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Effect of eyestalk removal on linear growth and water uptake during the molt cycle of the crab, Hemigrapsus… Baldwin, Mary Frances 1967

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E F F E C T OF EYES T A L K REMOVAL ON LINEAR GROWTH AND WATER U P T A K E DURING THE MOLT C Y C L E OF THE CRAB, Hemigrapsus nudus (DANA) by MARY FRANCES BALDWIN A.B., Duke University, 1962 A THESIS SUBMITTED IN P A R T I A L F U L F I L M E N T OF THE REQUIREMENTS FOR THE D E G R E E OF MASTER OF SCIENCE in the Department of Zoology We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA February, 1967 In presenting t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y avail able f o r reference and study, I furt h e r agree that permission...for extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s representatives, I t i s understood that copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n permission. Department of ^1 r^^t-ty >o CL The U n i v e r s i t y of B r i t i s h Columbia Vancouver 8, Canada ABSTRACT When eye stalks are removed from Hemigrapsus nudus at least 10 days before ecdysis, linear growth of the destalked animals, measured as an increase in carapace width after ecdysis, is significantly higher than the linear growth of the unoperated control animals. The amount of water that control and destalked H. nudus absorb during ecdysis (stage E) is not significantly different; however, during early postecdysis the destalked animals show a significantly greater increase in wet weight than the controls. The greater linear growth calculated for destalked animals must be realized during early postecdysis. To investigate one possible mechanism to account for the large increase in wet weight characteristic of destalked animals during post-ecdysis, total osmotic pressure of the blood was measured throughout the molt cycle for control and destalked animals. The osmotic pressure measured from destalked animals is not significantly different from the measurements of osmotic pressure from control animals during the short period of ecdysis, however determinations of osmotic pressure from destalked animals during pro- and postecdysis art significantly lower than those from control animals. There is postulated a water balance-regulating principle located in the eyestalk which regulates the early postecdysial absorption of water. The absorbent surface of the destalked crab may become more permeable to water just after ecdysis, thus causing an increase in wet weight and an increase in size. E R R A T A p. 2 P a r a 2 line 5 p. 24 P a r a 1 line 5 p. 43 P a r a 2 line 4 insert "and Bourquin" after 'Kleinholz' insert "of" after 'range' read "there is a greater difference" for 'here is greater different' i i i T A B L E OF CONTENTS P A G E INTRODUCTION 1 MAT E R I A L AND METHODS 6 RESULTS 14 1. Description of the molt cycle for control Hemigrapsus nudus 14 2. Linear growth values (lambda) calculated for control and destalked Hemigrapsus nudus 2<4 3. Wet weight changes during the molt cycle for control and destalked Hemigrapsus nudus 28 4. Total osmotic pressure measurements during the molt cycle for control and destalked Hemigrapsus nudus 38 DISCUSSION 48 SUMMARY 59 L I T E R A T U R E CITED 61 IV LIST OF T A B L E S T A B L E P A G E 1 The identifying characteristic of the pro-ecdysial, postecdysial, and intermolt stages described for control Hemigrapsus nudus. 16 2 Relative hardness of nine areas of the carapace recorded for control Hemigraphsus nudus. 22 3 Lambda values calculated for a l l control Hemigrapsus nudus molting during the summers of 1965 and 1966. 25 4. Lambda values calculated for destalked Hemigrapsus nudus molting during two consecutive years and for animals maintained in two salinities. 27 5 Comparison of mean lambda values for control and destalked Hemigrapsus nudus molting after 10 days in the laboratory. P value states the probability from a t-table of no difference between control and destalked lambda values. 29 6 Average lambda values for control and destalked Hemigrapsus nudus followed through two molts. 31 7 Orthogonal comparisons of per cent weight changes during the three main time periods of the molt cycle. SSq is tested over the E r r o r Mean Square from a one way analysis of variance. P values state the probability of no difference between control and destalked values at the three specified time periods. 34 8 Weight of water absorbed during exuviation, R, for control and destalked Hemigrapsus nudus, expressed as per cent of absorbant mass (Drach, 1939). 35 Orthogonal comparisons of osmotic pressure measurements during the three main time periods of the molt cycle for control and destalked Hemigrapsus nudus collected during the summer and maintained in 100% salinity. SSq is tested over the E r r o r Mean Square from a one way analysi of variance. P values state the probability from an F table of no difference between control and destalked values. Orthogonal comparisons of osmotic pressure measurements during three main time periods of the molt cycle for summer destalked animals (from Table 9) and winter destalked animals maintained in 100% salinity. SSq is tested over the E r r o r Mean Square from a one way analysis of variance. P values state the probability from an F table of no difference between summer and winter values. vi LIST OF FIGURES FIGURE P A G E Outline drawing of the dor sal carapace of Hemigrapsus nudus showing areas of the carapace and width measurement (arrow). Per cent change in wet weight, expressed as per cent of the intermolt wet weight, during the molt cycle of control and destalked Hemigrapsus nudus. Each point represents the mean of the measurements of at least 10 animals for each time period. 32 Absolute wet weight change during ecdysis calculated from Figure 2 for a 10 gram destalked animal. Time periods on the abcissa are as follows: P n = wet weight 12 - 24 hours before ecdysis, P n10-1 = wet weight 1 0 - 1 minutes before ecdysis, P n+0 = wet weight at time zero after ecdysis, P n - f l = wet weight 12 - 24 hours after ecdysis. 37 Osmotic pressure measurements during the molt cycle for summer control and destalked Hemi-grapsus nudus in 100% sea water. . Each point represents the mean of the measurements of at least 10 animals for each time period. The solid and dashed lines marked at 110.6 and 113.6 per cent sea water on the graph represent control and destalked intermolt baselines calculated as explained in the text. The solid lines above and below these baselines represent confidence inter-vals (P=0.01). Time periods during ecdysis are specified periods in terms of the amount of weight the animal has gained: 0-10, 1 - 10% of the proecdysial wet weight; 20 - 30, 20 - 30% of the proecdysial wet weight. Osmotic pressure is expressed as per cent sea water. 40 v i i F I G U R E P A G E O s m o t i c p r e s s u r e measurements during the molt c y c l e f o r winter destalked H e m i g r a p s u s nudus maintained i n 100% s a l i n i t y . E a c h point r e p r e s e n t s the mean of the measurements of approx i m a t e l y 5 a n i m a l s f or each time p e r i o d . O s m o t i c p r e s s u r e i s e x p r e s s e d in per cent sea water. 45 O s m o t i c p r e s s u r e measurements during the molt c y c l e f o r winter destalked H e m i g r a p s u s nudus m a i n t a i n e d i n 7 5 % sea water. E a c h point r e p r e s e n t s the mean of the measurements of approximately 6 animals f or each time p e r i o d . O s m o t i c p r e s s u r e i s e x p r e s s e d in per cent sea water. 47 v i i LIST OF P L A T E S P L A T E P A G E 1 Hemigrapsus nudus at the beginning of the passive phase of stage E when the animal has gained about 1% of the initial proecdysial wet weight (X2). 18 Hemigrapsus nudus during the passive phase of stage E showing the branchiostegite with the broken epimeral suture (x2) 18 Hemigrapsus nudus after the animal has gained 10 - 2 0 % of the initial wet body weight. Swollen pericardial sacs and the newly secreted dorsal carapace can be seen. 19 Hemigrapsus nudus during the active phase of stage E . The animal now pulls itself out of the old exoskeleton. Swollen pericardial sacs can stil l be seen and the abdomen has already been pulled out of the old exoskeleton ( x 2 ) . 19 ACKNOWLEDGMENT The author wishes to express appreciation and thanks to Dr. Paul Dehnel for his patient super-vision and advice throughout the course of this research. My thanks to Doctors J. E. Phillips, H. D. Fisher, and B. M. Bary for c r i t i c a l reading of the thesis and to Mr. S. Borden for advice on statistical usage. INTRODUCTION Removal of both eyestalks from a crab causes a precocious molt and an increase in size (Abramovitz and Abramovitz, 1940; Smith, 1940). This increase in size has been explained in terms of water uptake by Passano (1953), Car l i s l e (1955), and others. There has been postulated a water balance-regulation principle, located in the optic ganglion of the crab eyestalk, which regulates the amount of water that an animal takes in at ecdysis. Presumably, an animal without this water balance-regulation principle would take in more water and therefore become larger. However, molting animals without eyestalks have never been compared with control molting animals to see if, in fact, the animals without eyestalks do take in more water. Nor has the mechanism for the action of this principle been investigated. Megusar (1912) was the fi r s t to report that ecdysis occurred at more frequent intervals in a crayfish without eyestalks. The impor-tance of this work was not fully realized until nearly 30 years later when Brown and Cunningham (1939) reported the f i r s t direct evidence for an endocrine activity of the sinus gland located in the optic ganglion of the crustacean eyestalk. They found that sinus gland implants into destalked animals delayed the expected precocious molt and postulated 2 the existence of a molt-inhibiting hormone produced in the sinus gland. This hypothesis was generally accepted until Passano (1951a, b) and Bliss (1951) simultaneously gave unequivocal evidence that the molt-inhibiting hormone was produced in the X-organ cells of the medulla terminalis and transported along axonal tracts to the sinus gland where the hormonal material was merely stored and released. Abramovitz and Abramovitz (.1940} noted that removal of the eye-stalks of Uca lead to an increased size in conjunction with the precocious molt. They thought the increased size was probably the result of an increased number of molts and an increased appetite among the operated animals. Subsequently, Smith (1940) and Kleinholz (1941) reported that destalked animals were larger than comparable control animals but gave no data. Bauchau (1948) stated that linear growth values for destalked Eriocheir sinensis were four times larger than growth values calculated for control animals. This abnormal size increase was found for both fed and unfed animals. These data were confirmed later by several studies: Scudamore (1947); Koch (1952); Passano (1953); and Carlisle (1955). Since the swelling seen at ecdysis (Plate 3) has been shown to be caused by an uptake of sea water (Robertson, I96O) it has been assumed previously that destalked animals showing an increased size should take up more water than control molting animals. Scudamore (1947) stated that bilateral eyestalk excision produced an increase in body weight due to increased water content but his data 3 were compared with control non-molting animals, not control molting animals. Bl i s s (1953) also stated that she had evidence for a water balance-regulation principle located in the eyestalk which was responsible for the increase in weight during proecdysis in eyestalk -less. crabs. Again, her results are not compared with control proecdysial animals. Edwards (1950) presented some data which showed both weight increases and decreases following eyestalk ablation but the molting condition was not stated for these destalked animals nor were these weight changes followed through an ecdysis. Guyselman (1953) was not able to show any changes in weight for destalked Uca weighed for 12 days after removal of the eyestalks. Similarly he showed that his data, for the increase in weight accompanying ecdysis for normal animals, were approximately the same as thojtfound for destalked Uca by Kleinholz and Bourguin (1941). Conclusive data on eyestalk control of water balance clearly do not exist. That this size increase is not the result of an increased synthesis of protein was shown by Koch (1952). After molting, crabs without eyestalks were deficient in nitrogen by an amount corresponding to their greater increase in linear growth. The total N content before ecdysis for destalked animals was not different from control animals before ecdysis. Koch concluded that the observed increase in volume after the firs t molt following eyestalk removal was the result of modified water balance and changed osmotic conditions. He suggested, as did Passano (I960), that altered elasticity of the newly formed 4 integument may play an important part in this increase in volume. Passano (1953) made the assumption that water balance-regulation and the molt-inhibition were two expressions of the same hormone. Accordingly, water balance might be accomplished by a low blood titre of the molt-inhibiting hormone. This hormone presumably comes from both the medulla interna and medulla externa neurosecretory cells, and the X-organ-sinus gland complex, since the former showed a molt delay effect when implanted into eyestalkless crabs. Animals without the X-organ-sinus gland complex showed an intermediate size increase so that the blood concentration of this hormone was too low to have a molt inhibiting effect but lessened the increased size seen when both eyestalks are removed. Carlisle (1955) gave evidence for two separate hormones, a water balance-regulation principle and a molt-inhibiting hormone. In Carcinus, an injection of sinus gland extract at stages D 3 or D reduced the size increase of both eyestalkless and normal crabs. 4 Since the extracts were prepared from animals that were molting frequently and, therefore, did not have molt inhibiting hormone present in the sinus gland, then the sinus gland factor concerned was not the molt-inhibiting hormone. To date, there has been no conclusive evidence that there is a principle in the eyestalk that controls the amount of water that an animal takes up at ecdysis. This investigation was undertaken to test for the presence of a water balance-regulating principle in the molting crab, Hemigrapsus nudus. It was decided that the molt cycle characteristic for this animal must fi r s t be investigated before any data on the control of water uptake during ecdysis were taken. Linear growth values for control and destalked crabs were determined following the firs t molt in the laboratory. Changes in wet body weight throughout the molt cycle were compared for control and destalked animals. A possible mechanis for the increased body weight found for destalked animals was investi-gated by measuring total osmotic pressure of blood from destalked and control animals during the molt cycle. 6 MA T E R I A L AND METHODS The crab? Hemigrapsus nudus, used in this study were collected from Spanish Bank, Vancouver, B r i t i s h Columbia. Only male crabs ranging in size from 5 - 2 5 grams were used in this study. The animals were kept in plastic containers (10 animals/container), separated by plexiglass dividers, immersed in either 100% or 75% sea water. Salinity is expressed as per cent of a standard sea water o arbitrarily set at 100%. This standard sea water contains 31.88 /oo salinity and 17.65°/oo chlorinity at 25 C. The animals were fed every day a mixture of cooked liver and oatmeal (Dall, 1964) which was frozen but defrosted when needed. Summer conditions with respect to water temperature and photoperiod were maintained in the laboratory throughout the year. The water temperature was maintained at 17°C 1°C and the animals were subjected to a photoperiod of 16 hours light and 8 hours dark. For the description of the molt cycle of H. nudus only control animals, animals which have not had their eyestalks removed, were used. The hardness of nine prescribed areas (Fig. 1) of the carapace (Hiatt, 1948) was recorded each day for approximately 30 unfed animals by touching each area of the carapace with a probe under a dissecting microscope. The length and distinguishing characteristics of the post-ecdysial and intermolt stages were thus determined. To establish the length of the proecdysial stages, the exoskeleton from 7 the dactylopodite of a thoracic appendage was removed and the distinguishing characteristics of the new exoskeleton being laid down were determined. There is a basic arrangement of the exoskeleton throughout the Arthropoda. The nomenclature of the various layers was proposed by Richards (1951) and a modification of his scheme will be followed here: 1. epicuticle - - nonchitinous calcified outer layer of proteins and lipids, proecdysial. 2. procuticle -- chitin-protein (i) exocuticle (pigmented layer of Brach, 1939), proecdysial, pigmented, and calcified. (ii) endocuticle -- postecdysial a. calcified portion (principle layer of Drach, 1939). b. uncalcified portion (membranous layer of Drach, 1939). Drach (1939) described five successive molt stages (A, B, C, D, E). A somewhat simplified version of Drach's scheme proposed by Carlisle and Dohrn (1952) will be used in this paper for purposes of discussion. Proecdysis, the stage which precedes the shedding of the exoskeleton, is the first of the four stages proposed by Carlisle and Dohrn. This stage corresponds to Drach's stage D. The next stage, ecdysis ("to cast off"), corresponds to Drach's stage E and consists of the actual shedding of the exoskeleton. The third stage, postecdysis, immediately follows, and corresponds to Drach's stage A, B, and Cj_3 The fourth stage is termed intermolt and corresponds to Drach's stage C4. 8 FIGURE 1 Outline drawing of the dorsal carapace of Hemigrapsus nudus showing areas of the carapace and width measurement (arrow). 9 To determine if animals without eyestalks increase in linear growth after the f i r s t molt in the laboratory, a linear growth value, lambda (Drach, 1939) was calculated for 34 control animals and 72 destalked animals. Width was measured to the nearest hundreth of a centimeter (S.D. + 0.04) with a vernier caliper between the most posterior dorsal spines of the carapace (Fig. 1). The width of each animal was measured before ecdysis (W ) and at least one day following ecdysis (Wn^^) and a lambda value calculated as follows: Wn+1 - W n x 100= lambda W v vn A l l lambda values will be expressed as per cent increase in cm. Changes in wet body weight throughout the molt cycle for control and destalked animals were obtained by carefully drying the animal with Kleenex tissue before weighing on a Mettler Multi-Purpose Balance, accurate to 0. 1 mg. When weighing molting animals (from 12 hours tj before ecdysis to 2 hours after ecdysis), it was necessary to lump the weight measurement into six time periods before ecdysis (720 - 361, 360 - 121, 120 - 60, 60 - 21, 20 - 11, 10 - 1 min before ecdysis) and four time periods after ecdysis (0, 1 - 30, 31 - 60, 61 - 120 min) since the precise time of ecdysis could not be determined until the animal actually shed the old exoskeleton. Weight measurements are expressed as per cent change in weight based upon a proecdysial weight determined several days after bringing the animal into the laboratory. 10 Baumberger and Olmsted (1928) showed that there was a large increase in total osmotic pressure just before ecdysis in Pachygrapsus  cfassipes. Both Koch (1952) and Passano (1953) suggested that the increased linear growth values seen for destalked animals may be the result of a modified water balance and of changed osmotic conditions. Since this phenomenon has never been analyzed in destalked animals, total osmotic pressure of the blood was measured for control and destalked animals by freezing point determinations according to the method described by Ramsay and Brown (1955). The accuracy of this technique was tested by measuring the freezing point depression on 15 samples of a lOOOniiliiosmole Fiske Standard, Bethel, Connecticut (mean freezing point depression, 1.929, S. D. jr_ 0.018). Osmotic pressure is expressed as per cent sea water. The freezing point de-pression of 25, 50, 75, and 100% sea water was determined and a regression line fitted through zero was calculated. A l l determinations of freezing point depression of blood serum were then read off this regression line in per cent sea water. Generally, blood was sampled from both destalked and control animals every second day for 10 days before ecdysis and 15 days after ecdysis. During the passive phase of stage E (when the animal gains 30 - 50% of its initial wet weight), blood was sampled at three specified periods in terms of the amount of weight the animal had gained: the first blood sample taken when the animal had gained 1 - 10% of the proecdysial wet weight; the second taken when the animal had gained 11 20 - 30% of the p r o e c d y s i a l wet weight; and the t h i r d sample taken when the a n i m a l had just e m e r g e d f r o m the old exoskeleton. The time p e r i o d between these three blood samples v a r i e d f r o m one a n i m a l to another but g e n e r a l l y the f i r s t two samples were approximately one hour apart and the t h i r d sample was taken approximately f i f t e e n minutes after the second sample. B l o o d was sampled by puncturing the damp-dried a r t h r o d i a l membrane p r o x i m a l to the coxopodite of either the f o u r t h or f i f t h walking l e g . A p p r o x i m a t e l y 30-60 NP of blood was p u l l e d into a drawn gla s s c a p i l l a r y tube (1.5 mm. I.D.) by means of a rubber mouth tube connected to the c a p i l l a r y tube. The sample was i m m e d i a t e l y t r a n s f e r r e d to a 250 Y~ polyethylene centrifuge tube and centrifuged for 30 m i n at 3000 g. One to two y of s e r u m was then c o l l e c t e d f r o m the c e n t r i f u g e d sample and the f r e e z i n g point d e t e r m i n a t i o n made. B l o o d f r o m these animals coagulates v e r y qu i c k l y but one to two y of s e r u m could always be c o l l e c t e d f r o m the cen t r i f u g e d sample. Both eyestalks were r e m o v e d by means of a s i l k t h r e a d l i g a t u r e a pplied around the base of the eyestalk and the wound was c a u t e r i z e d i m m e d i a t e l y after r e m o v a l . M o r t a l i t y after this o peration was n e g l i g i b l e . S t a t i s t i c a l a n a l y s i s of the data ;onl changes i n weight and osmotic p r e s s u r e during the molt c y c l e were c a r r i e d out. Orthogonal c o m p a r i s o n s were made between c o n t r o l and destalked a n i m a l s at thre e m a i n tim e p e r i o d s ( p r o e c d y s i s , e c d y s i s , p o s t e c d y s i s ) during the 12 molt cycle for both sets of data. A treatment sum of squares from a one way analysis of variance with t - 1 degrees of freedom may be partitioned to give t - 1 independent comparisons, each based on one degree of freedom. The comparison, Q, and the sum of squares for Q with unequal replication is defined as (Snedecor, p. 333): ss_ = (C. T. ) In. C 2 I I where C. = numerical constant when S n• C• = 0 i '— l l n^ = replicate totals for each comparison T^ = total over replicates for each treatment T^ SSq = sum of square for Q Two comparisons, andQ^, are orthogonal if §^ n^ = 0. The sum of squares for Q (with 1 degree of freedom) is the numerator and the error mean square from the analysis of variance is the denominator for an F test. Significance is considered at the 0.01 level of probability. Data on linear growth w«rc analyzed by a one way analysis of variance and Scheffe's test (1933) was performed to detect significance among any two of the treatment means. When an experiement involved only two treatment means, the data were analyzed by Student's t-test, and s i g n i f i c a n c e was c o n s i d e r e d at the 0. 01 l e v e l of p r o b a b i l i t y . 14 RESULTS 1. Description of the molt cycle for control Hemigrapsus nudus. The identifying characteristics and average length in days of the proecdysial stages (D^, I>2' ^3» ^4) a r e s u m m a r i z e d in Table 1. The identification of Stage D^, the first stage in preparation for ecdysis, is easily made by removal of the exoskeleton from the dactylopodite of a thoracic appendage and observing, in sea water under a microscope, "bumps" which push out the uncalcified membranous layer overlying them. According to Drach (1939) and Hiatt (1948) spines are secreted during this stage but because only the nonchitinized epicuticle is formed during D]_, these spines are closely applied to the surface of the dacty-lopodite. The length of this stage is difficult to determine since it is impossible to know just when these "bumps" were f i r s t formed. In at least two animals the length of D-^  was recorded to be six days. . Stage D2 is readily identified since the exocuticle (containing chitin-protein) has been secreted and both the spines and "bumps" of the dactylopodite are chitinized. Stage D3 is identified by the beginning of reabsorption or localized breakdown of the organic matrix along the epimeral suture in that part of the carapace above the leg base, the branchiostegites. Towards the latter part of this stage another well defined area of re-absorption becomes apparent in the dorsal side of the meropodite and 15 basipodite of the chelae. This area of reabsorption does not really become soft until 12 - 24 hours before ecdysis (stage D^). The epimeral suture splits along its entire length in Stage D^. The actual ecdysis or stage E was observed in approximately 30 animals. This phenomena in H. nudus does not appear to differ greatly from that described by Hiatt (1948) for Pachygrapsus or by Drach (1939) for Maia. The majority of the control animals molted in the laboratory between 12 noon and 9 p.m. Stage E can be divided into two parts; the passive phase, when the volume of blood is increasing by absorption of sea water (Robertson, I960), and the active phase, which is identified by the muscular activity of the animal pulling itself out of the old exoskeleton. There are many reports which state that animals preparing to molt (one to two days before) "will move but slightly" (Drach, 1939; Hiatt, 1948). This has not been found to be true for H. nudus, for even towards the end of the passive phase of stage E (when the animal has increased in weight by 10 - 20% of initial wet body weight) the animal moves readily in the plastic container and becomes excited if handled. The passive phase begins approximately 6 hours before ecdysis when the animal has increased in weight by 1 - 10% of the initial wet weight (Fig. 2). In an animal just at the beginning of the passive phase, the posterior edge of of the carapace is raised (Plate 1) to expose the first abdominal segment of the old exoskeleton. After the animal has increased in weight by 5 - 10% of the initial wet weight (Plate 2) the bright kelly-green exoskeleton of the new branchiostegite can be seen 16 T A B L E 1 The identifying characteristics of the proecdysial, postecdy sial, and intermolt stages described for control Hemigrapsus nudus. Stage No. of animals Av. length in days Identifying characteristics F i r s t stage in preparation for ecdysis; remove the dactylopdite, "bumps" can be seen forming. 3.3 Second stage in preparation for ecdysis; remove dactylopodite and chitinized spines can be seen pushing up through the membrane layer. B, 30 40 '5.4 12 - 24 hours Third stage in preparation for ecdysis; the beginning of reabsorp-tion along the epimeral suture of the branchiostegite. Weight increase of 1% of initial wet weight. E 30 Passive phase-12 hrs. Active phase-7 min. 42 2.4 F i r s t stage after ecdysis, all areas of the new exoskeleton can be easily depressed (++}. 28 3.3 Second stage after ecdysis, proto-gastric area of new carapace and exoskeleton of the chelae have hardened but easily depressed (+}. 17 4.3 Protogastric area of carapace and exoskeleton of chelae become rigid (-). B, 17 3.7 The anterior branchial region of the carapage becomes rigid but can be depressed (- to •&•). 17 T A B L E 1 (cont.) Stage No. of Av. length Identifying characteristics animals in days C i 17 7.7 Anterior branchial region becomes rigid (.-). C2 7 - A l l regions of the carapace are rigid except for the cardiac. - - A l l regions of carapace rigid C. 4 130 Control animals 4 L 0 28 Destalked animals Formation of the membranous layer of the exoskeleton. Hemigrapsus nudus at the beginning of the passive phase of Stage E when the animal has gained about 1% of the initial proecdysial wet weight. (x2) Hemigrapsus nudus during the passive phase of Stage E showing the branchio-stegite with the broken epimeral suture (x2) 19 Plate 3. Hemigrapsus nudus after the animal has gained 10-20% of the initial wet body-weight. Swollen pericardial sacs and the newly secreted dorsal carapace can be seen. (x2) Plate 4. Hemigrapsus nudus during the active phase of Stage E. The animal now pulls itself out of the old exoskeleton. Swollen pericardial sacs can sti l l be seen and the abdomen has already been pulled out of the old exoskeleton. (x2). 20 and there is a considerable fissure in the old brachiostegite caused by-rupture along the epimeral suture. Also, the fi r s t abdominal segment of the new exoskeleton can be seen at this time. Absorption of sea water continues, and approximately one hour prior to ecdysis the animal has increased in weight by 10 - 20% of the initial wet weight. Two to three millimeters of the new dark-colored dorsal carapace (Plate 3) can be seen at this time and the two pericardial sacs (extensions of the pericardial membrane) become markedly swollen. The abdomen usually is deflected down presumably to aid the animal in its removal from the old exoskeleton. The old carapace, raised along the epimeral suture by the swelling of the animal, makes an angle of approximately 30° with its initial position. The active phase of stage E now ensues. The duration of this period for H. nudus is approximately seven minutes. Blood is moved from one side of the body to the other. Drach (1939) states that these alternating depressions are the result of muscle contractions of the coxopodites and basipodites of the thoracic appendages which recur throughout the active phase as rhythmic depressions of the new dorsal carapace. As a result of these contractions the abdomen is fi r s t pulled out of the old exoskeleton along with the fourth and fifth thoracic appendages (Plate 4). As can be seen in Plate 4 the pericardial sacs are still quite swollen but as the thoracic appendages are pulled out these sacs deflate. The remaining thoracic appendages are pulled out, then the chelae, and the animal backs out of its old exoskeleton (the exuvium). Not only are all the appendages including the legs, gills, mouth-parts, and antennae withdrawn from the old exoskeleton but also the exoskeletal lining of the fore- and hind-gut which remain attached to the exuvium. The animal for the firs t 12 - 24 hours is "j e l l y - l i k e " and is supported only by the buoyancy of the water. To determine the average length and distinguishing charact-eristics of the postecdysial stages, the hardness of nine prescribed areas of the carapace was recorded for approximately 25 days after ecdysis according to a scheme used by Drach (1939), Hiatt (1948), and Kincaid and Scheer (1952). These results are summarized in Table 2. The identifying characteristics of the seven postecdysial stages (A^ £» ^1 2 3^  a n c ^ ^he intermolt stage (C4) and their average lengths are stated in Table 1. Stage A^ is easily identified since the parts of the exoskeleton laid down before ecdysis (the spicuticle and exocuticle) have not been mineralized and the animal is ++(the new exoskeleton very soft). In the latter part of stage A 2 the protogastric area of the new carapace and the exoskeleton of the chelae become more hardened but are not rigid (- to +). As a general rule the carapace regions, , mesogastric and urogastric, anterior branchial, and posterior branchial are + (new exoskeleton hardened but easily depressed) in stage B^ but, in 6 of the 17 animals, these regions were are firs t ++ and then towards the latter part of this stage became +. In stage B 2 the posterior branchial region is usually +, T A B L E 2 Relative hardness of nine areas,of the carapace recorded for '.control Hemigrapsus nudus. Stage Av. length in days proto gastric meso, uro gastric ant. bran. post, bran. cardiac chela walk, leg branchi-ostegite sternite A l 2 . 4 tt tt tt + + ++ tt tt + + A 2 3 . 3 . + + + - t t + + + ++ + + ++ B ; 4 . 3 — . + + + + to ++ ++; later ++; later + ++; later + 3 . 7 - + - to + + + + C i 7 . 7 - -to + - -to + + + + - to + c 2 ? - - - - - to + - - -c 3 ? - - - - - - -++, exoskeleton very soft; +, exoskeleton hardened but easily depressed; -, exoskeleton rigid however in 7 of the 17 animals recorded this region became more hardened but not rigid ( - to + ). The only morphological difference between stage C3 and stage C4 is the secretion of the thin noncal-cified layer of the endocuticle termed the membranous layer by Drach (1939). Four control animals and 6 destalked animals were observed to undergo two molts in the laboratory. The four animals averaged 160 days between the two molts (including the length of time in post-ecdysis plus the length of time in intermolt, stage G4) whereas the six destalked animals averaged 58 days between the two molts. If it is assumed that the average number of days between ecdysis and stage C3 is approximately 30 days, then the length of stage C4 for control and destalked animals is 130 days and 28 days respectively (Table 1). Data on control animals molting in the laboratory were obtained exclusively during the summer months. The four control animals observed to undergo two molts in the laboratory were collected in the early part of June, 1965, as proecdysial animals and then molted again approximately five months later. No control animals collected during the winter and early spring months (November - March) would initiate proecdysis in the laboratory even though the summer temperature (17°C), long photoperiod, and regular feeding periods were maintained. 24 2. Linear growth values (lambda) calculated for control and destalked Hemigrapsus nudus. Linear growth values calculated for many Decapods after each successive molt decrease with increasing initial width (Olmstead and Baumberger, 1923; Drach, 1939). Therefore, small crabs increase in wjdth at each molt by a larger amount than a large crab. The size range H. nudus used in these experiments was not large enough to show this relationship. A regression line calculated for linear growth values (lambda) as a function of initial width (W ) for control animals showed the slope to be not significantly different from zero. There was no decrease in the lambda values with increasing initial width. Initial width of control animals used in these calculations ranged from 2.33 cm to 2.90 cm (mean, 2.67 cm). Similarly, a regression line calcu-lated for lambda values as a function of initial width for the destalked animals also was not significantly different from zero, Initial width for the destalked animals ranged from 2. 05 cm to 2 .92 cm (mean 2.54 cm). Lambda values for control animals decrease significantly when maintained in the laboratory for varying lengths of time before ecdysis (Table 3). These results indicate that the lambda values from control animals molting during the fi r s t 10 days in the laboratory (Table 3, group 1) are significantly higher than for those animals molting anytime after the first 10 days (Scheffe's test). Lambda values calculated for T A B L E 3 Lambda values calculated for all control Hemigrapsus nudus molting during the summers of 1965 and 1966. Group Days in lab Year Mean lambda n before ecdysis value 1 1 - 10 1965 11.33 24 2 11 - 20 1965 8.88 13 3 11 - 20 1966 7. 61 21 control animals molting during the summer of 1965 and 1966 are not significantly different. The number of days in the laboratory before ecdysis does not significantly affect the lambda values calculated for destalked animals. The mean lambda values for those animals molting after 0-15 days in the laboratory is 11.49 (n = 8); the mean lambda value for those animals molting between 16 - 35 days in the laboratory is 12. 68 (n = 14). Animals were destalked during the summer of 1965 at three different stages of the molt cycle, C4, D^, and D 3 . The mean lambda values calculated for these three groups of animals (Table 4) are not significantly different (P - 0. 05). Linear growth also does not signif-icantly differ in destalked animals collected during summer and winter, or in animals maintained in 100% and 75% sea water. Lambda values calculated for all destalked animals in Table 4, therefore, can be pooled and the overall mean is 12.38 (Table 5). Comparison of lambda values from a l l destalked animals with lambda values from control animals molting after 10 days in the lab-oratory (Table 3, pooling groups 2 and 3) shows the difference to be significant, lambda values from destalked animals being higher (Table 5). If lambda values calculated for those control and destalked animals molting during the first 10 days in the laboratory are compared, there is no significant difference. Removal of both eyestalks in molting H. 27 T A B L E 4 Lambda values ca lcula ted for destalked Hemigrapsus nudus mol t ing during two consecutive yea rs and for animals mainta ined in two sa l in i t i e s . Group Stage of Yea r Sal ini ty M e a n Lambda n Des ta lking Value 1 C 4 summer 1965 100% 12.82 12 2 D l summer 1965 100% 12.90 5 3 D 3 summer 1965 100% 11.12 7 4, C4 summer 1966 100% 12.44 29 5 c 4 winter 1965 100% 11. 91 11 6 C 4 winter 1965 75% 12.93 8 2 8 nudus leads to an i n c r e a s e i n s i z e only when it is p e r f o r m e d at le a s t 10 days before e c d y s i s . P r i o r to that, the destalked animals act as con t r o l s i n t e r m s of l i n e a r growth. The lambda values for both c o n t r o l and destalked animals d e c r e a s e after two mol t s i n the l a b o r a t o r y . The average lambda value f o r the f i r s t and second molt i s stated i n Table 6 for both c o n t r o l and destalked a n i m a l s . 3. Wet weight changes during the molt c y c l e f o r c o n t r o l and destalked H e m i g r a p s u s nudus. D a i l y v a r i a t i o n i n wet weight f o r non-molting animals was det e r m i n e d f o r c o n t r o l and destalked H. nudus. A group of a p p r o x i -mately 15 i n t e r m o l t c o n t r o l a n i m a l s were weighed every day f o r 10 days and these data were e x p r e s s e d as per cent change i n wet weight f r o m Day 1 i n the l a b o r a t o r y . The mean per cent change c a l c u l a t e d f o r each day never exceeded 1% and the confidence i n t e r v a l s were not sig n i f i c a n t l y d i fferent f r o m z e r o . E y e s t a l k s were r e m o v e d f r o m another group of 10 i n t e r m o l t animals and weighed every day for 10 days. The data were c a l c u l a t e d i n the same manner as for c o n t r o l a n i m a l s . The mean per cent change f or each day never exceeded 1% and confidence i n t e r v a l s around the mean of Day 1 and Day 10 were not c o n s i d e r e d different f r o m z e r o . Neither c o n t r o l nor destalked a n i m a l s show any significant change i n weight during intermolt, at le a s t over the 10 day e x p e r i m e n t a l p e r i o d . 2 9 T A B L E 5 Comparison of mean lambda values for control and destalked Hemigrapsus nudus molting after 10 days in the laboratory. P value states the probability from a t-table of no difference between control and destalked lambda values. Control lambda Destalked lambda P value value mean 8. 10 12.38 0. 01 n 34 72 30 Approximately 10 control and 10 destalked animals were weighed each day during the molt cycle: for 8 days before ecdysis, during ecdysis, and for 15 days after ecdysis. The mean per cent change in wet weight (based on an intermolt wet weight measurement) for each time period during the molt cycle is plotted in Figure 2. Since it has been shown that destalked animals have a significantly higher lambda value? than control animals if the eyestalks are re-moved prior to 10 days before the firs t molt in the laboratory, it was desired to establish if destalked animals absorbed more water during ecdysis. The per cent change in wet weight for control and destalked animals during proecdysis is essentially zero, the mean values for destalked animals always being a little higher (Fig. 2). An orthogonal comparison of values from destalked and control animals during proecdysis shows the difference to be insignificant (Table 7). The majority of the change in weight during the molt cycle occurs during the short period before and after ecdysis. It can be observed in Figure 2 that during ecdysis, the values for the per cent change in weight for destalked animals are consistently higher than the values for control animals. The per cent change in wet weight for destalked animals during the passive phase of stage E (720 - 1 min before ecdysis) and after ecdysis (0 - 120 min) is significantly higher than the values for changes in weight calculated 31 T A B L E 6 Average lambda values for control and destalked Hemigrapsus nudus followed through two molts. Experimental Av. lambda value Av. lambda value Days between condition for molt 1 for molt 2 molt 1 and molt 2 control 10.09 8.02 160 destalked 11. 12 7.65 38 32 FIGURE 2 Per cent change in wet weight, expressed as per cent of the intermolt wet weight, during the molt cycle of control and destalked Hemigrapsus nudus. Each point represents the mean of the measurements of at least 10 animals for each time period. \ Per Cent Change In Wet Weight for control animals (Table 7). Most of the loss in weight observed in Figure 2 just after ecdysis is due to the loss of the exuviae. Although the mean weight of the exuvium (P n) for control and destalked animals (expressed as a per cent of the wet weight 10-1 min. before ecdysis) is 45.8% and 33.6% respectively (Table 8), they were not found to be signif-icantly different. Control animals show a 36% loss in weight after Ecdysis (Fig. 2), the calculated loss in weight due to the exuvium, 46%. The control animals therefore must have increased in weight by 10% just before emerging from the old exoskeleton. Destalked animals show a 52% loss in weight after ecdysis (Fig. 2), the mean exuvium weight accounting for 34% of the drop (Table 8). These animals must have lost 18% of their already "absorbed water" when emerging from the old exoskeleton. It has been previously suggested (Carlisle, 1955) that animals without eyestalks have a greater uptake of water during ecdysis than control animals. To get a measure of the amount of water that control and destalked animals absorb at ecdysis, an R value (Drach, 1939) was calculated for those control and destalked animals that had been weighed before and after ecdysis (Table 8). The weight of the animal one day after ecdysis (P ) has been taken minus the weight of the n + 1 animal one day before ecdysis (P n) plus the rejected exoskeleton (P n). An R value is then calculated expressed as per cent of the T A B L E 7 Orthogonal comparisons of per cent weight changes during the three main time periods of the molt cycle. SS is tested over the E r r o r Mean Square from a one way analysis of variance. P values state the probability of no difference between control and destalked values at the three specified time periods. Time period of Control Destalked molt cycle C i T i (% wt. C i S T i (% wt. SS P change in change in (EMS=58.44) grams grams Proecdy sis +144 113 -53. 0 -113 144 +71. 6 0. 05 0.10 (Day 8 - Day 1 before ecdysis) Ecdysis 1. Passive phase +67 47 772.4 - 47 67 1425.0 645.62 0. 01 of stage E (12 hours before ecdysis) 2. Just after +32 35 47.3 - 35 32 293.4 1021.54 0. 01 ecdysis ( 0 - 2 hours after) Postecdysis +85 106 3276.0 -106 85 3544.0 2745.23 0. 01 (Day 1 - Day 15 after ecdysis) T A B L E 8 Weight of water absorbed during exuviation, R, for control and destalked Hemigrapsus nudus, expressed as per cent of absorbant mass (Drach, 1939). Experimental P n P n l O - 1 Pn+1 Pn Pn as R condition ( g m ) { g m ) ( g m ) ( g m ) % of P 1 0 - l Control range 3.01 - 17.38 4.34 - 21.66 3.31 - 17.12 1.86 - 11.35 30.2 - 59.6 87.1 - 309.5 n 21 9 21 21 9 21 x 8.52 10.29 9.24 5.58 45.8 214.3 Destalked range 3.60 - 10.24 6.99 - 12.87 4.48 - 13.87 1.77 - 6.78 22.0 - 53.5 150.0 - 362.0 n 17 6 17 17 6 17 x 6.95 10.04 8.23 4.26 33.6 205.9 P n = wet weight 12 - 24 hours before ecdysis •^nlO-l = wet weight 10-1 minutes before ecdysis Pn+1 = wet weight 12 - 24 hours after ecdysis p n = wet weight of exuvium ~ Pn = absorbent mass R = ( P n * l - P n • Pn) 1 0 0  P n " P n absorbant mass, P n - (Drach, 1939). Comparison of R values for control and destalked animals is not significant, therefore, destalked animals do not have a greater uptake of water at ecdysis. This is verified by comparing the wet weight measurements of control and destalked animals one day after ecdysis (Figs. 2 and 3). Again, the change in wet weight for destalked animals one day after ecdysis is not significantly different from that of the controls. It would appear in Figures 2 and 3, however, that the destalked animals are taking up more water than control animals at the different time periods during ecdysis; and, indeed, these differences have been shown to be signif-icant. There are two factors mentioned above which can explain this apparent discrepancy. F i r s t , the destalked animals lose 18% of their "already absorbed" water just before ecdysis, whereas, the control animals have been shown to gain 10% more water just before ecdysis. Secondly, the control animals absorb more water than the destalked animals after ecdysis (Figs. 2 and 3, up to 24 hours after). Therefore, it is actually the time during which the water is absorbed that differs in these two groups of animals. Destalked animals absorb more water before ecdysis, control animals absorb more water after ecdysis, up to 24 hours after. Am orthogonal comparison was made between the data on per cent change in weight during the postecdysial period for control and destalked animals (Table 7). The per cent change in weight for destalked 3 7 FIGURE 3 Absolute wet weight change during ecdysis calculated from Figure 2 for a 1 0 gram control and a 1 0 gram destalked animal. Time periods on the abcissa are as follows: P n = wet weight 1 2 - 2 4 hours before ecdysis, P n ]_Q_I = w e ^ weight 1 0 - 1 minutes before ecdysis, Pn^ .Q = w e t weight at time zero after ecdysis, Pn^ .]_ = wet weight 1 2 - 2 4 hours after ecdysis. Time Intervals in Days animals is shown to be significantly higher than that of the control animals. Regression lines were calculated for the data on per cent change in weight from control and destalked animals during this postecdysial period. Both regressions are significantly different from zero; therefore, both control and destalked animals increase in weight with increasing time after ecdysis. Both the slope and the intercept of the destalked regression were calculated and are signif-icantly higher than those of the control regression line. Therefore, the greater linear growth values for destalked animals must be realized during this postecdysial period. 4. Total osmotic pressure measurements during the molt cycle for control and destalked Hemigrapsus nudus. The increased size and weight observed during postecdysis in destalked animals may be due to changed osmotic conditions (Koch, 1952). Osmotic pressure of blood, therefore, was measured for at least 10 control and 10 destalked animals during the molt cycle (Fig. 4). In order to get a measure of the daily variation of osmotic pressure during intermolt it was decided to determine a baseline osmotic pressure for control and destalked animals in the intermolt stage. Blood from 20 control animals, having been maintained in the laboratory for four days at 100% salinity, was sampled five times over a two day period: 0, 4 hours, 12 hours, 24 hours, and 48 hours. There was no significant change in osmotic pressure over this two day period. An overall mean and confidence interval were calculated from these 100 measurements of osmotic pressure, and this baseline for control animals is represented if Figure 4. Approximately 20 intermolt animals were destalked immediately after collection and maintained in the laboratory at 100% salinity for four days. Measurements of osmotic pressure were made at the same intervals used for the control group, and there was found a significant decrease is osmotic pressure over the two day interval. This decrease in osmotic pressure with time may be due to the destalked condition of the animals and will be discussed later. Since it was necessary to have a baseline osmotic pressure for destalked animals in intermolt, these 96 measurements of osmotic pressure were pooled and an overall mean and confidence interval were calculated (Fig. 4). The osmotic pressure baseline for destalked animals in intermolt is significantly higher than the osmotic pressure baseline for control animals during intermolt. There is a trend for the measurements of osmotic pressure from control animals during proecdysis to be above the osmotic pressure baseline for control animals (Fig. 4), however, most of the means are not significantly higher, due to the variability in osmotic pressure found for animals in a molting condition. Osmotic pressure measure-ments made on destalked animals during proecdysis are generally lower than the intermolt baseline for these animals. Again, the Yariation around each point is large and only four of the means during the proecdysial period are significantly lower than the osmotic pressure baseline calculated for destalked animals. Comparison of osmotic 40 FIGURE 4 Osmotic pressure measurements during the molt cycle for summer control and destalked Hemigrapsus nudus in 100% sea water. Each point represents the mean of the measurements of at least 10 animals for each time period. The solid and dashed lines marked at 110. 6 and 113. 6 per cent sea water on the graph represent control and destalked intermolt baselines calculated as explained in the text. The solid lines above and below these baselines represent confidence intervals (P = 0.01). Time periods during ecdysis are specified periods in terms of the amount of weight the animal has gained: 0-10, 1 - 10% of the proecdysial wet weight; 20 - 30, 20 - 30% of the proecdysial wet weight. Osmotic pressure is expressed as per cent sea water. pressure for control and destalked animals during proecdysis by orthogonal comparisons (Table 9) shows the difference to be significant, the destalked osmotic pressure is consistently lower than that of control animals. A large increase in osmotic pressure is characteristic of animals preparing for ecdysis (Fig. 4). Blood was sampled from seven control animals during a time period 12-5 hours before ecdysis and the osmotic pressure was essentially the same as Day 1 before ecdysis. The first sample of blood during the ecdysial period was taken when the animal had gained 1 - 10% of the proecdysial wet weight, 6-1 hours before shedding the old exoskeleton. The increase in osmotic pressure seen in Figure 4 therefore must occur some time after five hours before ecdysis. A decrease in osmotic pressure during the ecdysial period can be seen in Figure 4. To check the possibility that the technique of removing blood from the animal at the three already specified time intervals during ecdysis may itself have-changed the osmotic pressure of the blood, 10 intermolt control animals and 10 intermolt destalked animals were sampled at zero, one hour, and one hour and fifteen minutes. Analysis of the data from control animals showed that there was no significant change in osmotic pressure during the three periods. Analysis of the data obtained from destalked animals showed that there was a significant decrease in the osmotic pressure of blood during the three experimental time periods. Possible reasons for these results T A B L E 9 Orthogonal comparisons of osmotic pressure measurements during the three main time periods of the molt cycle for control and destalked Hemigrapsus nudus collected during the summer and maintained in 100% salinity. SS^ is tested over the E r r o r Mean Square from a one way analysis of variance. P values state the probability from an F table of no difference between control and destalked values. Time period of molt cycle Control Destalked Zn^ S T j ( M i l l i - Ci 2. Tj ( M i l l i osmoles) oSmoles) SS EMS=1017.68) Proecdysis (Day 10-day 1 before ecdysis) +21 102 103769 •51 42 41337 32651.24 0.01 Ecdysis (Passive and active phase of stage E) + 37 28 29660 •28 37 38643 3529.15 0.10 Postecdysis (Day 1-day 15 after ecdysis) +110 117 172508 •177 110 105574 14941.82 0.01 43 from destalked animals will be discussed later. The decrease seen in osmotic pressure during the ecdysial period for control animals and probably for destalked animals results from sea water that is absorbed during ecdysis. Osmotic pressure from destalked animals during ecdysis is not significantly different from control animals during ecdysis (Table 9). It should be noted, however, that the osmotic pressure from destalked animals is generally lower than the osmotic pressure from control animals. One day after ecdysis there is a large fal l in osmotic pressure for both groups of animals due to absorbed water which is approximately 200% of the absorbent mass Pj^ - f^n (Table 8). Osinotic pressure measured during the postecdysial period for both control and destalked animals is significantly lower for most of the time periods than the respective intermolt baselines (Fig. 4) . When determinations of osmotic pressure for control and destalked animals during the post-ecdysial period are compared by orthogonal comparisons (Table 9), the osmotic pressure of blood from destalked animals is significantly lower than that from control animals. In summary, the osmotic pressure values from destalked animals are consistently lower than those from control animals, and the differ-ence is significant in the pro- and postecdysial stages. It should also be noted that there is greater different between osmotic pressure values measured for control and destalked animals during proecdysis than during postecdysis. The postecdysial differences are significant, but they are not as large (Table 9). 4 4 Total osmotic pressure of the blood was measured during the molt cycle of destalked H. nudus molting during the winter in 100% sea water (Fig. 5). Comparison of the osmotic pressure data during the molt cycle for summer and winter destalked animals by orthogonal comparisons shows that the difference is not significant (Table 10). As was mentioned previously, control animals collected during the winter would not initiate proecdysis in the laboratory so that there are no data for control animals molting during the winter. Osmotic pressure was measured also for destalked winter animals maintained in 75% salinity throughout the molt cycle. (Fig, 6)'. The same general changes in osmotic pressure seen in destalked animals maintained in 100% salinity over the molt cycle are found in destalked animals in 75% salinity. If the mechanism for the increase in osmotic pressure just before ecdysis is to maintain the blood at a certain level relative to the external medium before absorbing water, then one might expect a smaller change in osmotic pressure just before ecdysis in an animal that is more hypertonic to the external medium. However, animals in 100% sea water and animals in 75% sea water increase the osmotic pressure of blood by 8% sea water just before ecdysis. 45 F I G U R E 5 O s m o t i c p r e s s u r e m e a s u r e m e n t s d u r i n g the m o l t c y c l e f o r w i n t e r d e s t a l k e d H e m i g r a p s u s nudus m a i n t a i n e d i n 100% s a l i n i t y . E a c h p o i n t r e p r e s e n t s the m e a n of the m e a s u r e m e n t s of a p p r o x i m a t e l y 5 a n i m a l s f o r e a c h t i m e p e r i o d , o O s m o t i c p r e s s u r e i s e x p r e s s e d i n p e r c e n t s e a w a t e r . 105 ' 7 6 5 4 3 2 I 0-20-Just I 2 3 4 5 © 7 10 30 out I Proecdysis i-Ecdysisn Posfecdysis-Time in Days T A B L E 10 Orthogonal comparisons of osmotic pressure measurements during three main time periods of the molt cycle for summer destalked animals (from Table 9) and winter destalked animals maintained in 100% salinity. SS^is tested over the E r r o r Mean Square from a one way analysis of variance. P values state the probability from an F table of no difference between summer and winter values. Time period of molt cycle C. I Control jn. S. T. (milli-oJmoles) C. I Destalked ^ £ T . (mil l i -oJmoles) SS P • (EMS=958.48) Proecdysis (Day 7-Day 1 before ecdysis) +30 31 30540 -31 30 29575 6.88 0.10 Ecdysis (Passive and active phase of stage E) +19 37 38643 -37 19 19821 17.92 0.10 Postecdysis (Day 1-Day 7 after ecdysis) +27 57 55038 -57 27 25944 403.01 0.10 4 7 FIGURE 6 Osmotic pressure measurements during the molt cycle for winter destalked Hemigrapsus nudus maintained in 7 5 % sea water. Each point represents the mean of the measurements of approximately 6 animals for each time period. Osmotic pressure is expressed in per cent sea water. 10 9 8 7 6 5 4 3 2 I 0- 20- Just 1 2 3 4 5 10 30 out Proecdysis i-Ecdysis-j-PostecdysisH Time in Days 48 DISCUSSION The effect of bilateral eye stalk removal at least 10 days before ecdysis in Hemigrapsus nudus is a significant increase in size after the firs t molt in the laboratory and an increase in wet weight during early postecdysis. This increase in wet weight after ecdysis is assumed to be due to an increased absorption of water since the animals are not feeding during the early part of postecdysis (Passano, I960), and i s , in all probability, a consequence of a modified water balance. These data are consistant with those of Koch, 1952; Passano, 1953; Carlisl e , 1955; and others, who postualted the existence of a water balance-regulation principle located in the eyestalk. The hormonal influence of the eyestalks regulates the early postecdyseal absorption of water (Koch, 1952). There are two possible mechanisms that could act to cause this increased absorption of water in destalked animals. F i r s t , there could be an increase in the osmotic pressure of blood during ecdysis. Or secondly, the absorbent surface of the destalked crab could become more permeable to sea water either during or after ecdysis. Comparison of osmotic pressure measurements show that there is no significant difference in the changes in blood osmotic pressure measured during ecdysis for control and destalked animals. The absorbent surface of the destalked animals therefore may become more permeable to sea water after ecdysis. The lower osmotic pressure measured on the blood of destalked animals during pro- and postecdysis suggests an increased permeability of the absorbent surface, however destalked animals do not show a significant increase in wet weight until after ecdysis. The difference between osmotic pressure meas-ured for control and destalked animals during postecdysis is not as large as the difference seen during proecdysis, again suggesting that destalked animals have absorbed more water during postecdysis. A change in size after ecdysis is measured by a linear growth value (Lambda), a per cent increase in width based on the initial width. These values for destalked animals are significantly larger only when the eyestalks are removed at least 10 days prior to ecdysis If the animals are destalked within 10 days before ecdysis (during stage or D3), the destalked animals act as controls in terms of linear growth. Passano (1953) found that eyestalks from Uca must be removed at least 6 days prior to ecdysis. Presumably, during this c r i t i c a l time period before ecdysis (10 days for H. nudus), which may be species specific, there is enough water balance-regulating hormone present in the destalked animals to prevent larger linear growth values. When comparing linear growth values for control and destalke animals, it is important that the animals have been maintained in the laboratory for the same length of time before ecdysis. Lambda 50 values calculated for control animals significantly decrease if the animals are maintained in the laboratory for more than 10 days before ecdysis. Due to the absence of the water balance-regulating hormone in the eyestalk, the destalked animals do not show this decrease in linear growth. (Drach: (1939) discusses the influence of captivity on linear growth at ecdysis in Maia squinado . After a certain time period, eight days for those animals molting in August, the lambda values for Maia begin to decrease and the longer the animals are maintained in the laboratory before ecdysis, the smaller the linear growth values. Control H. nudus used in the calculation of the data on changes in weight during the molt cycle (Fig. 2) had been in the laboratory an average of 10 days before ecdysis. Animals that had been in the laboratory a longer period of time (15 - 25 days before ecdysis) should have been used in order for these data to be comparable with the values calculated for destalked animals, main-tained in the laboratory an average of 23 days before ecdysis. Greater differences between the values for changes in weight for control and destalked animals may have resulted, if one assumes that animals with smaller lambda values gain less weight during and after ecdysis than animals with larger lambda values. Ecdysis results in an increase in size due to., at first, absorp-tion of sea water, tissue growth not occurring until late postecdysis [Ci - C 3,(Drach, 1939; Passano, I 9 6 0 ) . Relatively few data exist on the site of water uptake during and just after ecdysis. Control H. nudus absorb 33% of the intermolt wet weight in sea water at the peak of swelling during ecdysis. Drach (1939) has shown that most of the sea water that is absorbed by the animal during ecdysis enters through the lining of the foregut. He removed the entire dorsal carapace of the old exoskeleton from Carcinus maenas, Cancer pagurus and Maia squinado in late stage D^. Only a short time before ecdysis did the operated animals begin to swell; therefore, water must not be absorbed to any degree through the newly secreted exoskeleton. The possibility of water entering through the gill membranes also was rejected by Drach since the gills remain covered by the old exo-skeleton until the thoracic appendages are withdrawn from the exuvium. Robertson (I960) believes that water is absorbed in Carcinus maenas through the foregut and hepatopancreas. Several H. nudus were put into sea water with amaranth dye and allowed to gain about 20 - 30% of their initial weight.- The swollen animal was then sacrificed. The cardiac stomach was markedly swollen and amaranth dye was detected inside the stomach. Passano (I960) states that during ecdysis, water is swallowed, increasing the hydrostatic pressure in the lumen of the digestive tract (both stomach and hepatopancr eas), and the combined effects of filtration and osmotic uptake cause water to pass into the blood. Drach (1939) has pointed out that there must be a change in the permeability of the absorptive surface in order to explain the 52 beginning of the passive phase of stage E. He proposed that this change in permeability occurs when the old exoskeleton lining of the digestive tract is separated from the new lining which occurs just at the beginning of stage E. A histological study and permeability measurements should be made on the gastric lining before, during, and after ecdysis to verify this hypothesis. It would be interesting to test if eyestalk extracts act on the permeability of this membrane. L i m (1917) showed that intermolt Carcinus can tolerate distilled water for a longer length of time than postecdysial animals. This is consistent with Drach's hypothesis that the absorbent surface becomes more permeable during stage D 4 . The absolute amount of water that control and destalked H. nudus absorb during ecdysis has been shown to be not significantly different. The calculated R values for H. nudus are high when comparing them with those calculated for three species by Drach (1939). F o r Maia, which has an average linear growth of about 39%, the R value is 180. For Xantho floridus , which has a linear growth value similar to H. nudus (lambda, 15), the R value is approximately 106. Thus, for a linear growth of :15%, the absorbent mass ( P n - p n) is doubled after ecdysis. For H. nudus, with a linear, growth of approximately 10%, the absorbent mass increases by four times. Drach implies that the expected range for R values depends on the linear growth for a particular animal and that these values can not be predicted for any particular species. That the calculated R value for H. nudus is unreasonably high may be due to an error in the wet weight measure-ments of the exuviae. The exuvium was always damp dried with a Kleenex tissue and weighed immediately after ecdysis. A signif-icant amount of water could sti l l be present in the appendages causing an abnormally high weight. Travis (1954) and Guyselman (1953) have calculated the weight of water absorbed during ecdysis for Panulirus and Uca respectively. Travis, using the same method as Guyselman, arrives at a value of about 10% of the inital wet weight during intermolt. Her calculation is incorrect since the amount of water absorbed from Day 1 before ecdysis to 5 - 15 minutes before ecdysis is not taken into account. This time period represents an increase in wet weight of approxi-mately 10% so that the total amount of water that is absorbed during ecdysis for Panulirus should be 20% of the initial intermolt wet weight. Although Guyselman does not state the time before ecdysis that his initial wet weight was recorded, he calculates a value for the weight of water absorbed by Uca to be 34. 5% of the initial wet weight. Accord-ing to his method, H. nudus absorbs 65% of the initial wet weight in water. Robertson (1937) reports an uptake of 70% of body weight for Carcinus. The uptake of water at ecdysis is probably the result of an increase in osmotic pressure of the blood just before ecdysis. Baumberger and Olmsted (1928) and Baumberger and D i l l (1928) found that the osmotic pressure of tissue fluid from Pachygrapsus crassipes and Callinectes sapidus "about to molt" increased by 350 milliosmoles over the external medium. P a r r y (1953) also has shown an increase in osmotic pressure of blood from Ligia oceanica during ecdysis. H. nudus shows an increase of approx-imately 200 milliosmoles over that of the external medium. The major weakness in the work of Baumberger and Olmsted is the fact that the tissue fluid used in the freezing point determinations was obtained by squeezing the animal and therefore, the fluid examined consisted of a mixture of blood and juices fromthe hepatopancre as and muscle. The proportion of organic to inorganic molecules in the muscle must be different from that found in the blood (Shaw, 1955, 1958). Baumberger and D i l l (1928) did investigate a possible mechanism for the increase in osmotic pressure before ecdysis. It had been suggested previously by Baumberger and Olmsted (1928) that the conversion of stored glycogen to sugar and lactic acid at the time of ecdysis may be an important factor but they were not able to find any large increase in total sugar in C. sapidus about to molt either in the hepatopancreas or in whole crab minus hepatopancreas. Renaud (1949) states that the glycogen content of the hepatopancreas increases from 3% of dry weight at C4 to 6% of dry weight in in Cancer. In animals approaching or about to molt (stage D^ - D^) the glycogen content dimishes rapidly and he finds an increase in blood glucose which parallels the beginning of the passive phase of stage E. Drilhon (1935) shows an abrupt increase in the blood glucose level in Maia . Whether these increases in blood glucose are large enough to raise the osmotic pressure during proecdysis is not known. Both total blood protein and blood lipid increase prior to ecdysis in Maia (Drilhon, 1935) and the increase in osmotic pressure may be the result of greater total blood concentrations of these two materials. However, Travis (1955) has shown that total blood protein in Panulirus reaches a pea£Js increase four days before ecdysis, at least three days before any significant change in osmotic pressure. Robertson (I960) presents data to show that the plasma of C. maenas is hyperosmotic in the proecdysial stages by as much as 7.5% and that this increase in osmotic pressure is due mainly to an increase in Na, Ca, Mg, and CI ions over the intermolt values. He states that the contribution of organic substances such as proteins, free amino acids and lactic acid in the plasma is unimportant, yet he does not measure free amino acids or lactic acid in proecdysial animals Control H. nudus during proecdysis show blood which also is hyper-osmotic to the intermolt osmotic pressure baseline. It appears that the animals used by Robertson were animals in stage D^ - D^, whereas the greatest increase in osmotic pressure found in H. nudus occurs about 2 - 5 hours before the passive phase of stage E. It is possible that the increase in osmotic pressure seen in control H. nudus during p r o e c d y s i s (p o s s i b l y r e g u l a t e d by a p r i n c i p l e in the eyestalk) i s due to an i n c r e a s e d c o n c e n t r a t i o n of ions but the i n c r e a s e seen just p r i o r to e c d y s i s i s probably due to some v e r y different mechanism, not c o n t r o l l e d by the eyestalk. Data p r e s e n t e d i n this paper support this hypothesis. O s m o t i c p r e s s u r e f r o m destalked a n i m a l s during p r o e c d y s i s is s i g n i f i c a n t l y lower than osmotic p r e s s u r e f r o m c o n t r o l a n i m a l s , whereas the i n c r e a s e i n osmotic p r e s s u r e just before e c d y s i s is the same for both c o n t r o l and destalked a n i m a l s . C l e a r l y the m e c h a n i s m for the i n c r e a s e i n o s m o tic p r e s s u r e during p r o e c d y s i s and at e c d y s i s needs f u r t h e r investigation. T h e r e is some suggestion f r o m the data p r e s e n t e d here that a p r i n c i p l e i n the eyestalk may be inv o l v e d in maintaining a constant osmotic p r e s s u r e . D e s t a l k e d H. nudus i n i n t e r m o l t i n 100% sea water a r e not capable of maintaining a constant o s m o tic p r e s s u r e either when sampled at dai l y i n t e r v a l s or when sampled at i n t e r v a l s of zero, one hour, and one hour and fi f t e e n minutes. T h e r e is a significant d e c r e a s e i n osmotic p r e s s u r e with time when blood was sampled f r o m i n t e r m o l t destalked a n i m a l s . T h i s was never found for c o n t r o l a n i m a l s , Whether this p r i n c i p l e is the same as the water b a l a n c e - r e g u l a t i n g p r i n c i p l e can not be de t e r m i n e d f r o m the above r e s u l t s . The d e c r e a s e i n osmotic p r e s s u r e c h a r a c t e r i s t i c of destalked a n i m a l s may be due to a decrease in total i o n i c content of the blood or an i n c r e a s e i n the amount of water or both. However, a significant wet weight i n c r e a s e was not observed in destalked intermolt animals over a 10 day period. The water balance-regulation principle in H. nudus may or may not be the same as described in Carcinus by Ca r l i s l e (1955). He states that eyestalk ablation always leads to a greater and quicker uptake of water during the molt, which has not been found in H. nudus. These destalked Carcinus apparently increased in size 180% whereas the control animals only increased at each molt 80%. Carlisle further states "an extract of sinus glands of whatever provenance into a crab which is in stage D 3 or. invariably results in a reduced intake of water at the succeeding molt, whether the recipient is intact or eye-stalkless". Carlisle does not state what period during ecdysis he finds this decreased water uptake but he implies that he is using that period one day before to one day after ecdysis. If this assumption is correct, there does not seem to be a principle in the eyestalk of H. nudus which regulates the amount of water that the animal absorbs during ecdysis. These data do not rule out the possibility that the size increase observed in the destalked animals may be the result of an altered elasticity of the newly formed exoskeleton. The animals absorb water until the exoskeleton is completely stretched, the destalked exo-skeleton being more elastic and therefore absorbing more water. In order to prove conclusively that water balance after ecdysis is due to a hormonal mechanism located in the eyestalk, the optic ganglia or X-organ-sinus gland complex!: must be implanted into animals whose eye-58 stalks have been removed at least 10 days before ecdysis. These animals with implants should then act as control animals in terms of size increase and changes in wet weight after ecdysis. 59 SUMMARY 1. A description of the molt cycle for control Hemigrapsus  nudus has been made including the average lengths and identifying characteristics of the seven postecdysial stages ( A ] ( 2 j &1,2> G l 2,3)» the intermolt stage (C4), and the four proecdysial stages (Dj^ 2,3,4)• 2. Removal of both eyestalks in molting H. nudus leads to an increase in size only when it is performed at least 10 days before ecdysis. P r i o r to that, the destalked animal act as the controls in terms of size increase. 3. Salinity (100%, 75%) or season does not significantly affect linear growth values calculated for destalked animals. 4. The average lambda value for control and destalked animals decreases after the second molt in the laboratory. 5. Comparison of the amount of water (R value) that control and destalked H. nudus absorb at stage E (ecdysis) shows the difference to be not significant, control and destalked animals absorb the same amount of sea water at ecdysis. 6. Destalked animals show a significantly greater increase in wet weight during early postecdysis. The larger linear growth values calculated for destalked animals must be realized at this time. 7. Osmotic pressure measured during stage E (ecdysis) is the same for control and destalked animals. 60 8. Comparison of osmotic pressure data for control and destalked animals in 100% sea water during proecdysis and postecdysis show the difference to be greater during proecdysis, the osmotic pressure from destalked animals always being significantly lower than that of control animals during both pro- and postecdysis. 9. Osmotic pressure measured for destalked animals during the summer is not significantly different than osmotic pressure measured for destalked animals during the winter. 10. Destalked animals in 75% sea water show the same general changes throughout the molt cycle as those animals maintained in 100% sea water. 11. There is postulated a water balance-regulation principle located in the eyestalk which regulates the early postecdysial absorption of water. The absorbent surface of the destalked crab may become more permeable to water just after ecdysis, thus causing an increase in wet weight and an increase in size. The smaller difference in osmotic pressure between control and destalked animals seen during postecdysis is consistent with this hypothesis. 61 L I T E R A T U R E CITED Abramowitz, R. K. and A. A. Abramowitz, 1940. Moulting, growth and survival after eyestalk removal in Uca  pugilator. B i o l . B u l l . 78:179-188. Bauchau, A. G. , 1948. Phenomenes de croissance et glande sinusaire chez Eriocheir sinensis H. M. Edw.. Ann. soc. roy. zool. belg., 79:125-131. Baumberger, J. D. and D. B. D i l l , 1928. A study of the glycogen and sugar content and the osmotic pressure of crabs during the molt cycle. Physiol. Zool., 1:545-549. Baumberger, J. D. and J. M. D. Olmsted, 1928. Changes in the osmotic pressure and water content of crabs during the molt cycle. Physiol. Zool., 1:531-544. Bli s s , D. E. , 1951. Metabolic effect of sinus gland or eyestalk removal in the land crab, Gecarcinus lateralis. Anat. R e c , 111:502-503. Bl i s s , D. E. , 1953. Endocrine control of metabolism in the land crab, Gecarcinus lateralis (Freminville). I. Differences in the respiratory metabolism of sinus glandless and eystalkless crabs. B i o l . Bull. 104:275-296. Brown, F. A. and O. Cunningham, 1939. The influence of the sinus gland on normal viability and ecdysis. Bi o l . B u l l . , 77:104-114. Carlisle, D. B., 1955. On the hormonal control of water balance in Carcinus. Pubbl. staz. zool. Napoli, 27:227-231"! Carl i s l e , D. B. and P. F. R. Dohrn, 1952. Sulla presenza di un ormone d'accrescimento in un crostacea decapode, la Lysmata seticaudata Risso. Ric. s c i . , Torino, 23:95-100. Dall, W. , 1964. Studies on the physiology of a shrimp, Metapenaeus mastersii (Haswell) (Crustacea: Decapoda: Penaeidae). I. Blood constituents. Aus. J. Mar. Freshw. Res., 15:145-161. 62 Drach, P., 1939. Mue et cycle d'intermue chez les Crustaces Decapodes. Ann. inst. oceanog. (Paris) (N. S.), 19:103-391. Drilhon, A. , 1935. Etude biochimique de la mue chez les Crustaces Brachyoures (Maia squinado). Ann. Physiol. Physiochim. B i o l . , 11:301-326. Edwards, G. A., 1950. The influence of eyestalk removal on the metabolism of the fiddler crab. Physiol. Comp. et Oecol. , 2:34-50. Guyselman, J. B. , 1953. An analysis of the molting process in the fiddler crab, Uca pugilator. B i o l . B u l l . , 104:115-137. Hiatt, R. W. , 1948. The biology of the lined shore crab Pachygrapsus crassipes Randall. Pacific Sci., 2:135-213. Kincaid, F. D. and B. T. Scheer, 1952. Hormonal control of metabolism in crustaceans. IV. Relation of tissue composition of Hemigrapsus nudus to intermolt cycle and sinus gland. Physiol. Zool. , 25:372-380. Kleinholz, L. H. and E. Bourquin, 1941. Effects of eyestalk removal on decapod crustaceans. Proc. Nat. Acad. Sci. 27:145-149. Koch, H. J. A., 1952. Eyestalk hormones, post moult volume increase and nitrogen metabolism in the crab Eriocheir sinensis (M. Edw.). Mededel. Koninkl. vlaam. Acad. Wetenschap. Belg. , 14:3-11. Lim, R. K. S. , 1917. Period of survival of the shore crab (Carcinus maenas) in distilled water. Proc. Roy. Soc. Edinb., 38:14-23. Megusar, F. , 1912. Experimente uber den Farbwechsel der Crustaceen. Arch. Entwecklungsmech. Organ. , 33:462-665. Olmsted, J. M. D. and J. P. Baumberger, 1923. F o r m and growth of grapsiod crabs. A comparison of the form of three species of grapsoid crabs and their growth at molting. J. of Morph. , 38:279-294. 63 Parry, G. , 1953. Osmotic and ionic regulation in the isopod crustacean L i g i a oceanica. J. Exp. Bi o l . 30:567-574. Passano, L. M., 1951a. The X organ-sinus gland neurosecretory system in crabs. Anat. Rec. 111:502. Passano, L. M. , 1951b. The X organ, a neurosecretory gland controlling molting in crabs. Anat. Rec. 111:559. Passano, L. M. , 1953. Neurosecretory control of molting in crabs by the X-organ sinus gland complex. Physiol. Comp. et Oecol., 3:155-189. Passano, L. M. , I960. Molting and its control. In "The Physiology of Crustacea. " (T. Waterman, ed.}, Vol. I, Academic Press, New York. Ramsay, J. A. and R. H. J. Brown, 1955. Simplified apparatus and procedure for freezing-point determinations upon small volumes of fluid. , J. of Sci. Instrum. 32:372-375. Renaud, L. , 1949. Le cycle des reserves organiques chez les Crustaces Decapodes. Ann. inst. oceanogr. (Paris) (N. S.), 24:260-357. Richards, A. G. , 1951. The Integument of Arthropods. University of Minnesota Press, Minneapolis, p. 147. Robertson, J . D. , 1937. Some features of the calcium metabolism of the shore crab Carcinus maenas (Pennant). Proc Roy. Soc.B, 124:162-182"! Robertson, J. D. , I960. Ionic regulation in the crab Carcinus  maenas (L.) in relation to the moulting cycle. Comp. Biochem. Physiol., 1:183-212. Scheffe, H. , 1953. A method of judging a l l contrasts in the analysis of variance. Biometrika, 40:87-104. Scudamore, H. H. , 1947. The influence of the sinus glands upon molting and associated changes in the crayfish. Physiol. Zool. , 20:187-208. 64 Shaw, J. , 1955. Ionic regulation in muscle fibres of Carcinus maenas. I. Electrolyte composition of single fibres. J. Exp. B i o l . , 32:383-396. Shaw, J. , 1958. Osmoregulation in the muscle fibres of Carcinus maenas. J. Exp. B i o l . , 35:920-929. Smith, R. I. 1940. Studies on the effects of eyestalk removal upon young crayfish (Cambarus cl a r k i i Girard). Bi o l . B u l l . , 79:145-152. Snedecor, G. W., 1956. Statistical Methods. The Iowa State University Press, Iowa, 534 p. Travis, D. F. , 1954. The molting cycle of the spiny lobster Panulirus argus La t r e i l l e . I. Molting and growth in laboratory-maintained individuals. Bio. Bull. 107:433-450. Travis, D. F. , 1955. The molting cycle of the spiny lobster, Panulirus argus Lat r e i l l e . III. Physiological changes which occur in the blood and urine during the normal molting cycle. B i o l . Bull., 109:484-503. 

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