Influence of the sinus gland upon growth, molting, and general metabolism in crustaceans

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NAME AND ADDRESS

DATE

NORTHWESTERN UNIVERSITY

INFLUENCE OF THE SINUS GLAND UPON GROWTH, MOLTING, AND GENERAL METABOLISM IN CRUSTACEANS

A DISSERTATION SUBMITTED TO THE GRADUATE SCHOOL IN PARTIAL FULFILLMENT OF THE REQUIREMENTS for the degree

DOCTOR OF PHILOSOPHY

DEPARTMENT OF ZOOLOGY

BY HAROLD HUNTER SCUDAMORE EVANSTON, ILLINOIS JUNE, 1942

ProQ uest N um ber: 10101939

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ACKNOWLEDGMENT The author is grateful to Dr# Frank A# Brown, Jr* for suggesting this problem, for his guidance and inspiration during the experimental work, and for his advice and criti­ cisms in the preparation of this manuscript#

The author is

also Indebted to Dr* G* L* Turner for the use of field data and for assistance in the classification of the crayfish*

TABLE OP CONTENTS Page

Acknowledgment • • . • • • • • • • • • • • « • « • I*

1

Introduction • « • • • • • • • . • • • • • • • • •

1

II* Review of Literature * • • • • • • • • • • • • • •

2

A*

Color Change and the Incretory Properties of the Eyestalks* . . • • • • • • • •

..........

B*

The Discovery of the Sinus Gland • • • • • • •

C*

The Humoral Regulation of Molting, Growth, and Viability.

D*

...................

4

8

The Humoral Influences on General Metabolism • 15

III* Statement of the Problem * • * • • • • . • • . • * A*

2

26

Some Existing Problems of Crustacean Endo­ crine Mechanisms............................ 26

B*

Statement of the Specific Problem. . . . . . .

IV. Materials and General Methods. • • •

V*

27

.......... . 2 8

A*

The Experimental Animals • • • , . . • • • • • 2 8

B*

The Sinus Gland and the Eye s t a l k ........... . 2 9

C.

The Saline S o l u t i o n s ........................30

D.

Methods of Gland Removal • • • • • • • • • • • 3 1

E*

Methods of Replacing Glandular Products. . . .

32

Molting and Gastrolith Formation in the Crayfish • 34 A.

Introduction ...............

• • • . • • • *

34

B*

Experimental Methods • • • « • • • • • • • • •

37

C.

Description of the Life History of the Cray­ fish......................................... 38

D*

Description of the Molting Process* * • • « « •

E#

Experimental Induction of Molting and Gastro-

Page 40

lith Formation during Winter Months • • • . « • F*

Evidence for a Daily Rhythm in Gastrolith Formation *

VI*

41

. • • • . • • • • * . • •

G.

V i a b i l i t y ................

H*

Growth. • * » • • » •

I*

Molting and the Sexual Cycle.

55 60

• « • • •

62 65

The Influence of the Sinus Gland on Calcium Metabolism* • * • • • « • • • #

...................

71

• • • •

71

A*

Introduction. * • • • • • . *

5*

Experimental Methods.

C.

The Condition in the Molted Carapaces • • • • •

74

D*

The Rate of Deposition of Soluble Salts . • • •

76

E*

Summary*

78

.....................

73

/

...........

VII* The Influence of the Sinus Gland on Water Metabolism* . . .

• • • • • • * . • • •

79

A*

Introduction.

79

B.

Experimental Methods............

81

C*

Preliminary Weight Change Experiments........

83

D.

Experiments Involving Measurements of Both Changes in Body Weight and Water Content. . • •

E.

85

Changes in Weight under Different Experimental C o n d i t i o n s ...................................

87

F.

Changes in Water Content During the Molt Cycle *

88

G*

Viability of Eyestalkless Crayfish in Various Salt Solutions • • • • • . .

..........

. . . »

92

H. VIII.

Summary.

Page 93

..........

Influence of the Sinus Gland on Oxygen Con­ sumption . . . . . . . . . . .

.................

94

A.

Introduction.................................. 94

B.

Experimental Methods • • • • • • • . . . • «

97

C.

Experimental Results • • • • • • • • • • • •

99

D.

Variations in the Rate of Oxygen Consumption During the Molt C y c l e . .......................103

E.

IX.

Summary. . . . • • • • •

• • • • • • • • • •

105

Color Change and the Rate of Heart Beat in the Shrimp, Palaemonetes

.................... 106

A.

Introduction

106

B.

Experimental R e s u l t s ..........

107

C.

Conclusion

..............

110

X.

General Discussion . . .

XI.

Summary and Conclusions.

XII.

Literature Cited • • • • .........................121

........................

110

................ 116

Figures and Plates

131

Vita

153

I.

INTRODUCTION

Only fourteen years have lapsed since the dis­ covery of humoral regulating mechanisms in the invertebrates. Perkins (1928) and Roller (1928), independently, demon­ strated that the eyestalks of certain crustaceans produced blood-borne substances which controlled integumentary pigment migration.

This preliminary discovery of humoral agents in

crustaceans constitutes a landmark in the advancement of invertebrate endocrinology.

Extensive experimentation in

this new field has revealed several possible sources of hormones and, already, many functions have bgen adequately explained on the basis of an endocrine control.

Character­

istic of new fields of investigation, literature confirming and extending discoveries has accumulated rapidly.

Reviews

by Roller (1929, 1938), Hanstr^m (1937b, 1939), von der Wense (1938), Brown (1939, 1941), and Scharrer (1941) have been prepared to summarize the significant contributions which have been made thus far. Experimental invertebrate endocrinology has been complicated by the small size of the animals and by the tendency for localization of several functions in a few cells or tissues.

Thus, some of the agents involved in the coordi­

nation did not seem to fit the classical definition of a hormone, because many invertebrates lacked a well—developed circulatory system and definitely circumscribed glandular tissue.

Nevertheless, experimental evidence has been

presented describing several invertebrate glands, which are quite probably endocrine in function; among these are: the crustacean sinus glands, the insect corpora cardiaca and the insect corpora allata. This investigation is concerned with a study of the influence of the sinus gland upon molting, growth, and general metabolism in the crayfish, Cambarus« and in the shrimp, Palaemonetes.

II.

REVIEW OE THE LITERATURE The discovery of the incretory properties of the

sinus gland, together with a survey of the literature con­ cerning the functions of this gland, will be treated briefly. Literature describing specific functions studied in this investigation will be reviewed in greater detail at this time and in subsequent parts. A.

Color Change and the Discovery of the Incre­ tory Properties of the Eyestalks

It has long been known that certain crustaceans are able to change their color or pattern by chromatophore activity to correspond to that of the background.

This

adjustment involves a differential migration of the two to five different pigments present in the integumentary chromatophores (physiological color change) or a differential formation and destruction of the pigments (morphological color change).

It was while investigating the controlling

mechanisms of these pigment migrations that evidence for invertebrate endocrine mechanisms was discovered* As early as 1872, Pouchet had accurately described color changes and found that opaquing or extirpating the eye­ stalks resulted in a loss of color adaptation*

Pouchet

considered that these changes were controlled by nervous coordination although he failed to demonstrate innervation of the chromatophores. The first indication of an endocrine control was the discovery by Roller (1925, 1927) that, under certain conditions, blood transfusions carried substances which were capable of invoking chromatophore responses.

Later, Perkins

(1928), working with Palaemonetes vulgaris, demonstrated con­ clusively that color change depended upon substances produced in the eyestalks and circulated in the blood stream.

Re­

moval of both eyestalks resulted in a complete dispersion of the red and yellow pigments, while injections of an eyestalk extract produced a concentration of these pigments.

No other

tissues of the body were capable of producing these effects. Roller (1928), independently, reported a similar endocrine control of chromatophores in Crangon and Leander. In the next few years, Kropp and Perkins (1953a), as well as other investigators, examined many other stalk­ eyed crustaceans and found that all had such a chromatophorotropic substance.

Carlson (1935) and Abramowitz (1935,

1937b) discovered that, in Uca and Portunus, eyestalk removal

resulted in concentration of the black and red and the injection of eyestalk extracts of Uca or other crustaceans resulted in a dispersion of these pigments.

Further

studies have shown that these are the two fundamental types of color changes; all Macrura and Anomura behave like Palaemonetes. and all Brachyura behave like Uca. The presence of such pigment-activating substances seems to be of quite uniform occurrence in all orders of the higher stalk­ eyed decapods. B.

The Discovery of the Sinus Gland

In attempting to localize the source of these humoral substances within the eyestalks, Roller (1930) working with Crangon vulgaris and Hosoi (1934) working with Penaeus japonicus found that the distal portion had no pigment-activating effect.

Shortly thereafter, Hanstrtfm

(1933) described two gland-like bodies— the blood gland or sinus gland and the X-organ— in the eyestalks of several crustaceans. In Jater experiments, Hanstrfim (1935, 1937a) found, by partial extirpation of the distal one third and two thirds of the eye­ stalk of Palaemonetes vulgaris, that only when the middle third was present (or injected as an extract) was it possible to elicit chromatophore responses.

This middle third contains

both the sinus gland and the X-organ.

However, Carlson (1935,

1936) has shown by similar experiments that the middle third of the eyestalk of Uca also contained the source of pigmentactivating substances.

In Uca and certain other decapods the

X-organ has not been found in this portion of the eyestalk. Therefore, It seemed probable that the sinus gland was the source of these pigment-activating substances or hormones. The function of the X-organ still remains undetermined. Brown and Cunningham (1939) studied the histology of the sinus gland in Cambarus virilis and also discovered that the gland was a distinct organ which could be dissected from the rest of the eyestalk tissue.

These investigators

made implants of the Intact sinus gland into the abdominal musculature and found that this prolonged the life and delayed molt in eyestalkless crayfish.

Brown (1940) has described

the gross appearance of the sinus glands in seven additional marine crustaceans. A technique for the removal of the sinus gland of Palaemonetes vulgaris without loss of vision and removal of the nervous tissue in the eyestalk was developed by Brown, Ederstrom, and Scudamore (1939).

This method offered an

opportunity to study the effects of sinus gland removal and as­ certain functions limited to the sinus gland. Recently, Brown (1940) confirmed the hypothesis that the sinus gland was the source of the chroma tophorotropic substances.

A comparison between the chromatophoric

activity of extracts of isolated sinus glands, whole eyestalks, and sinus glandless eyestalk tissue indicated that the source of the active principle was the sinus gland. Finally, Welsh (1941) made methylene blue prepa-

rations of the sinus gland in freshly killed crayfish*

It

was found that the gland was not only innervated with nerves from the medulla terminalis (Hanstrom, 1937a) but also with a branch of the oculomotor nerve from the supra-oesophageal ganglia* The investigations of Hanstri'm (1933, 1937a), Brown and Cunningham (1939), and Welsh (1941) indicate that the sinus gland is a definitely circumscribed gland located in the eyestalks of decapod crustaceans.

Macroscopically,

the gland appears as a bluish, fibrous organ easily dis1 tinguished from the rest of the stalk— nervous and connective— tissue.

The sinus gland is located in the eyestalk between

the medulla interna and medulla externa in most decapods.

It

is richly innervated with nerves from the medulla terminalis and supra-oesophageal ganglia.

The surface of the gland is

bounded by the neurilemma on one side and by a structureless membrane on the other.

The gland is bathed in blood supplied

| by the optic arteries and the surrounding blood sinuses.

The

! endocrine nature of the gland is indicated by the presence i

of tiny secretory canals, eosinophilic secretory granules, and by the position of the nuclei. Hanstrfim (1939) and Scharrer (1941) have efficiently summarized the experimental work on the physico-chemical pro­ perties of eyestalk hormones, which suggests that specific chemical substances are present.

Up to the present time, no

investigator has isolated an active principle or hormone from

the sinus gland and determined its chemical composition; consequently, our information is based on physiological experiments of removal and replacement of the glandular material* Brown (1935a, b, 1939) suggested that several hormones or "modifiers” are involved in controlling the independent activity of the four different pigments (red, yellow, white, and blue) present in Palaemonetes.

On the

other hand, Abramowitz (1937b), with questionable inter­ pretations, considered that a single hormone in different concentrations could account for differential pigment mig­ rations.

Kleinholz (1938), on the basis of different

thresholds of response, suggested that a separate hormone was required to account for the retinal and integumentary pigment migrations.

The first attempt at a physiological

and chemical differentiation of two chromatophoric principles within the sinus gland of crustaceans was made by Brown and Scudamore \1940).

These investigators noted that by extrac­

tion in absolute ethyl alcohol, two separate fractions could be obtained which acted differently on the black pigment of eyestalkless Uca and on the red pigment of eyestalkiess Palaemonetes All of the discussion thus far has considered primarily the chromatophoric functions of the sinus gland. There are, in addition, several extra-chromatophoric functions associated with this gland.

The control of retinal

pigment migration was one of the first such functions to re­ ceive unequivocal evidence for a hormonal regulation.

The

differential migration of the pigments (iris, retinal, and reflecting) act to adjust the compound crustacean eye to different intensities of light.

Rleinholz (1934, 1936) re­

ported that injections of sea water extracts of eyestalks into dark-adapted Palaemonetes vulgaris caused the distal and reflecting pigments to assume the day or light position* Similar injections into light-adapted.animals produced no effects.

Welsh (1939a)confirmed these observations using

the crayfish;and later (1941) demonstrated that the sinus gland was probably the source of the active principle Involved.

Final evidence for this conclusion will involve

a study of the effects of sinus gland removal.

Subsequent

paragraphs summarize pertinent extra-chromatophoric functions of the sinus gland. C.

The Humoral Regulation of Molting. Growth, and Viability

Characteristic of all crustaceans is the periodic molting or shedding of their chitinous exoskeleton after which there is marked growth.

The series of complex changes

which occur at this time make this period a critical one in the life and development of decapods,

it has been only within

the last few years that any attempt to analyze a possible humoral control of molting has been undertaken. Molting. The first suggestion of a humoral control

of molting was the discovery by Megusar (1912) that not only the first but subsequent molts take place earlier in eyestalkless than in normal Astacus vulgaris. HanstrSm (1939) claims to have observed that eyestalkiess Eriocheir sinensis molt earlier than normal animals.

Brown and Cunningham (1939)

have shown that removal of eyestalks in Cambarus immunis increased the rate of molting in these animals.

This suggests

that the eyestalk of the crayfish is the source of a moltinhibiting substance.

Abramowitz and Abramowitz (1940) and

Kleinholz and Bourquin (1941b) have made similar observations on Uca pugilator.

Smith (1940) indicated that the removal of

eyestalks of young crayfish, Cambarus clarkii. shortened the intermolt period by more than thirty per cent.

Kyer (1942)

has indicated, since the completion of my own experiments in this regard, that the formation of gastroliths (a process which normally precedes molting in crayfish and other Macrurans) in the crayfish, Cambarus virilis, could be in­ duced by eyestalk removal in a non-molting season. Darby (1938) claimed that the increased rate of molting in eyestalkless Crangon armillatus was due to oper­ ative injury.

However, Smith (1940) demonstrated that this

was not true because the operative removal of retinas alone did not have a significant effect in increasing the rate of molting. Hess (1941) has investigated some of the factors influencing molting in Crangon armillatus, and found that

light was not an important factor but ttaat temperature was* In this southern species a temperature of 29° C. or greater was required for molting to ensue, while a lower temperature tended to check molting.

Smith (1940) indicated that higher

temperatures speeded up the rate of molting in young crayfish* Kyer (1942) reported that temperatures below 15° C. greatly retarded gastrolith formation in eyestalkless crayfish. Plankemann (1935) reported that such diverse factors as starvation, increased metabolism, color of background, and pH of sea water might affect the rate of molting, and sug­ gested that the molting cycle might be determined by carbo­ hydrate metabolism.

Kleinholz and Bourquin (1941a) were able

to lengthen the intermolt period in Uca pugilator by starvation. Further evidence that internal factors are involved was pre­ sented by Scudamore (1941a); namely, that there was an increase in the rate of oxygen consumption preceding molt and a decrease thereafter. Evidence presented thus far has suggested that the sinus gland is the source of a molt-inhibiting principle, and that environmental factors are involved in stimulating the onset of molt.

No investigator

has, to date, presented evi­

dence for the presence of a molt-accelerating principle in crustaceans, although Wigglesworth (1940) has reported the presence of a molt-accelerating or adult-producing hormone in the bug, Rhodnius prolixus.

Hess (1941) reported that the presence of eggs or embryos on the females also Inhibited molting in Crangon. This type of inhibition seemed to be dependent upon the attachment of the embryos to the female, for shortly after removal the females molted.

Therefore, Hess concluded that

the inhibition was due only to the presence of the embryos* longe (1937; had previously suggested that a hormone might be involved in the inhibition of molt. The source of the molt-inhibiting hormone. Brown and Cunningham (1939) were the first to test the possibility of a molt-inhibiting hormone by the implantation of sinus glands into eyestalkless crayfish.

They found that implan­

tation of entire sinus glands decreased the number of animals molting during the course of their experiments.

Their work

was somewhat complicated by the fact that the experiments were carried on during a normal molting season.

However, the re­

tardation of the onset of molting in eyestalkless animals by this technique was suggestive of an endocrine-controlling mechanism. Recently, Kyer (1942) confirmed these results and studied the influence of the sinus gland on gastrolith for­ mation.

Removal of both eyestalks of Cambarus virilis and £.

immunis induced the formation of gastroliths in a non—molting season.

The formation of the gastroliths was preceded by

changes in the stomach wall epithelium from columnar to pseudo-stratif led columnar cells by the end of the first day,

and the secretion of the gastroliths had commenced.

This is

the first indication of an influence of the presence or absence of humoral agents on tissue structure and secretory activity.

Eyer also noted that the transplantation (pro­

bably implantation) of sinus glands into the abdominal region of eyestalkless animals prevented the formation of gastroliths. The removal of a single eyestalk failed to induce gastrolith formation (Kyer, 1942).

Of interest in this regard

was the discovery of Brown and Cunningham (1939) that the re­ moval of one eyestalk produced molts in a smaller percentage of animals than eyestalkless animals during the spring molting season, when the average life span was considered. Evidence seems to indicate clearly the presence of a moIt-inhibiting principle originating in the sinus gland. It still remains to be shown that the removal of the sinus gland without the rest of the stalk tissue will induce gastro­ lith formation or molting. Growth.

It has long been known that an increase in

size accompanies molting (Huxley, 1906).

Olmsted

and Baum—

berger (1923) reported increases in weight following molting as great as 33.9% in Pachygrapsus erassipes and 44.9% in Hemigrapsus oregonensis; and Drach (1939) reported increases of 107.5% in Maia squinado and 73% in Cancer pagurus.

Van

Deventer (1937) and Tack (1941) found that crayfish do not increase markedly in size following the spring molt but in­ crease 10 to 30% in carapace length after the summer molt.

Abramowitz and Abramowitz (1940) have shown that at the end of a forty-eight day experiment, the eyestalkless Uca were much larger than the largest of normal animals observed at the same time.

This was due, in part, to an early

onset and increased number of molts.

No weighings were made,

but a photograph of size ranges was suggestive. Similarly, Smith (1940) found that eyestalkless young crayfish became distinctly larger than retinaless animals. This may have been due to the faster rate of molting in the former; however, it was mentioned that the eyestalkless animals ate much more food than did the control animals which also may have affected the growth rate. Kleinholz and Bourquin (1941b) have studied growth in Uca pugilator and found an increase of 30% in weight following molting of eyestalkless animals; however, no com­ parison was made with normal animals since none of these molted during the course of the experiments.

The reported

percentage increase in weight was not as great as that reported by other investigators for normal animals following molting. In this regard, it still remains to be shown that the sinus gland has a growth-retarding influence or that the increased rate of growth in eyestalkless animals is only a secondary result of an increased molting rate which results from removal of the source of a molt-inhibiting hormone. Viability.

Brown (1938) was the first to suggest

that the sinus gland might have a "viability effect" and therefore be essential for life.

The removal of eyestalks

greatly shortened the length of life of the crayfish, Cambarus

blandingii acutus, whereas implantation of whole eyestalk tissue extended longevity for a significant period of time. However, none of the animals lived longer than a few days due, possibly, to lack of food or high summer temperatures. About twenty per cent of the animals died within twenty-four hours from operative shock.

Brown and Cunningham (1939) have

proven that implantation of whole sinus glands in the abdomi­ nal musculature will significantly lengthen the life of eye­ stalkless animals.

These experiments indicated that the

action of the sinus gland in molt control appeared to be insufficient to explain tthe "viability effect" completely. However, other investigators, working with other genera, doubt the presence of a viability factor.

According

to Hanstrom (1939), Megusar (1912) and Roller (1930) were able to keep certain decapods alive for months without their eye­ stalks.

These were mostly marine crustaceans, wherein the

condition of water metabolism may be different.

However, the

marine shrimp, Palaemonetes, died sooner in the absence of the sinus gland than normal animals (Brown, 1940). Smith (1940) found that eyestalk removal in young crayfish always resulted in death.

It seemed possible to

distinguish between the factors influencing viability and molt, although there is an apparent correlation between molting and viability. in the molting process.

Many animals died about to molt or The operative loss was only about

two per cent. Abramowitz and Abramowitz (1940) found that in eye­ stalkless Uca pugilator there was a mortality of 89% in fortyeight days with no deaths among normal animals.

Of these

deaths, 75% occurred at approximately the time of molt and, therefore, seemed to be related to ecdysis.

Kleinholz and

Bourquin (1941b) found that molting Uca died because of insufficient water during the molting process.

With an ample

volume of sea water, the mortality rate was decreased.

In

forty days there was a mortality rate of only 22% among the experimental animals and a mortality rate of 10% in normal animals. The problem of viability remains somewhat contro­ versial.

Regardless of interpretations to the contrary, the

evidence suggests that the eyestalks (or sinus glands) are essential for life and maintenance of the normal physiological processes in many crustaceans.

Further evidence for this

concept will be presented in this thesis. D.

The Humoral Influences on General Metabolism

Although the influence of the sinus gland on growth, molting, and viability suggeststhat perhaps more fundamental processes are affected, the literature on the influences of the eyestalk hormones on general metabolism is somewhat limited. Calcium metabolism. The observation that changes occur in the calcium content of the exoskeleton and body tissues of crustacea during molting has prompted a few investi­

gators to determine whether calcium metabolism was also regulated in some manner by humoral substances. In some Brachyura, preceding molt, calcium is re­ sorbed from the exoskeleton and stored in the hepato-panereas and other soft tissues, and following molt this calcium is re­ deposited in the exoskeleton which, together with some absorbed from the sea water, serves to harden the exoskeleton. Paul and Sharpe (1916) and Robertson (1937) have presented evidence that considerable calcium is stored in the hepatopancreas of certain crabs.

However, Hecht (1914) working with

Callinectes and Kleinholz and Bourquin (1941a) working with Uca discovered that the amount of calcium in freshly molted crabs was insufficient for rebuilding the new exoskeleton and therefore concluded that additional calcium must be obtained from an external source. In Anomura and Macrura, according to Huxley (1906) and Robertson (1941), gastroliths are formed in the foregut prior to molt and probably represent a storage of calcium which is used in hardening the new exoskeleton.

Kyer (1942)

has presented evidence to indicate that the gastroliths are actually secreted by the stomach wall.

Maluf (1940a) has

shown that the integument, and not the gills, of the crayfish is permeable to calcium ions.

Furthermore, calcium was found

to be taken up only by premolt and postmolt crayfish, not by hard-cuticled animals.

During molting only 4% of the raw ash

and 2.3fo of organic material of the old cuticle was resorbed;

both, seemed to be stored in the gastroliths.

However, he

found that the gastroliths had only 3.16% of the total ash present in the hard exoskeleton and concluded that they were of no significance in hardening the shell after molting. Numanoi (1939) found the gastroliths of Sesarma haematocheir to enlarge as molting approached and to disappear after molt. The blood was more viscous and milky both during gastrolith formation and dissolution.

When the calcium reserve was

being dissolved after molting and precipitated in the integument, blood calcium was raised to over forty times the normal value.

Maluf (1940a) has suggested that the calcium

may be deposited directly into the cuticle after molt and does not enter directly into the blood, so the high calcium increases indicated by Numanoi were probably due to withdrawal of calcium from internal reservoirs or else calcium does enter through some other membranes* The first investigator to suggest a humoral control of calcium metabolism was Roller (1930) who reported the molted exoskeletons of eyestalkless Crangon vulgaris contained less calcium (soluble salts) than normal ones.

This suggested that

in the absence of eyestalks, calcium was resorbed more rapidly. From his results, Roller concluded that an eyestalk hormone was associated with calcium metabolism.

Plankemann (1935) drew a

similar conclusion when he found that the calcium content of eyestalkless cast exoskeletons was less than the calcium content of normal crayfish.

Kleinholz and Bourquin (1941a) repeated these experiments using a more refined technique for calcium analysis than mere solution by dilute HC1 (used by Koller and Plankemann) which removed more than just calcium salts* These experiments revealed no significant difference between the calcium content of the cast exoskeletons of eyestalkless and normal Palaemonetes vulgar is * They also found that the amount of deposition of calcium in the exoskeletons of eye­ stalkless Uca pugilator depended on the length of the intermolt period.

By extending the length of this period by

inanition the calcium content approached the normal value. Prom these results they concluded that an eyestalk hormone had no direct effects on calcium metabolism. On the other hand, Kyer (1943) has presented evi­ dence to indicate that the sinus glands are concerned in calcium metabolism during molting and suggested the possi­ bility of a common mechanism controlling both processes.

Re­

moval of eyestalks induced the formation of gastroliths in a non^molting season, whereas implantation of sinus glands into the abdominal region of eyestalkless animals prevented or re­ tarded gastrolith formation.

However, many of the details of

this mechanism remain unexplained. Koller (1930) suggested that the fundamental role of an eyestalk pigment-activating hormone was to regulate the distribution of calcium ions; and, that pigment migrations

might he explained by assuming changes in permeability of the chromatophore membrane.

Hanstrom (1939) has summarized evi­

dence in support of a theory for a single hormone regulating molt, pigment migration, and calcium metabolism; however, it has not been generally accepted by most investigators. Water metabolism. The term, water metabolism, is used to describe the changes involved in the maintenance of water balance in the body tissues of an organism; ionic and osmotic regulation are, undoubtedly, concerned in this regu­ lation.

In marine crustacea there is no problem of osmoregu­

lation.

In fresh water crustacea such as the crayfish, the

blood is hypertonic and there is a definite problem of osmo­ regulation (cf. Maluf, 1938b, and Krogh, 1939).

The problem

of water metabolism in fresh water crustacea is described in a later section of this thesis. Changes in water content during the molting process have already been cited in an earlier section of this review. Paul and Sharpe (1916), Olmsted

and Baumberger (1923),

Robertson (1937), and Drach (1939) have reported an increase In water content following molt in several different crus­ taceans.

Baumberger and Olmsted

(1928) found that both

osmotic pressure and water content increased preceding the molt cycle of Pachygrapsus crassipes and decreased following molt. Kleinholz and Bourquin (1941b) found that eyestalk­ less Uca increased in weight thirty per cent following molt.

Greater increases in size of eyestalkless than normal animals were reported by Abramowitz and Abramowitz (1940) and Smith (1940), although quantitative measurements were not made. These investigators made no mention of a possible part played by the eyestalk hormones in regulating water metabolism.

The

change in water content which accompanies molting indicates the possibility of hormonal factors influencing water metabolism as well as molt. Gray and Pord (1940) have given the only experimental evidence of a humoral regulation of water metabolism.

Extracts

of eyestalks of Uca pugilator and Oambarus blandingii acutus when injected into frogs produced changes in weight, due to water uptake, comparable to similar changes following injections of extracts of the hypophysis of certain vertebrates (frog, fish, and turtle)• Sugar metabolism.

Changes in blood sugar level are

important considerations in the metabolism of animals; however, very little work has been done on the blood sugars of crus­ taceans.

Hemmingsen (1924) has studied the blood sugar regu­

lation in the crayfish and revealed that the gills were not permeable to glucose even when the blood sugar was raised to 0.27fo.

Stott (1932) found that the blood sugar in starved

Careinus maenas was only 6 m g m a n d that the blood sugar con­ centration was raised to ten times this value after feeding. In low oxygen tension sea water, the blood sugar level was raised to that of well fed animals.

He concluded that there

was no regulation of upper blood sugar level in either feeding or anoxemia experiments* The changes in blood sugar level during the molt cycle were studied by Baumberger and Dill (1928).

These

investigators found an increase of the sugar in the hepatopancreas

from 1.2 - 1.5$ of the solids at the time of molt

to 3.5 - 4.5$ shortly after molt in Pachygrapsus crassipes. In the whole crab, the change was from 1.4$ to 2.1$.

Like­

wise, Drilhon (1933) found the amount of glucose in the blood of Maia squinado immediately before molt may rise to 18 mgm.$, while just after molt it may fall to 6 mgm.$. These changes in the blood sugar may be linked with the hormonal regulation of the molting process; however, there has been very little evidence presented to suggest this possi­ bility.

Welsh (1941) reported that one of his students found

a variation in blood sugar with rest and activity and suggested that an eyestalk hormone seemed to be involved in its regulation. In this connection, Hanstrflm (1939) has reviewed the literature concerning the influence of vertebrate hormones on blood sugars.

The field of opinion is divided with some results

showing that insulin and adrenaline alter the blood sugar level in decapods, while others indicate no influence on blood sugars by these substances. Oxidative metabolism. Although the effects of various environmental factors on oxygen consumption have been studied rather extensively in crustaceans, there has been very little mention made of any hormonal influences on metabolism.

Menke (1911) observed a diurnal rhythm in the migration of the integumentary pigments of the isopod, Idothea, which persisted under conditions of constant dark­ ness*

These pigmentary responses were accompanied by vari­

ations in oxygen consumption, which led Menke to suggest a connection between

pigment-activation and metabolism in

crustaceans. Maluf (1937b) mentioned, without evidence, the possi­ bility of some factors (nervous-secretory?) which may be responsi­ ble for the sudden decrease in the rate of oxygen consumption when crayfish are placed in water of various oxygen tensions# Finally, Scudamore (1941a) discovered that the rate of oxygen consumption increased for at least a week preceding molt and decreased for about a week to a new low rate following molt.

This discovery constituted one of the first pieces of

evidence to suggest humoral influences on oxygen consumption. Humoral influences on heart action.

The inverte­

brate heart has long been the object of much and varied investigation.

The classical experiments of Carlson (1906 and

later) on the comparative physiology of the Invertebrate heart gave us a clear picture of the structure and physiology of this organ.

Alexandrowicz (1932) has carefully worked out the

innervation of the heart of several decapods.

Both Carlson

and Alexandrowicz described neurons in the heart muscle and nerve fibers with both inhibitory and acceleratory effects. In addition, Alexandrowicz reported the presence of a local

"autonomic" nervous sytem from which the muscles of the heart received impulses necessary for regular contractions*

This

neuromuscular relationship suggested a pacemaker similar to that described in Limulus (Carlson, 1906).

Thus, any study

of humoral influences on the heart must consider the possible effects of these nervous elements. The action of various ions on the heart of the crayfish have been studied by Lindeman (1928, 1929) and Cole, Heifer, and Wiersma (1939).

The effect of temperature on the

heart beat of Camber us was reported by Corgan (1932). Gamble and Keeble (1900) observed that the pale or nocturnal phase was accompanied by a higher rate of heart beat (about 240 beats per minute) than the dark or day phase (150 beats per minute) in the prawn, Hippolyte varians.

These

investigators considered the changes in both coloration and heart beat to be controlled by the nervous system.

However,

since the discovery of the eyestalk hormones by Perkins (1928) and Koller (1928), it is now realized that the paling (due to concentration of the red and yellow pigments) is due to the presence of an eyestalk (sinus gland) hormone in the blood stream of Macrura.

This suggested that the increase in rate

of heart beat might be due to the presence of an eyestalk hormone. In preliminary tests on the crayfish, Cambarus, Welsh (1937a) indicated definitely the presence of a heartaccelerating principle in the crustacean eyestalk extracts.

The application of crayfish extracts to the exposed heart of a crayfish increased the "normal" rate of heart beat from thirty-nine beats per minute to sixty-five beats per minute* Application of muscle extract, as a control, caused only a slight change

in the rate.

Later, Welsh (1939a) repeated

these experiments on the intact heart of Leander serratus, with similar results, but added that alcohol extracts of eyestalks did not accelerate the heart although the usual color changes occurred.

From this he concluded that the heart-

accelerating principle in the water extracts was not the same as the pigment-activating principle. In the same paper, Welsh (1939a) tested the action of various agents on the perfused, isolated heart of Maia and Carcinus. Both adrenaline and acetylcholine accelerated the heart rate; the action of acetylcholine was similar to that of nerve extracts.

Large amounts of acetylcholine were found in

nervous tissue, especially ganglia, by bio-assay (Welsh, 1938b and Smith, 1939) from which he concluded that acetylcholine was the active principle involved in heart stimulation.

Again,

Welsh (1939c) found that both adrenaline and acetylcholine had an excitatory action on the isolated heart of Panulirus argus. The actions of eserine and atropine suggested that the local nervous system in the heart was cholinergic.

The difference

in the nature of response to adrenaline and acetylcholine suggested that these substances acted on different mechanisms within the heart.

Bain (1929) had previously indicated that

dilute solutions of adrenaline had an excitatory action on the heart of Carcinus. MacLean and Beznak (1933) had also studied the action of adrenaline and acetylcholine on the crayfish heart and obtained similar results. Additional evidence for a humoral influence on heart action was presented by Scudamore (1941b).

The state

of dispersion of the red pigment and the rate of heart beat were compared when the shrimp, Palaemonetes, was placed on white and black backgrounds. demonstrated.

A definite correlation was

When the red pigment was concentrated on a white

background (pigment-activating hormone in the blood stream) the rate of heart beat was high (293 beats per minute); and when the red pigment was dispersed on the black background the rate of heart beat was low (203 beats per minute).

Experiments

involving eyestalk removal, sinus gland extirpation, and injections of sinus gland extracts always gave parallel results for changes in pigment dispersion and heart rate.

These

results suggested the the red-pigment-activating hormone had an accelerating effect on the heart of Palaemonetes. Summary. The literature reviewed in the preceding pages indicates that the sinus gland hormones may have an influence on the general metabolism of crustaceans.

Evidence

for influences on calcium, water, sugar, and oxidative metabolism and heart action have been summarized.

In nearly

every case, the evidence is inadequate or merely suggestive; therefore, considerable additional investigation is in order.

III.

STATEMENT OF THE PROBLEM

A.

Some Existing Problems of Crustacean Endocrine Mechanisms

The review of literature on endocrine activities of the sinus glands and/or eyestalks reveals numerous gaps in our information in this field.

For example, much of the experi­

mental evidence was based on the removal of entire eyestalks and injection of eyestalk extracts.

Eyestalk extirpation

results in a loss of visual perception as well as four important ganglia, the X-organ, and other tissues in addition to the sinus gland; likewise, injections of an eyestalk extract includes not only the sinus gland material, but also nervous tissue, connective tissue, and pigments.

Therefore it was impossible to

distinguish between the effects produced by loss of vision or loss of nervous elements and loss of the sinus gland.

In

recent years, intact sinus glands have been removed without loss of vision and whole sinus glands have been implanted.

Many of

the earlier experiments bear re investigation writh these new techniques* Furthermore, much remains to be done in the way of separation, purification, standardization, and determination of the chemical composition of possible sinus gland hormones. Many functions, for which an endocrine control has been suggested, still lack sufficient experimental evidence; whereas other possible important functions doubtlessly remain un­ described.

Very little evidence has accumulated to explain

the fundamental role or mode of action of the sinus gland hormones.

And, in addition, the presence of other possible

sources of hormones in crustaceans and the endocrine inter­ relationships need further elucidation.

Finally, one should

be cautious when assigning too much importance to a single gland, in spite of all the functions apparently under its regulating influence. B.

Statement of the Specific Problem

The purpose of this investigation has been to study \

the role of the crustacean sinus glands or eyestalks in the possible humoral regulation of certain extra-chromatophor ic functions.

The particular activities studied include: molting

and gastrolith formation, calcium metabolism, water metabolism, respiratory metabolism (oxygen consumption), and rate of heart beat • In studying possible modifications of these functions the normal physiology of the experimental animals was always determined first.

The role of the sinus gland in regulating

or modifying the normal physiology was then studied by the classical method of, first, removing the suspected source of the controlling humoral agents and, secondly, replacing the glandular source or its products in an attempt to prevent the modifications resulting from a removal of the gland.

In some

experiments this involved the entire eyestalks but, in other instances, sinus glands were removed, implanted, or used as the source of glandular extracts. controlled.

All experiments were carefully

It was hoped that through these experiments, information would be added to our present knowledge of the endocrine function of the sinus gland, and to the problem of the fundamental mode of action of the sinus gland principles or hormones.

IV.

MATERIALS AND GENERAL METHODS *Pke Experimental Animals Several species of the fresh-water crayfish were

used in the experiments; these included: Cambarus immunis Hagen, Cambarus virilis Hagen, Cambarus blandingii acutis Girard, and Cambarus rusticus Girard.

The C. immunis were

obtained largely from the Grassyforks Fisheries, Martins­ ville, Indiana.

The other species were procured by seining

in streams near Evanston, Illinois. The crayfish was a convenient animal to use for the study of problems in crustacean endocrinology, because they were easy to obtain and to keep alive in the lead lined tanks filled with running water.

Occasionally, the animals

were fed small amounts of earthworm, liver, and crayfish muscle.

The animals were of a convenient size for experimental

work and the eyestalks and sinus glands were readily removed in an analysis of the function of the sinus gland.

The main

objection to these animals was the difficulty encountered in determining internal changes without sacrificing the animal.

A portion of the work was done during the summer of 1939 at the Marine Biological Laboratory, Woods Hole, Massachusetts.^

In these experiments, on heart rate and

color change, the shrimp, Palaemonetes vulgaris Stimpson, was used.

These animals were ideal for this study for the heart

and chromatophores were readily observed due to the trans­ parent cuticle. The Sinus Gland and the Eyestalk The sinus gland, which is the endocrine organ studied in this investigation, is located within the eye­ stalks.

Although the sinus gland has already been described

by earlier workers, the description should be repeated briefly. The position of the sinus gland in the eyestalk is shown diagrammatically in Pig. 18.

The gland is a definitely

circumscribed organ and is readily distinguished from the rest of the eyestalk tissue, when viewed by reflected light, for it appears as a bluish, granular and fibrous organ in deep contrast to the whitish appearance of the rest of the stalk tissue.

The sinus gland lies just beneath the neurilemma

between the medulla interna and medulla externa and is bathed by a rich blood supply.

The sinus gland innervation is partly

The author wishes to express his appreciation to Dr. F. A. Brown, Jr. for the opportunity to spend the summer of 1939 at the Woods Hole Marine Biological Laboratory as a re­ search associate.

visible and is composed of fibers from the supra-oesophageal ganglia and medulla terminalis. Saline Solutions The saline solution adopted for experimental use was one proposed by Van Harreveld (1936), and it will be called Van Harreveld's solution or merely saline in future references. Any physiological problem makes it essential to have a solution which does not differ greatly from the salt concentration and ionic ratios of the normal blood.

The ideal

method of preparing such a solution is to analyze the blood and attempt to duplicate its composition.

In selecting a

saline solution several suggested solutions were tested experimentally:

Frog Ringer's, Griffith's (Helff, 1931),

Lienemann's (1938), Lindeman's (1928), Prosser's (1940) and Van Harreveld's (1936).

As the result of injection experi­

ments, Van Harreveld*s solution was found to produce no significant change in oxygen consumption, while Ringer's or Griffith's produced an increase in rate of oxygen consumption. In addition, when tested on the chromatophores in isolated pieces of integument, only Van Harreveld's solution produced no pigment migration, while other solutions caused minor changes in the red or white pigments.

Cole, Heifer and

Wiersma (1939) had previously found Van Harreveld*s solution satisfactory for work on the crayfish heart.

D.

Methods of Gland Removal

Eyestalk removal. The eyestalks were extirpated with a sharp, fine pointed scalpel which severed all tissues cleanly. The open wound was seared with an electric cautery to prevent hemorrhage. In some experiments, the eyestalks were ligated at the basal membrane with fine, silk thread and then extirpated distal to the ligature.

This prevented loss of blood and

possible stimulation of the supra-oesophageal ganglion. Sinus gland extirpation.

In Palaemonetes it was

possible to remove the sinus gland, which was visible through the integument, by piercing the integument with a needle, inserting a capillary tube connected with pressure tubing to an aspirator, and sucking out the gland (Brown, Ederstrom, and Scudamore, 1939). It was also possible, by another technique developed by Brown (1942) to remove the sinus gland without extensive injury to the nervous tissue and ommatidia in Cambarus. The crayfish was secured to the stage of a dissecting microscope with rubber bands and the eyestalks brightly illuminated. The eyestalk was held in place with a glass capillary, attached to an aspirator with rubber tubing, while the chitinous covering of the ommatidia was torn away with a 3c watchmaker's forceps on the dorso-posterior margin.

The tissue in this

region was cleared away, with forceps and the sinus gland.

suction, revealing

The gland and a small portion of the

neurilemma were removed with the forceps; there was some

hemorrhage but the blood seemed to coagulate even more rapidly than in the case of eyestalk removal.

The success of the

operation was determined by examining the eyestalk tissues for fragments of the sinus gland at the end of the experiment.

In

most cases, the gland was completely removed with little injury to the nerve tissue or ommatidia. E.

Methods of Replacing Glandular Products

The active material of the eyestalks was replaced in eyestalkless animals by two different methods: implantation of whole sinus glands and injection of sinus gland (or eyestalk) extracts.. Implantation. The technique used was essentially that developed in our laboratories during the last few years. Eyestalks were removed and placed in a Syracuse watch glass containing a small quantity of the saline solution.

With the

aid of a strong beam of light and a dissecting microscope, the contents of the stalk were withdrawn from the open, proximal end using a pair of 3c watchmaker's forceps.

The neurilemma

was torn away from the nervous tissue and the sinus gland re­ moved from its position between the medulla interna and externa or from its place of attachment to the neurilemma. The gland was then drawn into the glass capillary tip of a specially prepared tuberculin syringe, Fig. 19. This was made by fastening the capillary tip to a hypodermic needle with a small amount of de IChotinsky cement.

It was

possible to draw the sinus gland, with a small quantity of

saline, into the tip of this capillary, where it was plainly visible. Implantations were made into the ventral abdominal sinus by inserting the capillary tip, between two sternites of the abdomen, for about a quarter of an inch anteriorly.

By

the precision of the plunger it was possible to place the sinus gland near the abdominal nerve cord where it would have ample blood supply and a chance to become incorporated in the muscle tissue.

Such implants retained their characteristic blue color

and were probably viable from one to three weeks. Injections and extracts. extracts of sinus glands were used.

In most cases, saline The sinus glands were

dissected from the eyestalks as indicated in the previous section and placed in a small glass mortar (the tip of pyrex test tube) and allowed to dry.

The dried material was tritu­

rated in a measured amount of saline solution.

The tube

containing the water extract was suspended for a few minutes in a boiling water bath.

Any remaining residue was removed by

filtration or, the extract was taken up immediately into a tuberculin syringe. In a few instances, extracts were prepared by alcohol extraction.

The dried eyestalks or sinus glands were tritu­

rated in 100$ ethanol and filtered.

The filtrate was placed

in a 25 cc. Erlenmeyer flask, with a capillary inlet and another outlet attached to an aspirator, and evaporated to dryness. This process was repeated.

The final residue was dissolved in

an appropriate amount of saline solution. Control extracts were prepared in a similar manner with muscle tissue and various parts of the central nervous system. The extracts were drawn into 1 cc. tuberculin syringes for immediate use or introduced into serum bottles and kept in the refrigerator for later use.

A twenty-six

gauge hypodermic needle was used for the injections.

The

amount injected was usually .05 cc. in average sized crayfish and between .02 and .03 cc. in small experimental animals. Usually, injections were made into the blood sinuses through the membrane at the base of the walking legs.

V.

MOLTING AND GASTROLITH FORMATION IN THE CRAYFISH A.

Introduction

Growth, by intussusception, is one of the funda­ mental attributes of all living organisms.

Furthermore, it

is a complex process involving many body functions, it is influenced by many factors, and, in the vertebrates, it is in part hormonally controlled.

In the crayfish, as in other

arthropods which have a hard, chitinous exoskeleton, growth is not a continuous process but is permitted by periodic ecdyses or moltings of the exoskeleton, which otherwise pre­ vents an increase in size.

The integument of the freshly

molted crayfish is soft and leathery, thus permitting growth

to take place before the exoskeleton becomes hardened again by impregnation with lime salts and secretion of chitin. It follows, therefore, that an analysis, of the physiological changes involved in molting and of the factors concerned in controlling this process, will reveal information regarding growth phenomena and some of the integral processes involved. The first experimental attempts to analyze the factors involved in the regulation of molting in the crayfish were made by Brown and Cunningham (1939).

These investi­

gators discovered a greater frequency of molts in animals with both eyestalks removed than in normal animals.

Homo­

plastic implants of sinus glands reduced this frequency of molts, whereas implants of sinus glandless eyestalk tissue did not influence this rate.

From this evidence they suggested

that the sinus gland was probably the source of a moltinhibiting hormone. Smith (1940) repeated these observations using young crayfish and found that bilateral eyestalk removal decreased the average length of the intermolt period by thirty per cent. Experimental ablation of retinas did not produce a signifi­ cant change in the length of the intermolt period. In these earlier experiments, the normal molt cycle was not given sufficient consideration.

In young crayfish,

the length of the intermolt period is much shorter than in older crayfish; consequently, the results are not comparable. The experiments of Brown and Cunningham (1939) were performed

during the spring end summer seasons which are normal molting periods.

However, when experiments were repeated in the fall,

all of the eyestalkless animals died before molting.

The

results were further complicated by the early death of many of the eyestalkless animals; therefore, it was necessary to calculate an index of per cent molts to represent the situation average life span clearly.

Although their results were conclusive, a better

method of analysis seemed desirable. In the crayfish, molting is preceded, along with other changes,by the formation of gastroliths in the anterior wall of the cardiac stomach (Figs. 20, 24).

These are white,

lenticular growths secreted by the stomach epithelium.

The

gastroliths are readily detected and removed from crayfish on autopsy.

Thus, the analysis of gastrolith size provided an

excellent method for studying the problem of molt control in the crayfish.

In the work which follows, a careful analysis

of the hormonal control of gastrolith formation and molting has been made using the technique of removing the source of the suspected hormone and then replacing it.

In addition,

information was obtained from these experiments concerning the physiology of gastrolith formation, viability, growth, and male sex hormones. Since the completion of my experiments, Kyer (1942) has published a paper dealing with the sinus gland and gastro­ lith formation.

He found that gastroliths were always present

in eyestalkless animals and absent in normal animals during

the winter.

Sinus gland implants suppressed or retarded the

formation of gastroliths.

Within twenty-four hours after

eyestalk removal, histological changes appeared in the gastric epithelium and the secretion of gastroliths was commenced. Nevertheless, many problems are

still not solved; in view of

the fact that my experiments contain further information and that confirmation of these observations is in order, it seems advisable to present my results in this thesis. B.

Experimental Methods

The general methods of eyestalk removal, of sinus gland extirpation, of extract preparation, and of implanting sinus glands have already been described. During the experiments, the crayfish were placed in individual dishes filled with tap water which was changed daily. The water was maintained at room temperature, 20° +1.5° C. Neither the experimental animals nor controls were fed during the observations; this was necessary to keep conditions uniform.

It had been observed previously that eyestalkless

crayfish invariably ate whenever food was offered, whereas normal animals usually refused to eat.

Complete records of

viability, sex, carapace length, and body weight of the animals were kept.

The carapace length was determined with a milli­

meter rule or with a vernier caliper by measuring the straight distance from the tip of the rostrum to the posterior margin of the carapace. Animals were sacrificed and examined for the presence

and size of the gastroliths at definite intervals of time. The eyestalkless animals were examined upon death in order that a record of viability and molting could be kept.

A

normal or implanted crayfish was killed and examined at the same time for comparison.

The gastroliths were dissected

from the anterior wall of the stomach, dried in an oven at 100° C. for twenty-four hours, and the weight of the pair determined to tenths of a milligram with an analytical chainomatic balance. C.

Description of the Life History of the Cray­ fish

Complete accounts of the life history of the cray­ fish are rare.

Van Deventer (1937) has written a complete

description of the life history of Cambarus propinquus and Tack (1941) has given an account of the life history of C_. immunis.

The writer's description is based on these -works

together with his own observations and the work of Turner (1926).

This description will deal primarily with £. immunis,

as this species was used in the majority of the experiments performed. The seasonal life history consists essentially of a fall mating season, winter hibernation, spring spawning season, spring molt, and summer molt. The young of £. immunis hatch in the early part of May and undergo two molts while still clinging to the pleopods of the female.

In the laboratory, the young may hatch as

early as February or March.

During the summer months there

are about eight additional molts.

These animals reach a

carapace length of thirteen to twenty-nine millimeters by the end of the first summer; some are sexually mature although others do not reach maturity until the second season.

There

are two sexual forms in male crayfish: Form I and Form II. The male sexual form may be readily identified by the shape of the gonopods as indicated in Figs. 31, 32.

Form I gono-

pods, sexually functional, in C. immunis are sharply curved and have pointed chitinous tips; Form II gonopods, not sexually

functional, are straighter and have blunt tips. Copulation takes place during the mating season,

from mid-June to late October, although a few crayfish may mate the

next April. Eggs are extruded in October and

November and are carried, attached to the pleopods of the female, until spring.

No growth or molting occurs during the

winter in the natural environment. During the second summer, there are two molts.

In

the spring molt, from mid-April through May, all immature crayfish and males molt.

Most of the males are Form I at this

time and change to Form II after molt.

A few Form II males

become Form I, while most of the juveniles become Form II. The females are carrying eggs at this time and, by some mechanism, molting is delayed until late May or June after the embryos have developed.

The spring intermolt period of

approximately two months follows. In the summer molt, mid-June to August, the males,

mainly Form II, become Form I and mating begins as soon as the exoskeleton hardens.

There are a very few Form I males

which become Form II at this molt.

Both Van Deventer (1937)

and Tack (1941) claim there is only a single molt in the mature females.

Although the author observed a second molt

in late July and August in the mature females. The same cycle of events is repeated during the second year.

Most crayfish live only two or three years;

many of them die during the molting periods or at the end of the summer.

A few crayfish may live to be three or four

years of age. Some of these events in the seasonal life cycle of the crayfish are summarized in Fig. 4.

This summary of the

life history of the crayfish has been presented in order to give a clear picture of the molting cycle, so that hormonal influences may be accurately interpreted. D • Description of the Molting Process Molting is a complex process which involves a series of changes over a long period of time and not just an event completed in a single day.

Baumberger and Olmsted

(1928)

and Baumberger and Dill (1928) have shown that a series of changes in water content, osmotic pressure, glycogen, and sugar content occur during the molt cycle of certain crabs. Drilhon (1933) and Drach (1939) also found variations in the blood sugar during the molt cycle.

Numanoi (1939) found

increases in the blood calcium during formation and dissolution

of gastroliths in Sesarma*

Scudamore (1941a) reported an

increase in oxygen consumption preceding molt and a decrease following molt* The resorption of carbonate salts and organic material from the exoskeleton and the deposition In the form of gastroliths constitutes another important change which precedes molting in the crayfish*

According to Huxley (1906),

in Astacus fluvlatilia which are four years old, gastroliths begin to form forty days before ecdysis but require only ten days to form in young crayfish the first summer after hatching* The spring intermolt period between the spring and summer molts is about sixty days long*

It Is probable that not more than

thirty days are spent In forming gastroliths for the summer molt*

Crayfish start to form gastroliths as early as the

March before the spring molt, which would occur at least thirty to forty days later* The process of shedding the exoskeleton has been described previously by many early workers (Huxley, 1906) and will not be repeated*

The gastroliths are cast off into the

stomach, dissolved, and utilized in the preliminary hardening of the cuticle*

Additional salts are obtained from the water

and chifcin is secreted by the hypodermal cells* E*

Experimental Induction of Molting and Gastro­ lith Formation during Winter Months

1.

Extirpation experiments

Winter is an ideal time to study the humoral factors regulating gastrolith formation and molting, because, in nature,

this is a long intermolt period during which molting does not normally occur*

Several experiments were conducted to determine

the role of the eyestalks in regulating these processes# In the first experiment several crayfish were divided into three groups and placed in individual finger bowls# In the first group, both eyestalks were removed; in the second group one eyestalk was removed, and In the third group there was no operation#

As the eyestalkless animals died, the gastro­

liths were removed and weighed#

Periodically, normal animals

and animals with a single eyestalk removed were also killed and examined for gastroliths# TABLE I Showing gastrolith formation and molting In the crayfish, C* immunis, induced by bilateral eyestalk removal during October and November. Length of carapace

Animal

Days after eyestalk removal

1 2

4 5

3 4 5

8

27*5 25*0 22*0 22.3 21*8 26.4 26*4 26*8 25*8 25.7 25.5 26*0 25*1 25*8 25*3 24.0

6 7 8 9

10 11 12 13 14 15 16 17 18 19 20

21

9 9 9 9

10 11 11 12 12 13 13 16 16 18 18 18 19 27

(mm#)

22*8 25*4 26.6 23*5 24*0

,

Weight of gastroliths .(pgS .0 -.

0.6 0.3 3.4 1.0 1.8 17*8 2.8 3*0

12.8 1*4 3*1 7.1

Molt Molt 11.0 Molt Molt 17*7 35*2

Molt '48.3

In eyestalkiess crayfish (Table 1) gastroliths were already present by the fourth day#

The gastroliths

remained small until the tenth day when they began to increase in size rapidly#

On the thirteenth, sixteenth,

eighteenth and nineteenth days, animals molted.

In every

one of the twenty-one animals examined, gastroliths were forming# TABLE 2 Showing condition of gastroliths in normal crayfish, Cambarus immunls, during the period from October to February# Asterisk (V) indicates that the animal molted during the experiment#

Animal

Days after observations were started

1 2 3 4 5 6 7 8 9 10 11 12* 13 14 15 16 17 18 19

8 9 10 11 11 12 16 18 20 22 22 46 63 63 63 70 81 90 91

Length of carapace (mm#) 25#7 22,4 25.6 27.0 25.8 27.0 28.2 24.5 24.6 26#3 24.2 20 #5 22.5 25.5 24.2 24.2 23.0 22.2 22#3

Weigjht of gastroliths (mgm#) 0 0 0 0 0 0 0 0 1.3 0 0 36.6 0 0 0 0 0 0 0

Normal animals (Table 2) examined during the same period usually did not show any gastroliths even though observed ninety-one days after the experiment was started#

Animals (9) and (12) were noticeable exceptions to the rest of the oases*

Animal (12) was a small, immature female and

therefore the molt was possibly a delayed molt of the several that occur during the first summer*

During the entire winter

some 600 animals were observed and only five normal animals in this lot molted*

It is not possible to state that animals

do not molt in the winter, but that as a general rule, they do not* TABLE 3 Showing gastrolith formation in crayfish, C* immunis, with one eyestalk removed, during the period from October to February*

Animal

1 2

Days after operation 9 16 22

3 4

22

5

44 63 63 63 70 70 70 82 83

6 7

8 9

10 11 12 13

Length of carapace (ami*) 25*0 23*2 24*5 24.5 20.0 21.8 24.4 24.6 22.0 21.0 24.0 23.7 25.0

Weight of gastroliths

(mgm.) 0 0 +

0 41.0

0 0 Molt 0

1.0 0 0

0

In the thirteen animals with only one eyestalk re­ moved (Table 3) none of the animals examined within three weeks after the operation had any gastroliths.

However,

animal (5) had large gastroliths and was ready to molt on the forty-sixth day.

This was another small female and an expla­

nation similar to that suggested for the normal animal, which

molted on the same date, might he offered*

Another animal

(8) molted on the sixty-third day; and still another animal (10) had small gastroliths on the seventieth day* these animals formed gastroliths is not clear*

Just why

One animal

was observed eighty-three days after operation and had not started to form gastroliths.

It appears that removal of one

eyestalk is not sufficient to induce gastrollth formation and molting, or at least it proceeds at a much slower rate than in animals with both eyestalks excised* The eyestalkiess animals shown in Tables 1 and 6 which molted, were permitted to live after molting to determine whether they would enter into a second molt as indicated by the formation of a second set of gastroliths*

None of the

animals were fed during this time* The results of the examination of such animal^ which lived longer than one week, are shown in Table 4. TABLE 4 Showing gastrolith formation in eyestalkless crayfish with an average 23*8 mm* carapace length, approaching a second molt*

Animal 1 2 3 4 5 6 7 8 9

First premolt period ___ (days) 13 14 15 15 16 16 17 19 22

Days after first molt

Weight of gastroliths (mgm*)

16 23 21 19 25 27 20 23 19

15*4 11*2 0*8 0.5 5.9 1.0 0.5 1.4 +

Animals living only a week still had the old gastroliths un-

dissolved*

In every instance, when the animals were examined

from two to three weeks after the previous molt, a second set of gastroliths were found to he present (Fig* 28)*

In one

case, animal (1), the crayfish appeared "ready-to-molt11 as evidenced by the loosened cuticle, although the gastroliths were only one quarter their usual size at the time of molt* In most instances, it probably would have required at least another seven days for a molt to take place, thus the length of the second premolt period would have been approximately twice the length of the first premolt period* In a final experiment, an attempt was made to isolate the factor in the eye stalks which inhibits molting and gastrolith formation*

If the sinus gland was the source of this

substance, sinus gland extirpation should also result in gastro lith formation and accelerated molting*

To test this possi­

bility both sinus glands were removed, using the technique previously described, from one group of animals; and, both eyestalks were extirpated from a second group* normal animals were used as controls*

A group of

The animals in each

group were of approximately the same size*

The results are

compared in Table 5* Sinus gland extirpation resulted in the formation of gastroliths, although at a slightly slower rate than In the case of bilateral eyestalk removal.

The experiments were

complicated by the fact that, normal animals, which had been In the laboratory for several months, were beginning to form

gastroliths for the spring molt*

One of the sinus glandless

animals molted twenty days after the operation indicating that sinus gland removal was sufficient to initiate gastrolith formation and molting*

In every instance, the eyestalk tissue

was not injured; however, small fragments of sinus gland material frequently were present*

This condition might account

for the retarded rate of gastrolith formation as compared to that of eyestalkiess animals* TABLE 5 Comparison of gastrolith size in normal, eyestalkless, and sinus glandless crayfish during February and March* Asterisk (#) indicates that the gastroliths had started to form before the experiment was undertaken. Days after )ration

________ Weight of gastroliths (mgm.)______ Normal Sinus glandless Eyestalkless 10*7* + 0*6 0.8 0.0 + + 0.0

2 4 7 10 11 13 17 20 2*

+ 0.2 2.6 2.1 12.6 ---

8.2 Molt

+ 0.8# 1.9* 10.7 23.1 25.5 ——

Replacement experiments

Having established the observation that the eye­ stalkless (sinus glandless) condition resulted in gastrolith formation and earlier molting, it seemed desirable to determine whether the replacement of sinus gland products would prevent gastrolith formation and molting. Sinus gland implants.

In this set of experiments a

comparison was made in the rate of gastrolith formation and molting between eyestalkless crayfish which received a sinus

gland Implant every three or four days, and eyestalkless animals which received implants of sinus glandless-eyestalk tissue at the same intervals#

The animals were placed in

individual finger bowls and sacrificed one at a time to examine for gastroliths at definite intervals* The implantation of sinus glands into the ventral abdominal sinus prevented or retarded the formation of gastro­ liths and, as long as fresh implants were added, prevented molting (Table 6)*

At a time when eyestalkless crayfish had

fully developed gastroliths and were molting, gastroliths were either entirely absent or of a very small size in the crayfish with implants* TABLE 6 Gastrolith formation in eyestalkless crayfish, which received a sinus gland implant every three to four days, during period from October to January# Asterisk (*) indicates that first implant was not made until four days after eyestalk extirpation.

Animal 1 2 3 4 5

Number of implants

Days after eyestalk removal

1 1 2 2 4

4 4 5 7 12

6

4

7 8 9 10 11 12 13* 14* 15* 16* 17* 18* 19*

3 4 4 2 4 4 2 2 4 5 5 6 6

Length of carapace (mm.)

Weight of gastroliths (mgm.)

24.2 23.3 24.6 25.0 23.8

0 0 0 0 +

12

21.6

0.8

12 13 14 14 15 15 12 12 19 22 22 37 37

25.0 23.0 24.3 24.4 24.2 21.7 23.4 22.6 25.4 23.8 25.0 24.2 24.5

0 0 0 1.5 0 0 0.9 0.8 2.0 + 1.0 1.0 3.8

Eight animals (Table 7) were permitted to continue in the eyestalkless condition for some time following the last implant.

Only three of these molted, although others

were in various stages of gastrolith formation.

In all but

two instances the period after the last implant was approxi­ mately twice as long as that required for the complete for­ mation of gastroliths and molting in eyestalkless crayfish. TABLE 7 Showing gastrolith formation in eyestalkless crayfish following the last sinus gland implant. Animals examined after death.

Animal

Number of implants

Days after eyestalk removal

Days after last implant

Length of carapace (mm.)

Weight of gastroliths (mgm.)

1 2 3 4 5 6 7 8

6 6 6 6 4 3 Z 3

37 39 57 48 43 42 35 42

15 17 25 26 27 27 29 32

22.6 25.2 24.7 25.4 24.0 25.4 25.6 24.8

Molt 42.4 13.0 8.7 Molt Molt 29.7 29.1

It appears that the sinus gland was active for several days after implantation.

Microscopic examination

revealed that the sinus gland implants retained the charac­ teristic bluish appearance, were attached to the muscular tissue or near nervous tissue, and bathed with blood from the blood sinus. In the control experiment (Table 8) it was found that sinus glandless-eyestalk tissue was unable to inhibit gastrolith formation and molting.

All the animals had molted

or died with large gastroliths during the period of twelve to twenty-two days.

This was about the same rate as that

observed in eyestalkless animals without implants. TABLE 8 Showing gastrolith formation in eyestalkless crayfish, which received implants of sinus glandless eyestalk tissue during the period from October to January.

Animal

1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18 19

20 21 22 23 24 25 26 27

Days after eyestalk removal

Length of carapace (mm.)

2

23.0 23.2 24.3 24.0 24 o2

2 5 7 7 8

12 12 12 13 14 14 14 15 15 15 15 16 16 16 17 17 19 19

20 22 22

Weight of gastroliths - _(-fligm.)

0 0

22.8

+ + + +

24.5 24.8 22.8 25.6 22 .0 24.0 25.0 23.0 24.6 22.5 21.9 24.5 23.4 25.0 23.4 25.0 23.5 23.6 25.6 24.8 24.2

56.7 26.9 37.6 Molt 17.2 Molt 6*6 30.3 Molt Molt Molt 46.9 36.9 Molt Molt 39.2 Molt 29.5 61.4 Molt

1.2

These experiments strongly suggest that the sinus gland is the source of a molt—inhibiting hormone which also prevents or retards the formation of gastroliths*

This con­

firms and extends the observations of earlier workers.

Sinus gland extracts.

In some preliminary experi­

ments it appeared that injections of eyestalk extracts were ineffective in preventing gastrolith formation.

All of the

animals died within ten days following eyestalk removal, but even then gastroliths had started to form. Two eyestalkless crayfish were injected daily with freshly prepared sinus gland extracts and later examined for the presence of gastroliths.

In one animal, examined at the

end of eleven days, there was a complete absence of gastro­ liths; another examined after fourteen days nan gastroliths, which weighed 4.0 milligrams.

The results of another experi­

ment, Table 9, indicated that daily injections of sinus gland extracts into eyestalkless crayfish retarded but did not prevent gastrolith formation.

These meager results suggest

that the molt-inhibiting principle of the sinus gland may be water soluble. 3.

Comparison of different replacement methods

Experiments were conducted to test for the presence of a molt-accelerating hormone in the central nervous system and to compare the relative ability of sinus gland implants and extracts to suppress gastrolith formation.

The crayfish

were divided into normal and eyestalkless control groups; the eyestalkless animals were further divided into those whieh received two sinus gland implants at the time of the operation and a third implant on the seventh day after the operation, those which received daily injections of .03 cc. of sinus gland extract (six sinus glands in 0.4 cc. of saline

solution) and, in a third group of eyestalkless animals, those which received daily injections of .03 cc. of a central nervous system extract ("brain” or supra-oesophageal ganglia and the thoracic ganglia in 1.0 cc. of saline).

The in­

jections were made at midnight. The importance of the results indicated in Table 9 are lessened due to the fact that even the normal animals had started to form gastroliths (in March) for the approaching spring molt.

For this reason, only those observations made

on the tenth day are entirely comparable.

These measurements

indicate that the gastroliths were much larger in the animals which had received the C. N. S. extract injections than were the gastroliths in any of the other crayfish (Fig. 29). Although the results varied too greatly to draw a definite conclusion, the possibility of a molt-accelerating principle was indicated. TABLE 9 Comparing gastrolith size under various experimental conditions during February and March. Asterisk (*) indicates that gastro­ liths had begun to form before this experiment was undertaken. Weight of gastroliths (mgm.)_________ _____________ Eyestalkless Animals Bays after oper at ion 2 4 7 10 11 13 16 17

Sinus gland implants (3) Normal 10.7* —

^

M

0.6 0.8

Sinus gland extracts daily

"Brain” and thoracic ganglia extract

___

--------

3.4* 3.2

1.2 2.2

5.2? 4.9* 2.0 16.4

--------

--------

4.4*

---------

16.2*

---------

--------

5100

18.7*

8.8* 2.9 ---

13.3 13*3 ----

No treat ment 0.8 1.9 10.7 23.1 25.5 —

—

---

Another interesting observation was the fact that sinus gland implants made at this time of the year did not prevent gastrolith formation completely. of either of two hypotheses:

This is suggestive

(1) that, once the molting

process is initiated, the sinus gland activity cannot suppress gastrolith formation altogether (cf. Table 6); (2) that one of the factors involved in the induction of molt may be a seasonal cycle in the secretory activity of the sinus gland. 4.

Analysis of the frequency of molts

While analyzing the possible hormonal regulation of gastrolith formation, a number of the crayfish molted. This is the first reported instance of eyestalkless crayfish molting in the winter.

Therefore, the observation gives

further support to the role played by the sinus gland in producing a molt-inhibiting principle.

The frequency of

molts under various experimental conditions is indicated in Table 10. The high percentage of molts in eyestalkless cray­ fish, reported by earlier workers, during normal molting seasons was verified; similarly, a low percentage of molts occurred in normal and eyestalkless animals with sinus gland implants.

The reason for the few molts in normal animals and

the single molt in a crayfish with one eyestalk removed was not apparent, although temperature was in all probability an important factor.

TABLE 10 Frequency of molts In crayfish, £♦ immunis, observed during the winter of 1941-42* Number of animals observed

Number of molts

600

5

0,83

Unilateral eyestalk removal

13

1

7*69

Bilateral eyestalk removal

58

24

41.38

Bilateral eyestalk removal with sinus gland implants

27

0

0,00

The same after discontinuance of implants

10

3

33,33

Experimental condition Normal

Per cent animals molting

The length of the premolt period (time from beginning of gastrolith formation to molt) in the nineteen eyestalkless animals, which molted during the course of the winter experi­ ments was 16*26 + 0*68 days'** but varied from eleven to twentytwo days.

This was much shorter than the summer premolt period

which is approximately thirty days.

As a rule, the span from

mid-August to mid-April constitutes a long winter intermolt 1

.

The first figure represents the mean of the premolt periods. This was calculated by dividing, the summation of the size classes times the frequencies, by the number of variables. The second figure represented the standard error of the mean. This was calculated by dividing the standard deviation by the square root of the number of variables, When n was small, the value n-1 was substituted. Wherever statistics are included, these methods were used in the computations.

period (time from one molt to the next) during which molting does not normally occur*

The length of the spring premolt

period probably varies from thirty to sixty days, because some normal crayfish had already started to form gastroliths in February and March for the spring molt beginning April 15th* These cfcservatfom further substantiate the conclusion that the sinus gland is the source of a molt-irihibiting hormone* Although there was no significant difference in the length of the premolt period in eyestalkless animals of different sexes (Table 11), egg-carrying females required a slightly longer period than did the others.

With the complete removal of

the source of the molt-inhibiting principle, it was not sur­ prising to note little significant variation. TABLE 11 Sex variation in the length of the premolt period induced by eyestalk extirpation. Male I Number of animals Length of premolt period

Male II

Female

Gravid Female

4

5

6

4

15.75 +1.86

16.25 +1.32

16.67 +1.43

17.50 +1.83

Evidence for a Dally Rhythm in Gastrolith Formation Rate of gastrolith formation.

In studying the

relationship of the sinus gland to gastrolith formation it was observed that the gastroliths began to form as very small discs and developed into large plano-convex growths*

The results of

an analysis of the rate of gastrolith formation are illus­ trated in Fig# 1* All of the animals were approximately the same size ranging in carapace length from 23.3 to 27*5 millimeters.

In

the eyestalkless animals It was found that for eight to ten days the gastrolith increased In size very slowly and that growth was extremely rapid during the last seven days preceding molt.

The weights of the pair of gastroliths in animals which

died molting were averaged to obtain the maximum size at the time of molt. Another experiment was performed to determine the rate of gastrolith formation under constant conditions.

Both

eyestalks were extirpated from seventeen crayfish of approxi­ mately the same carapace length (av., 26.9 mm.) and body weight (av., 5*5 gm.).

These animals were placed in individual finger

bowls in which the water was changed daily.

The temperature

of the water during these experiments was 19° + 1° C.

A cray­

fish was examined each day to determine the daily increment of growth of the gastroliths.

The results are represented graphi­

cally in Fig. 2 and are well illustrated in Fig. 27. In both this and the previous experiment, there was no appreciable increase in gastrolith size until the seventh day; then, following the tenth day, there was an extremely rapid rate of growth.

Unfortunately, all of the animals were

killed before any molted.

The last gastrolith measured was

approximately sixty to seventy-five per cent formed.

Therefore,

most of the gastrolith growth occurs during the last week before molting.

Manner of gastrolith formation and growth. The materials for the formation of the gastroliths are derived largely from the cuticle and probably carried by the blood to the stomach epithelium where the materials are concentrated and deposited in the form of gastroliths.

Huxley (1906) has

cited the work of Chantran (1874) who found the gastroliths to be composed of about 96$ soluble materials, mostly calcium salts with some organic matter, and 4% Insoluble chitin. The gastroliths are located on either side of the median frontal line of the anterior wall of the cardiac stomach and resemble two eyes (Fig* 24)*

They lie in a

pouch formed by the cuticular lining of the stomach and the gastric epithelium as shown in Figs. 20, 22.

The flat inner

surface, in the form of a chitinous disc, lies against the cuticle and the smooth or crenulated outer surface lies against the epithelium (Figs. 20 to 28).

According to Kyer

(1942), the epithelium, which secretes the gastroliths, thickens and apparently begins secretory activity within twenty-four hours after eyestalk removal.

Internally, the

gastrolith is composed of a series of concentric layers (Figs. 23, 26); the Inner layers are thin and parallel to the disc, while the outer layers are thicker and concentric to the convex outer surface.

Each of the major layers is composed

of three to twelve sub-layers. On close examination, it was noted that there were from eight to ten very thin inner layers and about seven thicker outer layers.

This distribution of layers corresponded

remarkably well with rate of growth and changes in size of gastrolith*

A similar examination of the gastroliths from

spring and summer molts revealed about twenty to forty thin layers in the oldest portion of the gastrolith and seven convex layers on the outside*

In later stages the layers

were often found to be fused and to have a spongy appearance making it almost impossible to distinguish the layers.

Some

gastroliths consisted of small mounds of layers covered with a second disc and additional layers*

This observation was

interpreted to mean that the gastroliths had commenced to form before eyestalk removal and afterwards a new disc and subsequent layers were secreted. The daily rhythm in layer formation.

Although the

presence of layers in the gastrolith has been known for many years, there have been no attempts to correlate the number of layers with the number of days since the onset of gastrolith formation.

The technique of inducing gastrolith formation by

eyestalk extirpation provided an opportunity to study the possibility of a rhythm in formation. In order to test this possibility, both eyestalks were extirpated from twenty crayfish at 5:00 P.M. and there­ after an animal was sacrificed and examined each day for a period of thirteen days.

The gastroliths were removed, cut

in half with a scalpel and observed under a dissecting micro­ scope using bright reflected light.

In most cases, the layers

stood out distinctly (Fig. 26) and were easily counted.

The

number of layers in the gastrolith corresponded perfectly with the number of days after eyestalk removal. Having established the possibility of a daily rhythm in gastrolith formation, it was desirable to determine the time of day at which there was activity in gastrolith secretion.

An examination of the animals in the morning as

well as in the afternoon revealed no difference in the number of layers (Table 12).

This suggested a nocturnal activity

in the secretion of the gastroliths.

In another group of

animals, the eyestalks were extirpated at 8:00 A.M. and, again in this instance, the evidence suggested a nocturnal period of deposition. TABLE 12 Analysis of the time of day at which the layers of salts and organic matter are deposited in the gastroliths*

Animal 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Time examined 5 5 9 5 5 9 5 9 5 5 9 12 9 1

P.M. P.M. A.M. P.M. P.M. A.M. P.M. A.M. P.M. P.M. A.M. Mid. A.M. A.M.

Days after eyestalk removal 1 2 2* 4 6 vi 8 ai 9 10 ni 12± 124 134

No. of No. of layers layers,if present in deposited gastroliths diurnally 1 2 3 4 6 8 8 9 9 10 12 12+ 13 13+

1 2 2 4 6 7 8 8 9 10 11 12 12 13

No. of layers,if deposited nocturnally 1 2 3 4 6 8 8 9 9 10 12 13 13 14

In a final experiment, both eyestalks were removed

from eight crayfish at 11:00 A.M. and the gastroliths permitted to develop for eight days.

At the end of this time, one animal

was killed every three hours during the day and night and the extend of layer formation in the gastroliths was determined# The deposition of a new layer in the gastrolith had commenced at 4:00 P#M# and was completed before noon the following day# This evidence seemed to suggest a daily rhythm in gastrolith layer formation with the greatest activity at night#

The daily

rhythm of layer formation in gastroliths represents another physiological rhythm (of. Welsh, 1938a and Park, 1940)# The crayfish also displays a nocturnality in motor activity (Roberts, 1936; Kalmus, 1938).

However, eyestalk

removal interrupts this rhythm and results in a decrease in total activity.

Therefore, the factors regulating these two

rhythms are probably different.

The internal factors controlling

the rhythm in gastrolith formation were not established, although the evidence suggested that the central nervous system might be involved.

This discovery of a daily rhythm in gastro­

lith formation is of greater Interest in view of the findings of Schour and Steadman