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Changes in fluid transport across the perfused midgut of the freshwater prawn, Macrobrachium rosenbergii during the molting cycle
CHANGES IN FLUID TRANSPORT ACROSS THE PERFUSED MIDGUT OF THE FRESHWATER PRAWN, ~~CR~~R~C~~U~ R~~EN~ER~~~, DURING THE MOLTING CYCLE DONALD L. MYKLES* and Department
of Zoology,
University
GREGORY
of Hawaii
at Manoa.
A. AHEARN
Honolulu.
HI 96822. U.S.A.
Abstract-l. Net water flux was measured across the perfused midgut of Mrrcrohrclc.hiun~ rosenhergii during different stages of the molting cycle. 2. Compared to fluid transport rates exhibited during intermolt. fluxes were elevated in animals approaching ecdysis. 3. However. fluxes were much reduced in postmolt individuals. 4. The si8nifi~ance of these findings in relation to salt and water balance of the organism is discussed.
INTRODUCTION
The phenomenon of water absorption at ecdysis has been described in many decapod crustaceans (Drach, 1939; Corrivault & Tremblay, 1948: Travis, 1954: Robertson, 1960: Haefner, 1964; Dali, 1974), which leads to an increase in hemolymph volume (Drach, 1939: Born. 1970; Dali, 1974). The major route of water absorption at ecdysis is believed to be across the lining of the digestive tract (Drach, 1939; Passano, 1960; Robertson, 1960). The u~trastructure of the midgut lining of the shrimp ~ac~o~~ac~~~rn ~ose~ber~ij and Penaeus marginatus (Cooke, 1976; Cooke & Ahearn, 1976; Ahearn & Maginniss, 1977) and the crab Pachygrapsus crassipes (Mykles, 1977) resembles that of other epithelia capable of active transport of ions and water (Berridge & Oschman, 1972). In addition, Ahearn et al. (1977) have shown that the perfused midgut of intermolt ~uero~~~c~j~~ rosenbergji actively transports a fluid from lumen to serosa. Thus it appears that the midgut is the principal part of the digestive tract where water is absorbed into the hemolymph at ecdysis. The purpose of this study is to investigate possible changes in net water transport across the perfused midgut of ~aero~racb~um rosetlbergii that may occur during the molting cycle.
at 23 f I C. The perfusate contained inulin (carboxYI-‘~C) (ICN. 1.4 pCi/mg) (0.25 pCi/ml) and 0.10 mM unlabelled inulin to eliminate absorption of radioactivlty to the walls of the tubing. Usually six 5-min collections of perfusate effluent were obtained from each preparation. Experiments were completed within 1 hr after removal of the midgut from the animal. An aliquot of serosal bath was taken after completion of the experimental period to check for leakage of label, which was usually much less than 1.5”,, of the perfusion rate. Effluent volumes were determined gravimetrically. I&tilled water (2 ml) and lOm1 Aquasol scintillation cocktail mix (NEN) were added to each sample before counting in a Beckman LS-230 liquid scintillation spectrometer. Classification of molt stages were based generally on the work of Drach (1939) and Scheer (1960). and more speclfitally on the criteria of Peebles (1977) as applied to Mornbruchium rosenhergii. In the present study. stage C, comprised hard intermolt animals. stage D were animals in proecdysis (D, to D,). stage A contained animals which had molted the night before measurement. and stage B animals were individuals 3-4 days postecdysis. Sixty-three effluent collections from 11 stage C, (intermolt) midgut preparations, 59 collections from 1 I stage D preparations. 23 collections from five stage A preparations and 17 collections from three stage B preparations were obtained in this investigation.
RESULTS
MATERIALS
AND METHODS
Measurement of net water flux employed the method of Burg & Orloff (19681as modified by Ahearn er ai. (1977) for ~ucr~br~~~i~~~ rosenbrrgii. Briefly. the change in perfusate concentration of a non-absorbed volume marker, in this case “C-inulin. after passage through the midgut yields information on both the magnitude and direction of net water transport. The midgut was mounted in a lucite chamber containing physiological saline (Ahearn & Maginniss, 1977; Ahearn ec al.. 1977) and perfused with saline containing the volume marker at a rate of 8~i~~l;min * Present address: Department of Zoology, University of California. Berkeley. CA 94720, U.S.A. and Bodega Marine Laboratory. Bodega Bay. CA 94923. U.S.A. <.R.P. 61 4~
H
Significant alterations in transepithelial net water transport were observed in the perfused midgut of Macrobruchium roserhergii during the molting cycle (Fig. 1). Preparations from intermolt (stage C,) animals had a net water flux of 35.3 + 2.0&m’ per hr from mucosa to serosa. This rose to 55.8 & 5.1 pi/cm’ per hr in preparations from individuals in proecdysis (stage D). Within 12 hr after ecdysis (stage A) net water flux was far below the intermolt level (7.9 _t 4.5 PI/cm2 per hr). By 3-4 days following ecdysis (stage B), net water flux had returned to intermolt levels (31.3 + 5.3 &cm2 per hr). Analysis of variance (Zivin & Bartko. 1976) indicates that the group means vary significantly with stage of the molting cycle (P -C 0.01). According to the Newman-Keuls 643
Mo.t
stage
Fig. 1. Net water Hux across the mrdgut of Mlrcrohrcicltiiof~ rowtrherqii during different stages of the molting cycle (mean f I SE.). Number of effluent collections for each group given III parentheses. Molt stases arc defined in the text test (Zivin & Bartko. 1976). al! the group means are significantly different from one another (P < 0.01). except the means of stage C, and stage B animals.
This is the first report of changes in net fluid transport across the decapod midgut during different stages of the molting cycle and provides evidence that this portion of the gut is a major site of water absorption at ecdysis. The increased net water flux in proecdysis by Mncrobrachium midgut appears to anticipate water absorption by the animal since swelling due to water uptake is not observed until late proecdysis (stage II,). Rapid increases in weight in other crustaceans approaching ecdysis are not observed until stage D4 (Drach, 1939: Travis, 1954). It is possible that even greater rates of Ruid transport may be obtained in preparations from animals undergoing actual ecdysis. What is remarkable is the low net water flux observed in midgut preparations from early postmoit (stage A) individuals. suggesting that water absorption had been completed by the time of measurement. Corrivault & Tremblay (1948) have shown that in the lobster. Hnrnurus umericanus. water absorption is completed four to six hours after ecdysis. Given the higher ambient temperature for Mac~ohruct~iu~t~, it is likeiy that absorption of water would be completed more rapidly. Thus it is possible that an even more elevated fluid transport rate immediately following ecdysis has been missed. It has been suggested that water enters at ecdysis through the gills (Travis. 1954) or through the genera! body ‘surface (Dandrifosse. 1966) as well as via the gut. Although these authors present no direct experimental evidence to support any of these routes, it is probable that some water enters the hemolymph by osmosis via gills, integument, and gut in crustaceans living in hypotonic media. Crangon vulgaris, maintained in lo”,, salinity (30”” seawater), shows a marked dechne of 33’2 in blood osmolality immediately following ecdysis (Hagerman & Larsen, 1977). Comparable declines in electrolytes are also observed
III the craytish Orcow~ IK\ l~uro.~r,~ (Andrcwb. iLKI and Porc~rnohiu~ (Huf. 1933). Hagerman 8.~ Larsen I 1’)771 demonstrated that urinary output IS greatest tn postmolt individua!s. suggesting that osmottc ttptnkc ol water is substantial in this stage of the molttng cycle. Integumenta! permeability to ions is constderable 111 postmolt animals. In the freshuatcr amphipod. (;tltnITILITUS cft&x,ni. Lock\bood R: Andre\vs I I0691 showed that both Inllux and &!uk of Na + art’ greatest immediately after the molt. then decline: iti intermolt levels by 3--4 days after ccdysis. Therefore. both d~ffusional loss of electrolytes and osmotic uptahc of water ma) hot h hc responsible for the drop in blood c~noiality after ecdysis. Interpretation of the preseni results obtnincd for .ttucw~hruchium rosrnh~qii is hampered bj thtz paucity of information on hemolymph osmolality and urinarv output during the molting cycle in this and other ‘freshwater species. Assuming that ?iltrt~oh~&iun~ also experiences a decline in blood osmo!a!~t~ following ecdysts. the I lrtua! shutdown of flutd transport tn the midgut in early postmolt animals ma) prevent additional uptake of water that must he eliminated by the antenna! gland. With few cyceptmns. the urine of decapods IS Iso-osmotic to the blood (Presser, I973). suggesting that considerable losses of ions may occur via this pathway when urine is being boided. Whet her Mtl(.Y(~hr~(.hiil))j produces a hypoosmotic urine has not been demonstrated. but another freshwater palaemonid. Ptrluemot~ mtruotitrc~~hrs. eliminates a urine which is iso-osmotic to the blood (Born, 19681. A reduction of postmolt urine production by hyper-regulating crustaceans would be adaptive by reducing loss of electrolytes \~a the urine. Therefore. reduced fluid transport by the midgut might reduce urinaq output at a time when the cuticle is most permeable and los5 of electrolytes most severe. .,l(,~,low(c,li!li,‘rlrnt.s---Thts work IS a result of research sponsored II> NOAA Office of Sea Grant. Department of Commerce. under Grant No. 04-6-I%-44ilO. The U.S. Government IS authorixd to produce and distribute reprints for governmental purpose, notwithstanding an> copyright notatmn that may appear hereon. In addition. t’armns aspects of this project were funded by a National Science Foundation grant (PCM 76-84105) to G AA
KEFEREWIS G. A. 8z MACZUNISS L. A 11977) Kinetics of glucose transport by the perfused midgut of the freshwater prawn Mucrohrachium rnsrnhergli. .I. Physrol. Loud. 271.
AHEARN
319-336. AHEARS G.
A.. MAGINNU L. .A.. S<>NCi Y. I(. & T~~RNQITSI A. (1977) Intestinal hater and fan transport In freshwater malacostracan prawns (Crustaceal. In Wrrrrr Reluriocu in Memhranr 7T-m1sporr it1 P/trm.\ oml Attimo/.\ (Edited by JUNGKIES A. M.. HODC~-sT. E;., KLEI~ZELLERA. & SHULTZS. G,). pp. 129- 142. Acadermc Press. New York. ANDREWSP. (1967) ijber den BIutchemismus des Flusskrabses Orconrcres limosus und seine Verinderung im Laufe des Jahres. Z. rurgl. Ph~sioi. 57, 7-43. BERRIDC;EM. J. & OSCHMA~J. L. (1972) Transporfmq Epirhelia. Academic Press, New York. BORNJ. W. (1968) Osmoregulatory capacities of two carrdean shrimps, Sync&s pac$ca (Atyidae) and Pulaenzon macrodacrylus (Palaemonidae). Biol. Bull. 134, 135-244.
Changes
in fluid transport
BORN J. W. (1970) Changes in blood volume and permeability associated with molting in a marine crab. Pugerria producrcc. Ph.D. dissertation. University of California, Berkeley. BURG M. B. & ORLOFF J. (1968) Control of fluid absorption in renal proximal tubule. J. c/in. Inrest. 47. 20162024. COOKE W. J. (1976) Comparative ultrastructure of intestinal epithelia from a freshwater prawn and a desert scorpion. PUCI$ Sci. 30. 213-214. COOKE W. J. & AHEARN G. A. (1976) Comparative cell morphology of freshwater and marine shrimp midgut epithelia. Ant. Zoo/. 16. 225. CORRIVAULT G. W. & TREMBLAYJ. L. (1948) Contribution a la biologie du homard (Homcrrus americtrnus MilneEdwards) dans la Baie-des-Chaleurs et le golfe SaintLaurent. Univ. Lava]. Contr. Sta. biol. Saint-Laurent, no. 19. DANDRIFOSSE G. (1966) Absorption d’eau au moment de la mue chez un Crustace Decapode. Maia syuinado. Archs int. Eiochim. 74, 3299331. DALL W. (1974) Indices of nutritional state in western rock lobster, Ptrnu/iru.t longiprs (Milne-Edwards). I. Blood and tissue constituents and water content. J. exp. mar. Biol. Ecol. 16. 167-180. DRACH P. (1939) Mue et cycle d’intermue chez les Crustaces Decapodes. Ann. Inst. ociunogr. Paris N.S. 19, 1033391. HAGERMAN L. & LARSEN M. M. Z. (1977) The urinary flow in Crungort rulgaris (Fabr.) (Crustacea. Natantia) during the moult cycle. Opheliu 16, 143-150. HAEFNER P. A. (1964) Hemolymph calcium fluctuations as
645
related to environmental salinity during ecdysis of the blue crab, Callinectes supidus. Physiol. Zod. 37, 247258.
HUF E. (1933) Uber die Aufrechterhaltung des Salzgehaltes bei Stisswasserkrebsen (Poramohius). Pjiigers Arch. yes. Physiol.
232. 55%573.
LOCKW~~ID A. P. M. & ANDREWS W. R. H. (1969) Active transport and sodium fluxes at moult in the amphipod, Gummaru.s dueheni. J. exp. Biol. 51, 591-605. MYKLES D. L. (1977) The ultrastructure of the posterior midgut caecum of Pachygrapsus crassipes (Decapoda. Brachyura) adapted to low salinity. Tissue Cell 9. 681-691. PA~SANO L. M. (1960) Molting and its control. In The Physiology of Crustacea (Edited by WATERMAN T. H.). pp. 473-536. Academic Press. New York. PEEBLES J. B. (1977) A rapid technique for molt staging in live Macrohrachium rosenhergii. Aquaculture 12. 173-180.
PROSSERC. L. (1973) Compararire Animal Physiology. W. B. Saunders. Philadelphia. ROBERTSONJ. D. (1960) Ionic regulation in the crab Carcirrus maenos (L.) in relation to the moulting cycle. Comp. Biochem. Physiol. I. 183-2 12. SCHEER B. T. (1960) Aspects of the intermoult cycle in natantians. Comp. Biochem. PhysioL I. 3-18. TRAVIS D. F. (1954) The molting cycle of the spiny lobster. Pan&-us argus Latreille. I. Molting and growth in laboratory-maintained individuals. Eiol. Bull. 107. 433450. ZIVIN J. A. & BARTKO J. J. (1976) Statistics for disinterested scientists. Lrye Sci. 18. 15-26.
of Zoology,
University
GREGORY
of Hawaii
at Manoa.
A. AHEARN
Honolulu.
HI 96822. U.S.A.
Abstract-l. Net water flux was measured across the perfused midgut of Mrrcrohrclc.hiun~ rosenhergii during different stages of the molting cycle. 2. Compared to fluid transport rates exhibited during intermolt. fluxes were elevated in animals approaching ecdysis. 3. However. fluxes were much reduced in postmolt individuals. 4. The si8nifi~ance of these findings in relation to salt and water balance of the organism is discussed.
INTRODUCTION
The phenomenon of water absorption at ecdysis has been described in many decapod crustaceans (Drach, 1939; Corrivault & Tremblay, 1948: Travis, 1954: Robertson, 1960: Haefner, 1964; Dali, 1974), which leads to an increase in hemolymph volume (Drach, 1939: Born. 1970; Dali, 1974). The major route of water absorption at ecdysis is believed to be across the lining of the digestive tract (Drach, 1939; Passano, 1960; Robertson, 1960). The u~trastructure of the midgut lining of the shrimp ~ac~o~~ac~~~rn ~ose~ber~ij and Penaeus marginatus (Cooke, 1976; Cooke & Ahearn, 1976; Ahearn & Maginniss, 1977) and the crab Pachygrapsus crassipes (Mykles, 1977) resembles that of other epithelia capable of active transport of ions and water (Berridge & Oschman, 1972). In addition, Ahearn et al. (1977) have shown that the perfused midgut of intermolt ~uero~~~c~j~~ rosenbergji actively transports a fluid from lumen to serosa. Thus it appears that the midgut is the principal part of the digestive tract where water is absorbed into the hemolymph at ecdysis. The purpose of this study is to investigate possible changes in net water transport across the perfused midgut of ~aero~racb~um rosetlbergii that may occur during the molting cycle.
at 23 f I C. The perfusate contained inulin (carboxYI-‘~C) (ICN. 1.4 pCi/mg) (0.25 pCi/ml) and 0.10 mM unlabelled inulin to eliminate absorption of radioactivlty to the walls of the tubing. Usually six 5-min collections of perfusate effluent were obtained from each preparation. Experiments were completed within 1 hr after removal of the midgut from the animal. An aliquot of serosal bath was taken after completion of the experimental period to check for leakage of label, which was usually much less than 1.5”,, of the perfusion rate. Effluent volumes were determined gravimetrically. I&tilled water (2 ml) and lOm1 Aquasol scintillation cocktail mix (NEN) were added to each sample before counting in a Beckman LS-230 liquid scintillation spectrometer. Classification of molt stages were based generally on the work of Drach (1939) and Scheer (1960). and more speclfitally on the criteria of Peebles (1977) as applied to Mornbruchium rosenhergii. In the present study. stage C, comprised hard intermolt animals. stage D were animals in proecdysis (D, to D,). stage A contained animals which had molted the night before measurement. and stage B animals were individuals 3-4 days postecdysis. Sixty-three effluent collections from 11 stage C, (intermolt) midgut preparations, 59 collections from 1 I stage D preparations. 23 collections from five stage A preparations and 17 collections from three stage B preparations were obtained in this investigation.
RESULTS
MATERIALS
AND METHODS
Measurement of net water flux employed the method of Burg & Orloff (19681as modified by Ahearn er ai. (1977) for ~ucr~br~~~i~~~ rosenbrrgii. Briefly. the change in perfusate concentration of a non-absorbed volume marker, in this case “C-inulin. after passage through the midgut yields information on both the magnitude and direction of net water transport. The midgut was mounted in a lucite chamber containing physiological saline (Ahearn & Maginniss, 1977; Ahearn ec al.. 1977) and perfused with saline containing the volume marker at a rate of 8~i~~l;min * Present address: Department of Zoology, University of California. Berkeley. CA 94720, U.S.A. and Bodega Marine Laboratory. Bodega Bay. CA 94923. U.S.A. <.R.P. 61 4~
H
Significant alterations in transepithelial net water transport were observed in the perfused midgut of Macrobruchium roserhergii during the molting cycle (Fig. 1). Preparations from intermolt (stage C,) animals had a net water flux of 35.3 + 2.0&m’ per hr from mucosa to serosa. This rose to 55.8 & 5.1 pi/cm’ per hr in preparations from individuals in proecdysis (stage D). Within 12 hr after ecdysis (stage A) net water flux was far below the intermolt level (7.9 _t 4.5 PI/cm2 per hr). By 3-4 days following ecdysis (stage B), net water flux had returned to intermolt levels (31.3 + 5.3 &cm2 per hr). Analysis of variance (Zivin & Bartko. 1976) indicates that the group means vary significantly with stage of the molting cycle (P -C 0.01). According to the Newman-Keuls 643
Mo.t
stage
Fig. 1. Net water Hux across the mrdgut of Mlrcrohrcicltiiof~ rowtrherqii during different stages of the molting cycle (mean f I SE.). Number of effluent collections for each group given III parentheses. Molt stases arc defined in the text test (Zivin & Bartko. 1976). al! the group means are significantly different from one another (P < 0.01). except the means of stage C, and stage B animals.
This is the first report of changes in net fluid transport across the decapod midgut during different stages of the molting cycle and provides evidence that this portion of the gut is a major site of water absorption at ecdysis. The increased net water flux in proecdysis by Mncrobrachium midgut appears to anticipate water absorption by the animal since swelling due to water uptake is not observed until late proecdysis (stage II,). Rapid increases in weight in other crustaceans approaching ecdysis are not observed until stage D4 (Drach, 1939: Travis, 1954). It is possible that even greater rates of Ruid transport may be obtained in preparations from animals undergoing actual ecdysis. What is remarkable is the low net water flux observed in midgut preparations from early postmoit (stage A) individuals. suggesting that water absorption had been completed by the time of measurement. Corrivault & Tremblay (1948) have shown that in the lobster. Hnrnurus umericanus. water absorption is completed four to six hours after ecdysis. Given the higher ambient temperature for Mac~ohruct~iu~t~, it is likeiy that absorption of water would be completed more rapidly. Thus it is possible that an even more elevated fluid transport rate immediately following ecdysis has been missed. It has been suggested that water enters at ecdysis through the gills (Travis. 1954) or through the genera! body ‘surface (Dandrifosse. 1966) as well as via the gut. Although these authors present no direct experimental evidence to support any of these routes, it is probable that some water enters the hemolymph by osmosis via gills, integument, and gut in crustaceans living in hypotonic media. Crangon vulgaris, maintained in lo”,, salinity (30”” seawater), shows a marked dechne of 33’2 in blood osmolality immediately following ecdysis (Hagerman & Larsen, 1977). Comparable declines in electrolytes are also observed
III the craytish Orcow~ IK\ l~uro.~r,~ (Andrcwb. iLKI and Porc~rnohiu~ (Huf. 1933). Hagerman 8.~ Larsen I 1’)771 demonstrated that urinary output IS greatest tn postmolt individua!s. suggesting that osmottc ttptnkc ol water is substantial in this stage of the molttng cycle. Integumenta! permeability to ions is constderable 111 postmolt animals. In the freshuatcr amphipod. (;tltnITILITUS cft&x,ni. Lock\bood R: Andre\vs I I0691 showed that both Inllux and &!uk of Na + art’ greatest immediately after the molt. then decline: iti intermolt levels by 3--4 days after ccdysis. Therefore. both d~ffusional loss of electrolytes and osmotic uptahc of water ma) hot h hc responsible for the drop in blood c~noiality after ecdysis. Interpretation of the preseni results obtnincd for .ttucw~hruchium rosrnh~qii is hampered bj thtz paucity of information on hemolymph osmolality and urinarv output during the molting cycle in this and other ‘freshwater species. Assuming that ?iltrt~oh~&iun~ also experiences a decline in blood osmo!a!~t~ following ecdysts. the I lrtua! shutdown of flutd transport tn the midgut in early postmolt animals ma) prevent additional uptake of water that must he eliminated by the antenna! gland. With few cyceptmns. the urine of decapods IS Iso-osmotic to the blood (Presser, I973). suggesting that considerable losses of ions may occur via this pathway when urine is being boided. Whet her Mtl(.Y(~hr~(.hiil))j produces a hypoosmotic urine has not been demonstrated. but another freshwater palaemonid. Ptrluemot~ mtruotitrc~~hrs. eliminates a urine which is iso-osmotic to the blood (Born, 19681. A reduction of postmolt urine production by hyper-regulating crustaceans would be adaptive by reducing loss of electrolytes \~a the urine. Therefore. reduced fluid transport by the midgut might reduce urinaq output at a time when the cuticle is most permeable and los5 of electrolytes most severe. .,l(,~,low(c,li!li,‘rlrnt.s---Thts work IS a result of research sponsored II> NOAA Office of Sea Grant. Department of Commerce. under Grant No. 04-6-I%-44ilO. The U.S. Government IS authorixd to produce and distribute reprints for governmental purpose, notwithstanding an> copyright notatmn that may appear hereon. In addition. t’armns aspects of this project were funded by a National Science Foundation grant (PCM 76-84105) to G AA
KEFEREWIS G. A. 8z MACZUNISS L. A 11977) Kinetics of glucose transport by the perfused midgut of the freshwater prawn Mucrohrachium rnsrnhergli. .I. Physrol. Loud. 271.
AHEARN
319-336. AHEARS G.
A.. MAGINNU L. .A.. S<>NCi Y. I(. & T~~RNQITSI A. (1977) Intestinal hater and fan transport In freshwater malacostracan prawns (Crustaceal. In Wrrrrr Reluriocu in Memhranr 7T-m1sporr it1 P/trm.\ oml Attimo/.\ (Edited by JUNGKIES A. M.. HODC~-sT. E;., KLEI~ZELLERA. & SHULTZS. G,). pp. 129- 142. Acadermc Press. New York. ANDREWSP. (1967) ijber den BIutchemismus des Flusskrabses Orconrcres limosus und seine Verinderung im Laufe des Jahres. Z. rurgl. Ph~sioi. 57, 7-43. BERRIDC;EM. J. & OSCHMA~J. L. (1972) Transporfmq Epirhelia. Academic Press, New York. BORNJ. W. (1968) Osmoregulatory capacities of two carrdean shrimps, Sync&s pac$ca (Atyidae) and Pulaenzon macrodacrylus (Palaemonidae). Biol. Bull. 134, 135-244.
Changes
in fluid transport
BORN J. W. (1970) Changes in blood volume and permeability associated with molting in a marine crab. Pugerria producrcc. Ph.D. dissertation. University of California, Berkeley. BURG M. B. & ORLOFF J. (1968) Control of fluid absorption in renal proximal tubule. J. c/in. Inrest. 47. 20162024. COOKE W. J. (1976) Comparative ultrastructure of intestinal epithelia from a freshwater prawn and a desert scorpion. PUCI$ Sci. 30. 213-214. COOKE W. J. & AHEARN G. A. (1976) Comparative cell morphology of freshwater and marine shrimp midgut epithelia. Ant. Zoo/. 16. 225. CORRIVAULT G. W. & TREMBLAYJ. L. (1948) Contribution a la biologie du homard (Homcrrus americtrnus MilneEdwards) dans la Baie-des-Chaleurs et le golfe SaintLaurent. Univ. Lava]. Contr. Sta. biol. Saint-Laurent, no. 19. DANDRIFOSSE G. (1966) Absorption d’eau au moment de la mue chez un Crustace Decapode. Maia syuinado. Archs int. Eiochim. 74, 3299331. DALL W. (1974) Indices of nutritional state in western rock lobster, Ptrnu/iru.t longiprs (Milne-Edwards). I. Blood and tissue constituents and water content. J. exp. mar. Biol. Ecol. 16. 167-180. DRACH P. (1939) Mue et cycle d’intermue chez les Crustaces Decapodes. Ann. Inst. ociunogr. Paris N.S. 19, 1033391. HAGERMAN L. & LARSEN M. M. Z. (1977) The urinary flow in Crungort rulgaris (Fabr.) (Crustacea. Natantia) during the moult cycle. Opheliu 16, 143-150. HAEFNER P. A. (1964) Hemolymph calcium fluctuations as
645
related to environmental salinity during ecdysis of the blue crab, Callinectes supidus. Physiol. Zod. 37, 247258.
HUF E. (1933) Uber die Aufrechterhaltung des Salzgehaltes bei Stisswasserkrebsen (Poramohius). Pjiigers Arch. yes. Physiol.
232. 55%573.
LOCKW~~ID A. P. M. & ANDREWS W. R. H. (1969) Active transport and sodium fluxes at moult in the amphipod, Gummaru.s dueheni. J. exp. Biol. 51, 591-605. MYKLES D. L. (1977) The ultrastructure of the posterior midgut caecum of Pachygrapsus crassipes (Decapoda. Brachyura) adapted to low salinity. Tissue Cell 9. 681-691. PA~SANO L. M. (1960) Molting and its control. In The Physiology of Crustacea (Edited by WATERMAN T. H.). pp. 473-536. Academic Press. New York. PEEBLES J. B. (1977) A rapid technique for molt staging in live Macrohrachium rosenhergii. Aquaculture 12. 173-180.
PROSSERC. L. (1973) Compararire Animal Physiology. W. B. Saunders. Philadelphia. ROBERTSONJ. D. (1960) Ionic regulation in the crab Carcirrus maenos (L.) in relation to the moulting cycle. Comp. Biochem. Physiol. I. 183-2 12. SCHEER B. T. (1960) Aspects of the intermoult cycle in natantians. Comp. Biochem. PhysioL I. 3-18. TRAVIS D. F. (1954) The molting cycle of the spiny lobster. Pan&-us argus Latreille. I. Molting and growth in laboratory-maintained individuals. Eiol. Bull. 107. 433450. ZIVIN J. A. & BARTKO J. J. (1976) Statistics for disinterested scientists. Lrye Sci. 18. 15-26.