at testing the existence of such an attraction was made by George 
Lester Kite, a pioneer in microdissection. In a lecture (unpublished) given dur- 
ing the summer of 1915 at the Marine Biological Laboratory, Woods Hole, Dr. 
Kite described his efforts at interposing the tip of a microneedle as an obstacle 
between the male and female pronuclei in the transparent egg of Lytechinus. then 
known as Toxopneustes. As he dramatically stated : "The pesky nuclei insisted 
in slipping around the obstacle and no efforts, short of destroying the egg, could 
prevent the nuclei from approaching one another and uniting." 

More recently, E. L. Chambers (1939) was able to offer an interpretation in 

1 Fol's 1879 paper is extraordinary for the abundance and accuracy of his extended observa- 
tions on the living Echinoderm egg. His assumption of extruded cytoplasmic filaments of the 
Asterias egg which serve to draw the blunt-nosed spermatozoa through the surrounding jelly 
to the surface of the egg had been largely discredited until fully substantiated many years later 
not only for Asterias but also for many of the Asteroidea. Even in regard to the quadrille des 
centres described by Fol in his paper of 1891 and attacked by Wilson and Mathews (1895), Fol 
had a case. In his 1879 paper (p. 210) Fol remarked that in heavy polyspermy the sperm asters 
assume identical distances from one another placed with their centers along a theoretical circle. 
This fits in with the findings of E. L. Chambers (1939) regarding the sperm aster as a growing 
spherical gelated body. Several sperm asters simultaneously growing in size would assume the 
positions ascribed to them by Fol. Such symmetrical positions of four sperm asters would 
explain Fol's quadrille des centres. It was unfortunate that Fol was not able to correct his 
one wrong hypothesis because of his untimely death soon after publication of his paper. 

270 



EGG NUCLEO-CYTOPLASMIC RELATIONS 271 

terms of physical changes in the cytoplasm. He showed that the movements could 
be ascribed to the growing sperm aster as a gelated body (R. Chambers, 1917), the 
sperm pronucleus lying in or close to the center of the aster. The progressive 
increase in size of the aster transfers the sperm pronucleus passively to a central 
position in the egg, while the egg pronucleus is carried to the sperm pronucleus 
by centripetal streaming in radial channels converging at the center of the aster. 
Fol (1879, pp. 105 and 194), who first described the aster, had already presented 
the idea that the astral radiations are due to streams of centripetal flow. 

The normal dissolution of the germinal vesicle of the fully grown oocyte initiates 
a gradual and prolonged process (R. Chambers, 1921) which converts the somatic 
cytoplasm of the ovarian egg into the karyocytoplasm of the maturing egg ready 
for fertilization. The experiments described in this paper, a brief account of which 
has been published (R. Chambers and E. L. Chambers, 1940), present the matter 
in detail with evidence concerning hitherto unsuspected causal interrelations be- 
tween the egg nucleus, the sperm pronucleus, and the egg cytoplasm during and 
after alteration of the cytoplasm by the spontaneous dissolution of the germinal 
vesicle. These interrelations constitute, as it were, the performances of a three 
ring circus in the maturation of the egg. 

The experiments stress features which are concerned with the egg and sperm 
nuclei during their earlier stages before the sperm aster has attained full expres- 
sion. They are not to be compared with the egg fragmentation studies of Delage 
(1899), Tennent, Taylor and Whitaker (1929) and Whitaker (1928), all of which 
were done on fully mature sea urchin eggs and with reconstituted female pronuclei, 
both polar bodies already having been eliminated. 

MATERIAL AND METHODS 

The starfish egg is admirably suited for the present study, since, commencing 
with the germinal vesicle stage, the eggs develop in sea water and insemination can 
take place at any time. 

Fol (1877) had observed that the eggs of Asterias glacialis normally are expelled 
into the sea water with the germinal vesicle still intact. In our work the fully 
grown germinal vesicle eggs were uniformly obtained by removing the ripe ovaries 
into finger bowls of sea water, where the eggs were immediately distributed in a 
large volume of sea water. Most of the work was done during the months of 
June and July. Only those batches of eggs were used in which over 90 per cent of 
samples of the eggs matured. All the bisecting operations on the eggs were done 
under oil and water immersion objectives. 

The fragmented eggs and their controls were maintained at a temperature of 
16 C. .in Syracuse watch glasses. The operations and observations were made 
in hanging drops suspended from a coverslip in the moist chamber of a micro- 
manipulator at room temperature. The eggs were transferred to the moist chamber, 
and several eggs immediately bisected. This required about three or four 
minutes. The eggs were then replaced in the watch glasses at 16 C., kept there 
until a few minutes before appearance of the sperm aster was expected, and then 
re-transferred to the moist chamber for observation. 

The bisections were performed on the eggs at varying intervals after dissolu- 
tion of the germinal vesicle, some before and others after insemination. The eggs, 



272 ROBERT CHAMBERS AND EDWARD L. CHAMBERS 

suspended from the roof of the moist chamber, were divided by compressing them 
with the horizontal shaft of a slender microneedle. The vitelline membrane of 
the unfertilized egg and the enveloping membrane of the fertilized egg are firm 
enough to remain more or less intact during the bisection. The two egg frag- 
ments, which immediately round up and are completely separated, tend to remain 
together. The cutting was generally done so as to have both fragments of about 
the same size, one fragment never being smaller than about one half the volume 
of the other. Such a difference in size had no appreciable effect on the time of 
appearance of the polar bodies or of the sperm aster. This is in accord with 
Tennent, Taylor, and Whitaker (1929) who had shown that the cleavage time of 
egg fragments is independent of size as long as the fragments, when fertilized, 
undergo segmentation. 

In all the experiments, every individual fragment was kept under observation 
simultaneously with its companion fragment in the same microscopic field. Hence, 
when a phenomenon was detected in one fragment it could be immediately com- 
pared with what might appear in the companion fragment. The time sequences 
and the phenomena looked for in each individual case w r ere so clear-cut that in- 
tervals as short as two minutes were significant. The phenomena observed were 
the appearance in the granular cytoplasm of a diminutive radiating star which 
represented the sperm aster, and the elevation of a hyaline nipple on the surface 
of the egg, the beginning of one or other of the polar bodies. 

Bisecting eggs with intact germinal vesicles confirmed the already recognized 
finding that fragments lacking the germinal vesicle are not fertilizable (Delage, 
1901). After normal dissolution of the germinal vesicle, both fragments are 
capable of being fertilized, one with a diploid (sperm and egg), and the other with 
a haploid (sperm) nucleus. 

The bisection of eggs already inseminated was done at varying times prior to 
first polar body formation. As was to be expected, only those fragments were 
capable of further development which contained the sperm pronucleus. Special 
attention was given to those eggs in which the sperm and egg nuclei were separated, 
one in each fragment. 

RESULTS 

The investigation is classified under two general headings. The first deals with 
observations on the sequence of events in whole eggs, and the second with bisected 
eggs. In the latter, attention was directed toward the reactions of the male and 
female nuclei when together and when isolated in the respective fragments of 
karyocytoplasm. 

/. Observations on the Whole Egg 
A. The unfertilised egg 

The first intimation of the dissolution of the germinal vesicle is the development 
of an irregular contour of the membrane and a fading from view of the prominent 
nucleolus. An irregularity in shape of the membrane is not necessarily related 
to impending dissolution of the germinal vesicle. A mere collapse of the mem- 
brane induced by shaking the eggs does not accelerate maturation. The one 



EGG NUCLEO-CYTOPLASMIC RELATIONS 



273 



visible change which consistently heralds dissolution is the disappearance of the 
nucleolus. This is followed by disappearance of the nuclear membrane and a dif- 
fusion of the nucleolar contents mixed with the hyaline karyoplasm of the nucleus 
into the granular cytoplasm. Within five to fifteen minutes, the region formerly 
occupied by the germinal vesicle is filled with cytoplasmic granules indistinguishable 
from the rest of the egg. In the granular cytoplasm it is possible to detect the 
diminutive, hyaline egg nucleus which later gives off the polar bodies. 

TABLE I 

Sequence of events in the maturing unfertilized eggs of Aster ias forbesii at 16-18 C. 



Time 

8'- 19' 
76'- 90' 



From time of deposition in sea water to: 

Disappearance of nucleolus (50% completion) 
Formation of 1st polar body (50% completion) 
Formation of 2nd polar body (50% completion) 

Table I gives the approximate times of the three most obvious events during the 
maturation of the unfertilized egg. The data were obtained from ten separate 
batches of eggs of at least 100 eggs in each, kept at a temperature of 16-18 C. 
The variations in the times recorded are due to the different batches. Within a 
single batch the variations did not exceed two to three minutes. The figures to the 
left denote the times, within a two minute range, recorded for seven of the batches. 
The figures to the right are of one batch. The times for the two remaining batches 
lie in between. 

B. The fertilised egg 

Table II, with data averaged from records of five batches of eggs, presents an 
analysis of the effect on the appearance of the first and second polar bodies and of 



TABLE II 

Effect of insemination on time of 1st and 2nd polar body formation, 
and of 1st cleavage in eggs of Asterias forbesii, at 16 C. 



(1) 


(2) 


(3) 


(4) 


(5) 


(6) 


Eggs inseminated 
at following in- 
tervals of time 
after deposition 


Time 50% 1st 
P.B. formation 
after deposition 
in sea water: 


Time 50% 2nd 
P.B. formation 
after deposition 
in sea water: 


Time 50% 1st 
cleavage after 
deposition in 
sea water: 


Time 50% 1st 
cleavage after 
2nd P.B. 

formation: 


Time 50% 1st 
cleavage after 
insemination: 


in sea water: 















78.2 


107.3 


. 





. 




(unfertilized) 


(unfertilized) 








25' 


70.0 


98.0 


169.0 


70.5 


144.5 


40' 


71.5 


101.0 


172.0 


71.0 


132.0 


50' 


74.0 


102.5 


173.0 


70.5 


123.0 


60' 


75.8 


104.0 


175.0 


71.0 


115.0 


70' 


77.5 


105.5 


178.0 


72.0 


108.0 


80' 





106.5 


184.5 


78.0 


104.5 


90' 





107.0 


193.5 


86.5 


103.5 


100' 





107.5 


203.5 


96.5 


103.5 


130' 








234.0 


127.0 


104.0 



274 ROBERT CHAMBERS AND EDWARD L. CHAMBERS 

the first cleavage by inseminating the eggs at successive intervals following break- 
down of the germinal vesicle. The first column gives the times of insemination. 
The figures in the second and third columns show that, up to a certain time, the 
earlier the insemination the more accelerated is the formation of the polar bodies. 
When the insemination is delayed to and beyond the time of first polar body forma- 
tion, there is no evidence of acceleration, whereupon, the time of appearance of 
the second polar body tends to coincide with that of its appearance in the unfertilized 

egg- 
Evidently it is only when the fertilization process is started early that the con- 
version of the egg nucleus into its pronucleus is accelerated. Later, when fertiliza- 
tion occurs at the time that the polar body formation has been initiated, there is no 
longer any appreciable accelerating action. 

A consideration of the cleavage times, presented in the fourth, fifth and sixth 
columns, brings out several significant features. From the fourth column it can 
be seen, as is to be expected, that cleavage time corresponds with the time the eggs 
are deposited in sea water. However, during the earlier stages, up to some time 
before first polar body formation (after 60 minutes), the lapse is not as great as 
during the later stages (cf. Fig. 1). This is brought out more clearly from the 
figures in the fifth column which give the times between those of first cleavage and 
of second polar body formation. They indicate that the time interval, irrespective 
of insemination time, is constant until about the time when the first polar body is 
being initiated. After this the cleavage time becomes directly proportional to the 
insemination time. 

The figures in the sixth column give the times between insemination and first 
cleavage. They show that the earlier the insemination up to the time when the 
first polar body is initiated (about 70 minutes), the longer is the time which elapses 
before cleavage occurs. After 70 minutes the time between insemination and 
cleavage becomes constant. 

These analyses indicate that the rate at which the fertilization events proceed 
depends upon the cytoplasmic maturation which is completed at about the time 
of first polar body formation. Prior to this, it would seem that the immature state 
of the karyocytoplasm has a delaying effect on the development of the sperm and 
its accompanying events. Upon initiation of first polar body formation, the matura- 
tion of the karyocytoplasm is complete, whereupon the development of the sperm 
from the time of its entry proceeds without delay and cleavage occurs within a 
constant period of time. 

A graphic presentation of Table II is given in Figure 1. The abscissae rep- 
resent the times of insemination ; the ordinates, the times when the various events 
occur. Concerning the unfertilized egg, the two vertical dotted lines and the two 
horizontal dotted lines intercept the X and Y axes respectively at the times when 
the first polar body forms (average of 78.2 minutes) and when the second polar 
body forms (average of 108.2 minutes). 

Concerning the fertilized egg, the three solid curves represent the times for the 
formation, respectively, of the first and of the second polar bodies, and of the first 
cleavage in eggs inseminated at different intervals after germinal vesicle break- 
down. The curves for the first and second polar body formation are parallel 
throughout and their upward slopes represent the acceleration due to insemination. 



EGG NUCLEO-CYTOPLASMIC RELATIONS 



275 



It is to be noted that when the insemination occurs at 78 minutes (time of first 
polar body formation) or later, the time of second polar body formation remains the 
same as that of the unfertilized egg. 

Let us now consider the dotted dash curve which represents the time of first 
appearance of the sperm aster and which was calculated from data obtained on 
about 100 eggs observed with an oil immersion objective. The sperm aster never 
appears until after the second polar body, no matter how early the eggs have 
been inseminated (the earliest recorded being at 25 minutes). During these earlier 



OJ 

I 

o 

<D 
CO 



O 



(/) 
o 
CL 

Q 

o 



c 

O) 



to 
.a 

CO 




c 
a> 

LJ 



135 
130 

120 
110 
100 
90 
80 
70 



<D 
P 




1st Cleavage 



Sperm Aster 



2nd Polar Bod 



1st Polar Body 




25 40 55 70 85 100 115 

Time of Insemination in Minutes 
Subsequent to Deposition in Sea Water 

FIGURE 1. Relation of time of insemination to time of appearance of first P.B., second 
P.B., and first cleavage (solid lines), and of sperm aster (dotted dash line). Faint dotted 
horizontal lines represent times of appearance in unfertilized eggs of first and second P.B. The 
observations were made on eggs of Asterias forbcsii at 16 C. 

stages of the developing egg, it appears at two to three minutes after the second 
polar body has been given off. In later stages, viz., after 70 minutes, this interval 
becomes progressively greater. It is to be noted that the interval between the 
time of appearance of the sperm aster and the time of first cleavage is always just 
about 68 minutes, whether the time of insemination is early or late. This is the 
normal time for the events following aster appearance. The length of the interval 
between the time of insemination and the time of sperm aster appearance varies, 
depending upon the state of maturation of the karyocytoplasm. It becomes con- 



276 ROBERT CHAMBERS AND EDWARD L. CHAMBERS 

stant only when the egg is fully mature, which also coincides with the time of first 
polar hody formation. Thus, when the eggs are inseminated as early as 25 minutes 
after being placed in sea water, a period of about 78 minutes must elapse before 
the sperm aster appears. When the egg is inseminated during or after the first 
polar body formation, the interval between insemination and appearance of the 
sperm aster is found to be constantly about 35 minutes. 

Insemination after formation of the first polar body results, as Lillie (1915) 
has shown, in a tendency toward a decline of fertilizability and of subsequent de- 
velopment. Abnormalities become pronounced when eggs are inseminated 30-60 
minutes after the formation of the second polar body. 

To summarize : there is a period of progressive ripening of the karyocytoplasm. 
Optimum ripening is heralded by the development of the polar asters and the initia- 
tion of the sperm pronucleus to form its aster. After this period there is a decline 
in the proper functional interrelations between the sperm and karyocytoplasm. 
The decline is made evident by the fact that sperm entry, subsequent to second 
polar body formation, results in an increasing abnormality of cleavage. 

//. Observations on Bisected Eggs 

A. Unfertilized eggs bisected and the fragments immediately inseminated 

1 . Early bisections up to about ten minutes before first polar body formation. 

Thirty pairs of fragments were studied. The cutting was done at 25, 40 and 60 
minutes after deposition of the eggs in sea water (ca. 10, 25 and 45 minutes re- 
spectively after the germinal vesicle had disappeared). Each pair of fragments 
was then inseminated immediately. Figure 2 is representative of all the cases. 
The sperm aster in the non-egg-nucleated fragment appeared earlier than in the 
egg-nucleated fragment. Its time of appearance was always after the companion 
fragment had formed its first polar body and two to three minutes before the forma- 
tion of the second polar body. On the other hand, in the egg-nucleated fragment, 
the sperm aster never appeared until two to three minutes after the second polar 
body had been formed. This difference between the two fragments was reflected 
in the earlier cleavage of the haploid fragment. 

2. Late bisections immediately before and during first polar body formation. 
Twenty pairs of fragments were studied. In all of them the sperm aster appeared 
simultaneously at about two to three minutes after formation of the second polar 
body in both non-egg-nucleated and egg-nucleated fragments of each pair. The 
cleavage time of both fragments was simultaneous. 

B. Eggs fertilised early and fragmented at varying times until shortly after first 
polar body formation 

The eggs were inseminated 25 minutes after deposition in sea water, that is, 
shortly after dissolution of the germinal vesicle. In many of the bisected eggs 
both male and female nuclei lay in the same fragment. These double nucleated 
fragments, regardless of the time of cutting, behaved exactly like the whole eggs in 
regard to the time of polar body formation, appearance of the sperm aster and 
sul (sequent cleavage. Attention was devoted to the few fragments in which the 



EGG NUCLEO-CYTOPLASMIC RELATIONS 



277 



sectioning had separated the sperm from the egg nucleus. Four fragments were 
of eggs cut at 35 minutes ; three at 50 minutes, and six at 74 minutes after deposi- 
tion in sea water. 

The results are shown in Figure 3. In the eggs (A a ) cut 35 minutes after 
deposition in sea water, the sperm aster (A 2 ) appeared after the first polar hody 
of the companion fragment and two to three minutes before the second polar body. 
In an egg (Bj) cut at 50 minutes, the sperm aster (B 2 ) appeared simultaneously 
with the second polar body of the companion fragment. In an egg (Q) cut at 
74 minutes, the sperm aster (C L> ) appeared after the second polar body in the 
companion fragment. D represents the first cleavage stage, at 170 minutes, of 
the eggs, A, B, and C. Cleavage occurred, as is to be expected, only in the frag- 
ment containing the sperm pronucleus. The egg nucleus in the other fragment 
produced the first and second polar bodies at the same rate as that of fertilized 
control whole eggs and, finally, moved to a central position in the fragment, where, 
as the female pronucleus, it enlarged somewhat but otherwise remained quiescent. 
The time of appearance of the polar bodies \vas thus seen to be the same, irrespective 
of when the sperm pronucleus had been separated from the egg nucleus by the cut- 
ting process. Evidently neither a brief nor a long sojourn of the sperm pronucleus 
in cytoplasmic continuity with the egg nucleus affects the hastening which the 
fertilization process induces in the formation of the polar bodies. 






Jst p.b. 



100 min. 



n 




s.a. 



s.a. 




105 min. 



165 min. 



FIGURE 2. Asterias egg bisected 40 minutes after deposition in sea water, both fragments 
inseminated simultaneously. 

A. At 40 minutes. Cutting of unfertilized egg with microneedle, n. 

B. At 100 minutes. Both fragments with fertilization membranes. Haploid fragment with 
sperm aster, s.a. Diploid fragment with first P.B. which had formed 25 minutes earlier. 

C. At 105 minutes. Haploid fragment with considerably enlarged sperm aster. Diploid 
fragment with beginning sperm aster and second P.B. w r hich had formed two minutes earlier. 

D. At 165 minutes. Haploid fragment just after completion of first cleavage. Diploid 
fragment still in amphiaster stage. 



278 



ROBERT CHAMBERS AND EDWARD L. CHAMBERS 









1st p b 




170 mm. 

FIGURE 3. Three eggs inseminated 25 minutes after deposition in sea water and then 
bisected at different times so as to have sperm pronucleus in one fragment and egg nucleus in 
the other. 

A. Cut at 35 minutes (10 minutes after insemination), A,. At 97 minutes, A 2 . Beginning 
sperm aster, s.a., appears in one fragment and first P.B. in companion fragment. 

B. Cut at 50 minutes (25 minutes after insemination), B,. At 99 minutes, B 2 . Beginning 
sperm aster appears in one fragment and beginning second P.B. in companion fragment. 

C. Cut at 74 minutes, just after the first P.B. has formed, d. At 101 minutes, C-. Be- 
ginning sperm aster appears in one fragment and completed P.B. in companion fragment. 

D. Condition of all three bisected eggs at 170 minutes. Sperm-haploid fragment has cleaved, 
while egg nucleus, c.n., of companion fragment has taken a central position and remained inactive. 



DISCUSSION 

The results presented in this paper stress two major features concerning the 
events after the material of the germinal vesicle has mixed with the cytoplasm of 
the egg. One deals w r ith the maturation of the karyocytoplasm ; the other, with 
the fertilization process of the male and female nuclear elements in their relations 
to the maturing karyocytoplasm. 

Delage (1899) had already surmised that the dissolution of the germinal vesicle 
is essential to maturation and fertilizability of the sea urchin egg, and confirmed 
it from his merogonic experiments (1901) on the immature eggs of Asterias 
glacial is. 

In our experiments with Asterias forbcsii the disappearance of the germinal 
vesicle and the mixing of its hyaline fluid with the granular cytoplasm lasts about 
10-15 minutes. The resulting karyocytoplasm contains the definitive egg nucleus. 



EGG NUCLEO-CYTOPLASMIC RELATIONS 279 

Any viable fragment of this karyocytoplasm is fertilizable. The significant feature 
is that the karyocytoplasm must undergo a protracted maturing process. The 
prime evidence for completed karyocytoplasmic maturation is the appearance of 
asters initiated in the egg either by the egg nucleus in forming the polar bodies, 
or by the spermatozoon in forming its sperm aster. 

It is of interest to note that when the fertilization process is initiated in eggs 
with still maturing karyocytoplasm, the activity of the egg nucleus starts earlier 
than it would if the egg were unfertilized. 2 On the other hand, the activity of 
the sperm pronucleus in forming its aster starts later when it is associated with the 
egg nucleus than it does when it is isolated in a separate body of karyocytoplasm. 
This indicates that the fertilization process accelerates the egg nucleus to polar body 
formation, while the presence of the egg nucleus delays the formation of the sperm 
aster. As an example of this, let us consider the situation in which an egg nucleus 
and a sperm pronucleus are lying together in karyocytoplasm which is still matur- 
ing. Upon completed maturation of the karyocytoplasm, there is initiated around 
either the male or the female nucleus a localized, centripetal cytoplasmic stream- 
ing which becomes evident to the eye as asters. The first asters to appear are those 
of the polar spindles of the egg nucleus. Not until the second polar body has been 
eliminated is there any sign of cytoplasmic streaming centered about the sperm 
pronucleus for the formation of the sperm aster. The course of these phenomena 
is of phylogenetic interest, viz., the fact that it is the egg nucleus rather than 
the sperm around which the radial streaming first occurs. 

In the development of the sex elements, the last step taken by the fully grown 
primary oocyte is to undergo two successive cleavages (equational and redtictional). 
In the early history of sex the resulting four egg cells may be equal in size or, 
in accordance with later evolutionary changes, they may be unequal, viz., the typical 
egg and its three polar bodies. In either event, growth of the mother cell, followed 
by two successive nuclear mitoses, has been repeated presumably over countless 
periods of time before the male sex cell came into being. This would establish a 
condition such that the maturation of the karyocytoplasm tends to lead directly to 
the formation of the two polar bodies. The sperm in the egg is a relatively late 
comer in evolution so that reactions concerned with it should come after, with the 
development of the sperm aster and eventually the amphiaster of the first cleavage 
spindle of the fertilized egg. This might be regarded as a case of evolutionary 
memory, colloidal or otherwise. 

When the sperm pronucleus is isolated in a non-egg-nucleated fragment of an 
egg with karyocytoplasm which has not yet become mature, maturation leads to 
cytoplasmic streaming and aster formation about the sperm pronucleus. There is 
no egg nucleus to assert priority, and the result is that the sperm aster appears 
before it otherwise would. 

When insemination occurs after the karyocytoplasm has completed its matura- 
tion, the conditions which now exist do not call for an interplay of the reactions 
described above. In a completely mature egg, the lapse of 30 to 35 minutes be- 

2 Recently, Lovelace (1947) was able by artificial means to accelerate the penetration of the 
spertnatozoan in the Nereis egg. She found that this induced earlier formation of the polar 
bodies than would have been the case if the sperm had penetrated later. Fol (1879, pp. 117 
and 335) had already noted that for the Asterias egg, polar body formation is accelerated by 
early insemination of the egg. 



280 ROBERT CHAMBERS AND EDWARD L. CHAMBERS 

tween sperm entry and appearance of the sperm aster is just about the time between 
the initiation of the first and completion of the second polar body. Therefore, if 
sperm entry occurs at the earliest moment of completed karyocytoplasmic matura- 
tion, i.e., just prior to the formation of the first polar body, the astral streaming 
of the polar body spindles will have been completed before the sperm aster begins 
to be appreciable. 

An indication of the necessity for proper time relations between the formation of 
the sperm aster and that of the polar body asters is given in extremely interesting 
experiments performed years ago by A. Brachet (1922). Brachet discovered a 
means of disturbing these time relations, and by doing so secured abnormal astral 
configurations. Brachet found that the immature eggs of Paracentrotus, on being 
removed from the ovary, could be stopped at various stages of their maturation by 
plunging the eggs into sea water. In the sea water these eggs readily became 
polyspermic, and the sperm which had entered continued' to develop and formed 
sperm asters. These asters either remained small or grew to larger dimensions 
according to the stage of the eggs they were in. The stages of special interest in 
this discussion were those of the eggs possessing egg nuclear polar spindles and 
their asters. Sperm asters present at the same time became intermingled with 
them and formed abnormalities such as tripolar mitoses, etc. Fol was also able 
to observe similar discrepancies in polyspermic Asterias eggs. In the event that 
several sperm asters appeared while the chromosomal vesicles of the egg nucleus in 
mitosis were still infused, Fol noted that one or more of the vesicles became in- 
corporated in the sperm asters, thus upsetting the normal course of events. 

The avoidance of such a phenomenon is ensured in the Asterias egg, which 
normally matures in sea water and which is fertilizable at any stage during its 
maturation. In monospermic eggs an appropriate time-spacing between the male 
and female nuclear events is occasioned by the following: On the one hand, the 
formation of the egg-nuclear polar bodies is accelerated by the fertilization process, 
while on the other, the appearance of the sperm aster is delayed by the presence of 
the egg nucleus. The two features combine to separate in time the formation of 
the polar bodies from the formation of the sperm aster. The result is that in the 
normal course of development, the cytoplasmic streaming, involved in the forma- 
tion of the polar body asters, reaches completion before the initiation of the stream- 
ing associated with the growing sperm aster. It appears, therefore, that the 
peculiar interrelations between karyocytoplasm, egg, and sperm nuclei are of service 
in preventing a possible interference between the reactions concerned in polar body 
formation and those concerned with preparation of the fertilized egg for its first 
cleavage. 

SUMMARY 

Full-sized germinal vesicle oocytes of Asterias forbcsii undergo normal matura- 
tion in sea water. At 16 C. the first polar bodies are formed in about 80 minutes, 
and the second, in 108 minutes. The eggs are sperm-fertilizable from the time of 
germinal vesicle breakdown until some time after elimination of the second polar 
bodies. Fol (1879, p. 204) indicated that the optimum time for insemination is 
after germinal vesicle breakdown up to the first polar body formation. In accord- 
ance with Fol, the earliest period for the sperm aster to appear was found to be 



EGG NUCLEO-CYTOPLASMIC RELATIONS 281 

always a few minutes after the formation of the second polar body. A feature to 
be stressed is the progressive change of the karyocytoplasm induced by the mixing 
of the contents of the germinal vesicle with the cytoplasm during maturation. 

Maturation of the karyocytoplasm 

1. When eggs are inseminated two to three minutes before first polar body for- 
mation or later, the time for the sperm aster to appear in the living egg is about 
35 minutes at 16 C. 

When the eggs are inseminated at any time prior to the above, the time taken 
for the sperm aster to appear is equal to 35 minutes plus the interval between the 
time of insemination and the time of initiation of the first polar body. 

Evidently, therefore, the time of appearance of the sperm aster is a function of 
the maturation of the karyocytoplasm. The maturation begins at the time of 
germinal vesicle breakdown and reaches completion two to three minutes prior 
to formation of the first polar body. In a fully mature karyocytoplasm the interval 
between sperm entry and the appearance of the sperm aster is constant. 

The egg nucleus 

2. Sperm-fertilization of whole eggs or of egg-nucleated fragments accelerates 
the egg nucleus in the formation of its polar bodies. The earlier the insemination 
the greater is the acceleration. 

3. The effect of the fertilization process in accelerating polar body formation 
persists after removal of the sperm pronucleus. This was ascertained by removing 
the sperm pronucleus, through bisection, at several intervals of time, the earliest 
being ten minutes after insemination. 

In other words, once given the impetus the egg nucleus maintains its hastened 
progress independently of the presence of the sperm pronucleus. 

The sperm aster 

4. In eggs bisected while undergoing maturation and then inseminated, the 
sperm aster appears earlier in the fragment lacking the egg nucleus than in the 
egg-nucleated fragment. 

In eggs fertilized while undergoing maturation and then bisected at different 
times, the sooner the sperm pronucleus has been isolated from the egg nucleus, the 
earlier the sperm aster appears. 

In other words, the presence of the egg nucleus has a delaying action on the 
development of the sperm aster. However, the earlier the egg nucleus has been 
removed through bisection of the egg, the less is the delaying action. 

GENERAL CONCLUSION 

There is a close interrelation between (a) the fertilization process, (b) the 
ripening of the karyocytoplasm, (c) the development of the sperm pronucleus, and 
(d) the activity of the egg nucleus in forming its polar bodies. The fertilization 
process, by hastening the maturation of the karyocytoplasm, accelerates the activity 
of the egg nucleus in forming its polar bodies. On the other hand, the egg nucleus 



282 ROBERT CHAMBERS AND EDWARD L. CHAMBERS 

exerts a lag effect on that feature of the maturation of the karyocytoplasm which 
is concerned with the development of the sperm aster. The net result is the attain- 
ment of an adequate spacing hetween the times of the cytoplasmic streaming activi- 
ties concerned with polar body formation and those concerned with the develop- 
ment of the sperm aster. This permits normal development of Asterias eggs 
fertilized at any time during their maturation. 

LITERATURE CITED 

BRACKET, ALBERT, 1922. Recherches sur la fecondation prematuree de 1'oeuf d'oursin. Arch. 

dc Biol., 32 : 205. 
CHAMBERS, E. L., 1939. The movement of the egg nucleus in relation, to the sperm aster in 

the echinoderm egg. Jour. E.rp. Biol., 16 : 409. 

CHAMBERS, R., 1917. Microdissection studies. II. The cell aster. A reversible gelation phe- 
nomenon. Jour. E.vp. ZooL, 23 : 483. 
CHAMBERS, R., 1921. Microdissection studies. III. Some problems in the maturation and 

fertilization of the echinoderm egg. Biol. Bull., 41 : 318. 
CHAMBERS, R. AND E. L. CHAMBERS, 1940. Interrelations between egg nucleus, sperm nucleus 

and cytoplasm of the Asterias egg. Biol. Bull., 79 : 340. 
CHAMBERS, R., 1949. Micrurgical studies on the kinetics of cell division. Annals N. Y. Acad. 

Sci. (In press.) 

DELAGE, YVES, 1899. fitudes sur la merogonie. Arch, dc Zoo], c.vp. ct gen., 3" serie, 7 : 383. 
DELAGE, YVES, 1901. fitudes experimentales sur la maturation cytoplasmique et sur la par- 

thenogenese artificielle chez les echinodermes. Arch, dc Zool. c.vp. ct gen., 3 C serie, 9 : 

285. 
FOL, HERMANN, 1877. Sur le commencement de 1'henogenie chez divers animaux. Arch, dc 

Zool. cxp. ct gen., 6 : 145. 
FOL, HERMANN, 1879. Recherches sur la fecondation, et le commencement de 1'henogenie chez 

divers animaux. Mem. dc la Socicte Physique ct Hist. nat. (Geneve), 26: 89. 
FOL, HERMANN, 1891. Le quadrille des centres, un episode nouveau dans 1'histoire de la 

fecondation. Arch, des sci. phys. et nat., 3 e serie, 25 : 393-420. 
LILLIE, R. S., 1915. Momentary elevation of temperature as a means of producing artificial 

parthenogenesis and the conditions of its action. Jour. E.vp. ZooL, 5 : 375. 
LOVELACE, ROBERTA, 1947. Precocious sperm entrance. Anat. Rcc., 99 : 655. 
TENNENT, D. H., C. V. TAYLOR, AND D. M. WHITAKER, 1929. An investigation on an organiza- 
tion in a sea urchin egg. Carnegie hist, of IV ash. Publ. 391 : 1. 
WHITAKER, D. M., 1928. Localization in the starfish egg and fusion of blastulae from egg 

fragments. Physiol. Zool., 1 : 55. 
WILSON, E. B. AND ALBERT P. MATHEWS, 1895. Maturation, fertilization, and polarity in the 

Echinoderm egg. New light on the "quadrille of the centers." Jour. Morph., 10: 319- 

342. 



FORM AND GROWTH IN THE DEVELOPMENT OF A 

SCYPHOMEDUSA 

N. J. BERRILL 
McGill University, Montreal 

The nature and development of the scyphistoma and strobila of certain Scypho- 
medusae have been described a number of times, from various points of view. 

Among the Semaeostomae, our knowledge of Aurelia and Chrysaora (including 
Dactylometra) is fairly complete, although correlations of form and size have not 
been emphasized. The other two forms that have been studied to some extent are 
Pelagia, the egg of which transforms directly into a medusa, and Cyanea. In the 
case of the Rhizostomae the developmental cycle is known for Cassiopea, Cotylorhiza 
and Nausithoe. 

The present account is based upon a collection of scyphistomae and strobilae 
tentatively identified as those of Cyanea capillata Eschscholtz. 

SOURCE OF MATERIAL 

The material was part of an unlabelled collection in the Zoology museum at 
McGill University, a circumstance that adds an uncertainty of original site to the 
usual uncertainty of parentage of scyphistomae found in their natural habitat. 

Fortunately, the internal evidence is decisive. The scyphistomae were attached 
to ascidians or to eel grass (Zostera marina) to w r hich the ascidians in turn were 
attached. Fastened between some of the ascidians were several very young 
specimens of Ciicumaria jrondosa. The presence of the holothurian places the 
locale on the Atlantic coast north of Cape Cod. The ascidian is definitely identified 
as Molgnla provisionalis Van Name, a species closely related to M. manhattensis 
and previously confused with it (cp. Van Name, 1945, p. 389). Molgnla provi- 
sionalis, however, is recorded only from waters in the general region of Eastport, 
Maine, from Passamaquoddy Bay to Mount Desert. Since it is known that col- 
lections of this species of Molgula, attached to eel grass, have been made at St. 
Andrews Point in Passamaquoddy Bay, there is little doubt that the material is 
part of such a collection, and in any case there appears to be no doubt that these 
scyphistomae came from shallow water near the mouth of the Bay of Fundy. 

IDENTIFICATION OF MATERIAL 

Identification of the genus and species is rather more difficult. The obvious 
suspects are Aurelia aurita and Cyanea capillata, since both of these are abundant 
in the region. Dactylometra qiiinqitecirrha (a "Chrysaora") reaches the shore- 
line at Cape Cod, but is not reported from inshore waters of northern New England. 
The most northerly occurring rhizostomid of the Atlantic coast is Rhopilema ver- 
rillii, a southern form that occasionally strays into Long Island sound. The only 

283 



284 N. J. BERRILL 

remaining form is Phacellophora ornata, another semaeostomid, which is known 
only from Eastport and the Bay of Ftmdy as two isolated records, by Verrill in 
1869 and Fewkes in 1888. 

The strobilae do not resemble those of Aurelia (cp. Percival, 1923) or Chrysaora 
(cp. Chuin, 1930), and while they are remarkably like those of the rhizostomids 
Cassiopea (cp. Bigelow, 1900) and Cotylorhiza (cp. Claus, 1892), it is not rea- 
sonable to assume the occurrence of an unknown rhizostomid in the region in ques- 
tion, nor to extend the range of Rhopilema from Long Island Sound through the 
five hundred miles of cold water north of Cape Cod. The alternatives remain 
Cyanea or Phacellophora, and the absence of any record of Phacellophora during 
the last sixty years makes it a most unlikely candidate. It is provisionally as- 
sumed, therefore, that our scyphistomae and strobilae belong to Cyanea, even though 
the somewhat brief earlier descriptions of the life cycle of Cyanea are significantly 
different from the account given here. 

The Cyanea of the western Atlantic is C. capillata Eschscholtz. According to 
Mayer (1910), C. arctica Peron and Lesueur and C. laniarckii Peron and Lesueur 
are synonymous, or at the most are varieties of doubtful stability. The embryonic 
and early larval stages have been intensively studied by Hyde (1894) as C. arctica. 
Young scyphistomae were reared by L. Agassiz (1862) as C. arctica and by Perez 
(1920) as C. capillata. Planulae were reared in aquaria through the scyphistoma 
to the strobila and ephyra stages by Hargitt (1902 and 1910) as C. arctica and by 
Delap (L905) as C. laniarckii. The scyphistomae described by the above investi- 
gators might well be of one and the same species, but the strobilae are very dif- 
ferently described and in neither case do they conform at all closely with the one 
given here. Both Hargitt and Delap obtained planulae directly from known 
medusae, and the difference expressed in their descriptions must be due either to 
differences in culture conditions or to a genetic difference in the parent organisms. 
These differences will be discussed following the description of the present ma- 
terial, which in spite of the element of doubt will be assumed to be that of Cyanea 
capillata. 

GROWTH OF THE SCYPHISTOMAE 

Since there is no indication that long lateral stolons are formed, as in Aurelia, 
that could produce buds at a considerable distance from a parent scyphistoma, the 
minute individuals found in scattered and very isolated positions are assumed to be 
newly attached planulae. The possibility of migratory buds, however, is not ex- 
cluded. Typical examples are shown in Figure 1, A-D. 

The planula apparently attaches by its narrow end, and in some cases at least 
sends out two or three root-like processes of attachment (Fig. 1, A, B). Four 
tentacles appear around the developing manubrium, while four more are added, 
bringing the number to eight without significant change in size from the original 
state (Fig. 1, C, D). Eight new tentacles appear, raising the total to sixteen, again 
with little increase in the size of the whole. 

At the same time a small bud protrudes from the wall of the hydroid at or near 
the junction of the body and stalk (Fig. IE). Similar buds, appearing at the 
same site, occur in scyphistomae of all sizes (Fig. 1, G-J), although many scyphis- 



DEVELOPMENT OF A SCYPHOMEDUSA 



285 



tomae equally representative of all sizes were found without buds (Fig. IF). The 
conclusion is that a series of such buds may be produced by an individual scyphis- 
toma. The first appearance of a bud in a minute scyphistoma is in itself an expres- 
sion of a local acceleration of growth, and it would be gratuitous to assume that 
this growth would become abruptly arrested and that the same bud would remain 



A 



H 




FIGURE 1. Growth and budding of scyphistomae of Cyanca capillata. A, B, attached 
planulae. C, 4-tentacle scyphistoma. D, 8-tentacle form. E, 16-tentacle scyphistoma with 
lateral bud. F, larger form without bud. G, H, I, J, older scyphistomae with buds. 

but little changed in relative proportions in the large scyphistomae. It is more 
reasonable to interpret the conditions illustrated as being either the production of 
several buds successively from one site, or the production of but one bud, though at 
different stages of growth among different individuals. 



286 



N. J. BERRILL 



In the great majority, the direction of growth of the bud is from the top of the 
stalk downwards towards the substratum. Growth of the bud is primarily stolonic, 
and is mainly by terminal proliferation of cells (cp. Fig. 1J). The largest scy- 
phistoma of this type is shown in Figure 2B. No indication that such outgrowths 
extend to any distance has been found, and the occurrence of associations such as 
that shown in Figure 1, A and E, suggests that the buds grow down to become 
attached to the substratum close to the base of the parent, and constrict off from 
the parent at the point of origin. 




FIGURE 2. Fully grown scyphistomae of C. capillata. A, commencement of strobilation 
B, with bud directed downwards. C, D, with buds directed anteriorly. E, detached and 
attached bud at base of parent. F, metamorphosing scyphistoma with late bud. 

In a minority of cases the bud grew upwards instead of downwards (Fig. 
2, C, D) and in one case grew from the top of a long tenuous stalk that was bearing 
a metamorphosing scyphistoma at its end. Conditions such as these probably lead 
to those shown in Figure 3, A and B. In fact. Figures 2C and 3 A might well be 
placed in sequence, the scyphistoma of Figure 2C having partially metamorphosed 
to become an ephyra in Figure 3A, the bud of Figure 2C having become a scyphis- 
toma in Figure 3 A, while the mutual relationship of the stalks remains unchanged. 



DEVELOPMENT OF A SCYPHOMEDUSA 



287 



On the other hand, the comparable stages of metamorphosis exhibited by the 
two heads of the individual shown in Figure 3B suggest the possibility that the 
division of the distal end preceded differentiation into scyphistomae, especially 
since the head that is somewhat the smaller is actually the more advanced, for only 
the eight interlobular tentacles remain. Such a condition seems more likely to arise 



B 




FIGURE 3. Retention and division of buds of C. capillata. A, bud forming scyphistoma 
attached to stalk of parent. B, double-headed strobila. C, strobila with three-headed scyphis- 
toma attached to stalk base. 

at the point of detachment of a bud from its parent than at the distal end of a newly 
attached planula. This is somewhat forcibly indicated by the example shown in 
Figure 3C. The parent scyphistoma is well advanced in its metamorphosis into an 
ephyra. The associated stalk may possibly have arisen from a bud similar to that 
seen in Figure 2D, but one arising even more proximally, or equally, if not more 



288 N. J. BERRILL 

likely, from a hud that grew downward from the usual site to become attached at 
the base of the parental stalk. In any case its distal end has given rise to three 
scyphistomae of approximately equal size. It does not seem possible that any one 
of the three could have given rise to the other two by budding, for there is too 
close an identity of size and form. In one of the three individuals a bud is growing 
downward, almost like a regeneration of an additional stalk to compensate for the 
multiplicity of heads. 

FORMATION AND DEVELOPMENT OF STROBILAE 

During the process of growth, the scyphistoma becomes progressively differ- 
entiated into stalk and head as in Figure II. In many cases metamorphosis into an 
ephyra occurs in a typical manner and purely as a monodisk. The head shortens 
and widens, eight of the sixteen tentacles resorb during the formation of the eight 
rhopalia, while somewhat later the eight interlobular tentacles are also resorbed. 
At the same time, the outer margin of the scyphistoma divides into eight 
lobes corresponding to the lappets of the future ephyra. 

While in many cases a single ephyra may form from the head of a scyphistoma, in 
as many others, if not more, two or three ephyrae are produced in series. Whether 
one or more are to be formed is discernible from the contour of the scyphistoma 
before there is any other metamorphic indication, as in Figure 1J and 2A. In 
most cases, if not all, the interlobular tentacles are retained until shortly before the 
ephyra is set free (Fig. 3A). In no case have tentacles been seen in a developing 
ephyra that is second in line. 

Three stages in the later development are illustrated by Figure 3, A, C, and E, 
representing the eight-tentacle stage (3A), all tentacles resorbed (3C), and the fully 
developed ephyra on the point of liberation (3C). 

Cases such as the one shown in Figure 3B, in which two ephyra are almost at 
the same advanced stage of development, suggest that the ephyra probably grows 
to a certain critical size, when its development is functionally complete and it is 
ready to be set free, even though greater differences in size may be more evident 
at an earlier stage (cp. Fig. 3A). The individual shown in Figure 3D probably 
represents a second ephyra, the first having been liberated, and the same may be 
true for the primary individual in Figure 2C. Otherwise there is considerable 
variation in the time or size at which all tentacles become resorbed. 

In all of the individuals with ephyrae, shown in Figure 3, there is present a 
relatively small basal swelling at the junction with the stalk, suggestive of a third 
ephyra. Marginal lobes tend to develop, though not in relation to any particular 
size (cp. Fig. 3 A, 7D), and it is possible that an ephyra would have developed. 
The fact, however, that no individual has been found with three unmistakable 
ephyrae in process of formation may mean one of two things ; either the third effort 
remains abortive, or else the first ephyra is _ always liberated before the third is 
definitely established. 

It is notable that these third attempts at annular growth usually bear short 
tentacles in the lobular position (e.g. Fig. 3, C and E), possibly indicative of the 
re-establishment of the scyphistoma state. 

A number of isolated stalks were found, of the same size as the largest bearing 
ephyrae, which possessed four distal tentacles as in Figure 3F. These may rep- 



DEVELOPMENT OF A SCYPHOMEDUSA 289 

resent a return to the scyphistoma condition as is generally the case in Aurelia and 
Chrysaora, giving rise to another crop of ephyrae at some later time. On the 
other hand no scyphistoma was found that had a fully grown stalk and a head with 
either eight or sixteen tentacles. In our opinion such stalks as that illustrated are 
merely the final differentiation of the residual stumps after the ephyrae have been 
liberated, and in this form they do not give rise to further generations. 

DISCUSSION 

The essentially, monodisk character of strobilation just described is much more 
reminiscent of the strobilae of the rhizostomids Cotylorliiza tubcrciilata (Claus, 
1892) and Cassiopea xamanchu (Bigelow, 1900) than the polydisk strobilation 
described for Cyanea lainarckii by Delap (1905) at Valencia, and much more ex- 
treme than that of Cyanea arctica as described by Hargitt (1910) from Woods 
Hole. The question arises whether the differences indicate different parentage 
or a varying response to different conditions of growth. 

Both size and shape appear to determine the type of strobilation, and since 
there is the possibility that the type may vary greatly with external conditions, it 
may be well to exclude Aurelia as a candidate somewhat more definitely. In the 
first place, a freshly liberated ephyra of Aurelia has a relatively shorter manubrium, 
gastral filaments much more remote from the manubrial base, and less sugges- 
tion of inter-rhopalial tentacles, than the ephyra of our present form shortly before 
liberation. Secondly, the manner of budding of the scyphistomae is markedly dif- 
ferent. If the choice lies between Aurelia and Cyanea, as it appears, there is little 
doubt that Cyanea is the parent form. 

The growth of a scyphistoma up to the time of liberation of an ephyra is divisible 
into three phases. The first concerns the transformation of the planula into a 16- 
tentacle scyphistoma. This phase has been intensively studied in relation to the 
manner of origin of the stomach pouches and the order in which the tentacles arise. 
Neither of these features greatly concerns us here ; our main interest lies in the 
manner of growth and budding of the scyphistoma, and in the strobilation to form 
ephyrae. 

The second phase, the growth of the 16-tentacle scyphistoma, is associated with 
the production of buds. In both the rhizostomids, Cotylorhiza and Cassiopea, buds 
arise one at a time from the scyphistoma body wall above the apex of the stalk. 
The buds break free, are ciliated and free-swimming, but they eventually settle and 
become attached by their original outer end. 

In the semaeostomids Aurelia and Chrysaora, buds are formed initially as lateral 
outgrowths from the body wall near the base of the scyphistoma. They grow out 
as stolons for a considerable distance before becoming attached (Fig. 5B) either to 
give rise to a new scyphistoma at the point of attachment, or to one or two scyphis- 
tomae at some place between origin and attachment. The connection with the 
parent is finally broken. 

In our Cyanea the buds arise from a site equivalent to the point of origin in 
Cotylorhiza and Cassiopea, but grow longer and downward to become attached 
basally by the time separation from the parent takes place. In both types, how- 
ever, the scyphistoma head grows from the upper end of the bud. It is therefore 
intermediate in character between that of Aurelia and Cassiopea. The three kinds 



290 



N. J. BERRILL 



A 




FIGURE 4. Strobilae of C. capillata. A, strobila with two ephyra and possible third. B, 
strobila with two equalized ephyrae. C, strobila with advanced ephyra and a potential second 
bearing scyphistoma tentacles. D, strobila with second ephyra well developed and a potential 
third. E, ephyra on point of liberation. F, post-strobila stalk with four tentacles. 



DEVELOPMENT OF A SCYPHOMEDUSA 291 

of buds are essentially the outcome of two variables, the direction of outgrowth 
and the intensity of growth. Subsequent development depends upon the orienta- 
tion of the outgrowth, and a new scyphistom'a always arises from an upper surface, 
whether it be the distal or proximal end of an outgrowth or from some point on its 
side wall. 

The question of monodisk or polydisk strobilation concerns both size and shape, 
both of which are expressions of growth. In monodisk development, growth in the 
basal part becomes progressively linear and apparently becomes arrested, while 
anterior growth becomes progressively transverse. Between the two regions there 
is a steep growth gradient producing a comparatively abrupt transition from head 
to stalk. 

In contrast to this, the scyphistoma of Aurelia exhibits no such differentiation, 
and both transverse and linear growth occur throughout, so that while growth in 
length of the whole is the greater, transverse growth continues in basal as well as 
anterior regions. A large scyphistoma is therefore not very different in shape 
from a small one. 

Shape is probably one of the main factors in determining the nature of strobila- 
tion. Constrictions carve off the shallow saucer-like discs of the scyphistoma to 
form ephyrae, and whether one, two, or many such discs can be produced is mainly 
a matter of the shape of the whole and the extent of growth occurring at the various 
levels. In this light, the difference between monodisk and polydisk strobilation is 
primarily a difference in the extent to which significant transverse growth can be 
maintained along the antero-posterior axis of the scyphistoma (cp. Fig. 2A, 5C). 
This activity may well vary with different conditions of temperature and food 
supply. 

The scyphistomae reared by Delap grew steadily through summer months, ap- 
parently without producing buds, in each of two successive years, and in each year 
strobilated to form eight to eleven ephyra in late winter when the temperature fell 
below 45 C. The scyphistomae were abundantly fed with small planktonic organ- 
isms throughout the whole period. Those reared by Hargitt were fed even more 
concentratedly, at relatively high temperatures, and grew to the strobila condition 
with astonishing rapidity. One to five ephyrae were produced, with an average of 
three to four. Hargitt states that buds were seen but were extremely rare. For- 
tunately Delap gives the scale of her drawing of the strobila, so that a comparison 
of actual size is possible. Her polydisk strobilae are approximately three times 
the height of ours, and have no sharp division into stalk and head. 

Our own scyphistomae were without doubt collected during the summer or 
late spring, and in Passamaquoddy waters would accordingly be developing at 
low temperatures (below 50 C.), even though maximum for the region. Growth 
would be relatively slow at the prevailing temperature and the food supply would 
probably fall far short of the degree of forced feeding employed by Hargitt and 
Delap. 

The form of the sessile phase of the Hydromedusae responds sharply to varying 
conditions of temperature and food supply (Berrill, 1948, 1949) and it would be 
expected that the scyphomedusae would also react, in their own way. Differences 
in relative growth rates, however, may very well be inherited within the limits of a 
single species, and different races of Cyanca capillata may vary in the quantitative 
growth response their respective scyphistomae make to changing external conditions. 



292 N. J. BERRILL 

SUMMARY 

The developmental cycle of a scyphomedusa, probably Cyanea capillata Esch- 
scholtz, is described, with emphasis upon the correlation of size and form. 

The nature of the budding process, giving rise either to free buds or to double- 
headed forms, is described. 

An analysis of monodisk and polydisk strobilation is given in terms of growth, 
size and shape. 

LITERATURE CITED 

AGASSIZ, L., 1862. Contributions to the natural history of the United States. 4. Boston. 
BERRILL, N. J., 1948. A new method of reproduction in Obelia. Biol. Bull., 95: 94-99. 
BERRILL, N. J., 1949. Growth and form in Bougainvillid Hydroids. I. Polymorphic develop- 
ment in Bougainvillia and Aselomaris. Jour. Morph., 84 : 1-30. 
BIGELOW, R. P., 1900. The anatomy and development of Cassiopea Xamancha. Mem. Biol. 

Lab. Johns Hopkins Univcr., 4: 191-233. 
CnuiN-T. T., 1930. Le cycle evolution de scyphistome de Chrysaora. Trav. Stat. Biol. 

Roscoff, 5: 1-180. 
CLAUS, C., 1892. Entwicklung der scyphistoma von Cotylorhiza, Aurelia and Chrysaora. Arb. 

Zoo/. lust, ll'icn, 10 : 1-70. 
DELAP, M. J., 1905. Notes on the rearing in an aquarium of Cyanea lamarckii P. & L. Rep. 

Fisheries Ireland Sci. Invest, (for 1902), pp. 20-22. 

FEWKES, J. W., 1888. Report U. S. Expedition to Lady Franklin Bay, 2 : 40. 
HARGITT, C. W., 1902. Notes on the coelenterate fauna of Woods Hole. Amcr. Nat., 36 : 

549-560. 
HARGITT, C. W. AND G. T. HARGITT, 1910. Development of scyphomedusae. Jour. Morph., 

21 : 217-262. 

HYDE, I., 1894. Entwicklungsgesichte einigen scyphomedusen. Zcit. wiss. Zoo/., 58: 531-566. 
MAYER, A. G., 1910. Medusae of the world. 3. Publ. Carneg. Inst. Washington. 1910. 
PERCIVAL, E., 1923. On the strobilization of Aurelia. Quart. Jour. Micr. Sci., 67 : 85-100. 
PEREZ, C. L., 1920. Un elevage de scyphistome de Cyanea capillata. Bull. Biol. Fr. ct Bclg., 

59: 167-178. 
VAN NAME, W. G., 1945. The North and South American Ascidians. Bull. Amcr. Mus. Nat. 

Hist., 84: 1-476. 
VERRILL, A. E., 1869. Description of a remarkable new jellyfish and two Actinians from the 

coast of Maine. Amcr. Jour. Sci., ser. 2, 48: 116-118. 



FUNDAMENTAL PRINCIPLES IN OXIDATION-REDUCTION ' - 

L. MICHAELIS 
From the Laboratories of The Rockefeller Institute for Medical Research, New York 

Oxidation of organic compounds is the source of energy for living organisms. 
This mechanism of supply of energy is made possible by the fact that the organic 
compounds used as food, as well as the oxidizing agent, molecular oxygen, are 
inert and can, at the proper time, be activated by catalysts so as to interact with 
each other. This inertia is due to the principle of "compulsory univalent oxida- 
tion" or of "single-electron transfer." This may be explained as follows. Oxida- 
tion is, primarily, the withdrawal of electrons. It is unessential for the process 
proper of oxidation whether a proton is withdrawn together with the electron. If so, 
oxidation is the same as dehydrogenation. Reduction is the reversal of oxidation. 
The principle just mentioned states that any bivalent (or polyvalent) oxidation or 
reduction has practically no other chance to proceed than in successive transfers of 
a single electron, or, in "univalent steps" of oxidations or reductions. These steps 
may overlap, and often to such an extent that the nature of the two-step process is 
difficult to recognize. In this way, "bivalent" oxidation or reduction of such sub- 
stances as quinones and dyestuffs was conceived until recently as a bivalent process 
occurring by the simultaneous transfer of a pair of electrons. 

The experimental evidence for the principle of single-electron transfer can be 
furnished essentially by two methods : measurement of redox-potentials, and meas- 
urement of magnetic susceptibility. 

In a reversible redox system, if it is a univalent one, such as Fe ++i - Fe ++ , the 
molecular species involved can exist on two levels of oxidation. If it is a bivalent 
one, according to the principle of single-electron transfer, it can exist on three 
oxidation levels : the reduced form, R ; the semioxidized, S ; and the totally oxidized, 
T, which are related to each other as follows (c is the electron) : 

R^S + e 
S^T + e 

The equilibrium 2S ^ R + T is always established with immeasurably high speed, 
just as the equilibrium of electrolytic dissociation, in contrast to most other reactions 
in organic chemistry which usually are relatively slow, the rate being measurable 
and strongly dependent on temperature. 

All valence-saturated organic compounds have an even number of electrons, 
each chemical bond being represented by an electron pair. So, any S compound 

1 Paper presented as part of a Symposium of the Society of General Physiologists, Woods 
Hole, September, 1948. 

This paper was originally presented with the aid of about fifty lantern slides representing 
experimental evidence and a few demonstrations of experiments. The abstract given here is 
made up in a form supposed to be understandable without this aid. 

293 



294 L. MICHAELIS 

must have an odd number of electrons ; it must be a free radical. Two molecules 
of a free radical may or may not combine to form a dimer, D, which again has an 
even number of electrons. In most cases, among organic dyestuffs of the type 
of methylene blue, or the flavine dyes of the yellow respiration enzymes, the inter- 
mediate form is a free radical, S, and not D. The existence of an intermediate 
form, be it S or D, can be easily recognized on oxidizing R or reducing T, because 
the intermediate form usually has a color of its own. Whether it is S or D can be 
recognized as follows. When a solution of R is titrated with an oxidizing agent, 
and the potential at a blank platinum electrode is plotted against the percentage 
of oxidation, the curve obtained in this plot is independent of the initial concentra- 
tion of R, if the intermediate form is S ; in contrast, its shape strongly depends on 
the initial concentration if the intermediate form is D. The experiment shows that 
in most cases there is S ; in some cases, at higher concentraton, some D may be 
in equilibrium with S. Furthermore, the equilibrium constant, called the semi- 
quinone formation constant 



X[T] 

can be measured with this method : The slope of the titration curve depends on k. 
One can calculate k from this slope. If k is very large (> 16) the titration curve 
is not simply S-shaped but shows a steepening around 50 per cent oxidation, which 
directly manifests the two steps of the oxidation. 

The magnitude of k depends on the chemical nature of the redox system and on 
pH. For cationic redox systems (say, basic dyestuffs) k increases with decreas- 
ing pH ; sometimes the separation into two steps is clearly recognizable in ex- 
tremely acid solution, e.g. in methylene blue. For anionic redox systems (such as 
quinone systems), k increases with increasing pH. However, k never becomes 
vanishingly small for reversible redox systems. The fact that S at any pH is 
capable of existence in a finite concentration, is the condition sine qua non for the 
reversibility of the redox system. If k is utterly small, it means that the S state is 
capable of existence only in infinitely small concentration. Since the oxidation 
has to pass through the S-state, it means that the rate of the oxidation or reduc- 
tion is slow, that a high activation energy is required both in the direction R T 
as well as T R. For instance, the oxidation of ethanol to acetaldehyde is ir- 
reversible and needs a high activation energy because the S form (which would be 
CH 3 -CH-OH, a free radical with "tervalent" carbon), is utterly unstable. If 
this process has to be made reversible, as it is in the living organism, some means 
must be provided to increase the stability of the S form. 

Another method of demonstrating a free radical, S, during the reduction of 
T S is the measurement of magnetic susceptibility. Since the uncompensated 
spin of the odd electron in a free radical must produce paramagnetism, free radicals 
can be recognized, and their concentration determined, by the measurement of 
magnetic susceptibility. When the solution of a suitable quinone is slowly re- 
duced by glucose in an alkaline solution, the magnetic susceptibility changes first in 
a direction indicative of the appearance of a free radical, later in a direction to 
indicate its disappearance again. 



PRINCIPLES IN OXIDATION-REDUCTION 295 

Not only does the oxidation of organic compounds as used for food need activa- 
tion, but also molecular oxygen needs activation for its reduction. The successive 
steps of the reduction of O, are : 

O 2 -> O 2 ~ - O 2 = - Or - O 2 m 



or, in presence of water, which can furnish protons : 

O 2 -* O 2 H -> O 2 H 2 -> OH + OH 2 - 2OH 2 

The harrier for this reaction is represented by the high energy content of the 
radicals O 2 H and OH. A high activation energy is required to reduce oxygen to 
hydrogen peroxide, and also to reduce hydrogen peroxide to water. 

Enzymes concerned with oxidation-reduction exhibit their function in lowering 
the activation energy. The enzyme forms a reversible compound with the sub- 
strate. In such enzymes there are always two substrates : the electron acceptor 
(such as Oo, or Fe +++ , or a flavine dye), and the electron donor (such as glucose or 
lactic acid). One of the two "substrates" represents a coenzyme or a prosthetic 
group such as heme. It is over and over again reduced and oxidized reversibly in 
the course of metabolism. The other "substrate" is the substrate proper. Al- 
though probably all oxidation-reduction processes are conducted in a reversible man- 
ner by enzymes, the whole process, as far as the "substrate" proper is concerned, 
goes one way only because the reaction product is immediately removed and 
shuttled on to another enzyme which will cause a further step in its metabolic 
change. 

The problem as to how the enzyme brings about the lowering of the activation 
energy may be answered as follows. The attractive force exerted by an enzyme 
to its substrate, resembling that of the force between a protein and its immunological 
antibody, brings about the enzyme-substrate compound, and even the ternary com- 
pound consisting of apoenzyme (the protein part of the enzyme), prosthetic group 
and substrate proper. The specific shape of the protein surface forces the sub- 
strate molecule into a shape not attainable spontaneously. The energy released by 
the formation of the compound is not entirely dissipated as heat but used to distort 
the substrate in such a manner as to ease the making of the free radical which is 
the necessary intermediate step of the oxidation or reduction. The free radical 
does not exist, under these conditions, in a free state, but as an intramolecular con- 
stituent of the enzyme-substrate complex only. The case is comparable to the 
"activation" of hydrogen by platinum black. The attraction of Pt towards H 2 is 
strong enough to squeeze the H 2 molecule into the lattice of the Pt atoms in which 
it does not entirely fit. The H 2 is hereby stretched so that it behaves now almost 
as though it consisted of two H atoms. An H atom is the analogue of an S form ; 
it contains an odd number of electrons, namely one. The difference is that the 
enzyme is specific, sometimes for the electron donor, sometimes for the acceptor, 
and often for both. 



PLANT HORMONES, GROWTH AND RESPIRATION * 

KENNETH V. THIMANN - 
Harvard Biological Laboratories, Cambridge 38, Mass. 

One of the greatest values of the discovery of the auxins as growth hormones 
in plants was that they made it possible to control growth. From the study of 
growth as produced under controlled conditions by auxins came a number of ex- 
periments on the interrelation between respiration and growth. Some of these, 
and the conclusions to which they lead, will be reported in this paper. Problems 
concerned with the rate of formation, use, and inactivation of auxin in the intact 
plant represent another field of endeavor which will not be considered herein. 
It should also be made clear at the outset that the precise chemical nature of the 
auxin of higher plants need not concern us here. Most of the experiments below 
were carried out with indole-acetic acid, which is a natural auxin of widespread 
occurrence in both higher and lower plants. For this type of work the auxin 
is regarded merely as a tool to produce growth at will. 

Now the central problem in regard to auxin and the growth of plants is an old 
one ; namely, how it is that one substance can produce many different kinds of 
effects. Visible growth in plants, such as stem elongation, is mainly growth by cell 
enlargement, while the formation of roots or fruits rests in the first stages on a 
great stimulation of cell division, which only later is followed by enlargement. 
Yet both these processes are controlled by the supply of auxin to the tissues. The 
direct effect of auxin on the cambium is also stimulation of cell division. Elsewhere, 
as in lateral buds, auxin, in physiological concentrations, causes complete inhibition 
of growth. Such a diversity of the ultimate effects of one hormone suggests 
strongly that the results observed are remote from the initial action, and that this 
initial action of auxin on the cell is a fundamental one exerted on some process of 
metabolism. From this hypothetical change in metabolism the visible effects ensue, 
according to the age and location of the cell or tissue, the supply of water and of 
both plastic and catalytic materials, and perhaps also the interaction of auxin with 
other specific substances. 

The purpose of this paper is to consider the evidence that auxin brings about 
growth through causing a change in metabolism. Now it is known in a general 
way that growth of higher plants is aerobic and does not take place in nitrogen. 
This was first shown for a specialized growth reaction, namely geotropic curvature, 
by van Amejden in 1917. More than ten years ago J. Bonner found (1936) that 
growth of the oat coleoptile is directly dependent on respiration and is inhibited by 
cyanide to the same extent that respiration is. On this account van Hulssen tried 

1 Paper presented as part of a Symposium of the Society of General Physiologists, Woods 
Hole, September, 1948. 

- 1 wish to acknowledge the assistance and contributions (many of them unpublished) of 
Dr. Walter D. Bonner, Jr., and of several students, past and present, including Dr. Schneider, 
Dr. Commoner, Dr. Sweeney, and Mr. Christiansen. 

296 



PLANT HORMONES, GROWTH, RESPIRATION 297 

to detect an influence of auxin on respiration, using oat coleoptiles, but found no 
effect. Others also obtained negative results, although subsequently it has been 
found that there are some conditions under which auxin may produce an increased 
respiration. The absence of any necessary increase in respiration to accompany 
the increase in growth rate, however, indicates that growth by cell enlargement 
does not involve any considerable overall expenditure of energy. This has been 
shown in another way by the calculations of Goddard (1948) and of Frey-Wyssling 
(1948) whose figures indicate that the actual energy involved in growth is probably 
not over one per cent of the total energy available to the cell from respiration. 

Cyanide, of course, acts on the terminal oxidase and thus inhibits the respira- 
tion of all kinds of metabolites. Its effect, therefore, is not specific. It was thought 
that a fresh approach might be made through studying the effects on growth of the 
somewhat more specific dehydrogenase inhibitors. This at once proved to be 
fruitful and has led to numerous metabolic experiments with growth inhibitors. 

THE EXPERIMENTAL MATERIAL 

At this point mention should be made of the experimental objects often used in 
growth studies. The requirement for strictly uniform plants available in large 
numbers makes it essential to use seedlings, and, because light influences the 
production of auxin and also causes curvature and other complications, the seed- 
lings are almost invariably grown in the dark, and worked on only in red light. 
Of such dark-grown (etiolated) seedlings the most widely used is the oat, the 
coleoptile of which completes its growth in about five days at 25 C., and in which 
all cell division ceases after the first 10 mm. is reached. This provides an ideal 
experimental object, in which the growth involves only cell elongation. Sections 
cut from such coleoptiles grow well in simple auxin solutions, and much better if 
sucrose is added. The optimum concentrations are about 1 per cent sucrose and 
1 to 5 mg. per liter of indole-acetic acid (= 0.6 to 3.0 X 10~ 5 M). Another very 
satisfactory etiolated seedling is that of the pea ; we use sections cut from the apex 
of the third internode. The growth of these sections is small (about 50 per cent 
of their initial length) but very reproducible ; it is not appreciably affected by the 
addition of sugar, and hence this material is convenient for chemical studies. The 
same internodes when slit lengthwise give a large curvature in auxin solutions which 
has been extensively used in assaying synthetic substances for their auxin activity; 
this response has the advantage of not being nearly so limited in its applicability as 
that of the well known "Avena test" of Went, in which the auxin is applied in agar 
to one side of the decapitated coleoptile. 

The growth of seedling sections in pure auxin solution with sucrose added in 
the case of the oat coleoptile is of course a highly limited growth process. 
Shorn of the complications due to cell division, mineral nutrition, light and nitro- 
gen supply, these sections represent about the simplest system which can still be 
regarded as growing. The detailed analysis of such a simplified system should, 
however, be the first step towards an understanding of the whole complex of 
growth reactions which takes place under natural conditions. 

The growth of coleoptile sections is highly aerobic. Even submergence beneath 
1 mm. depth of solution retards it about 50 per cent ; aeration of such lightly sub- 
merged sections restores the rate to its full value (Thimann and Bonner, 1948). 
In most of our experiments the sections are arranged so as just to break surface. 



298 



KENNETH V. THIMANN 



GROWTH INHIBITION BY IODOACETATE 



The growth is reduced or prevented by inhibitors of dehydrogenases. The effect 
of iodoacetate was studied in detail by Commoner and Thimann (1941), who 
showed that growth of coleoptile sections is strongly inhibited by this substance. 
However, concentrations of the inhibitor which reduce growth practically to zero 
have only a very small effect on respiration. (The data on respiration are in excel- 
lent agreement with the later measurements of J. Bonner, 1948). Here again it 
follows that of the total energy released by respiration only a few per cent can 
be needed for growth, for otherwise the complete inhibition of growth could be 
achieved only with a substantial inhibition of respiration. This confirms the ex- 
periments and calculations mentioned above ; most of the energy of metabolism 
evidently goes for maintenance. However, it is not excluded that growth might 
involve an appreciable fraction of the respiration, but that when this process is 
inhibited another type of respiration might take its place, so that the total oxygen 
consumption would show little change. We shall return to this important con- 
sideration below. It is also necessary to point out that in the pea stem the relations 
are not quite the same, for here auxin does produce a slight increase of oxygen 
consumption, and growth inhibition is accompanied by a definite respiration de- 
crease (see below). 

An interesting and important effect was observed with iodoacetate ; the sensi- 
tivity to this inhibitor varies with the age of the coleoptile. This is shown in 
Figure 1. Young coleoptiles show an incomplete inhibition, as well as a marked 



160 



BREAKING SURFACE 




2345678 
CONCN. OF IODOACETATE X IO" 5 M 



10 



FIGURE I. 3 The total growth, after 48 hours, as per cent of that of the controls, plotted as 
a function of iodoacetate concentration. Curve A, sections from 74 hr. coleoptiles ; curve B, 
from 64-66 hr. coleoptiles ; curve C, from 54-56 hr. coleoptiles ; and curve D, from 96 hr. 
coleoptiles. All solutions contained 1 mg. indole-acetic acid and 10 grams sucrose per liter. 

3 Figures 1, 2, 3, and 5 are from Thimann and Bonner (1948 and 1949). 



PLANT HORMONES, GROWTH, RESPIRATION 



299 



acceleration at low inhibitor concentrations ; with the oldest coleoptiles, on the 
other hand, the concentrations necessary for threshold and for 50 per cent inhibi- 
tion are much lower, and the maximum inhibition is very high. Two explanations 
are possible for this effect : 

(a) the amount of enzyme with which the iodoacetate has to combine decreases 
with increasing age ; 

(b) the young plant contains substances which oppose the inhibition and which 
decrease in amount with increasing age. 

Evidence for the latter view will be presented below. First, however, it will be 
convenient to consider in more detail the nature of the enzyme system. 

SULFHYDRYL NATURE OF THE "GROWTH ENZYME" 

It is known that iodoacetate (or iodoacetamide, which behaves similarly) reacts 
with sulfhydryl groups, although as Michaelis and Schubert have shown (1934) 
it is not strictly specific for these. Since the enzyme system which controls growth 
is of great importance, it seemed worth while to determine definitely whether it is 
of sulfhydryl nature or not. The action of a number of other inhibitors has given 
clear-cut evidence on this (Thimann and Bonner, 1949). 

Arsenite and the organic arsenical mapharsen inhibit growth strongly. The 
effective concentrations are lower than for iodoacetate. The extent of inhibition 
by arsenite, unlike that by iodoacetate, does not vary with the age of the coleoptile 
(Fig. 2). The growth of pea stems is also inhibited by arsenite, showing exactly 



120 



A= 72-74 HOURS 



B= 54-56 HOURS 




23456789 10 
ARSENITE CONG. X IO~ 6 M 



20 



30 



FIGURE 2. Data similar to Figure 1 but for arsenite. Sections from the three ages of coleoptiles 
show no significant difference in sensitivity to the inhibitor. 



300 



KENNETH V. THIMANN 



the relationship with arsenite concentration to be expected of a titration curve 
(Fig. 3). Such inhibitions can, of course, be regarded as titration of the enzyme 
with the inhibitor. The "titration curves" of iodoacetate are complicated by the 
promotion of growth at low iodoacetate concentrations ; arsenite does not produce 
this effect. 

A more specific sulfhydryl reagent is parachloromercuribenzoate, introduced 
by Hellerman et al. (1943). This also inhibits growth and, like arsenite, the 
effective concentrations do not vary appreciably with age. Again the growth and 
slit stem curvatures of peas are also inhibited by this reagent. 



(9 



70 

I 
60 

50 
40 



</>20 - 

< 



'oh 

o 

K 
O 



H 2 



I 
10-5 



I 



10-3 3X 

CONCN. OF ARSENITE 



3XIO" 5 I0~ 4 



3X10 



-4 



10 



-3 



FIGURE 3. Growth, after 24 hrs., of 20 mm. sections of the uppermost internode of 7-day- 
old etiolated pea stems. All solutions contained indole-acetic acid 10 mg. per liter and arsenite 
as shown, but no sucrose. Growth of controls in water is shown at left. 



Finally the phenyl-mercuric salts inhibit growth strongly. The concentra- 
tion for 50 per cent growth inhibition of coleoptile sections is very lo\v, about 
7 X 10" M. However, there is reason to believe that these substances are not 
so specific as those above, since they definitely inhibit respiration. At concentra- 
tions which produce 50 per cent inhibition of growth, arsenite, iodoacetate and 
parachloromercuribenzoate exert no detectable effect on coleoptile respiration, 
as shown in Figure 4. The phenyl-mercuric salts thus inhibit somewhat in the 
same way as cyanide. 

Taking the data together it is clear that the growth-controlling enzyme is of 
sulfhydryl composition. This conclusion holds far beyond the higher plants on 
which this work was done, for long ago Hammett, Voegtlin, Chalkley and others 
adduced evidence for the importance of the SH -group in the growth of inverte- 
brates, and recently, Ryan, Tatum and Giese (1944) showed that the growth of 
Neurospora is inhibited by iodoacetate in a manner both quantitatively and qualita- 
tively similar to that of coleoptile sections. This clearly is an aspect of General 
1 'hysiology. 



PLANT HORMONES, GROWTH, RESPIRATION 



301 



THE ROLE OF ORGANIC ACIDS IN GROWTH 

Some years ago Commoner and Thimann (1941) found that the inhibition by 
iodoacetate is prevented by malate, succinate, fumarate and pyruvate. More 
recently we have confirmed this and refined the technique of such experiments. 
Isocitrate has a similar effect ; so also, unexpectedly, do maleate and malonate. The 
growth system behaves in this respect like succinic dehydrogenase, which has been 
shown by Hopkins, Morgan and Lutwak-Mann (1938) to be "protected" against 
iodoacetate by these acids. However, while maleic and malonic acids bring the 
growth rate in presence of iodoacetate back to normal, they have no further effect ; 
malate, succinate, fumarate and isocitrate bring the growth rate considerably above 
normal (Fig. 5). In other words these acids accelerate growth. There are thus 
two phenomena to be distinguished : protection against iodoacetate, and promotion 
of growth. Maleate and malonate, which protect the SH group against iodoacetate, 
do not promote growth, and indeed in higher concentrations (0.05 M) actually 
inhibit it. Growth promotion is limited to the acids mentioned above, with the 




40 80 

TIME IN MINUTES 



120 



160 



200 



240 



FIGURE 4. The respiration of sections from 72-hour-old coleoptiles in indole-acetic acid 
1 mg., sucrose 10 gins., per liter. Two experiments are shown ; the uppermost curve being the 
controls in each case. The inhibitors were added at the arrow, in concentrations which cause 
50 per cent decrease in growth. As: arsenite 1.10" 5 M As,O 3 . IODA: 5.10^ M iodoacetamide. 
CIHgB : 4.10" 5 M parachloromercuribenzoate. PhHgCl : 1.10 5 M phenyl-mercuric chloride. 



302 



KENNETH V. THIMANN 



addition of pyruvate (which is very active when pure), of citrate for young plants 
only, and of acetate. 

These acids active in growth are those of the Krebs cycle. The importance 
of this cycle in respiration is well known, and recently J. Bonner (1948) has made 
it probable that the same or a similar cycle occurs in the respiration of coleoptile 
tissue. The experiments above, and several others, make it clear that malate and 
other acids of the cycle actually participate in the normal growth process. 



100 




HOURS 



FIGURE 5. The effects of malonate, maleate and malate in preventing the iodoacetate inhibi- 
tion of growth. Sections cut from 66-hour-old coleoptiles, breaking surface, in solutions con- 
taining 1 mg. indole-acetic acid and 10 grams sucrose per liter. The iodoacetate was added 7 
hrs. after placing the sections in organic acids. While all three acids act against iodoacetate, 
only the malate increases growth above that of the controls. 

For one thing, treatment with malate makes possible a direct demonstration 
that auxin does influence respiration in the coleoptile. For if coleoptile sections, 
which show no increase in respiration when auxin is added, are first soaked for 
some hours in malate or fumarate, then the addition of auxin at once increases 
their respiration rate. This means, of course, that malate (etc.) is required for 
auxin to exert its full effect, and that when this effect is exerted on growth it is by 
\vny of a respiratory system. 

The role of the organic acids in facilitating the action of auxin also makes 
possible an explanation of the effect of age on growth. Some years ago, Mrs. 
Sweeney and I (1942) made a study of the effect of auxin on the rate of proto- 
plasmic streaming in the epidermal cells of the coleoptile. The usual effect of 



PLANT HORMONES, GROWTH, RESPIRATION 



303 



physiological concentrations of indole-acetic acid or other auxins is to accelerate 
the rate of streaming; the process requires both oxygen and sugar, and from its 
dependence on auxin concentration and other features, we deduced that it is 
closely related to the promotion of growth. Now when the coleoptiles are very old 
(120 hours) this acceleration of streaming no longer occurs, but it was found that 
if the old coleoptiles are soaked in malate for some hours, the acceleration is rein- 
stated. This would suggest that in old coleoptiles the concentration of malate has 
greatly decreased. A priori this would seem unlikely since it is known from ex- 
periments on the excystment of protozoa that in fully grown grasses the opposite is 
the case organic acids increase markedly with increasing age. However, W. 
Bonner has recently shown by direct microanalysis that the amount of malate and 
other organic acids in the coleoptile does decrease with increasing age (Table I). 



TABLE I 

Organic acids of A vena coleoptiles and pea stems 
All figures per gram dry wt. 



AVENA SECTIONS 


TOTAL ETHER-SOLUBLE 
ACIDS 

micro-equivalents 


CITRIC 

mg. 


MALIC 

mg. 


54 hours 


432 


1.44 


13.7 


72 hours 


329 


1.05 


10.2 


96 hours 


284 


1.02 


5.7 


PISUM STEMS 


616 


1.29 


13.7 



Another interesting point shown in the table is that the pea stems contain more 
organic acids than any age of coleoptile. Now the pea stems do not show increased 
growth with malate. Furthermore, they differ from the coleoptiles in another 
important respect : the addition of auxin causes a direct increase in respiration rate. 
This increase is about 15 per cent and is maintained for 24 hours or throughout 
the whole period during which growth of the sections takes place. In other words, 
the pea stems behave like coleoptiles pretreated with malate. The correlation 
between this behavior and their content of malate and other acids shows how the 
age effect in coleoptiles is to be interpreted ; the increasing sensitivity to iodoacetate 
with increasing age is due to a decreasing content of the organic acids, which "pro- 
tect" against iodoacetate. Correspondingly, aging causes no increase in sensi- 
tivity to arsenite or parachloromercuribenzoate, because the organic acids do not 
protect against these reagents. 

There are other substances whose decreasing concentrations play a role in 
growth. Preliminary determinations of the keto-acids indicate that they behave 
in a similar way. J. Bonner (private communication) has recently shown that 
arginine also plays a role in growth of coleoptiles, which can be accelerated by 
adding arginine and inhibited by adding the related substance canavanine. In our 
experiments the promotion of growth by arginine is limited to older coleoptiles, so 
that the amount of this substance appears to decrease with age, like the organic 
acids. Doubtless still other materials behave similarly. However, to be able to 



304 KENNETH V. THIMANN 

explain even one aspect of aging in terms of a decrease in concentration of known 
substances in the plant is a definite step forward. 

Why should the amount of these substrates decrease with age? To some ex- 
tent the reason might be merely that the amount contributed by the seed is limited, 
so that the substances are essentially diluted by the increasing volume of the seed- 
ling. But in the case of some of the organic acids there is evidence, which will not 
be presented here, that they are actually used up in the growth process, and when 
growth is inhibited, the rate of their disappearance slows down. This brings us to 
the last part of the subject, namely the phenomena occurring during inhibition. 

METABOLIC CHANGES ASSOCIATED WITH GROWTH INHIBITORS 

Since the pea stem grows very well in auxin solution without sucrose, and 
shows little increase in growth when sugar is added, it seemed to us excellent ma- 
terial for a study of the fate of carbohydrates during growth and inhibition. Sev- 
eral conclusions can be drawn from this work. It appears, first, that the amount 
of reducing sugar which disappears during growth is exactly the same in auxin 
as in water. When it is considered that the growth in auxin is three or four 
times that in water, and that the R.Q. in both cases is close to 1, this result is sur- 
prising. Changes in non-reducing sugars are small and only minute amounts of 
metabolizable polysaccharides are present. It follows that the consumption of 
sugar in growth, both as a metabolic substrate and as a constituent of cell-walls, 
is negligibly small. 

Secondly, when growth is inhibited, reducing sugars do not pile up as might be 
expected, but instead their amount decreases. With 50 per cent growth inhibi- 
tion by iodoacetate, the reducing sugar decreases about 25 per cent more than when 
growing without inhibitor. The same effect is shown by other inhibitors. Typical 
results are shown in Table II (Christiansen et al, 1949). Fluoride has not been 
mentioned above as an inhibitor ; its effect in lowering the reducing sugar is some- 
what greater than that of the sulfhydryl-combining inhibitors. This, however, 
may be due to the fact that it slightly increases respiration after some hours, instead 
of decreasing it as do the others. 

Naturally the fate of the reducing sugar which disappears when growth is in- 
hibited is of the greatest interest. It is not respired away, since respiration is (ex- 
cept with fluoride) decreased. It is not converted to detectable amounts of starch, 
and chemical analyses show that it is not deposited as cellulose or any other wall 
materials. It is not converted to phosphate esters either, since the amounts of 
these, both in inhibited and uninhibited sections, are very small in comparison to the 
amounts of sugar involved. 

In brief it appears that, though the effect is qualitatively the same with different 
inhibitors, the ultimate fate of the sugar differs in each case. In arsenite the sugar 
is converted to neutral ether-soluble material, i.e. fats. In fluoride the same con- 
version occurs but some of the material is respired away, while in iodoacetate, 
surprisingly enough, it is excreted into the solution in the form of organic acids, or, 
more strictly, a quantity of organic acid equal in weight to the sugar which has 
disappeared is excreted into the solution. It should be noted that growing tissues 
normally accumulate solutes vigorously from solution so that an excretion (or 
exosmosis) in quantity is most unusual. The nature of this stem exudate is now 
under investigation ; it contains some fructose and 1 5 per cent of asparagine. 



PLANT HORMONES, GROWTH, RESPIRATION 



305 



In general the experiments show that inhibitors not only inhibit one process 
(which leads to growth), but they also promote another, which consumes sugars. 
In this respect the phenomena are suggestively similar to the "uncoupling" of 
phosphorylation by dinitrophenol (Loomis and Lipmann, 1948) which also leads to 
increased disappearance of sugar. Indeed dinitrophenol does inhibit growth, though 
we have not yet studied it in detail. Space does not permit the detailed presenta- 
tion of data on other inhibitors but it may be mentioned (a) that fluoroacetate, 
which specifically inhibits acetate metabolism, causes a partial growth inhibition 
which can be reversed by adding acetate, and (b) that poisoning the enzyme which 
decarboxylates pyruvic acid does not inhibit, but actually promotes growth. 

TABLE II 

Growth, respiration and reducing sugar of 20 mm. sections 
of etiolated pea stems after 24 hrs. in solution 





GROWTH 

as per cent 
elongation 


REDUCING 
SUGAR 

as per cent 
of fresh \vt. 


RESPIRATION 
as Q 02 
(after 12 hrs.) 


PER CENT DECREASE 


of growth 
in length 


of reducing 
sugar 


of 
respiration 


Initial 





1.12 














In water 


20.0 


0.83 


3.34 











In auxin 
(1 mg./l.) 


50.9 


0.86 


3.82 











In auxin plus: 
lodoacetate 
6 X 10~ 4 M 


25.6 


0.64 


3.32 


50 


26 


13 


1 X 10~ 3 M 


18.2 


0.54 





64 


37 





Arsenite 
1 X 10-" M 


26.5 


0.71 


2.89 


48 


17 


24 


1 X 10-3 M 





0.51 





100 


41 





Fluoride 
5 X 1C- 3 M 


25.3 


0.48 


4.47 


50 


44 


17 


1 X 10~ 2 M 


17.8 


0.42 





65 


51 






The relation between respiration and growth is thus not a simple one. The 
process mediated by the SH-enzyme appears to be a major limiting factor. It 
either consumes only a small part of the total oxygen and sugar used, or else it is 
readily replaced by an equivalent reaction not causing growth, and it probably is 
a step in the oxidation of pyruvic acid via the Krebs cycle or some modification 
of it. The SH-enzyme is almost certainly one of the dehydrogenases of this cycle. 
It may be noted in passing that attempts to demonstrate an iodoactetate-inhibited 
enzyme of this type in coleoptile brei, by Berger and Avery (1944), failed, but 
this would be expected from the data presented above, because the test has to be 



306 KENNETH V. THIMANN 

made in presence of malate or other dicarboxylic acid, and these very substrates 
protect the enzyme fully against the inhibition by iodoacetate. It also appears that 
the simple decarboxylation of pyruvic acid does not lead to growth, but its oxidative 
decarboxylation and the resulting metabolism of acetate does d6 so. The inhibi- 
tion of growth in presence of auxin is not the same as the mere absence of growth 
in sections not supplied with auxin; it is a positive process and leads to the diver- 
sion of sugar metabolism into other pathways. The action of an inhibitor is 
that of a switch, diverting the stream of traffic into a direction which does not lead to 
growth. Correspondingly the action of auxin itself may also be that of a switch, 
causing the metabolism of carbohydrate, or perhaps more specifically that of 
pyruvate, to go via the growth-promoting system instead of by another. It may 
be, therefore, that the apparently small fraction of metabolism involved in growth is 
a misleading phenomenon and that the true picture is rather one of alternative 
routes involving approximately equivalent amounts of respiration. However, such 
considerations are at present only speculative. 

LITERATURE CITED 

BERGER, J. AND G. S. AVERY, JR., 1944. Glutamic and isocitric acid dehydrogenases in the 

Avena coleoptile and the effects of auxins on these enzymes. Amcr. Jour. Bot., 31 : 

11-19. 
BONNER, J., 1936. The growth and respiration of the Avena coleoptile. Jour. Gen. Physiol., 

20: 1-11. 
BONNER, J., 1948. Biochemical mechanisms in the respiration of the Avena coleoptile. Arch. 

Biochem., 17: 311-326. 
CHRISTIANSEN, G. S., L. J. KUNZ, W. D. BONNER, JR., AND K. V. THIMANN, 1949. The action 

of growth inhibitors on carbohydrate metabolism in the pea. Plant Physiol., 24: 178- 

181. 
COMMONER, B. AND K. V. THIMANN, 1941. On the relation between growth and respiration 

in the Avena coleoptile. Jour. Gen. Physiol., 24 : 279-296. 
FREY-WYSSLING, A., 1948. Uber die Dehnungsarbeit beim Streckungswachstum pflanzlicher 

Zellen. Vierteljahrsschr. Naturf. Ges. Zurich, 93 : 23-34. 
GODDARD, D. R., 1948. Metabolism in relation to cell growth. Gro-wth Suppl. (Proc. Symposium 

Soc. Groivtli and Development), 12: 17-46. 
HELLERMAN, L., F. P. CHINARD, AND V. R. DEITZ, 1943. Protein sulfhydryl groups and the 

reversible inactivation of the enzyme urease. The reducing groups of egg albumin and 

of urease. Jour. Biol. Chem., 147 : 443^462. 
HOPKINS, F. G., E. J. MORGAN, AND C. LUTWAK-MANN, 1938. The influence of thiol groups 

in the activity of dehydrogenases. II. Biochem. J our., 32 : 1829-1848. 
LOOMIS, W. F. AND F. LIPMANN, 1948. Reversible inhibition of the coupling between phos- 

phorylation and oxidation. ] our. Biol. Chem., 173 : 807-808. 
MICHAELIS, L. AND M. P. SCHUBERT, 1934. The reaction of iodoacetic acid on the mercaptans 

and amines. Jour. Biol. Chem., 106: 331-341. 
RYAN, F. J., E. L. TATUM, AND A. C. GIESE, 1944. The four-carbon respiratory system and 

growth of the mold Neurospora. Jour. Cell. Comp. Physiol., 23 : 83-94. 
SWEENEY, B. M. AND K. V. THIMANN, 1942. The effect of auxins on protoplasmic streaming. 

III. Jour. Gen. Physiol., 25: 841-854 (and previous papers cited therein). 
THIMANN, K. V. AND W. D. BONNER, JR., 1948. Experiments on the growth and inhibition of 

isolated plant parts. I. Amcr. Jour. Bot., 35: 271-281. 
THIMANN, K. V. AND W. D. BONNER, JR., 1949. Experiments on the growth and inhibition 

of isolated plant parts. II. Amcr. Jour. Bot. 36 : 214-222. 



INDEX 



^BELSON, PHILIP H. AND WILLIAM R. 
DURYEE. Radioactive sodium permeabil- 
ity and exchange in frog eggs, 205. 

Actomyosin, free-energy relations and con- 
traction of, 140. 

Algae, Woods Hole region bryozoa associated 
with, 32. 

Anaphase movement of chromosomes in the 
spermatocytes of the grasshopper, 90. 

ANDERSON, WILLIAM W., JOSEPH- E. KING, 
AND MILTON J. LINDNER. Early stages 
in the life history of the common marine 
shrimp, Penaeus setiferus (Linnaeus), 168. 

Arbacia punctulata, stratification and deforma- 
tion of eggs of, centrifuged in caffeine 
solutions, 70. 

Asterias egg, nuclear and cytoplasmic inter- 
relations in fertilization of, 270. 

gEAMS, H. W. Some effects of centri- 
fuging upon protoplasmic streaming in 
Elodea, 246. 

BERRILL, N. J. Form and growth in the 
development of a Scyphomedusa, 283. 

Blood-sugar concentration, studies in regula- 
tion of, in crustaceans, 218. 

BODINE, JOSEPH HALL AND LAURENCE ROCK- 
WELL FITZGERALD. Effect of urea, thio- 
urea, phenylthiourea and thiouracil on the 
oxygen consumption of blocked and active 
embryonic cells, 1. 

BORBIRO, M. AND SzENT-GYORGYI. On the 

relation between tension and ATP in 
cross-striated muscle, 162. 

BOREI, HANS. Independence of post-fertiliza- 
tion respiration in the sea-urchin egg from 
the level of respiration before fertilization, 
117. 

BOREI, HANS AND SIGVAR LYBING. Tempera- 
ture coefficients of respiration in Psamme- 
chinus eggs, 107. 

BOVEE, EUGENE C. Studies on the thermal 
death of Hyalella azteca Saussure, 123. 

BOWMAN, THOMAS E. Chromatophorotropins 
in the central nervous organs of the crab, 
Hemigrapsus oregonensis, 238. 

BROWN, FRANK A., JR. AND GWENN M. 
JONES. Ovarian inhibition by a sinus- 
gland principle in the fiddler crab, 228. 



Bryozoa (marine), Woods Hole region, asso- 
ciated with algae, 32. 

Buffers (non-toxic), piperazine dihydrochlo- 
ride and glyclyglycine as, in distilled 
water and sea water, 233. 

BUTLER, PHILIP A. Gametogenesis in the 
oyster under conditions of depressed salin- 
ity, 263. 

(CAFFEINE solutions, stratification and de- 
formation of Arbacia punctulata eggs cen- 
trifuged in, 70. 

Cells (embryonic), effect of urea and urea 
compounds on oxygen consumption of, 1. 

Cells (foam), mitochondrial arrangement in, 
of mouse lungs, 173. 

Central nervous organs, Chromatophorotropins 
in, of the crab Hemigrapsus oregonensis, 
238. 

Centrifuging, some effects of, upon proto- 
plasmic streaming in Elodea, 246. 

CHAMBERS, ROBERT AND E. L. CHAMBERS. 
Nuclear and cytoplasmic interrelations in 
the fertilization of the Asterias egg, 270. 

Characeae of the Woods Hole region, Massa- 
chusetts, 179. 

CHENEY, RALPH HOLT. Stratification and 
deformation of Arbacia punctulata eggs 
centrifuged in caffeine solutions, 70. 

Chromatophorotropins in the central nervous 
organs of the crab, Hemigrapsus orego- 
nensis, 238. 

Chromosomes, anaphase movement of, in grass- 
hopper spermatocytes, 90. 

Colpidium campylum, presence of the tricar- 
boxylic acid cycle in, 257. 

Contraction, free-energy relations and, of acto- 
myosin, 140. 

Crab (fiddler), ovarian inhibition in, by sinus- 
gland principle, 228. 

Crab (Hemigrapsus oregonensis), Chromato- 
phorotropins in central nervous organs of, 
238. 

CROASDALE, HANNAH. See MARY D. ROGICK, 
32. 

r\URYEE. WILLIAM R. See PHILIP H. 
^ ABELSON, 205. 



307 



308 



INDEX 



"p ARLY stages in the life history of the 
' common marine shrimp, Penaeus setiferus 
(Linnaeus), 168. 

Earthworm (Eisenia foetida Savigny, 1826), 
regeneration in, 129. 

Effect of urea, thiourea, phenylthiourea and 
thiouracil on the oxygen consumption of 
blocked and active embryonic cells, 1. 

Effects of electrolytes and sugars on erythro- 
cytes of the turtle, Chelydra serpentina, 9. 

Egg (Asterias), nuclear and cytoplasmic in- 
terrelations in fertilization of, 270. 

Egg (sea urchin), independence of post- 
fertilization respiration in, from level of 
respiration before fertilization, 117. 

Eggs (Arbacia), stratification and deforma- 
tion of, centrifuged in caffeine solutions, 
70. 

Eggs (frog), radioactive sodium permeability 
and exchange in, 205. 

Eggs (Psammechinus), temperature coeffi- 
cients of respiration in, 107. 

Electrolytes, effects of, on turtle erythrocytes, 
9. 

Elodea, some effects of centrifuging upon pro- 
toplasmic streaming in, 246. 

Embryonic cells, effects of urea and urea 
compounds on oxygen consumption of, 1. 

Epicytes (alveolar), mitochondrial arrange- 
ment in, of mouse lungs, 173. 

Erythrocytes, effects of electrolytes and sugars 
on, of turtle, Chelydra serpentina, 9. 

Experiments on the determination and differ- 
entiation of sex in the bopyrid Stego- 
phyryxus hyptius Thompson, 17. 



Glycylglycine, piperazine dihydrochloride and, 
as non-toxic buffers in distilled water and 
sea water, 233. 

Growth, plant hormones, respiration, and, 296. 

"LJ YALELLA azteca Saussure, thermal 

death of, 123. 
Hyperglycemia (experimental), normal values 

and, in Libinia emarginata, 218. 

JNDEPENDENCE of post-fertilization res- 
piration in the sea-urchin egg from the 
level of respiration before fertilization, 117. 

TONES, GWENN M. Sec FRANK A. BROWN, 
JR., 228. 



, JOSEPH E. Sec WILLIAM W. AN- 
DERSON AND MILTON J. LINDNER, 168. 

KLEINHOLZ, L. H. AND BARBARA CHASE 
LITTLE. Studies in the regulation of 
blood-sugar concentration in crustaceans. 
I. Normal values and experimental hy- 
perglycemia in Libinia emarginata, 218. 

KRAUSS, MAX. A mucin clot reaction with 
sea-urchin fertilizin, 74. 

T IBINIA emarginata, experimental hyper- 

glycemia in, 218. 
LINDNER, MILTON J. Sec WILLIAM W. AN- 

DERSON AND JOSEPH E. KING, 168. 
LITTLE, BARBARA CHASE. Sec L. H. KLEIN- 

HOLZ, 218. 
LYBING, SIGVAR. Sec HANS BOREI, 107. 



pERTILIZATION, nuclear and cytoplasmic 
interrelations in, of Asterias egg, 270. 

Fertilizin, sea-urchin, mucin clot reaction with, 
74. 

FITZGERALD, LAURENCE ROCKWELL. Sec JO- 
SEPH HALL BODINE, 1. 

Form and growth in the development of a 
Scyphomedusa, 283. 

Free-energy relations and contraction of acto- 
myosin, 140. 

Fundamental principles in oxidation-reduction, 
293. 



QAMETOGENESIS in the oyster under 
conditions of depressed salinity, 263. 

GATES, G. E. Regeneration in an earthworm, 
Eisenia foetida (Savigny) 1826. I. An- 
terior regeneration, 129. 



JyfACKLIN, CHARLES C. Mitochondrial 
arrangement in alveolar epicytes and foam 
cells of mouse lungs, particularly as in- 
duced by vacuoloids, 173. 

MICHAELIS, L. Fundamental principles in 
oxidation-reduction, 293. 

Mitochondrial arrangement in alveolar epicytes 
and foam cells of mouse lungs, particu- 
larly as induced by vacuoloids, 173. 

Mucin clot reaction with sea-urchin fertilizin, 
74. 

Muscle (cross-striated), relation between ten- 
sion and ATP in, 162. 



N 



O 



UCLEAR and cytoplasmic interrelations 
in the fertilization of the Asterias egg, 270. 

N the relation between tension and ATP 
in cross-striated muscle, 162. 



INDEX 



309 



Ovarian inhibition by a sinus-gland principle 
in the fiddler crab, 228. 

Oxidation-reduction, fundamental principles in, 
293. 

Oxygen consumption, effect of urea and urea 
compounds on, of blocked and active em- 
bryonic cells, 1. 

Oyster, gametogenesis in, under conditions of 
depressed salinity, 263. 



pHENYLTHIOUREA, effect of, on oxy- 
gen consumption of blocked and active 
embryonic cells, 1. 

Piperazine dihydrochloride and glycylglycine 
as non-toxic buffers in distilled water and 
in sea water, 233. 

Plant hormones, growth and respiration, 296. 

Presence of the tricarboxylic acid cycle in the 
ciliate Colpidium campylum, 257. 

Protoplasmic streaming, some effects of cen- 
trifuging upon, in Eleodea, 246. 

Psammechinus eggs, temperature coefficients 
of respiration in, 107. 



DADIOACTIVE sodium permeability and 
exchange in frog eggs, 205. 

Regeneration in an earthworm, Eisenia foetida 
(Savigny) 1826. I. Anterior regenera- 
tion, 129. 

REIN HARD, EDWARD G. Experiments on the 
determination and differentiation of sex in 
the bopyrid Stegophryxus hyptius Thomp- 
son, 17. 

Respiration, independence of post-fertilization, 
from level before fertilization, in sea- 
urchin egg, 117. 

Respiration, plant hormones, growth and, 296. 

Respiration, temperature coefficients of, in 
Psammechinus eggs, 107. 

Ris, HANS. The anaphase movement of 
chromosomes in the spermatocytes of the 
grasshopper, 90. 

ROGICK, MARY D. AND HANNAH CROASDALE. 
Studies on marine bryozoa, III. Woods 
Hole region bryozoa associated with algae, 
32. 



gALINITY, depressed, gametogenesis in the 
oyster under conditions of, 263. 

Scyphomedusa, form and growth in the de- 
velopment of, 283. 

Sea-urchin egg, independence of post-fertiliza- 
tion respiration in, from level of respira- 
tion before fertilization, 117. 



Sea-urchin fertilizin, mucin clot reaction with, 
74. 

SEAMAN, GERALD R. The presence of the 
tricarboxylic acid cycle in the ciliate Col- 
pidium campylum, 257. 

Sex, determination and differentiation of, in 
Stegophryxus hyptius Thompson, 17. 

Shrimp (Penaeus setiferus Linnaeus), com- 
mon marine, early stages in life history 
of, 168. 

Sinus-gland principle, ovarian inhibition by, 
in the fiddler crab, 228. 

SMITH, LYN\VOOD B. Sec MARSHALL E. 
SMITH, 233. 

SMITH, MARSHALL E. AND LYNWOOD B. 
SMITH. Piperazine dihydrochloride and 
glycylglycine as non-toxic buffers in dis- 
tilled water and in sea water, 233. 

Sodium (radioactive), permeability and ex- 
change in frog eggs, 205. 

Some effects of centrifuging upon proto- 
plasmic streaming in Elodea, 246. 

Spermatocytes (grasshopper), anaphase move- 
ment of chromosomes in, 90. 

Stegophryxus hyptius Thompson, determina- 
tion and differentiation of sex in, 17. 

Stratification and deformation of Arbacia 
punctulata eggs centrifuged in caffeine 
solutions, 70. 

Studies in the regulation of blood-sugar con- 
centration in crustaceans. I. Normal 
values and experimental hyperglycemia in 
Libinia emarginata, 218. 

Studies on marine bryozoa, III. Woods Hole 
region bryozoa associated with algae, 32. 

Studies on the thermal death of Hyalella az- 
teca Saussure, 123. 

Sugars, effects of, on turtle erythrocytes, 9. 

SZENT-GYORGYI, A. Free-energy relations 
and contraction of actomyosin, 140. 

SZENT-GYORGYI, A. Sec M. BORBIRO, 162. 

^EMPERATURE coefficients of respira- 
tion in Psammechinus eggs, 107. 

Tension, relation between ATP and, in cross- 
striated muscle, 162. 

Thermal death of Hyalella azteca Saussure, 
studies on, 123. 

THIMANN, KENNETH V. Plant hormones, 
growth and respiration, 296. 

Thiouracil, effect of, on oxygen consumption 
of blocked and active embryonic cells, 1. 

Thiourea, effect of, on oxygen consumption of 
blocked and active embryonic cells, 1. 



310 



INDEX 



Tricarboxylic acid cycle, presence of, in the 
ciliate Colpidimn campylum, 257. 

Turtle, (Chelydra serpcntina), effects of elec- 
trolytes and sugars on erythrocytes of, 9. 

TJREA, effect of, on oxygen consumption of 
blocked and active embryonic cells, 1. 

WACUOLOIDS, mitochondrial arrangement 
in epicytes of mouse lungs as induced by, 
173. 



KARL M. Sec R. TERRELL 

WlNGFIELD, 9. 

WINGFIELD, R. TERRELL AND KARL M. WIL- 
BUR. The effects of electrolytes and 
sugars on the erythrocytes of the turtle, 
Chelydra serpentina, 9. 

WOOD, R. D. Characeae of the Woods Hole 
region, Massachusetts, 179. 

Woods Hole region, Characeae of, 179. 

Woods Hole region, marine bryozoa of, asso- 
ciated with algae, 32. 



Volume 96 Number 1 



THE 



BIOLOGICAL BULLETIN 



PUBLISHED BY 

THE MARINE BIOLOGICAL LABORATORY 



Editorial Board 



E. G. CONKLIN, Princeton University CARL R. MOORE, University of Chicago 

DONALD P. COSTELLO, University of North Carolina GEORGE T. MOORE, Missouri Botanical Garden 

E. N. HARVEY, Princeton University G. H. PARKER, Harvard University 

LEIGH HOADLEY, Harvard University A. C. REDFIELD, Harvard University 

L. IRVING, Swarthmore College F. SCHRADER, Columbia University 

M. H. JACOBS, University of Pennsylvania DOUGLAS WHITAKER, Stanford University 



H. B. STEINBACH, University of Minnesota 
Managing Editor 



Marine Biological laborato 

X.IS3R A-J^Y 

WAR 4 -1949 

WOOOS HOLE, ASS. 



FEBRUARY, 1949 



Printed and Issued by 

LANCASTER PRESS, Inc. 

PRINCE &. LEMON STS. 

LANCASTER, PA. 



INTERNATIONAL DEPOSITORY 
OF CYTOLOGICAL SLIDES 

created by 

THE INTERNATIONAL UNION OF BIOLOGICAL SCIENCES. 

An Appeal to Cytologists. 

In 1939, the International Union of Biological Sciences requested Prof. 
P. MARTENS, Director of the CARNOY INSTITUTE in LOUVAIN, 
BELGIUM, to resume the project of an International Depository of Cyto- 
logical Slides, Zoological and Botanical. This proposition had previously 
been submitted to the late Prof. V. GREGOIRE by the Union; but, owing 
to his poor health at the time, he was unable to bring it into operation. 
On the other hand, the state of war and the international situation have 
delayed until now the announcement of the creation of this organism. 

The Union's purpose is to group in an easily accessible center, the 
Cytological Laboratory of the CARNOY INSTITUTE, at LOUVAIN 
(BELGIUM), preparations entrusted by numerous research centers and 
used as a basis for previously published research. Every scientist interested 
in a definite problem would thus be able to compare his own documentation 
with the original cytological documentation of other authors in his field of 
research. It is hardly necessary to emphasize the considerable value a de- 
pository of this kind would acquire in the future, the extent to which better 
understanding amongst workers would be aided, and how many'difificulties 
and vain contestations, presently filling scientific literature, would thus be 
avoided. 

But this result can only be attained by the broadest understanding and 
collaboration from the largest possible number of Cytologists. The I. U. B. S. 
invites them to send, from now on, to the above address, several of the 
slides used as a basis for their published work, and to repeat such deposits 
in the future. It is also desirable that areas thought to be especially demon- 
strative or actually used as published illustrations be marked on the slides, 
as clearly as possible, and that a separate of the published paper be attached. 

Any Biologist known for his publications, or. any other person in pos- 
session of authorized recommendation, will be able to consult or study, as 
long as convenient, any preparation entrusted to the collection. The con- 
sultant will have free use of the laboratory and of all necessary instrumenta- 
tion. All work shall be done within the depository, except if written permis- 
sion for outside use be granted by the depositor of the slides. 

The slides will remain the private property of the depositors who may, 
at any time, have them returned to them, postage to be paid by the institu- 
tion. 

The General Secretary of the I. U. B. S. The Administrator of the Depository 
Prof. P. VAYSSIERE Prof. P. MARTENS 

(Paris) (Louvain) 



BIOLOGICAL ABSTRACTS 

COVERS THE WORLD'S BIOLOGICAL LITERATURE 

How do you keep abreast of the literature in your field? No individual 
possibly could accumulate and read all of the biological contributions in the 
original yet some relatively obscure journal might publish a revealing paper 
on the very subject in which you are most interested. 

Biological Abstracts now publishes concise, informative abridgments of all 
the significant contributions from more than 2,500 journals. As well as the 
complete edition, it also is published in nine low-priced sectional editions which 
are specially designed for individuals who are interested only in one or more 
closely related fields. 

Production costs have increased to such an extent that the active support 
of all biologists is needed to maintain this important service. Write for full 
details and a sample copy of the sectional edition covering your field. 

BIOLOGICAL ABSTRACTS 

UNIVERSITY OF PENNSYLVANIA 

PHILADELPHIA 4, PA. 



MICROFILM SERVICE 



The Library of The Marine 
Biological Laboratory can 
supply microfilms of ma- 
terial from periodicals in- 
cluded in its list. Requests 
should include the title of 
the paper, the author, peri- 
odical, volume and date of 
publication. 



Rates are as follows: $1.00 for 
papers up to 50 pages, and $.10 
for each additional 10 pages or 
fraction thereof. 



LANCASTER PRESS, Inc. 

LANCASTER, PA. 



THE EXPERIENCE we have 
gained from printing some 
sixty educational publica- 
tions has fitted us to meet 
the standards of customers 
who demand the best. 

We shall be happy to have workers at 
the MARINE BIOLOGICAL LABORATORY 

write for estimates on journals or 
monographs. Our prices are moderate. 



INSTRUCTIONS TO AUTHORS 

The Biological Bulletin accepts papers on a variety of subjects of biologi- 
cal interest. In general, a paper will appear within three months of the date of 
its acceptance. The Editorial Board requests that manuscripts conform to the 
requirements set below. 

Manuscripts. Manuscripts should be typed in double or triple spacing on 
one side of paper, 8% by 11 inches. 

Tables should be typewritten on separate sheets and placed in correct 
sequence in the text. Explanations of figures should be typed on a separate 
sheet and placed at the end of the text. Footnotes, numbered consecutively, 
may be placed on a separate sheet at the end of the paper. 

A condensed title or running page head of not more than thirty-five letters 
should be included. 

Figures. The dimensions of the printed page, 5 by 7% inches, should be 
kept in mind in preparing figures for publication. Illustrations should be large 
enough so that all details will be clear after appropriate reduction. Explana- 
tory matter should be included in legends as far as possible, not lettered on the 
illustrations. Figures should be prepared for reproduction as line cuts or half- 
tones; other methods will be used only at the author's expense. Figures to be 
reproduced as line cuts should be drawn in black ink on white paper or blue- 
lined co-ordinate paper; those to be reproduced as halftones should be mounted 
on Bristol board and any designating letters or numbers should be made di- 
rectly on the figures. The author's name should appear on the reverse side of 
all figures. The desired reduction should be specified on each figure. 

Literature cited. The list of literature cited should conform to the style set 
in this issue of The Biological Bulletin. Papers referred to in the manuscript 
should be listed on separate pages headed "Literature Cited." 

Mailing. Manuscripts should be packed flat. Large illustrations may be 
rolled in a mailing tube, but all illustrations larger than 9 by 12 inches must 
be accompanied by photographic reproductions or tracings that may be folded 
to page size. 

Reprints. Authors will be furnished, free of charge, one hundred reprints 
without covers. Additional copies may be obtained at cost; approximate 
figures will be furnished upon request. 



THE BIOLOGICAL BULLETIN 

THE BIOLOGICAL BULLETIN is issued six times a year at the Lancaster 
Press, Inc., Prince and Lemon Streets, Lancaster, Pennsylvania. 

Subscriptions and similar matter should be addressed to The Biologi- 
cal Bulletin, Marine Biological Laboratory, Woods Hole, Massachusetts. 
Agent for Great Britain: Wheldon and Wesley, Limited, 2, 3 and 4 
Arthur Street, New Oxford Street, London, W. C. 2. Single numbers, 
$1.75. Subscription per volume (three issues), $4.50. 

Communications relative to manuscripts should be sent to the Manag- 
ing Editor, Marine Biological Laboratory, Woods Hole, Massachusetts, 
between June 15 and September 1, and to the Department of Zoology, 
University of Minnesota, Minneapolis, Minnesota, during the remainder 
of the year. 



Entered as second-class matter May 17, 1930, at the post office at Lancaster, Pa., 

under the Act of August 24, 1912. 



BIOLOGY MATERIALS 

The Supply Department of the Marine Biological Labora- 
tory has a complete stock of excellent plain preserved and 
injected materials, and would be pleased to quote prices on 
school needs. 



PRESERVED SPECIMENS 

for 

Zoology, Botany, Embryology, 
and Comparative Anatomy 

LIVING SPECIMENS 

for 
Zoology and Botany 

including Protozoan and 
Drosophila Cultures, and 
Animals for Experimental and 
Laboratory Use. 

MICROSCOPE SLIDES 

for 

Zoology, Botany, Embryology, 
Histology, Bacteriology, and 
Parasitology. 

CATALOGUES SENT ON REQUEST 



Supply Department 

MARINE 
BIOLOGICAL LABORATORY 

Woods Hole, Massachusetts 



CONTENTS 



Page 
BODINE, JOSEPH HALL AND LAURENCE ROCKWELL FITZGERALD 

Effect of urea, thiourea, phenylthiourea and thiouracil on the 
oxygen consumption of blocked and active embryonic cells ... 1 

WINGFIELD, R. TERRELL AND KARL M. WILBUR 

The effects of electrolyes and sugars on the erythrocytes of 
the turtle, Chelydra serpentina 9 

REINHARD, EDWARD G. 

Experiments on the determination and differentiation of sex 
in the bopyrid Stegophryxus hyptius Thompson 17 

ROGICK, MARY D. AND HANNAH CROASDALE 

Studies on marine bryozoa, III. Woods Hole region bryozoa 
associated with algae 32 

CHENEY, RALPH HOLT 

Stratification and deformation of Arbacia punctulata eggs cen- 
trifuged in caffeine solutions 70 

KRAUSS, MAX 

A mucin clot reaction with sea-urchin f ertilizin 74 

Ris, HANS 

The anaphase movement of chromosomes in the spermatocytes 
of the grasshopper 90 



Volume 96 



Number 2 



THE 

BIOLOGICAL BULLETIN 



PUBLISHED BY 

THE MARINE BIOLOGICAL LABORATORY 

Editorial Board 



E. G. CONKLIN, Princeton University CARL R. MOORE, University of Chicago 

DONALD P. COSTELLO, University of North Carolina GEORGE T. MOORE, Missouri Botanical Garden 

E. N. HARVEY, Princeton University G. H. PARKER, Harvard University 

LEIGH HOADLEY, Harvard University A. C. REDFIELD, Harvard University 

L. IRVING, Swarthmore College F. SCHRADER, Columbia University 

M. H. JACOBS, University of Pennsylvania DOUGLAS WHITAKER, Stanford University 

H. B. STEINBACH, University of Minnesota 

Managing Editor _____ 

Marine Biological b*"^; 



9 -1949 

WOODS HOLE, Mtf S. 



APRIL, 1949 



Printed and Issued by 

LANCASTER PRESS, Inc. 

PRINCE K. LEMON STS. 

LANCASTER, PA. 



INTERNATIONAL DEPOSITORY 
OF CYTOLOGICAL SLIDES 

created by 

THE INTERNATIONAL UNION OF BIOLOGICAL SCIENCES. 

An Appeal to Cytologists. 

In 1939, the International Union of Biological Sciences requested Prof. 
P. MARTENS, Director of the CARNOY INSTITUTE in LOU VAIN, 
BELGIUM, to resume the project of an International Depository of Cyto- 
logical Slides, Zoological and Botanical. This proposition had previously 
been submitted to the late Prof. V. GREGOIRE by the Union; but, owing 
to his poor health at the time, he was unable to bring it into operation. 
On the other hand, the state of war and the international situation have 
delayed until now the announcement of the creation of this organism. 

The Union's purpose is to group in an easily accessible center, the 
Cytological Laboratory of the CARNOY INSTITUTE, at LOUVAIN 
(BELGIUM), preparations entrusted by numerous research centers and 
used as a basis for previously published research. Every scientist interested 
in a definite problem would thus be able to compare his own documentation 
with the original cytological documentation of other authors in his field of 
research. It is hardly necessary to emphasize the considerable value a de- 
pository of this kind would acquire in the future, the extent to which better 
understanding amongst workers would be aided, and how many difficulties 
and vain contestations, presently filling scientific literature, would thus be 
avoided. 

But this result can only be attained by the broadest understanding and 
collaboration from the largest possible number of Cytologists. The I. U. B. S. 
invites them to send, from now on, to the above address, several of the 
slides used as a basis for their published work, and to repeat such deposits 
in the future. It is also desirable that areas thought to be especially demon- 
strative or actually used as published illustrations be marked on the slides, 
as clearly as possible, and that a separate of the published paper be attached. 

Any Biologist known for his publications, or any other person in pos- 
session of authorized recommendation, will be able to consult or study, as 
long as convenient, any preparation entrusted to the collection. The con- 
sultant will have free use of the laboratory and of all necessary instrumenta- 
tion. All work shall be done within the depository, except if written permis- 
sion for outside use be granted by the depositor of the slides. 

The slides will remain the private property of the depositors who may, 
at any time, have them returned to them, postage to be paid by the institu- 
tion. 

The General Secretary of the I. U. B. S. The Administrator of the Depository 
Prof. P. VAYSSIERE Prof. P. MARTENS 

(Paris) (Louvain) 



BIOLOGICAL ABSTRACTS 

COVERS THE WORLD'S BIOLOGICAL LITERATURE 

How do you keep abreast of the literature in your field? No individual 
possibly could accumulate and read all of the biological contributions in the 
original yet some relatively obscure journal might publish a revealing paper 
on the very subject in which you are most interested. 

Biological Abstracts now publishes concise, informative abridgments of all 
the significant contributions from more than 2,500 journals. As well as the 
complete edition, it also is published in nine low-priced sectional editions which 
are specially designed for individuals who are interested only in one or more 
closely related fields. 

Production costs have increased to such an extent that the active support 
of all biologists is needed to maintain this important service. Write for full 
details and a sample copy of the sectional edition covering your field. 

BIOLOGICAL ABSTRACTS 

UNIVERSITY OF PENNSYLVANIA 

PHILADELPHIA 4, PA. 



MICROFILM SERVICE 

* 

The Library of The Marine 
Biological Laboratory can 
supply microfilms of ma- 
terial from periodicals in- 
cluded in its list. Requests 
should include the title of 
the paper, the author, peri- 
odical, volume and date of 
publication. 



Rates are as follows: $1.00 for 
papers up to 50 pages, and $.10 
for each additional 10 pages or 
fraction thereof. 



LANCASTER PRESS, Inc. 

LANCASTER, PA. 



THE EXPERIENCE we have 
gained from printing some 
sixty educational publica- 
tions has fitted us to meet 
the standards of customers 
who demand the best. 

We shall be happy to have workers at 
the MARINE BIOLOGICAL LABORATORY 

write for estimates on journals or 
monographs. Our prices are moderate. 



INSTRUCTIONS TO AUTHORS 

The Biological Bulletin accepts papers on a variety of subjects of biologi- 
cal interest. In general, a paper will appear within three months of the date of 
its acceptance. The Editorial Board requests that manuscripts conform to the 
requirements set below. 

Manuscripts. Manuscripts should be typed in double or triple spacing on 
one side of paper, 8Vz by 11 inches. 

Tables should be typewritten on separate sheets and placed in correct 
sequence in the text. Explanations of figures should be typed on a separate 
sheet and placed at the end of the text. Footnotes, numbered consecutively, 
may be placed on a separate sheet at the end of the paper. 

A condensed title or running page head of not more than thirty-five letters 
should be included. 

Figures. The dimensions of the printed page, 5 by 7% inches, should be 
kept in mind in preparing figures for publication. Illustrations should be large 
enough so that all details will be clear after appropriate reduction. Explana- 
tory matter should be included in legends as far as possible, not lettered on the 
illustrations. Figures should be prepared for reproduction as line cuts or half- 
tones; other methods will be used only at the author's expense. Figures to be 
reproduced as line cuts should be drawn in black ink on white paper or blue- 
lined co-ordinate paper; those to be reproduced as halftones should be mounted 
on Bristol board and any designating letters or numbers should be made di- 
rectly on the figures. The author's name should appear on the reverse side of 
all figures. The desired reduction should be specified on each figure. 

Literature cited. The list of literature cited should conform to the style set 
in this issue of The Biological Bulletin. Papers referred to in the manuscript 
should be listed on separate pages headed "Literature Cited." 

Mailing. Manuscripts should be packed flat. Large illustrations may be 
rolled in a mailing tube, but all illustrations larger than 9 by 12 inches must 
be accompanied by photographic reproductions or tracings that may be folded 
to page size. 

Reprints. Authors will be furnished, free of charge, one hundred reprints 
without covers. Additional copies may be obtained at cost; approximate 
figures will be furnished upon request. 



THE BIOLOGICAL BULLETIN 

THE BIOLOGICAL BULLETIN is issued six times a year at the Lancaster 
Press, Inc., Prince and Lemon Streets, Lancaster, Pennsylvania. 

Subscriptions and similar matter should be addressed to The Biologi- 
cal Bulletin, Marine Biological Laboratory, Woods Hole, Massachusetts. 
Agent for Great Britain : Wheldon and Wesley, Limited, 2, 3 and 4 
Arthur Street, New Oxford Street, London, W. C. 2. Single numbers, 
$1.75. Subscription per volume (three issues), $4.50. 

Communications relative to manuscripts should be sent to the Manag- 
ing Editor, Marine Biological Laboratory, Woods Hole, Massachusetts, 
between June 15 and September 1, and to the Department of Zoology, 
University of Minnesota, Minneapolis, Minnesota, during the remainder 
of the year. 



Entered as second-class matter May 17, 1930, at the post office at Lancaster, Pa., 

under the Act of August 24, 1912. 



A.H.T. CO. SPECIFICATION 



BARCROFT-WARBURG APPARATUS 

Operating constancy 0.03 C; suitable for continuous service 




3604-A. 



BARCROFT-WARBURG APPARATUS, A.H.T. Co. Specification. Incorporating suggestions of Dr. Eric G. Ball, 
while in the Laboratory of Physiological Chemistry, Johns Hopkins School of Medicine. For the measurement of 
cell respiration and simijar processes which depend on reactions wherein a gas is either absorbed or evolved under 
carefully controlled conditions, but also suitable for almost any macro or micro analytical procedure involving kinetic 
gas exchanges. 

Consisting of sets of seven or fourteen Barcroft- Warburg Manometers of glass with standard taper interchangeable 
ground joints; mounted on improved type aluminum supports with white background behind the graduations and with 
nickel-plated clamp for convenient removal of the manometer without the use of tools; Monel metal water bath, 24$ 
inches long x 15 inches wide x 10 inches deep, with mercury-in-glass type thermo-regulator adjusted for 37C and sensi- 
tive to changes of 0.03C, equipped with electric shaking and stirring devices, immersion heater, special thermometer 
36 to 40C in 1/20 divisions, reading lamp on extension cord,-and slotted wooden base for supporting seven manometers 
in vertical position when not in use. Manometers are shaken in a vertical position on ball-bearing rollers. The speed 
of the shaking device is controlled by a rheostat, as is also the speed of the stirring unit. 

3603. Barcroft-Warburg Apparatus, A.H.T. Co. Specification, Seven-Unit, complete as above described, adjusted 
for 37C, including seven manometers on improved aluminum supports, constant temperature bath with 
shaking and stirring devices, immersion heater, thermo-regulator, special thermometer reading to 1/20C, 
reading lamp, one slotted base for seven manometers, cord and plug, and directions for use. For 115 

volts a.c 573.40 

NOTE Can be converted into a fourteen-unit assembly by addition of accessories offered separately. 

3604-A. Ditto, Fourteen-Unit, identical with above but with fourteen manometers and two slotted wooden bases, each 
for seven manometers. For 115 volts a.c. . ."V 859.30 

3612-A. Constant Temperature Bath, only, seven-unit, without manometers, thermometer, manometer supports or 
slotted base. For 1 15 volts a.c 326.50 

3612-L. Ditto, 14-unit, for 115 volts a.c 379.50 

More detailed description of above Apparatus, together with information regarding Summerson 

Differential Manometer, for interchangeable use in above bath, and reaction 

vessels of various types, sent upon request. 



ARTHUR H. THOMAS COMPANY 

RETAIL WHOLESALE EXPORT 

LABORATORY APPARATUS AND REAGENTS 

WEST WASHINGTON SQUARE PHILADELPHIA 5, PA., U. S. A 

Cable Address, "BALANCE", Philadelphia 



CONTENTS 



Page 

BOREI, HANS AND SlGVAR LYBING 

Temperature Coefficients of respiration in Psammechinus 
eggs 107 

BOREI, HANS 

Independence of post-fertilization respiration in the sea- 
urchin egg from the level of respiration before fertilization. . 117 

BOVEE, EUGENE CLEVELAND 

Studies on the thermal death of Hyalella azteca Saussure . . . 123 

GATES, G. E. 

Regeneration in an earthworm, Eisenia foetida (Savigny) 
1826. I. Anterior regeneration 129 

SZENT-GYORGYI, A. 

Free-energy relations and contraction of actomyosin 140 

';"--' '* 

BORBIRE, M. AND A. SZENT-GYORGYI 

On the relation between tension and ATP in cross-striated 
muscle 162 

ANDERSON, WILLIAM W., JOSEPH E. KING, AND MILTON J. 
LINDNER 

Early stages in the life history of the common marine shrimp, 
Penaeus setiferus (Linnaeus) 168 

MACKLIN, CHARLES C. 

Mitochondrial arrangement in alveolar epicytes and foam 
cells of mouse lungs, particularly as induced by the vacuo- 
loids 173 

WOOD, R. D. 

The Characeae of the Woods Hole region, Massachusetts. . 179 



Volume 96 Number 3 



THE 



BIOLOGICAL BULLETIN 



PUBLISHED BY 

THE MARINE BIOLOGICAL LABORATORY 

Editorial Board 



E. G. CONKLIN, Princeton University CARL R. MOORE, University of Chicago 

DONALD P. COSTELLO, University of North Carolina GEORGE T. MOORE, Missouri Botanical Garden 

E. N. HARVEY, Princeton University G. H. PARKER, Harvard University 

LEIGH HOADLEY t Harvard University A. C. REDFIELD, Harvard University 

L. IRVING, Swarthmore College F. SCHRADER, Columbia University 

M. H. JACOBS, University of Pennsylvania DOUGLAS WHITAKER, Stanford University 

H. B. STEINBACH, University of Minnesota 
Managing Editor 



Marine Bioioyttal \.<-i 

1u I B *< A It -* 

JUL 181949 

W60DS HOLE, M^S 



JUNE, 1949 



Printed and Issued by 

LANCASTER PRESS, Inc. 

PRINCE &. LEMON STS. 

LANCASTER, PA. 



A.H.T. CO. SPECIFICATION 



TISSUE FLOTATION BATH 

WITH AUTOMATIC TEMPERATURE REGULATION 




7200. 



TISSUE FLOTATION BATH, ELECTRIC, A. H. T. Co. Speci- 
fication, with automatic temperature regulation. For convenient floating 
and spreading of tissue sections embedded in paraffin, preparatory to mount- 
ing. Bath temperatures can be controlled to within lC in the range 
from room temperature to 60 C. 

Consisting of a Pyrex brand glass tray, 10 inches long X 6 inches wide 
X 2 inches deep, and a low, insulated, Monel metal stand with flat copper 
top and built-in heating unit, thermo-regulator and pilot lamp. Top has 
raised edges, and a black oxidized finish which aids in the inspection of 
translucent sections. The glass tray is readily removable for cleaning. 

Overall dimensions of stand, 10^ inches long X 6 inches wide X 2f inches 
high; power consumption, 225 watts; net weight of stand and tray, 5 Ibs. 
Shipping weight, 8 Ibs. 



7200. 



9717-E. 



Tissue Flotation Bath, Electric, A.H.T. Co. Specification, as above de- 
scribed, complete with Pyrex brand glass tray. With 5 ft. connecting 

cord, switch and plug; for use on 115 volts a.c. only 25.20 

Code Word -Kezzj 

Tray, only, of Pyrex brand glass, as supplied with 7200 Tissue Flotation 
Bath. Overall dimensions 10 inches long X 62 inches wide X 2 inches 

deep . - 69 

Code Word Oxqes 



ARTHUR H. THOMAS COMPANY 

RETAIL WHOLESALE EXPORT 

LABORATORY APPARATUS AND REAGENTS 

WEST WASHINGTON SQUARE PHILADELPHIA 5, PA., U. S. A 

Cable Address, "BALANCE", Philadelphia 



BIOLOGICAL ABSTRACTS 

COVERS THE WORLD'S BIOLOGICAL LITERATURE 

How do you keep abreast of the literature in your field? No individual 
possibly could accumulate and read all of the biological contributions in the 
original yet some relatively obscure journal might publish a revealing paper 
on the very subject in which you are most interested. 

Biological Abstracts now publishes concise, informative abridgments of all 
the significant contributions from more than 2,500 journals. As well as the 
complete edition, it also is published in nine low-priced sectional editions which 
are specially designed for individuals who are interested only in one or more 
closely related fields. 

Production costs have increased to such an extent that the active support 
of all biologists is needed to maintain this important service. Write for full 
details and a sample copy of the sectional edition covering your field. 

BIOLOGICAL ABSTRACTS 

UNIVERSITY OF PENNSYLVANIA 

PHILADELPHIA 4, PA. 



MICROFILM SERVICE 

+ 

The Library of The Marine 
Biological Laboratory can 
supply microfilms of ma- 
terial from periodicals in- 
cluded in its list. Requests 
should include the title of 
the paper, the author, peri- 
odical, volume and date of 
publication. 



Rates are as follows: $1.00 for 
papers up to 50 pages, and $.10 
for each additional 10 pages or 
fraction thereof. 



LANCASTER PRESS, Inc. 

LANCASTER, PA. 



THE EXPERIENCE we have 
gained from printing some 
sixty educational publica- 
tions has fitted us to meet 
the standards of customers 
who demand the best. 

We shall be happy to have workers at 
the MARINE BIOLOGICAL LABORATORY 
write for estimates on journals or 
monographs. Our prices are moderate. 



INSTRUCTIONS TO AUTHORS 

The Biological Bulletin accepts papers on a variety of subjects of biologi- 
cal interest. In general, a paper will appear within three months of the date of 
its acceptance. The Editorial Board requests that manuscripts conform to the 
requirements set below. 

Manuscripts. Manuscripts should be typed in double or triple spacing on 
one side of paper, 8Ya by 11 inches. 

Tables should be typewritten on separate sheets and placed in correct 
sequence in the text. Explanations of figures should be typed on a separate 
sheet and placed at the end of the text. Footnotes, numbered consecutively, 
may be placed on a separate sheet at the end of the paper. 

A condensed title or running page head of not more than thirty-five letters 
should be included. 

Figures. The dimensions of the printed page, 5 by 7% inches, should be 
kept in mind in preparing figures for publication. Illustrations should be large 
enough so that all details will be clear after appropriate reduction. Explana- 
tory matter should be included in legends as far as possible, not lettered on the 
illustrations. Figures should be prepared for reproduction as line cuts or half- 
tones; other methods will be used only at the author's expense. Figures to be 
reproduced as line cuts should be drawn in black ink on white paper or blue- 
lined co-ordinate paper; those to be reproduced as halftones should be mounted 
on Bristol board and any designating letters or numbers should be made di- 
rectly on the figures. The author's name should appear on the reverse side of 
all figures. The desired reduction should be specified on each figure. 

Literature cited. The list of literature cited should conform to the style set 
in this issue of The Biological Bulletin. Papers referred to in the manuscript 
should be listed on separate pages headed "Literature Cited." 

Mailing. Manuscripts should be packed flat. Large illustrations may be 
rolled in a mailing tube, but all illustrations larger than 9 by 12 inches must 
be accompanied by photographic reproductions or tracings that may be folded 
to page size. 

Reprints. Authors will be furnished, free of charge, one hundred reprints 
without covers. Additional copies may be obtained at cost; approximate 
figures will be furnished upon request. 



THE BIOLOGICAL BULLETIN 

THE BIOLOGICAL BULLETIN is issued six times a year at the Lancaster 
Press, Inc., Prince and Lemon Streets, Lancaster, Pennsylvania. 

Subscriptions and similar matter should be addressed to The Biologi- 
cal Bulletin, Marine Biological Laboratory, Woods Hole, Massachusetts. 
Agent for Great Britain: Wheldon and Wesley, Limited, 2, 3 and 4 
Arthur Street, New Oxford Street, London, W. C. 2. Single numbers, 
$1.75. Subscription per volume (three issues), $4.50. 

Communications relative to manuscripts should be sent to the Manag- 
ing Editor, Marine Biological Laboratory, Woods Hole, Massachusetts, 
between June 15 and September 1, and to the Department of Zoology, 
University of Minnesota, Minneapolis, Minnesota, during the remainder 
of the year. 



Entered as second-class matter May 17, 1930, at the post office at Lancaster, Pa., 

under the Act of August 24. 1912. 



BIOLOGY MATERIALS 

The Supply Department of the Marine Biological Labora- 
tory has a complete stock of excellent plain preserved and 
injected materials, and would be pleased to quote prices on 
school needs. 



PRESERVED SPECIMENS 

for 

Zoology, Botany, Embryology, 
and Comparative Anatomy 

LIVING SPECIMENS 

for 
Zoology and Botany 

including Protozoan and 
Drosophila Cultures, and 
Animals for Experimental and 
Laboratory Use. 

MICROSCOPE SLIDES 

for 

Zoology, Botany, Embryology, 
Histology, Bacteriology, and 
Parasitology. 

CATALOGUES SENT ON REQUEST 



Supply Department 

MARINE 
BIOLOGICAL LABORATORY 

Woods Hole, Massachusetts 



CONTENTS 



Page 

ABELSON, PHILIP H. AND WILLIAM R. DURYEE 

Radioactive sodium permeability and exchange in frog eggs . 205 

KLEINHOLZ, L. H. AND BARBARA CHASE LITTLE 

Studies in the regulation of blood-sugar concentration in 
crustaceans. I. Normal values and experimental hyper- 
glycemia in Libinia emarginata 218 

BROWN, FRANK A., JR. AND GWEN M. JONES 

Ovarian inhibition by a sinus-gland principle in the fiddler 
crab , 228 

SMITH, MARSHALL E. AND LYNWOOD B. SMITH 

Piperazine dihydrochloride and glycylglycine as non-toxic 
buffers in distilled water and in sea water 233 

BOWMAN, THOMAS E. 

Chromatophorotropins in the central nervous organs of the 
crab, Hemigrapsus oregonensis 238 

BEAMS, -H. W. 

Some effects of centrifuging upon protoplasmic streaming in 
Elodea 246 

SEAMAN, GERALD R. 

The presence of the tricarboxylic acid cycle in the ciliate 
Colpidium campylum 257 

BUTLER, PHILIP A. 

Gametogenesis in the oyster under conditions of depressed 
salinity 263 

CHAMBERS, ROBERT AND EDWARD L. CHAMBERS 

Nuclear and cytoplasmic interrelations in the fertilization of 
the Asterias egg 270 

BERRILL, N. J. 

Form and growth in the development of a Scyphomedusa . . . 283 

MlCHAELIS, L. 

Fundamental principles in oxidation-reduction 293 

THIMANN, KENNETH V. 

Plant hormones, growth and respiration 296 



MBL WHO! LIBRARY 



UH lAYU / 



HI