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A comparison of osmolality and specific ion concentrations in the fluid compartments of the regular sea urchin Lytechinus variegatus Lamarck (Echinodermata: Echinoidea) in varying salinities
Camp. Biochem. Physiol. Vol.lOBA,No.4, pp.497-502.1994 Elsevier Science Ltd Printed in Great Britain 0300-9629/94 $7.00+ 0.00
Pergamon 0300-%29(94)EOOO3-B
A comparison of osmolality and specific ion concentrations in the fluid compartments of the regular sea urchin Ly~ec~in~~ uariegatus Lamarck (Echinode~ata: Echinoidea) in varying salinities Charles D. Bishop, Kara J. Lee and Stephen A. Watts of Biology, University of Alabama at Birmingham, University Station, Birmingham, AL 35294-l 170, U.S.A. Department
LytecGnus unriegatus were exposed to four salinity treatments (20,25,30 and 35 ppt S) for 32 days. All compartmental fluids showed osmoconformity at all salinities. Sodium levels were slightly, hut significantly, higher in the lumenal fluids of the stomach and intestine as compared to the external medium at all salinities. Chloride levels were sign~cantly lower in the coelomic fluid in comparison to the seawater and the stomach and intestinal lumenai fluids. We hypothesize that chloride moves from the lumen of the stomach and intestine into the coelom, then into the external medium, generating an ion flux aiding in the co-transport of nutrients or other molecules. Potassium levels in the lumen of the stomach and intestine remained significantly higher and similar in all salinity treatments, while potassium levels in other compa~ment fluids varied directly with salinity. At low salinity, relatively high stomach and intestine lumenal fluid levels of potassium may represent a stress response and ultimately reflect physiological mechanisms leading to the death of the individual. Key words: Echinoderm;
Osmotic
stress; Ion regulation;
Coelomic fluid.
Camp. Biochem. Physiol. 108A, 497-502, 1994. -
introduction Echinoderms are generally stenohaline marine organisms that osmoconform to changes in ambient salinity and are found in salinities ranging from 7.7 to 60 ppt (Stickle and Diehl, 1987). In most species the osmoiality of the extracellular medium is similar to the external fluid (Binyon,
1966). Their inability to osmoregulate is due largely to the lack of any formidable excretory organ (Stickle and Diehl, 1987) and a semipermeable body wall (Binyon, 1972). However, it has been reported in several studies that some species of echinoderms exhibit slight differences in ion concentrations between the external medium and extracellular fluids (see Binyon review, -_ 1972). CorresFo~dence fo: Charles Bishop, Department of Differences in ion concentrations among Biotogy, University of Alabama at Birmingham, extracellular fluid compartments may exist University Station, Birmingham, AL 35294- 1170, as an effort by cells of internal tissues to U.S.A. Fax: 205-975-0637. maintain intracellular osmotic and ionic Received 27 October 1994; accepted 19 December 1994. concentrations by altering extracellular 497
498
Charles D. Bishop et al.
ionic concentrations (Donnan equilibrium). During osmotic stress, specific ion channels or transport proteins may be inactivated and disrupt the steady-state ion equilibrium. Consequently, the loss of one or more of the specific ion concentrations may initiate mortality in some species of echinoderms. Very little information has been reported on the osmotic and ionic concentrations of the extracellular fluids of echinoids in response to exposure to various salinities. In this study we report these parameters in the regular echinoid, Lytechinus variegatus. L. variegatus has been found in a wide range of habitats, some in which salinity fluctuations occurred regularly. Roller and Stickle (1993) observed that the LG,~ for L. variegatus occurs between 20 and 18 ppt S; below this threshold all individuals expired within 4 days. For this study, individuals from a permanent population of L. variegatus were collected from a shallow sea grass bay at Port Saint Joe, Florida, U.S.A. where salinity can fluctuate from 20 to 35 ppt S depending on the amount of rainfall or run-off (Valentine, personal communication). Animal mortality from exposure to decreased salinity in this population may be limited since normal salinity (32-34 ppt S) is usually restored within 24 hr, but may be longer during abnormal weather patterns. In periods of osmotic/ionic stress, the changes in the extracellular fluid compartments of this echinoid are unknown. The purpose of this study was to expose L. variegatus to a series of reduced salinities and report the physiological response by comparing the total osmolality and specific ion concentrations within the fluids of extracellular fluid compartments to the ambient seawater. Changes in ion concentrations with respect to lethal and sub-lethal hyposmotic stress will be discussed.
Materials and Methods In September 1992, 60 Lytechinus variegatus were collected from Port Saint Joe Wildlife Refuge, Port Saint Joe, Florida, U.S.A. (85”.24” Lat., 29O.47” Long.). Ambient water was 25°C and averaged 30 ppt S at the time of collection. The individuals were transported to the
University of Alabama at Birmingham. All individuals were placed in a 400 1 glass aquarium containing artificial seawater at 23°C and 30ppt S for 2 days before initiating salinity treatments. Treatment tanks were organized 2 weeks prior to animal collection. Four salinity treatments (20, 25, 30 and 35 ppt S) were prepared by mixing artificial sea salt (Tropic Marin) with deionized water; all treatments were maintained at 21°C and 12 light : 12 dark photoperiod with constant aeration. Biological filtration was maintained by recirculating water filters containing oyster shell hash seeded with the nitrifying bacteria Nitrosomonas and Nitrobacter. Essential trace elements (Fritz Aquaculture) were added to each aquarium (1 drop/4 1) 2 weeks from the initiation of feeding. Four groups of 13 randomly chosen individuals from the 400 1 aquarium were transferred step-wise (5 ppt/day) into their respective treatment salinity. All individuals remained undisturbed for 1 day prior to initiation of feeding. Food consisted of a 2.5% fish-seaweed meal embedded in 2.5% non-nutrient agar. Agar was prepared using artificial seawater at 32 ppt salinity. Each individual received a 2-3 g block of food placed on the aboral surface to minimize handling of the individuals. Blocks of food were exchanged daily to minimize decomposition; food was proffered ad libitum. Thirty days after the initiation of feeding, the fish meal was removed and pure agar (2.5%) blocks were fed to all treatments for 2 days to clear the gut of any remaining food. Thirty-two days after initiation of feeding, 10 individuals from each treatment were removed and the total wet weight (g) and diameter were measured. The individuals were then dissected by removing a small area around the periproct and carefully removing pieces of test towards the oral surface while separating suspensory ligaments holding the stomach and intestine to the test wall. Care was taken not to disrupt the gut wall. The test dissection ended when the top half of the test had been removed. Coelomic fluid samples were removed using a 5 cm3 syringe with a 20 gauge needle (separate syringes for each individual) and
Osmolality and ion con~ntration
placed in a labeled microfuge vial on ice. Hemostats were immediately placed at junctures between the esophagus and stomach, stomach and intestine, and intestine and rectum to ensure isolation of gut compartment fluids. Fluids were carefully removed using separate 3 cm3 syringes and 20 gauge needles from each gut compartment for each individual and placed in labeled microfuge vials on ice. The fluids collected were capped and centrifuged in a Costar (Model IO) centrifuge at high speed for 5 min to remove cells and debris. The cell-free fluids were then transferred to alternate labeled microfuge vials and placed on ice. After preparation of the cell-free fluid, the fluid osmolality of the treatment water, coelomic fluid, and lumenal fluid of the stomach and intestine was determined using a vapor pressure osmometer (Wescor 5100C). Subsamples were then placed in a - 80°C freezer for future specific ion determination. Chloride ion concentration was measured using a chloridometer (BuchlerCotlove) and sodium and potassium were measured concentrations simultaneously using a flame photometer (Radiometer FLM3). Statistical differences were tested by using a two-way ANOVA with salinity and fluid compartment as independent variables and ion osmolality levels within each fluid compartment as dependant variables; Tukey’s multiple comparison test was used to reduce the chance of committing type I errors. Differences were considered statistically significant at P -C0.05. All analyses were performed with the statistical package SyStat.
Results Organism activity and feeding
Initially all 13 individuals in the 25, 30 and 35 ppt salinity treatments were active, migrating up the sides of the aquarium and feeding. The individuals in the 20 ppt salinity treatment did not begin to eat until day 6 of the experiment. Individuals in two treatments; 30 and 35 ppt S, remained active and fed throughout the study. After 2 weeks, individuals in the 25 ppt S treatment were less active; only 2-4 individuals migrated up the sides of the aquarium.
in Lyteehin~ ~u~~egurus
499
There was, however, no noticeable change in feeding behavior in any of these individuals. The individuals in the 20 ppt S treatment showed the most obvious changes in activity and feeding. By the end of the first week, all individuals in the 20 ppt S treatment were on the bottom of the aquarium and at no time attempted to climb the sides of the aquarium. The feeding activity of these individuals decreased compared to the individuals in the other treatments by day 14, with many individuals leaving almost whole blocks of food intact. Upon collection of the individuals from the 20 ppt S treatment for dissection, distinct differences in animal response were noted compared to the remaining three treatments. Spine movement and rigidity (attachment site of the spine to the test was easily movable with little or no resistance; in healthy or low stressed individuals the spine is rigid and firmly held to the test) were decreased. Individuals in this treatment were losing small numbers of primary spines, an initial sign of an unhealthy or stressed animal. Artificial seawater osmolality was significantly lower in the fluid compartments of Lytechinus variegatus in the 20, 25 and 35 ppt S treatments. There was no significant difference between artificial seawater osmolality and the fluid compartments in the 30ppt S treatment (Fig. IA). In all treatments, sodium ion levels (mmoljl) of the lumenal fluid compartments of the stomach and intestine were slightly, but significantly, higher than in the artificial seawater (Fig. IB). Coelom sodium levels varied slightly from the artificial seawater; no trends were observed. Treatment sodium levels fluctuated among the fluid compartments of the coelom, stomach and intestine. The coelom fluid compartment was hyperionic in the 25 and 35 ppt S treatments and iso-ionic in the 20 and 30 ppt S treatments; no significant trends were observed. In all treatments, chloride ion levels (mmol/l) of all fluid compartments were equal to or less than artificial seawater (Fig. 1C). In salinities below 35 ppt S, the fluid compartments of the stomach and intestine had significantly lower Cl- ion levels than
Charles D. Bishop et al.
500
’ 2oo 1 e t3 0 P)
ARTIFICIALSEAWATER
ARTIFICIALSEAWATER COELOM STOMACH INTESTtNE
-_ 20 PPt
25 PP~
30 PPt
30 PPt
35 ppt
SALINITY TREATMENT
SALINITY TREATMENT
20 PPt
25 PPt
35 ppt
SALINITY TREATMENT
20 PPt
25 Ppt
30 PPt
35 ppt
SALINITY T~~TMENT
Fig. 1. (A) Osmolality (mmohkg), (B) sodium ion levels (mmol/l), (C) chloride ion levels (mmol/l), (D) potassium ion levels (mmol/l) of artificial seawater, coelomic fluid and stomach and intestinal lumenal fluids of Lytechinus uariegutus held for 32 days in four salinity treatments (N = 5 sub-samples for Artificial seawater; IV = 10 individuals for coelomic fluid, stomach and intestinal fluids: columns represent means, vertical lines represent standard error). Statistical significance is denoted by letters. Compartments within each salinity treatment with identical letters are not statistically significant, while those with different letters are statistically significantly different (P < 0.05). Statistical significance between salinity treatments within each ion group is discussed in the results. No significant difference was observed between the osmolality of the artificial seawater and the fluid compartments at any salinity.
artificial seawater. Chloride levels in the coelomit fluid treatments were also significantly lower than artificial seawater in all salinity treatments. Chloride levels were significantly lower in the coelomic fluid in relation to the lumenal fluid compartments of the stomach and intestine in all treatments. Chloride levels were similar in the stomach and intestine at all salinity treatments. In all salinity treatments, potassium levels (mmol/l) in the lumenal fluid of the stomach remained relatively stable, varying from 7.5 to 9.8 mmol/l, while other compartments varied directly with salinity
(Fig. 1D). In all treatments, there appeared to be an accumulation of potassium in the lumenal fluid of the stomach and intestine, while the IS+ level of the coelom (except for the 25 ppt S treatment) was equal to or lower than the artificial seawater. The most significant difference in potassium levels was observed in the 20 ppt S treatment, with lumenal stomach levels (and to a lesser degree, the intestine) well above the artificial seawater and coelomic fluid. In all salinity treatments, the stomach maintained significantly higher potassium levels than the intestine.
Osmolality and ion concentration
Discussion Lytechinus variegatus is an osmoconformer, showing no significant differences in the osmolalities among the fluid compartments compared to artificial seawater at any salinity. Stickle and Diehl (1987) indicated that most echinoderms are osmoconformers and that changes in the osmolarity of fluid compartments coincide directly with changes in salinity of the external medium. However, significant differences were observed in specific ion levels (Na+, Cl- and K+ ) among fluid compartments. Sodium levels did not vary among any fluid compartments at all salinities other than a slight elevation in the lumen of the stomach and intestine compared to the external medium. This, however, may be a consequence of Na+ leaching from the enterocytes lining the stomach and intestinal lumen. Chloride levels were significantly lower in the coelomic fluid than in the external medium or the stomach and intestinal lumenal fluids. This suggests that chloride was selectively moved from the coelomic fluid to another fluid compartment, surrounding cells or to the external medium. We hypothesize that chloride is transported from the gut lumen through the apical cell membrane of the mucosae and out the basal membrane and into the coelomic fluid. Chloride ions could then be transported into the surrounding seawater via the external epithelium or concentrated in the ampullae and removed through the tube feet as observed in many asteroids (Stickle and Diehl, 1987). This movement of’ chloride may be essential in the establishment of ion gradients necessary for the absorption or transport of nutrients and other ions across the gut epithelium. This is supported by the fact that Cl- levels in the gut were lower than in the artificial seawater. Alternatively, Cl- may not be taken up at the same rate as cations from the medium. Potassium in the lumenal fluids of the stomach and intestine was maintained at levels significantly hypertonic to the coelomic fluid and, to a lesser degree, the external seawater. The mechanism by which these levels are maintained or the physio-
in LyMzinus uariegufus
501
logical significance of these elevated levels of potassium is not known. Potassium in the lumenal fluids of the stomach and intestine may be selectively concentrated from the seawater obtained during feeding. Alternatively, potassium may be leaching into the lumen of the gut from cells lining the gut, since intracellular potassium is approximately 16 times greater within the mucosal cells of similar echinoids (Stickle and Diehl, 1987). Binyon (1972) described similar trends in the gut of holothuroids which maintained increased potassium levels within the gut relative to the external medium. In addition, elevated potassium levels in the lumen of the stomach and intestine may be necessary to establish ion gradients and/or be essential in the physiology of nutrient transport across the gut mucosae. At low salinities, relatively high stomach and intestine lumenal potassium levels may represent a stress response in an attempt to maintain established ion gradients and ultimately reflect physiological mechanisms leading to the death of the individual. Due to chronic low salinity exposure, individuals in the 20 ppt S treatment may have been further exhibiting this stress response as a cessation of feeding, movement and loss of primary spines. This is the first report utilizing echinoids which examines changes in specific ion levels of various fluid compartments at varying salinities. Although overall osmolarity of the fluids did not appear to change, specific ion levels vary within the fluid compartments, suggesting intracellular and perhaps extracellular ion regulation. These variations may be essential in the ability of an individual to survive salinity stress and maintain ionic homeostasis which is essential to basal physiological functions. Further work is required on specific ion flux and movement within the compartments of echinoids.
Acknowledgements-I would like to thank Dr Ray Henry at Auburn University for technical assistance, Dr William Stickle and Dr David Kraus for their comments and technical information in preparation of this manuscript and Dr Robert Angus for his help with the statistical analysis. This project was funded in part by the Department of Biology at the University of Alabama at Birmingham.
502
Charles D. Bishop et al.
References Binyon J. (1966) Salinity tolerance and ionic regulation In Physiology of Echinodermata (Edited by R. A. Boolootian), pp. 359-377. Interscience Publ., New York. Binyon J. (1972) Physiology of Echinoderms, pp. 264. Pergamon Press, Oxford.
Roller R. A. and Stickle W. B. (1993) Effects of temperature and salinity acclimation of adults on larval survival, physiology, and early development of Lytechinus variegatus (Bchinodermata: Echinoidea). Mar. Biol. 116, 583-591. Stickle W. B. and Diehl W. J. (1987) Effects of salinity on echinoderms. In Echinoderm Studies II (Edited by Jangoux M. and Lawrence J. M.), pp. 235-285. A. A. Balkema, Rotterdam.
Pergamon 0300-%29(94)EOOO3-B
A comparison of osmolality and specific ion concentrations in the fluid compartments of the regular sea urchin Ly~ec~in~~ uariegatus Lamarck (Echinode~ata: Echinoidea) in varying salinities Charles D. Bishop, Kara J. Lee and Stephen A. Watts of Biology, University of Alabama at Birmingham, University Station, Birmingham, AL 35294-l 170, U.S.A. Department
LytecGnus unriegatus were exposed to four salinity treatments (20,25,30 and 35 ppt S) for 32 days. All compartmental fluids showed osmoconformity at all salinities. Sodium levels were slightly, hut significantly, higher in the lumenal fluids of the stomach and intestine as compared to the external medium at all salinities. Chloride levels were sign~cantly lower in the coelomic fluid in comparison to the seawater and the stomach and intestinal lumenai fluids. We hypothesize that chloride moves from the lumen of the stomach and intestine into the coelom, then into the external medium, generating an ion flux aiding in the co-transport of nutrients or other molecules. Potassium levels in the lumen of the stomach and intestine remained significantly higher and similar in all salinity treatments, while potassium levels in other compa~ment fluids varied directly with salinity. At low salinity, relatively high stomach and intestine lumenal fluid levels of potassium may represent a stress response and ultimately reflect physiological mechanisms leading to the death of the individual. Key words: Echinoderm;
Osmotic
stress; Ion regulation;
Coelomic fluid.
Camp. Biochem. Physiol. 108A, 497-502, 1994. -
introduction Echinoderms are generally stenohaline marine organisms that osmoconform to changes in ambient salinity and are found in salinities ranging from 7.7 to 60 ppt (Stickle and Diehl, 1987). In most species the osmoiality of the extracellular medium is similar to the external fluid (Binyon,
1966). Their inability to osmoregulate is due largely to the lack of any formidable excretory organ (Stickle and Diehl, 1987) and a semipermeable body wall (Binyon, 1972). However, it has been reported in several studies that some species of echinoderms exhibit slight differences in ion concentrations between the external medium and extracellular fluids (see Binyon review, -_ 1972). CorresFo~dence fo: Charles Bishop, Department of Differences in ion concentrations among Biotogy, University of Alabama at Birmingham, extracellular fluid compartments may exist University Station, Birmingham, AL 35294- 1170, as an effort by cells of internal tissues to U.S.A. Fax: 205-975-0637. maintain intracellular osmotic and ionic Received 27 October 1994; accepted 19 December 1994. concentrations by altering extracellular 497
498
Charles D. Bishop et al.
ionic concentrations (Donnan equilibrium). During osmotic stress, specific ion channels or transport proteins may be inactivated and disrupt the steady-state ion equilibrium. Consequently, the loss of one or more of the specific ion concentrations may initiate mortality in some species of echinoderms. Very little information has been reported on the osmotic and ionic concentrations of the extracellular fluids of echinoids in response to exposure to various salinities. In this study we report these parameters in the regular echinoid, Lytechinus variegatus. L. variegatus has been found in a wide range of habitats, some in which salinity fluctuations occurred regularly. Roller and Stickle (1993) observed that the LG,~ for L. variegatus occurs between 20 and 18 ppt S; below this threshold all individuals expired within 4 days. For this study, individuals from a permanent population of L. variegatus were collected from a shallow sea grass bay at Port Saint Joe, Florida, U.S.A. where salinity can fluctuate from 20 to 35 ppt S depending on the amount of rainfall or run-off (Valentine, personal communication). Animal mortality from exposure to decreased salinity in this population may be limited since normal salinity (32-34 ppt S) is usually restored within 24 hr, but may be longer during abnormal weather patterns. In periods of osmotic/ionic stress, the changes in the extracellular fluid compartments of this echinoid are unknown. The purpose of this study was to expose L. variegatus to a series of reduced salinities and report the physiological response by comparing the total osmolality and specific ion concentrations within the fluids of extracellular fluid compartments to the ambient seawater. Changes in ion concentrations with respect to lethal and sub-lethal hyposmotic stress will be discussed.
Materials and Methods In September 1992, 60 Lytechinus variegatus were collected from Port Saint Joe Wildlife Refuge, Port Saint Joe, Florida, U.S.A. (85”.24” Lat., 29O.47” Long.). Ambient water was 25°C and averaged 30 ppt S at the time of collection. The individuals were transported to the
University of Alabama at Birmingham. All individuals were placed in a 400 1 glass aquarium containing artificial seawater at 23°C and 30ppt S for 2 days before initiating salinity treatments. Treatment tanks were organized 2 weeks prior to animal collection. Four salinity treatments (20, 25, 30 and 35 ppt S) were prepared by mixing artificial sea salt (Tropic Marin) with deionized water; all treatments were maintained at 21°C and 12 light : 12 dark photoperiod with constant aeration. Biological filtration was maintained by recirculating water filters containing oyster shell hash seeded with the nitrifying bacteria Nitrosomonas and Nitrobacter. Essential trace elements (Fritz Aquaculture) were added to each aquarium (1 drop/4 1) 2 weeks from the initiation of feeding. Four groups of 13 randomly chosen individuals from the 400 1 aquarium were transferred step-wise (5 ppt/day) into their respective treatment salinity. All individuals remained undisturbed for 1 day prior to initiation of feeding. Food consisted of a 2.5% fish-seaweed meal embedded in 2.5% non-nutrient agar. Agar was prepared using artificial seawater at 32 ppt salinity. Each individual received a 2-3 g block of food placed on the aboral surface to minimize handling of the individuals. Blocks of food were exchanged daily to minimize decomposition; food was proffered ad libitum. Thirty days after the initiation of feeding, the fish meal was removed and pure agar (2.5%) blocks were fed to all treatments for 2 days to clear the gut of any remaining food. Thirty-two days after initiation of feeding, 10 individuals from each treatment were removed and the total wet weight (g) and diameter were measured. The individuals were then dissected by removing a small area around the periproct and carefully removing pieces of test towards the oral surface while separating suspensory ligaments holding the stomach and intestine to the test wall. Care was taken not to disrupt the gut wall. The test dissection ended when the top half of the test had been removed. Coelomic fluid samples were removed using a 5 cm3 syringe with a 20 gauge needle (separate syringes for each individual) and
Osmolality and ion con~ntration
placed in a labeled microfuge vial on ice. Hemostats were immediately placed at junctures between the esophagus and stomach, stomach and intestine, and intestine and rectum to ensure isolation of gut compartment fluids. Fluids were carefully removed using separate 3 cm3 syringes and 20 gauge needles from each gut compartment for each individual and placed in labeled microfuge vials on ice. The fluids collected were capped and centrifuged in a Costar (Model IO) centrifuge at high speed for 5 min to remove cells and debris. The cell-free fluids were then transferred to alternate labeled microfuge vials and placed on ice. After preparation of the cell-free fluid, the fluid osmolality of the treatment water, coelomic fluid, and lumenal fluid of the stomach and intestine was determined using a vapor pressure osmometer (Wescor 5100C). Subsamples were then placed in a - 80°C freezer for future specific ion determination. Chloride ion concentration was measured using a chloridometer (BuchlerCotlove) and sodium and potassium were measured concentrations simultaneously using a flame photometer (Radiometer FLM3). Statistical differences were tested by using a two-way ANOVA with salinity and fluid compartment as independent variables and ion osmolality levels within each fluid compartment as dependant variables; Tukey’s multiple comparison test was used to reduce the chance of committing type I errors. Differences were considered statistically significant at P -C0.05. All analyses were performed with the statistical package SyStat.
Results Organism activity and feeding
Initially all 13 individuals in the 25, 30 and 35 ppt salinity treatments were active, migrating up the sides of the aquarium and feeding. The individuals in the 20 ppt salinity treatment did not begin to eat until day 6 of the experiment. Individuals in two treatments; 30 and 35 ppt S, remained active and fed throughout the study. After 2 weeks, individuals in the 25 ppt S treatment were less active; only 2-4 individuals migrated up the sides of the aquarium.
in Lyteehin~ ~u~~egurus
499
There was, however, no noticeable change in feeding behavior in any of these individuals. The individuals in the 20 ppt S treatment showed the most obvious changes in activity and feeding. By the end of the first week, all individuals in the 20 ppt S treatment were on the bottom of the aquarium and at no time attempted to climb the sides of the aquarium. The feeding activity of these individuals decreased compared to the individuals in the other treatments by day 14, with many individuals leaving almost whole blocks of food intact. Upon collection of the individuals from the 20 ppt S treatment for dissection, distinct differences in animal response were noted compared to the remaining three treatments. Spine movement and rigidity (attachment site of the spine to the test was easily movable with little or no resistance; in healthy or low stressed individuals the spine is rigid and firmly held to the test) were decreased. Individuals in this treatment were losing small numbers of primary spines, an initial sign of an unhealthy or stressed animal. Artificial seawater osmolality was significantly lower in the fluid compartments of Lytechinus variegatus in the 20, 25 and 35 ppt S treatments. There was no significant difference between artificial seawater osmolality and the fluid compartments in the 30ppt S treatment (Fig. IA). In all treatments, sodium ion levels (mmoljl) of the lumenal fluid compartments of the stomach and intestine were slightly, but significantly, higher than in the artificial seawater (Fig. IB). Coelom sodium levels varied slightly from the artificial seawater; no trends were observed. Treatment sodium levels fluctuated among the fluid compartments of the coelom, stomach and intestine. The coelom fluid compartment was hyperionic in the 25 and 35 ppt S treatments and iso-ionic in the 20 and 30 ppt S treatments; no significant trends were observed. In all treatments, chloride ion levels (mmol/l) of all fluid compartments were equal to or less than artificial seawater (Fig. 1C). In salinities below 35 ppt S, the fluid compartments of the stomach and intestine had significantly lower Cl- ion levels than
Charles D. Bishop et al.
500
’ 2oo 1 e t3 0 P)
ARTIFICIALSEAWATER
ARTIFICIALSEAWATER COELOM STOMACH INTESTtNE
-_ 20 PPt
25 PP~
30 PPt
30 PPt
35 ppt
SALINITY TREATMENT
SALINITY TREATMENT
20 PPt
25 PPt
35 ppt
SALINITY TREATMENT
20 PPt
25 Ppt
30 PPt
35 ppt
SALINITY T~~TMENT
Fig. 1. (A) Osmolality (mmohkg), (B) sodium ion levels (mmol/l), (C) chloride ion levels (mmol/l), (D) potassium ion levels (mmol/l) of artificial seawater, coelomic fluid and stomach and intestinal lumenal fluids of Lytechinus uariegutus held for 32 days in four salinity treatments (N = 5 sub-samples for Artificial seawater; IV = 10 individuals for coelomic fluid, stomach and intestinal fluids: columns represent means, vertical lines represent standard error). Statistical significance is denoted by letters. Compartments within each salinity treatment with identical letters are not statistically significant, while those with different letters are statistically significantly different (P < 0.05). Statistical significance between salinity treatments within each ion group is discussed in the results. No significant difference was observed between the osmolality of the artificial seawater and the fluid compartments at any salinity.
artificial seawater. Chloride levels in the coelomit fluid treatments were also significantly lower than artificial seawater in all salinity treatments. Chloride levels were significantly lower in the coelomic fluid in relation to the lumenal fluid compartments of the stomach and intestine in all treatments. Chloride levels were similar in the stomach and intestine at all salinity treatments. In all salinity treatments, potassium levels (mmol/l) in the lumenal fluid of the stomach remained relatively stable, varying from 7.5 to 9.8 mmol/l, while other compartments varied directly with salinity
(Fig. 1D). In all treatments, there appeared to be an accumulation of potassium in the lumenal fluid of the stomach and intestine, while the IS+ level of the coelom (except for the 25 ppt S treatment) was equal to or lower than the artificial seawater. The most significant difference in potassium levels was observed in the 20 ppt S treatment, with lumenal stomach levels (and to a lesser degree, the intestine) well above the artificial seawater and coelomic fluid. In all salinity treatments, the stomach maintained significantly higher potassium levels than the intestine.
Osmolality and ion concentration
Discussion Lytechinus variegatus is an osmoconformer, showing no significant differences in the osmolalities among the fluid compartments compared to artificial seawater at any salinity. Stickle and Diehl (1987) indicated that most echinoderms are osmoconformers and that changes in the osmolarity of fluid compartments coincide directly with changes in salinity of the external medium. However, significant differences were observed in specific ion levels (Na+, Cl- and K+ ) among fluid compartments. Sodium levels did not vary among any fluid compartments at all salinities other than a slight elevation in the lumen of the stomach and intestine compared to the external medium. This, however, may be a consequence of Na+ leaching from the enterocytes lining the stomach and intestinal lumen. Chloride levels were significantly lower in the coelomic fluid than in the external medium or the stomach and intestinal lumenal fluids. This suggests that chloride was selectively moved from the coelomic fluid to another fluid compartment, surrounding cells or to the external medium. We hypothesize that chloride is transported from the gut lumen through the apical cell membrane of the mucosae and out the basal membrane and into the coelomic fluid. Chloride ions could then be transported into the surrounding seawater via the external epithelium or concentrated in the ampullae and removed through the tube feet as observed in many asteroids (Stickle and Diehl, 1987). This movement of’ chloride may be essential in the establishment of ion gradients necessary for the absorption or transport of nutrients and other ions across the gut epithelium. This is supported by the fact that Cl- levels in the gut were lower than in the artificial seawater. Alternatively, Cl- may not be taken up at the same rate as cations from the medium. Potassium in the lumenal fluids of the stomach and intestine was maintained at levels significantly hypertonic to the coelomic fluid and, to a lesser degree, the external seawater. The mechanism by which these levels are maintained or the physio-
in LyMzinus uariegufus
501
logical significance of these elevated levels of potassium is not known. Potassium in the lumenal fluids of the stomach and intestine may be selectively concentrated from the seawater obtained during feeding. Alternatively, potassium may be leaching into the lumen of the gut from cells lining the gut, since intracellular potassium is approximately 16 times greater within the mucosal cells of similar echinoids (Stickle and Diehl, 1987). Binyon (1972) described similar trends in the gut of holothuroids which maintained increased potassium levels within the gut relative to the external medium. In addition, elevated potassium levels in the lumen of the stomach and intestine may be necessary to establish ion gradients and/or be essential in the physiology of nutrient transport across the gut mucosae. At low salinities, relatively high stomach and intestine lumenal potassium levels may represent a stress response in an attempt to maintain established ion gradients and ultimately reflect physiological mechanisms leading to the death of the individual. Due to chronic low salinity exposure, individuals in the 20 ppt S treatment may have been further exhibiting this stress response as a cessation of feeding, movement and loss of primary spines. This is the first report utilizing echinoids which examines changes in specific ion levels of various fluid compartments at varying salinities. Although overall osmolarity of the fluids did not appear to change, specific ion levels vary within the fluid compartments, suggesting intracellular and perhaps extracellular ion regulation. These variations may be essential in the ability of an individual to survive salinity stress and maintain ionic homeostasis which is essential to basal physiological functions. Further work is required on specific ion flux and movement within the compartments of echinoids.
Acknowledgements-I would like to thank Dr Ray Henry at Auburn University for technical assistance, Dr William Stickle and Dr David Kraus for their comments and technical information in preparation of this manuscript and Dr Robert Angus for his help with the statistical analysis. This project was funded in part by the Department of Biology at the University of Alabama at Birmingham.
502
Charles D. Bishop et al.
References Binyon J. (1966) Salinity tolerance and ionic regulation In Physiology of Echinodermata (Edited by R. A. Boolootian), pp. 359-377. Interscience Publ., New York. Binyon J. (1972) Physiology of Echinoderms, pp. 264. Pergamon Press, Oxford.
Roller R. A. and Stickle W. B. (1993) Effects of temperature and salinity acclimation of adults on larval survival, physiology, and early development of Lytechinus variegatus (Bchinodermata: Echinoidea). Mar. Biol. 116, 583-591. Stickle W. B. and Diehl W. J. (1987) Effects of salinity on echinoderms. In Echinoderm Studies II (Edited by Jangoux M. and Lawrence J. M.), pp. 235-285. A. A. Balkema, Rotterdam.