Neuroendocrine involvement in osmoregulation in the grapsid crab Metopograpsus messor

Comp. Biochem. Physiol., 1969, Vol. 28, pp. 665 to 674. Pergamon Press. Printed in Great Britain

NEUROENDOCRINE INVOLVEMENT IN OSMOREGULATION IN T H E GRAPSID CRAB METOPOGRAPSUS MESSOR* KENNETH

N. K A T O t and F R E D I. K A M E M O T O

Department of Zoology, University of Hawaii, Honolulu, Hawaii, U.S.A. (Received 9 3uly 1968)

Abstract--1. The grapsid crab Metopograpsus messor regulates both hypo- and hyperosmotically in sea-water (SW) concentrations ranging from 25 to 125%. Isosmoticity is approached at approximately 80% SW. The urine osmotic concentration is slightly higher than that of blood in all salinities, the urine/ blood ratios ranging from 1'03 to 1.07. 2. The ligation of the eyestalks causes a rapid decrease in the blood osmotic concentration when the animals are placed in 25~o SW. This drop can be prevented in part by the injection of eyestalk homogenates. 3. The decrease in the blood osmotic concentration in eyestalk-ligated animals is presumably due to an increased water influx. 4. The results support the idea of an eyestalk-ventral ganglion involvement in osmoregulation in crabs. INTRODUCTION SCUDAMORE (1947) demonstrated that the disturbed ecdysial water metabolism of eyestalkless crayfish was attributable to the absence of the sinus glands. Since then, a neuroendocrine regulation of ecdysial water balance in crustaceans has been suggested by a n u m b e r of investigators (Bauchau, 1948; Guyselman, 1953; Carlisle, 1956; Passano & Jyssum, 1963; Rangaro, 1965). Bliss et al. (1966) have proposed that in the crab Gecarcinus lateralis ecdysial water volume is controlled by a non-nervous antidiuretic hormone and a diuretic hormone from the brain and ventral ganglionic mass. Recently, K a m e m o t o et al. (1966) have provided evidence for a neuroendocrine regulation of salt and water "homoeostasis" in two species of crustaceans, the crayfish Procambarus clarkii and the grapsid crab Metopograpsus messor. Unlike the other investigators, they have centered their interests on the mechanisms which provide for the maintenance of intermolt animals in environmental media which markedly differ from that of the animals' blood. In the crab M . messor, they demonstrated that eyestalk ablations resulted in decreased blood osmotic concentrations when the animals were placed in hypo-osmotic media, and in increased * Supported by Grant GB-673 from the National Science Foundation and Public Health Service PredoctoraI Fellowship 1-F1-Gm-32-613-01 from the National Institute of General Medical Sciences. )t Predoctoral Fellow. Present address: Department of Physiology and Biophysics, University of Illinois, Urbana, Illinois, U.S.A. 665

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KENNETH N. KATOAND FRED I. KAMEMOTO

blood osmotic concentrations when the animals were placed in hyperosmotic media. M t h o u g h the apparent alteration in the osmotic concentrations of the blood was effected by the bilateral removal of the eyestalks, they suggested that a substance from the ventral ganglionic mass was the causative agent for these changes. T h e y postulated that the eyestalk component was effective in inhibiting the production or the secretion of a ventral ganglionic permeability factor thereby maintaining the permeability characteristic of the normal animals; eyestalk removal resulted in the release of the permeability factor which increased the permeability of the body surfaces. T h e i r hypothesis was based on the finding that the injection of ventral ganglionic homogenates or the implantation of whole ventral ganglia into normal animals resulted in changes of the blood osmotic concentrations toward that of the ambient medium; responses which were identical to those resulting from bilateral eyestalk ablations. I n this paper, an attempt is made to substantiate the earlier findings and to elucidate the neuroendocrine regulation of water metabolism in M . messor. MATERIALS AND METHODS The grapsid crab Metopograpsus messor (Forskfil) is abundant in the coastal waters of Oahu, Hawaii, and was collected from under rocks and debris in the intertidal zones. All animals were maintained in holding tanks at least 24 hr before experimental use. Although M . messor is a semiterrestrial

crab, the experimental animals were kept completely submerged in recirculating sea water. This was found to be necessary to control the varying degrees of desiccation the animals would have undergone had they had the opportunity to be out of water for any length of time. The animals were provided with adequate aeration and were maintained under this condition for many weeks without apparent ill effects. The animals were placed under a light rtgirne of 12 hr light and 12 hr dark. Only animals in the intermolt stage weighing within a range of 5-8 g were used. Animals were randomly selected without discrimination to sex. Blood samples were collected in calibrated microliter capillary pipets by puncturing the arthrodial membrane between the fourth and fifth thoracic appendages. For all quantities of blood reported herein, each sample was withdrawn from a single animal. For the collection of urine, the animals were wrapped in gauze to remove water from the body surfaces. An absorbent gauze plug was placed against the mouth to absorb all of the water ejected from the branchial chambers. The operculum covering t h e nephropore was gently lifted with a fine hooked needle and the flow of urine was collected with a finely drawn capillary tube. The urine was then expelled onto a piece of parafilm and the desired volume was taken up by capillarity into the calibrated capillary pipets. Eyestalk ablation is easily accomplished by first ligating the eyestalk at its base, followed by the severing of the stalk distal to the ligature. Because complete eyestalk ablations invariably resulted in some mortality, such ablations were avoided. Instead, the eyestalk was securely ligated with cotton thread and left as such. The ligated animals were 100 per cent fatality free, and displayed effects similar to those of the ablated animals. Ligated animals were placed individually in small perforated plastic slide-coverslip boxes to restrain them and to discourage cannibalism. Eyestalk-ligated animals were immediately returned to 100% sea water (sea water with an osmotic concentration approximately equal to the osmotic concentration of 550 mequiv. NaC1/l) and kept for at least 12 hr before further experimental use. Sea-water dilutions were made by adding appropriate amount of tap water to 100% sea water (SW). The concentrated medium was made by the addition of calculated amounts of

O S M O R E G U L A T I O N I N T H E G R A P S I D CRAB M E T O P O G R A P S U S M E S S O R

667

rock salt to 100~o SW. The osmotic concentrations of the varying concentrations of sea water were determined with an osmometer and were found to be within the desired dilutions or concentrations. Osmotic concentrations were measured on a Fiske osmometer. The instrument was calibrated against standard NaCI solutions to operate within a range of 0-1000 mOsm. All samples of blood and urine were diluted accordingly to fall within this range. Blood osmotic concentrations were determined on solutions made by diluting 20/~1 of blood with 180/~1 of distilled water. The diluted samples were thoroughly mixed and chilled. The relatively small sample size was found to be convenient for serial sampling of individual animals. Urine samples were also diluted and treated as the blood. A single animal usually provided a sufficient volume of urine for a single determination; however, in some cases it was necessary to pool urine samples from two or three animals. To estimate the influx of water under varying experimental conditions, the nephropores were bilaterally sealed by carefully applying a drop of Eastman 910 adhesive (Armstrong Cork Co.) under each operculum. In all cases, the adhesive provided a permanent impervious seal. Weight changes were then determined at the desired intervals after the animals were placed in 25% SW. Eyestalk homogenates were made of the dissected neural components of the eyestalk. The retinal pigments were removed as completely as possible. The soft tissue from twenty eyestalks were homogenized in crab physiological saline (Robertson, 1953 ; Lockwood, 1962) to yield a total volume of 0"5 ml. The sample was sonicated with an ultrasonic probe for 10 sec. The probe and the sample test tubes were chilled in ice packs before each sonication period. The tissue homogenate was then centrifuged at 8000 g for 5 min. Fifty /xl (an equivalent of two eyestalks) of the supernatant were then injected into the thoracic sinus of each animal through the arthrodial membrane separating the fourth and fifth thoracic appendages. The control animals were injected with 50/xl of crab physiological saline. RESULTS M . messor regulates b o t h h y p o - and hyperosmotically in sea-water concentrations ranging f r o m 25% to 125% (Fig. 1). I n 100% and 125% SW, the animal regulates hypo-osmoticaUy. Below the concentration of 80% SW, M . messor regulates hyperosmotically. Within the range of 25-125% SW, the osmotic concentration of the blood fluctuates b y 14%. T h e urine osmotic concentration is slightly higher than the osmotic concentration of the blood for all environmental salinities. Serial sampling of blood and urine of a group of animals subjected to 25% S W reveals that a new "steady state" in the blood osmotic concentration is achieved within 6-12 hr (Fig. 2). T h e new "steady state" is maintained approximately 5 - 6 % lower than the normal blood osmotic concentration for up to 48 hr. T h e data for serial sampling of bilaterally eyestalk-ligated animals are presented in Fig. 3. Within the first 6-12 hr after transfer to 25% SW, the blood osmotic concentration drops approximately 11 per cent below that of ligated animals in 100% SW. At 36 hr, the ligated group sustained 40 per cent mortality; the osmotic concentrations of the surviving animals are scattered over a fairly wide range. T h e ligated animals did not survive 48 hr in 2 5 % SW. T h e blood osmotic concentrations of bilaterally eyestalk-ligated animals subjected to 25% S W for 12 hr are significantly lower than those of normal animals subjected to the same treatment (Table 1). Injections of eyestalk

600

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Urine (n=lO)

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Blood (n=lO)

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Water)

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450

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N

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i

25

50

75

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FIG. 1. Osmotic concentrations of blood and urine of M . messor in varying sea-water concentrations. Mean and standard error presented.

500

NORMAL

(25 % S.W.)

Z

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~

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(n = IO)

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12

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1

36

48

FIo. 2. Osmotic concentrations (mean + S.E.) of blood and urine of normal animals after transfer from 100% to 25% SW. 668

669

O S M O R E G U L A T I O N I N T H E G R A P S I D CRAB M E T O P O G R A P S U S M E S S O R

L IGATED

(25

% S.W.)

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S.W.)

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FIO. 3. Osmotic concentrations (mean +_S.E.) of blood and urine of eyestalk-ligated animals after transfer from 100% to 25% SW.

TABLE

1----OsMoTIC

CONCENTRATIONS

OF

BLOOD

IN

NORMAL~

EYESTALK-LIGATED

EYESTALK-LIGATED--EXTRACT-INJECTEDM . messor arrEa 12 hr IN 25% SW Osmotic concentration (mequiv. NaC1/I) No.

Treatment

of

animals

Mean

S.E.

Normal

9

454

+ 2.3

Eyestalk-ligated

8

421

+ 2"6

P

0.001 0.005 0.025 Eyestalk-ligatedextract-injected

10

436

+ 4.6

AND

670

KENNETHN. KATOAND

FRED

I. KAMEMOTO

homogenates into ligated animals placed in 25% SW significantly retard the decreases in blood osmotic concentrations. T h e increases in weights of normal and eyestalk-ligated animals placed in 25% S W after the occlusion of the nephropores are illustrated in Fig. 4. T h e r e are greater increases in weights of the ligated animals as compared to normal 25%

Sea

Water

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cm

!

o rn

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1

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Llgated

(n=5)

0

0

Normal

(n=5)

1

I

!

I

I

3

6

12 Hours

FIG. 4. Weight change in normal and eyestalk-ligated animals after occlusion of nephropores and transfer from 100% to 25% SW. TABLE

2--WEIGHT

INCREASE

OF NORMAL, EYESTALK-LIGATED EXTRACT-INJECTED M . messor

AND

EYESTALK-LIGATED--

% Weight increase No.

Treatment

of

animals

Mean

S.E.

Normal

12

6"0

+ 0-50

Eyestalk-ligated

12

9-1

+ 0"50

7

8'9

+ 1"34

P 0"001 0'025

Eyestalk-ligatedextract-injected

Nephropores sealed and animals placed in 25% SW for 12 hr.

OSMOREGULATIONIN THE GRAPSIDCRABMETOPOGRAPSUS MESSOR

671

controls. After 12 hr, the weight increases of ligated animals are significantly higher than those of the controls (Table 2). Eyestalk homogenate injections into ligated animals are ineffective in preventing these weight increases. DISCUSSION The results presented in this paper demonstrate that M . messor is an excellent osmoregulator. When animals are transferred from 100°/0 to 125% SW, the mean blood osmotic concentration is increased by 8% whereas there is only a 6% change in animals transferred from 100% to 25% SW. This may suggest that M . messor is a better hyperosmotic regulator than it is a hypo-osmoregulator. The ability of this animal to regulate both hyper- and hypo-osmotically does not necessarily suggest that the one process is the reciprocal process of the other. The osmotic concentration of the urine is slightly higher than the osmotic concentration of the blood, but follows the same trend with either the concentration or the dilution of the medium. The production of the apparent hyperosmotic urine may not be a significant osmoregulatory function of the antennary gland, inasmuch as the urine/blood (U/B) ratios are not much greater than one (Table 3). TABLE 3--URINE/BLOOD RATIOS OF OSMOTIC CONCENTRATIONS OF M . messor SUBMERGED IN VARYING SEA WATER CONCENTRATIONS FOR 24 h r

Sea-water concentrations

Urine/blood

25%

50%

100%

125%

1-03

1'04

1"06

1"07

Ten animals/group. In most species of crabs, the urine is isomotic with the blood (Nagel, 1934; Prosser et al., 1955; Beadle, 1957; Gross, 1963, 1964; Potts & Parry, 1964; Gross et al., 1966). According to Dehnel & Stone (1964) the antennaI glands of H e m i grapsus nudus and H. oregonensis serve an osmoregulatory function only in the wintering condition. It appears, however, that the antennal glands of brackishwater species function in ionic regulation, removal of waste matter and possibly the control of body fluid volume. No clear evidence for an osmotic function of the antennal glands has been presented except for the specific situation reported by Dehnel & Stone (1964). Serial sampling of animals subjected to 25% SW reveals that a new "steady state" is attained within a relatively short time. This rapidity in the adjustment to a "new" environment is not uncommon, but is rather a general pattern of the osmotic changes observed in a number of animals (Prosser & Brown, 1961). This suggests that the greatest activity in the adaptation of an animal to cope with a

672

KENNETH N . KATO AND FRED I. KAMEMOTO

change in environmental salinity must occur within the first few hours and perforce necessitates the investigation on osmoregulatory mechanisms to be focused at this time. Bilateral eycstalk ligations affect the osmotic concentration of the blood. Animals which are returned to 100~o SW after ligation have increased blood and urine osmotic concentrations tending toward that of the external concentration. When the ligated animals are placed directly into 25°/~ SW, there is a sharper drop in the osmotic concentration of the blood as compared to that of non-ligated animals. Eyestalk homogenate injections into ligated animals prevent this great decrease. The difference between the blood osmotic concentrations of the ligated controls and ligated-eyestalk-injected animals is statistically significant (P = 0.025). Complete maintenance of the osmotic concentrations of ligated animals injected with eyestalk homogenates, however, is not obtained. It appears then that the greater and more rapid decrease in the blood osmotic concentration of ligated animals placed in 25~/o SW is primarily due to the removal of the eyestalk components. Physiologically, a decrease in the blood osmotic concentration of ligated animals may result from an alteration of the permeability characteristics of the animals resulting in a greater influx of water and/or greater loss of salts, a decrease in the active uptake of salts, and/or an increase in urine production resulting in a greater net loss of salts. Alteration in the permeability characteristics of an animal may be demonstrated by measurements of water influx and efllux. The change in water flux of an animal is manifested in either an increase or decrease in the total body weight if urine flow is controlled and there is no change in the volume of the digestive system. An evaluation of the changes in the permeability characteristics of M. messor is made by recording the percentage changes in the body weights of eyestalk-ligated and normal animals placed directly into 25% SW. The contribution of the antennal glands to water balance is eliminated by the bilateral obstruction of the nephropores. It is assumed that for these short-term experiments, ligation has no effect on the drinking and defecating capabilities of the animals. Ligated animals with sealed nephropores show greater weight changes (water influx) when placed in 25% SW than do normal controls. After 12 hr, the mean of the weight changes of ligated animals is 9 per cent as contrasted to a 6 per cent increase shown by the normal animals. The difference is statistically significant at the 0.001 level of probability. The rate of urine production in M . messor is not known. Attempts at the direct continuous collection of urine to determine urine flow were unsuccessful. Urine production, however, might be extrapolated from the data presented in Fig. 4. The increase in weight resulting from the apparent influx of water with the occlusion of the nephropores might represent the amount of urine normally eliminated by the animal inasmuch as there is no weight increase in animals if the nephropores are not occluded. In normal animals, there is approximately a 3 per cent increase in weight in the first 3 hr after the animals aFe placed in 25% SW. This might be interpreted as the amount of urine produced by the animal in

O S M O R E G U L A T I O N I N THE G R A P S I D CRAB ~ I E T O P O G R A P S U S M E S S O R

673

3 hr in terms of percentage of body weight. In the eyestalk-ligated animal, there is a 5 per cent increase in the body weight in 3 hr under similar conditions. This suggests that the ligation of the eyestalk also results in the increase in urine formation, perhaps in response to the increased influx of water as has been suggested for the crayfish by Kamemoto et al. (1966). Kamemoto & Ono (1967) found, by a continuous collection of urine in the crayfish, that the urine flow rate increased by twofold after the bilateral ligation of the eyestalks. Similar results were reported for the crab Eriocheir sinensis by de Leersnyder (1967). Such an increase in urine flow after eyestalk ligation might also contribute to the decreased osmotic concentration of the blood, for, as was demonstrated (Table 3), blood and urine are essentially isosmotic. The present data corroborate the hypothesis of Kamemoto et al. (1966). Presumably, the imposition of ligatures at the bases of the eyestalks isolate the eyestalk inhibitor (neural or humoral) which inhibits the ventral ganglion from elaborating or releasing a permeability substance. The removal of this inhibition results in the expression of the ventral ganglionic substance, thereby increasing the permeability of the body surfaces to water. Thompson (1967) has found that in the fresh-water crab Pseudothelphusa jouyi the removal of the eyestalks results in an increased permeability to tritiated water. The effect of the ventral ganglion on the permeability of the foregut of Gecarcinus lateralis has been demonstrated in vitro by Mantel (1967). She has noticed that in in vitro preparations of the foregut, the addition of ventral ganglionic extract to the hemolymph side results in an increased permeability of the foregut to water and salts. Although these data fit within the eyestalk-ventral ganglion pathway scheme, they do not preclude the possibility that the eyestalk component functions directly as an antagonist to the ventral ganglionic factor. There remains the possibility that the eyestalk might also contain a substance which acts directly on the body surfaces of the animal in decreasing permeability. If such is the case, the permeability characteristic of the animal may be determined by the factor which has the highest titer in the blood. REFERENCES BAUCHAUA. G. (1948) Intensit6 du m~tabolisme et grande sinusaire chez Eriocheir sinensis. Ann. Soc. roy. Zool. Belg. 79, 73-86. BEADLE L. C. (1957) Comparative physiology: osmotic and ionic regulation in aquatic animals. A. Rev. Physiol. 19, 329-358. BLISS D. E., WANG S. M. E. ~¢ MARTINEZE. A. (1966) Water balance in the land crab, Gecarcinus lateralis, during the intermolt cycle. Am. Zool. 6, 197-212. CARLISLED. B. (1956) On the hormonal control of water balance in Carcinus. Pubbl. Staz. Zool. Napoli 28, 227-231. DEHNEL P. A. & STONE D. (1964) Osmoregulatory role of the antennary gland in two species of estuarine crabs. Biol. Bull., Woods Hole 126, 354-372. DE LEERSNYDERM. (1967) Le milieu int~rieur d'Erioeheir sinensis H. Milne-Edwards et ses variations--II, t~tude exp6rimentale. Cahiers de Biologie Marine 8, 295-321. GROSSW. J. (1963) Cation and water balance in crabs showing the terrestrial habit. Physiol. Zool, 36, 312-324.

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GROSS W. J. (1964) Trends in water and salt regulation among aquatic and amphibious crabs. Biol. Bull., Woods Hole 127, 447-466. GROSS W. J., LASIEWSKIR. C., D~NNIS M. & RUDY P. (1966) Salt and water balance in selected crabs of Madagascar. Comp. Biochem. Physiol. 17, 641-660. GUYSELMANJ. B. (1953) An analysis of the molting process in the fiddler crab, Ucapugilator. Biol. Bull., Woods Hole 104, 115-137. KAMEMOTO F. I., KATO K. N. & TUCKER L. E. (1966) Neurosecretion and salt and water balance in the Annelids and Crustacea. Am. Zool. 6, 213-219. KAMEMOTO F. I. & ONO J. K. (1967) T h e effects of eyestalk ligation on salt and water balance in the crayfish, Procambarus clarkii. Am. Zool. 7, 723. LOCKWOOD A. P. M. (1962) Osmoregulation of Crustacea. Biol. Rev. 37, 257-305. MANTEL L. H. (1967) T h e foregut of Gecarcinus lateralis as an organ of water balance. Am. Zool. 7, 765. NACEL H. (1934) Die Aufgaben der Exkretionsorgane und der Kiemen bei der Osmoregulation yon Carcinus maenas. Z. vergl. Physiol. 21, 468-491. PASSANO L. M. & JYSSUM S. (1963) T h e role of the Y-organ in crab proecdysis and limb regeneration. Comp. Biochem. Physiol. 9, 195-213. PORTS W. T. W. & PARRY G. (1964) Osmotic and Ionic Regulation in Animals. Pergamon Press, Oxford, England. PROSSEa C. L. & BROWN F. A. (1961) Comparative Animal Physiology. W. B. Saunders, Philadelphia. PROSSER C. L., GREEN J. & CHOW T. J. (1955) Ionic and osmotic concentrations in blood and urine of Pachygrapsus erassipes acclimated to different salinities. Biol. Bull., Woods Hole 109, 99-107. RANCARO K. (1965) Isolation and partial characterization of the molt-inhibiting hormone of the crustacean eyestalk. Experentia 21,593-594. ROBERTSON J. E. (1953) Further studies on ionic regulation in marine invertebrates, ft. exp. Biol. 30, 277-296. SCUDAMORE n . H. (1947) T h e influence of the sinus gland upon molting and associated changes in the crayfish. Physiol. Zool. 20, 187-208. THOMPSON L. C. (1967) Osmotic regulation in the Mexican fresh water crab, Pseudothelphusa jouyi Rathbun. Am. Zool. 7, 735.