GENERAL
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COMPARATIVE
Neuroendocrine the Hemolymph
ENDOCRINOLOGY
84,
16-26 (1991)
Regulation of Osmotic and Ionic Concentrations in of the Freshwater Shrimp Macrobrachium olfersii (Wiegmann) (Crustacea, Decapoda)
JOHN C. MCNAMARA,LUIS
C. SALOMAO,* AND ELAINE A. RIBEIRO*
Departamento de Biologia, Faculdade de Filosojia, Ci@ncias e Letras, Vniversidade de Stio Paulo, Ribeireo Preto, 14049 SP, Bras& and *Departamento de Fisiologia Geral, Institute de Bioci&ncias, Vniversidade de Sdo Paula, Sbo Paul0 05508 SP, Brasil Accepted October 12, 1990 Putative neuroendocrine mediation of osmotic and ionic responses to acute exposure to high salinity medium was investigated in the freshwater shrimp Macrobrachium oljksii (Wiegmann). Homogenates of supra-esophageal or thoracic ganglia, prepared from shrimps exposed to seawater of 21X0 S for 6 hr, were injected into the abdominal musculature of shrimps previously exposed to freshwater and subsequently exposed to either freshwater or seawater (21X0 S). Osmotic, sodium, chloride, potassium, magnesium, and calcium concentrations were determined in hemolymph samples removed by intracardiac puncture at time = 0, 1,3, or 6 hr after homogenate application. Control shrimps were injected with faltered seawater, isosmotic to the hemolymph, and treated similarly. In control shrimps, the osmotic, Na+, Cl-, K+, Mg2+, and Ca2’ concentrations in the hemolymph increased (P ~0.05) after I-hr exposure to seawater. In shrimps injected with homogenates of supraesophageal ganglion and exposed to seawater, osmotic and ionic concentrations in the hemolymph did not vary with exposure time; in injected shrimps exposed to freshwater, Na+, Cl-, K+, and Mg2+ concentrations decreased (P Q 0.05) with time. In shrimps injected with homogenates of thoracic ganglion and exposed to seawater, hemolymph osmotic, K+, and Mg2+ concentrations increased (P s 0.05); Na+, Cl-, and CaZ+ concentrations remained unchanged. In injected shrimps exposed to freshwater, hemolymph osmotic concentration alone increased (P G 0.05) after 1 hr, all other ionic concentrations remaining unchanged. These data suggest that neurofactors apparently located within the ganglia of the central nervous system of M. orfersii may alter the apparent ionic permeabilities of this shrimp, depending on the salinity characteristics of the external medium. The data support the notion that invasion of the freshwater biotope by estuarine crustaceans has necessitated the evolution of specific physiological mechanisms capable of compensating for the osmotic dilution and ion loss typically encountered by such organisms. Q 1591 Academic Press. Inc.
trations in the face of typically low external ionic concentrations. Such systems include high affinity ion uptake mechanisms located in the gills (Shaw, 1961; Taylor and Harris, 1986; Harris and Bayliss, 1988) and intestine (Ahearn and Tornquist, 1977; Ahearn, 1978; Chu, 1987), reduction in body and organ osmotic and ionic permeabilities (Ehrenfeld and Isaia, 1974; Berlind and Kamemoto, 1977; Cantelmo, 1977), and efficient processes of solute reabsorp-
Implicit in the ongoing penetration and adaptation of marine and estuarine Crustatea to the freshwater biotope is the evolution of physiological mechanisms responsible for the maintenance of osmotically and ionically stable extra- and intracellular fluids. Essentially, successful invaders of the freshwater environment must possess effector systems capable of maintaining fairly high intra- and extracellular ionic concen16 001~6480/91 $1.50 Copyright 0 1991 by Academic Press, Inc. AU rights of reproduction in any form reserved
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tion, resulting in the excretion of a urine hypotonic to the hemolymph (Mantel and Farmer, 1983). These physiological adaptations, specifically the ion uptake and excretory mechanisms, are apparently under the control of neuroendocrine regulatory mechanisms originating in the central nervous system, particularly the optic ganglia (see Charmantier et al., 1984, for references), the supraesophageal ganglion (Kamemoto and Tullis, 1972; Norfolk and Craik, 1980; Kamemoto and Oyama, 1985), and the thoracic ganglion (Mantel, 1968; Kamemoto and Tullis, 1972; Tullis and Kamemoto, 1974; Norfolk and Craik, 1980; Kamemoto and Oyama, 1985) and the pericardial organs (Kamemoto and Oyama, 1985). There is also evidence that putative neurofactors released into the hemolymph affect ion transport mechanisms (Kamemoto, 1982; Savage and Robinson, 1983; Mantel, 1985). It would be of considerable interest to examine such neuroendocrine control in decapods still in the process of adapting to the limnetic environment; the freshwater palaemonid shrimp genus Macrobrachium represents such a group, encompassing species like M. potiuna, entirely independent of saline water for development, to those like M. olfersii, obligatorily dependent on saline water for the initiation of larval development and subsequent metamorphosis (see McNamara et al., 1986). M. olfersii is a prominent constituent of the coastal freshwater fauna of southern Brazil and spends its adult life in freshwater; zoeae 1 eclose in freshwater, are carried to estuarine and coastal saline waters where they develop until metamorphosis into postlarvae, then migrate to freshwater. The adult shrimp is an efficient hyperosmotic regulator, maintaining high osmotic and ionic concentrations in the hemolymph in this medium and exhibiting a strong tolerance of acute exposure to elevated salinity (McNamara, 1987). Eyestalk ablation
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experiments have indicated the presence of a factor that stimulates sodium uptake in this species (McNamara et al., 1990) while exposure to increased salinity activates granule synthesis in the neurosecretory cells of the medial protocerebrum (McNamara and Sesso, 1985). In the only study involving direct neuroendocrine involvement of extra-optic ganglia in osmotic and ionic regulation in Macrobrachium, Kamemoto and Tullis (1972) have shown that homogenates of the supra-esophageal ganglion from M. rosenbergii produce elevated chloride concentrations in the hemolymph and increased rates of sodium influx when injected into the freshwater crayfish Procambarus
clarkii.
Little information is available regarding the role of neurofactors in the control of osmotic and ionic regulation in such a dynamic group as the freshwater palaemonids. The present study thus investigates the effects of the administration of homogenates of the supra-esophageal and thoracic ganglia on the osmotic and ionic responses of M. olfersii acutely exposed to a high salinity medium or maintained in freshwater. MATERIAL
AND METHODS
Intermoult, female specimens of the freshwater shrimp M. olfersii were collected from the marginal vegetation of the Guaeca River (0% S, approximately 20”) in the State of Sao Paulo, Brazil. In the laboratory, shrimps were maintained unfed, in tanks containing about 10 liters river water, at approximately 25”, for 1 or 2 days prior to use. To obtain homogenates, groups of about 25 shrimps were exposed to aerated seawater of 21% S for 6 hr in a constant temperature chamber at 25”. The supraesophageal and thoracic ganglia were then exposed as described by McNamara and Moreira (1987); after the various connectives were cut, the ganglia were removed with iridectomy scissors and jeweler’s forceps and placed separately in filtered seawater, isosmotic to the hemolymph (300 mOsm), on ice. Twenty ganglia of each type were then triturated separately in 100 ul of filtered, isosmotic seawater with a glass pestle and a pinch of fine carborundum powder, in a I-ml polyethylene tube. The resulting homogenates were centrifuged at 10,OOOgfor 15 mitt, the supematants providing
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MCNAMARA,
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water-soluble extracts of each ganglion, containing 2 ganglion equivalents/IO ~1. Groups of 10 shrimp, previously exposed to aerated river water for 6 hr at 25”, each received 10 ul of either homogenate, injected ventrally into the abdominal musculature by means of a microsyringe. The injected animals were then exposed to aerated river water or to seawater of 21X0 S for 1, 3, or 6 hr in a constant temperature chamber at 25”. Controls for the injected animals exposed to high salinity medium consisted of shrimps injected with 10 ~1 filtered, isosmotic seawater and likewise exposed to seawater of 21%0. For shrimps injected with homogenate and exposed to freshwater alone, controls consisted of animals injected with isosmotic seawater and exposed to freshwater for up to 3 hr. After each exposure period, a single hemolymph sample of approximately 70-100 ~1 was removed from each shrimp by intracardiac puncture with a 25-8 needle and plastic insulin syringe, placed in a 1.5ml stoppered, polyethylene vial and frozen at *- 20”. At the end of the experiments, hemolymph samples were thawed at room temperature, centrifuged at 6,000 rpm for 15 min, and subsamples taken for osmotic and ionic analyses. Osmotic concentration was determined by the freezing point depression method (Salomao, 1980) and chloride concentration with a Beckman/ Spinco microtitrator, using mercuric nitrate as the titulant and diphenylcarbazone as the indicator. After appropriate dilution, cation concentrations were determined by flame spectrophotometry, Na+ (1:SOOO)and K+ (1:150) by emission, and Mg’+ (1:150) and Ca” (1:50) by absorption, using a Zeiss PMQII flame spectrophotometer. Data are presented as the mean * SEM and were analyzed statistically using one- or two-way analyses of variance to determine whether exposure time or homogenate injection or the interaction between these two factors influenced osmotic and ionic responses (P s 0.05). The Student-Newman-Keels procedure (Zar, 1984) and a t test were used to locate significant differences among means (P s 0.05). All statistical analyses were performed using the Statgraphics statistical package (Statistical Graphics Corporation).
RESULTS Summary of Analyses of Variance for Major Eficts on Hemolymph Parameters
The two-way analysis of variance treatment for control and injected M. olfersii exposed to a high salinity medium showed that both the application of homogenate of supra-esophageal ganglion and exposure
AND RIBEIRO
time affected the osmotic concentration and the concentrations of sodium, chloride, potassium, and magnesium in the hemolymph. The interaction factor was significant for magnesium concentration alone. In shrimps injected with homogenate of thoracic ganglion and subsequently exposed to high salinity medium, the concentrations of sodium, chloride, and potassium were affected by both exposure time and homogenate application. In this group, exposure time alone influenced the osmotic and magnesium concentrations while homogenate injection alone affected calcium concentrations. There was an interaction effect between exposure time and homogenate application for all ionic species. The one-way analysis of variance treatment for shrimps injected with homogenate of supra-esophageal ganglion and subsequently exposed to freshwater indicated that the concentrations of sodium, chloride, potassium, and magnesium in the hemolymph were altered during the postinjection interval. In shrimps injected with homogenates of thoracic ganglion, only the osmotic concentration of the hemolymph was influenced during the postinjection period of exposure to freshwater. Specific Effects on the Time Course of Changes in Hemolymph Parameters
Specific data on the effect of injection of homogenates of either supra-esophageal or thoracic ganglia on the osmotic and ionic concentrations of the hemolymph of M. olfersii subsequently exposed to either a high salinity gradient or freshwater are given in Figs. 1 to 6. Control shrimps. In control shrimps exposed to high salinity medium, the osmotic concentration (Fig. 1) and those of sodium (Fig. 2), chloride (Fig. 3), potassium (Fig. 4), and calcium (Fig. 6) in the hemolymph increased markedly after 1 hr, subsequent values not differing. Magnesium concentration (Fig. 5) likewise increased after 1 hr
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200-
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FIG. 1. Osmotic concentration (moan/kg water) of the hemolymph of the freshwater shrimp Macrobruchium olfersii in freshwater, after exposure to a high salinity medium (21%0 S, 608 mOsmkg water), or after the injection of 10 ul of homogenate of either supra-esophageal (SEC) or thoracic ganglion (TG) and subsequent exposure to either freshwater (0) or high salinity medium (21) for specific time periods. Data are given as the mean 2 SEM (8 < N > 17). ‘Significantly different from value for noninjected shrimps maintained in freshwater (time = 0), or immediately preceding value, at P < 0.05 (Student-Newman-Keuls test).
exposure, decreasing however from 3 to 6 hr exposure. The time course of alteration in hemolymph parameters in control shrimps maintained in freshwater was monitored only at time = 0 and 3 hr, the values not being significantly different. Values for the other intervals were assumed to be unchanged since no alteration in external osmotic or ionic gradients was applied, other experimental conditions being virtually constant. Shrimps injected with homogenate of supra-esophageal ganglion. In shrimps injected with homogenate of supra-esophageal ganglion and subsequently exposed to high salinity medium, the osmotic concentration of the hemolymph (Fig. 1) and those of sodium (Fig. 2), chloride (Fig. 3), potassium (Fig. 4), and calcium (Fig. 6) did not vary with exposure time; magnesium concentration increased after 3 hr exposure (Fig. 5). Ionic concentrations in the hemolymph of these shrimps were significantly lower than those for the control
shrimps in the case of sodium and chloride for all exposure intervals, for potassium after l-hr exposure and for magnesium up to 3-hr exposure. In shrimps injected with homogenate and exposed to freshwater, the concentrations of sodium (Fig. 2) and chloride (Fig. 3) in the hemolymph decreased after 3 hr while those of potassium (Fig. 4) and magnesium (Fig. 5) decreased after 6 hr. Osmotic and calcium concentrations remained constant. Shrimps injected with homogenate of thoracic ganglion. In shrimps injected with homogenate of thoracic ganglion and subsequently exposed to high salinity medium, the osmotic concentration of the hemolymph (Fig. 1) increased after 6 hr exposure. The concentrations of sodium (Fig. 2) and chloride (Fig. 3) in the hemolymph did not vary with exposure time. Potassium concentration (Fig. 4) increased from 3- to 6-hr exposure while magnesium concentration (Fig. 5) increased after I-hr exposure. Calcium concentration (Fig. 6) decreased
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6 Exposure Time, h FIG. 2. Concentration of sodium (meqil) in the hemolymph of the freshwater shrimp Macrobruchium olfersii in freshwater, after exposure to a high salinity medium (21X0 S, 280 meqkter Na+), or after the injection of 10 ul of homogenate of either supra-esophageal (SEG) or thoracic ganglion (TG) and subsequent exposure to either freshwater (0) or high salinity medium (21) for specific time periods. Data are given as the mean 2 SEM (8 < N > 16). ‘Significantly different from value for noninjected shrimps maintained in freshwater (time = 0), or immediately preceding value, at P s 0.05 (StudentNewman-Keuls test). *Significantly different from respective value for noninjected shrimps exposed to high salinity medium, at P < 0.05 (t test).
after I-hr exposure, subsequently increasing to initial values. The ionic concentrations in the hemolymph of these shrimps were significantly lower than those for the control shrimps in the case of sodium and calcium for all exposure periods, for chloride up to 3-hr exposure and for potassium after I-hr exposure. Magnesium concentrations were significantly higher than those in control shrimps for all exposure periods. In shrimps injected with homogenate and exposed to freshwater, the osmotic concentration of the hemolymph (Fig. 1) increased after l-hr exposure, decreasing after 3-hr exposure. No alteration was noted in the concentration of any ionic species. DISCUSSION The present study demonstrates that homogenates of supra-esophageal and thoracic ganglia taken from freshwater shrimps exposed to a strong salinity gradient induce
alterations in hemolymph osmotic and ionic concentrations when injected into other shrimps subsequently exposed either to freshwater or high salinity medium. Administration of homogenate of supraesophageal ganglion reduces the osmotic, sodium, chloride, potassium, magnesium, and calcium concentrations in the hemolymph of shrimps exposed to high salinity medium when compared to salineinjected controls exposed to the same high salinity. A decrease occurs in the sodium, chloride, potassium, and magnesium concentrations of the hemolymph of shrimps exposed to freshwater on comparison with saline-injected controls maintained in freshwater. These data do not coincide with those presented by Kamemoto and Tullis (1972) showing that injection of homogenates of supra-esophageal ganglion from Macrobrachium rosenbergii into the freshwater crayfish P. clarkii exposed to 0.1 M NaCl results in an increase in hemolymph chlo-
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6 Exposure Time, h FIG. 3. Concentration of chloride (meqil) in the hemolymph of the freshwater shrimp Mucrobrachium olfersii in freshwater, after exposure to a high salinity medium (21%0 S, 330 meqkter Cl-), or after the injection of 10 ~1 of homogenate of either supra-esophageal (SEG) or thoracic ganglion (TG) and subsequent exposure to either freshwater (0) or high salinity medium (21) for specific time periods. Data are given as the mean f SEM (8 < N > 16). ‘Significantly different from value for noninjected shrimps maintained in freshwater (time = 0), or immediately preceding value, at P 6 0.05 (StudentNewman-Keuls test). 2Signifcantly different from respective value for noninjected shrimps exposed to high salinity medium, at P s 0.05 (r test).
ride concentration and sodium influx but no change in water movements within 2.5 hr. The data are also in conflict with the demonstration by these authors that a brain homogenate from P. clarkii increases hemolymph chloride concentration and sodium influx similarly to that from M. rosenbergii when assayed on the crayfish itself. However, McNamara et al. (1990) have shown that eyestalkless M. olfersii, exposed to seawater for up to 24 hr, exhibit reduced concentrations of sodium in the hemolymph when compared to intact, control shrimps likewise exposed to seawater, suggesting the existence of a factor in the optic ganglia which stimulates sodium uptake in freshwater. This factor may be similar to that found in the supra-esophageal ganglion of M. rosenbergii by Kamemoto and Tullis (1972). The discrepancy noted in comparison with the data of Kamemoto and Tullis (1972) may reflect the fact that in the present experiments, the shrimps providing
the homogenates were previously exposed to seawater, thereby increasing the synthesis of granules in the neurosecretory cells of the medial protocerebrum (McNamara and Sesso, 1985). In contrast, the brain homogenates used by Kamemoto and Tullis (1972) were derived from M. rosenbergii and P. clarkii apparently maintained in freshwater and thus may not contain the same factor or the same proportion of factors as found in supra-esophageal homogenates from M. olfersii. McNamara and Moreira (1987) have demonstrated an increase in the rates of oxygen consumption by isolated supra-esophageal ganglia after removal from intact M. oifersii exposed to high salinity gradients, which also suggests augmented ganglionic neurosynthetic activity associated with exposure to high salinity. The oxygen consumption rate of M. elfersii maintained in freshwater increases differentially after the injection of homogenate of supra-esophageal ganglia derived from shrimps exposed to either freshwater or sa-
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MCNAMARA,
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6
Exposure Time, h FIG. 4. Concentration of potassium (meq/l) in the hemolymph of the freshwater shrimp Macrobruchhm olfersii in freshwater, after exposure to a high salinity medium (21%0 S, 6.2 meq/liter K+), or after the injection of 10 ~1 of homogenate of either supra-esophageal (SEG) or thoracic ganglion (TG) and subsequent exposure to either freshwater (0) or high salinity medium (21) for specific time periods. Data are given as the mean f SEM (6 < N > 14). ‘Significantly different from value for noninjected shrimps maintained in freshwater (time = 0), or immediately preceding value, at P < 0.05 (Student-Newman-Keuls test). 2Signiticantly different from respective value for noninjected shrimps exposed to high salinity medium, at P s 0.05 (t test).
line water (Souza and Moreira, 1987), reproducing the increases in rate of oxygen consumption demonstrated for this shrimp in freshwater and at high salinities (McNamara and Moreira, 1987)which possibly reflect the metabolic consequences of acute osmotic adaptation (McNamara, 1987). The data obtained in the present study are consistent, however, with those obtained by Kamemoto and Tullis (1972) on injecting homogenates of supra-esophageal ganglion derived from the marine crabs Metapograpsus messor (semi-terrestrial/ estuarine) and Thalamita crenata (marine/ estuarine) into the freshwater crayfish P. clarkii, provoking a decrease in hemolymph chloride concentration and sodium and water influxes. A supra-esophageal homogenate from the marine shrimp Spirontocaris marmoratus assayed on P. clarkii reduces water influx alone. Thus, on exposure to seawater, the freshwater shrimp M. oifersii apparently synthetizes a neurofactor in the supra-esophageal ganglion which induces
modifications in hemolymph ionic concentration similar to those provoked by supraesophageal homogenates from marine crabs, although not present in the marine shrimp S. marmoratus. Viewed then, in terms of the invasion and colonization of the freshwater biotope by the freshwater decapods, M. ofirsii appears to exhibit osmotic and ionic responses to neurofactor application more closely identifiable with those of marine/estuarine crustaceans rather than with those of a truly freshwater form such as the freshwater astacid crayfish, substantiating the notion that this species is indeed a recent invader of the freshwater habitat (McNamara, 1987). Similarly to the action of homogenate of supra-esophageal ganglion, the injection of thoracic ganglion homogenate in M. olfersii likewise produces a reduction in sodium, chloride, potassium, magnesium, and calcium concentrations in the hemolymph of shrimps exposed to high salinity medium, compared to saline-injected control
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Exposure Time, h FIG. 5. Concentration of magnesium (meq/l) in the hemolymph of the freshwater shrimp Macrobrachium olfersii in freshwater, after exposure to a high salinity medium (21X0 S, 33.4 meq/liter Mg2+), or after the injection of 10 ~1 of homogenate of either supra-esophageal (SEG) or thoracic ganglion (TG) and subsequent exposure to either freshwater (0) or high salinity medium (21) for specific time periods. Data are given as the mean 2 SEM (8 < IV > 13). ‘Significantly different from value for noninjected shrimps maintained in freshwater (time = 0), or immediately preceding value, at P s 0.05 (Student-Newman-Keuls test). ZSignificantly different from respective value for noninjected shrimps exposed to high salinity medium, at P s 0.05 (t test).
shrimps similarly exposed to high salinity; however, in shrimps exposed to freshwater, injection of thoracic ganglion homogenate, while causing an increase in hemolymph osmotic concentration, does not alter ionic concentrations. Few comparative data are available concerning the effects of homogenates of thoracic ganglia. Kamemoto and Tullis (1972) have shown that there is a decrease in chloride concentration in the hemolymph of the marine crab Grapsus sanguineus and in Metapograpsus messor after the injection of homogenates of thoracic ganglion compared to controls when both crabs are placed in 25% seawater. A decrease in water influx but no change in sodium influx occurs after injection of homogenate of thoracic ganglion from Thalamita crenafa into P. clarkii maintained in 0.1 M NaCl or into the crab itself on exposure to 50% seawater (Tullis and Kamemoto, 1974). This tendency does not appear to be consistent with that noted after the injection of thoracic
ganglion homogenate into M. olfersii exposed to high salinity medium where a decrease in ionic concentrations of the hemolymph is found. Clearly, alterations in water and ion permeabilities, in addition to specific osmolyte concentrations and hydration levels, must be considered when interpreting data on the effects of putative neurofactors present in the crustacean central nervous system. Although such data are still lacking for M. olfersii, a possible mechanism accounting for the decreased ionic concentrations of the hemolymph encountered in M. olfersii, injected with homogenates of either supraesophageal or thoracic ganglia, and subsequently exposed to high salinity medium, would be a decrease in apparent ionic permeability. It is unlikely that a net flux of water into the animal (hemolymph osmotic concentration = 387 mOsm after 6 hr) would occur against its concentration gradient (osmotic concentration of external medium = 608 mOsm); likewise, it is im-
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MCNAMARA,
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6 Exposure
Time, h
FIG. 6. Concentration of calcium (meq/l) in the hemolymph of the freshwater shrimp Mucrobruchium olfersii in freshwater, after exposure to a high salinity medium (2110 S, 6.2 meqkter Ca”), or after the injection of 10 pl of homogenate of either supra-esophageal (SEG) or thoracic ganglion (TG) and subsequent exposure to either freshwater (0) or high salinity medium (21) for specific time periods. Data are given as the mean f SEM (3 < N > 11). ‘Significantly different from value for noninjected shrimps maintained in freshwater (time = O), or immediately preceding value, at P s 0.05 (StudentNewman-Keuls test). ‘Significantly different from respective value for noninjected shrimps exposed to high salinity medium, at P s 0.05(t test).
probable that a net eftlux of the principal ions involved (Na+, Cl-) would take place against their concentration gradients, (i.e., hemolymph [Na+] = approximately 150 meq/liter after 6 hr vs external medium [Na+] = 280 meqfliter). Thus, the reduced ionic concentrations in the hemolymph most probably reflect a decrease in the apparent permeability to ion intlux rather than an increase in ion efflux. In the case of shrimps injected with homogenate of supra-esophageal ganglion and exposed to freshwater, clearly net water influx or net Na+ and Cl- efflux must be considered. The reduced ionic concentrations in the hemolymph can only have resulted from osmotic dilution, i.e., increased rates of water influx (see Tullis and Kamemoto, 1974) and/or ion efflux, either in the urine as the result of decreased reabsorption, or through increased apparent permeability of the body surfaces, possibly the gills. It is feasible that the homogenate may have reduced the efftciency of the inwardly directed, Na+ transporting system,
which subsequently becomes unable to compensate for diffusional effluxes, resulting in a net decrease in hemolymph Na+ concentration. The rapid increase in osmotic concentration of the hemolymph in M. olfersii injected with homogenate of thoracic ganglion and exposed to freshwater, which is not reflected in the concentrations of the principal ions measured, may result from an increase in the quantity of organic osmolytes in the hemolymph, possibly free amino acids. Tan and Choong (1981) have demonstrated that gradual acclimation (3day steps) of M. rosenbergii from freshwater to varying dilutions of seawater up to 30% S induces a decrease in hemolymph protein content and an increase in muscle ninhydrin positive substances and free amino acids. Neurofactors associated with the thoracic ganglion thus may be involved in the regulation of hemolymph protein hydrolysis and free amino acid concentration. Curiously, Kamemoto and Tullis (1972) have shown that the hemolymph chloride
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concentration of the freshwater crab Potamon dehaani maintained in freshwater increases after injection of thoracic ganglion homogenate from the crab itself. The present study thus has revealed the presence of neurofactors located in the ganglia of the central nervous system of the freshwater shrimp M. olfersii which are apparently involved in ionic regulation, under conditions frequently encountered in the normal biotope of the species. The existence of such capability substantiates the hypothesis that the establishment of successful limnetic species requires specific physiological mechanisms capable of compensating for the phenomena of osmotic dilution and ion loss typically encountered by such organisms. ACKNOWLEDGMENTS The authors thank Alice G. Lima and Alexandre C. Coutinho for skillful technical assistance. This study, partially conducted at the Centro de Biologia Marinha, Universidade de S&o Paul0 in Szlo Sebastiao, SP, was financed by research grants from the Conselho Nacional de Desenvolvimento Cientifico e TecnoMgico (303282/84,403353&t, 403635/84) to J. C. McNamara.
REFERENCES Ahearn, G. A. (1978). Allosteric cotransport of sodium, chloride, and calcium by the intestine of freshwater prawns. J. Membr. Biol. 42, 281-300. Ahearn, G. A., and Tomquist, A. (1977). Aliosteric cooperativity during intestinal cotransport of sodium and chloride in freshwater prawns. Biochim. Biophys. Acta 471, 273-279. Berlind, A., and Kamemoto, F. I. (1977). Rapid water permeability changes in eyestalkless euryhahne crabs and in isolated, perfused gills. Comp. Biothem. Physiol. 58A, 383-385. Cantelmo, A. (1977). Water permeability of isolated tissues from decapod crustaceans. 1. Effect of osmotic conditions. Comp. Biochem. Physiol. 58A, 343-348. Charmantier, G., Charmantier-Daures, M., and Aiken, D. E. (1984). Neuroendocrine control of hydromineral regulation in the American lobster Homarus americanus H. Mime-Edwards 1837 (Crustacea, Decapoda). 1. Juveniles. Gen. Comp. Endocrinol. 54, 8-19.
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Chu, K. H. (1987). Sodium transport across the perfused midgut and hindgut of the blue crab, Callinectes sapidus: The possible role of the gut in crustacean osmoregulation. Comp. Biochem. Physiol. 87A, 21-25. Ehrenfeld, J., and Isaia, J. (1974). The effect of ligaturing the eyestalks on the water and ion permeabilities of Astacus leprodactylus. J. Comp. Physiol. 93, 105-l 15. Harris, R. R., and Bayliss, D. (1988). Gill (Na+ + K+)-ATPases in decapod crustaceans: distribution and characteristics in relation to Na+ regulation. Comp. Biochem. Physiol. 9OA, 303-308. Kamemoto, F. I. (1982). Crustacean neuropeptides and osmoregulation. In “Neurosecretion: Molecules, Cells, Systems” (D. S. Famer and K. Lederis, Eds.), pp. 329-335. Plenum, New York. Kamemoto, F. I., and Oyama, S. I. (1985). Neuroendocrine influence on effector tissues of hydromineral balance in crustaceans. In “Current Trends in Comparative Endocrinology” (B. Lofts and W. N. Holmes, Eds.), pp. 883-886. Hong Kong Univ. Press, Hong Kong. Kamemoto, F. I., and Tullis, R. E. (1972). Hydromineral regulation in decapod Crustacea. Gen. Comp. Endocrinol. Suppl. 3, 299-307. Mantel, L. H. (1%8). The foregut of Gecarcinus lateralis as an organ of salt and water balance. Am. Zool. 8, 433-442. Mantel, L. H. (1985). Neurohormonal integration of osmotic and ionic regulation. Am. Zool. 25, 253263. Mantel, L. H., and Farmer, L. (1983). Osmotic and ionic regulation. In “The Biology of Crustacea” (L. H. Mantel, Ed.), Vol. 5, pp. 54-161. Academic Press, New York. McNamara, J. C. (1987). The time course of osmotic regulation in the freshwater shrimp Macrobrachium o/fersii (Wiegmann) (Decapoda, Palaemonidae). J. Exp. Mar. Biol. Ecol. 107, 245-251. McNamara, J. C., and Moreira, G. S. (1987). 0, consumption and acute salinity exposure in the freshwater shrimp Macrobrachium olfersii (Wiegmann) (Crustacea: Decapoda): Whole animaI and tissue respiration. J. Exp. Mar. Biol. Ecol. 113, 221-230. McNamara, J. C., and Sesso, A. (1985). Estudo ultraestmtural em tortes Bnos e criorreplicas de cclulas neurossecretoras no g&nglio supra-esofagico do camarao de agua dote Macrobrachium olfersii (Crustacea, Palaemonidae) exposto a salinidade. Resumes, XII Congress0 Brasileiro de Zoologia, pp. 63-64. McNamara, J. C., Moreira, G. S., and Souza, C. R. (1986). The effect of salinity on respiratory metaboIism in selected ontogenetic stages of the
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SALOMaiO,
freshwater shrimp Macrobrachium olfersii (Decapoda, Palaemonidae). Comp. Biochem. Physiol. 83A, 359-364. McNamara, J. C., Salomao, L. C., and Ribeiro, E. A. (1990). The effect of eyestalk ablation on haemolymph osmotic and ionic concentrations during acute salinity exposure in the freshwater shrimp hfucrobrachium olfersii (Wiegmann) (Crustacea, Decapoda). Hydrobiologiu 199, 193-199. Norfolk, J. R., and Craik, J. C. (1980). Investigation of the control of urine production in the shore crab Carcinus maenas L. Comp. Biochem. Physiol. 67A, 141-148. Salomao, L. C. (1980). Determina@o da concentra@o osm6tica de micro-amostras de thlidos atravds do ponto de congelamento. Bolm Fisiol. Anim. USP 4, 133-141. Savage, J. P., and Robinson, G. D. (1983). Inducement of increased gill Na + K-ATPase activity by a hemolymph factor in hyperregulating Callinectes sapidus. Comp. Biochem. Physiol. 75A, 65-69. Shaw, J. (1961). Sodium balance in Eriocheir sinensis
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RIBEIRO
(M. Edw.). The adaptation of the Crustacea to fresh water. .I. Exp. Biol. 38, 153-162. Souza, C. R., and Moreira, G. S. (1987). Salinity effects on the neuroendocrine control of respiratory metabolism in Macrobrachium olfersii (Wiegmann) (Crustacea, Palaemonidae). Comp. Biothem. Physiol. 87A, 399-403. Tan, C. H., and Choong, K. Y. (1981). Effect of hyperosmotic stress on hemolymph protein, muscle ninhydrin-positive substances and free amino acids in Macrobrachium rosenbergii (De Man). Comp. Biochem. Physiol. 7OA, 48549. Taylor, P. M., and Harris, R. R. (1986). Osmoregulation in Corophium curvispinum (Crustacea: Amphipoda), a recent coloniser of freshwater. I. Sodium ion regulation. J. Comp. Physiol. 156B, 32F 329. Tullis, R. E., and Kamemoto, F. I. (1974). Separation and biological effects of CNS factors affecting water balance in the decapod crustacean Thalamita crenata. Gen. Comp. Endocrinol. 23, 19-28. Zar, J. H. (1984). “Biostatistical Analysis.” Prentice Hall, Englewood Cliffs, NJ.
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Neuroendocrine the Hemolymph
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16-26 (1991)
Regulation of Osmotic and Ionic Concentrations in of the Freshwater Shrimp Macrobrachium olfersii (Wiegmann) (Crustacea, Decapoda)
JOHN C. MCNAMARA,LUIS
C. SALOMAO,* AND ELAINE A. RIBEIRO*
Departamento de Biologia, Faculdade de Filosojia, Ci@ncias e Letras, Vniversidade de Stio Paulo, Ribeireo Preto, 14049 SP, Bras& and *Departamento de Fisiologia Geral, Institute de Bioci&ncias, Vniversidade de Sdo Paula, Sbo Paul0 05508 SP, Brasil Accepted October 12, 1990 Putative neuroendocrine mediation of osmotic and ionic responses to acute exposure to high salinity medium was investigated in the freshwater shrimp Macrobrachium oljksii (Wiegmann). Homogenates of supra-esophageal or thoracic ganglia, prepared from shrimps exposed to seawater of 21X0 S for 6 hr, were injected into the abdominal musculature of shrimps previously exposed to freshwater and subsequently exposed to either freshwater or seawater (21X0 S). Osmotic, sodium, chloride, potassium, magnesium, and calcium concentrations were determined in hemolymph samples removed by intracardiac puncture at time = 0, 1,3, or 6 hr after homogenate application. Control shrimps were injected with faltered seawater, isosmotic to the hemolymph, and treated similarly. In control shrimps, the osmotic, Na+, Cl-, K+, Mg2+, and Ca2’ concentrations in the hemolymph increased (P ~0.05) after I-hr exposure to seawater. In shrimps injected with homogenates of supraesophageal ganglion and exposed to seawater, osmotic and ionic concentrations in the hemolymph did not vary with exposure time; in injected shrimps exposed to freshwater, Na+, Cl-, K+, and Mg2+ concentrations decreased (P Q 0.05) with time. In shrimps injected with homogenates of thoracic ganglion and exposed to seawater, hemolymph osmotic, K+, and Mg2+ concentrations increased (P s 0.05); Na+, Cl-, and CaZ+ concentrations remained unchanged. In injected shrimps exposed to freshwater, hemolymph osmotic concentration alone increased (P G 0.05) after 1 hr, all other ionic concentrations remaining unchanged. These data suggest that neurofactors apparently located within the ganglia of the central nervous system of M. orfersii may alter the apparent ionic permeabilities of this shrimp, depending on the salinity characteristics of the external medium. The data support the notion that invasion of the freshwater biotope by estuarine crustaceans has necessitated the evolution of specific physiological mechanisms capable of compensating for the osmotic dilution and ion loss typically encountered by such organisms. Q 1591 Academic Press. Inc.
trations in the face of typically low external ionic concentrations. Such systems include high affinity ion uptake mechanisms located in the gills (Shaw, 1961; Taylor and Harris, 1986; Harris and Bayliss, 1988) and intestine (Ahearn and Tornquist, 1977; Ahearn, 1978; Chu, 1987), reduction in body and organ osmotic and ionic permeabilities (Ehrenfeld and Isaia, 1974; Berlind and Kamemoto, 1977; Cantelmo, 1977), and efficient processes of solute reabsorp-
Implicit in the ongoing penetration and adaptation of marine and estuarine Crustatea to the freshwater biotope is the evolution of physiological mechanisms responsible for the maintenance of osmotically and ionically stable extra- and intracellular fluids. Essentially, successful invaders of the freshwater environment must possess effector systems capable of maintaining fairly high intra- and extracellular ionic concen16 001~6480/91 $1.50 Copyright 0 1991 by Academic Press, Inc. AU rights of reproduction in any form reserved
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tion, resulting in the excretion of a urine hypotonic to the hemolymph (Mantel and Farmer, 1983). These physiological adaptations, specifically the ion uptake and excretory mechanisms, are apparently under the control of neuroendocrine regulatory mechanisms originating in the central nervous system, particularly the optic ganglia (see Charmantier et al., 1984, for references), the supraesophageal ganglion (Kamemoto and Tullis, 1972; Norfolk and Craik, 1980; Kamemoto and Oyama, 1985), and the thoracic ganglion (Mantel, 1968; Kamemoto and Tullis, 1972; Tullis and Kamemoto, 1974; Norfolk and Craik, 1980; Kamemoto and Oyama, 1985) and the pericardial organs (Kamemoto and Oyama, 1985). There is also evidence that putative neurofactors released into the hemolymph affect ion transport mechanisms (Kamemoto, 1982; Savage and Robinson, 1983; Mantel, 1985). It would be of considerable interest to examine such neuroendocrine control in decapods still in the process of adapting to the limnetic environment; the freshwater palaemonid shrimp genus Macrobrachium represents such a group, encompassing species like M. potiuna, entirely independent of saline water for development, to those like M. olfersii, obligatorily dependent on saline water for the initiation of larval development and subsequent metamorphosis (see McNamara et al., 1986). M. olfersii is a prominent constituent of the coastal freshwater fauna of southern Brazil and spends its adult life in freshwater; zoeae 1 eclose in freshwater, are carried to estuarine and coastal saline waters where they develop until metamorphosis into postlarvae, then migrate to freshwater. The adult shrimp is an efficient hyperosmotic regulator, maintaining high osmotic and ionic concentrations in the hemolymph in this medium and exhibiting a strong tolerance of acute exposure to elevated salinity (McNamara, 1987). Eyestalk ablation
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experiments have indicated the presence of a factor that stimulates sodium uptake in this species (McNamara et al., 1990) while exposure to increased salinity activates granule synthesis in the neurosecretory cells of the medial protocerebrum (McNamara and Sesso, 1985). In the only study involving direct neuroendocrine involvement of extra-optic ganglia in osmotic and ionic regulation in Macrobrachium, Kamemoto and Tullis (1972) have shown that homogenates of the supra-esophageal ganglion from M. rosenbergii produce elevated chloride concentrations in the hemolymph and increased rates of sodium influx when injected into the freshwater crayfish Procambarus
clarkii.
Little information is available regarding the role of neurofactors in the control of osmotic and ionic regulation in such a dynamic group as the freshwater palaemonids. The present study thus investigates the effects of the administration of homogenates of the supra-esophageal and thoracic ganglia on the osmotic and ionic responses of M. olfersii acutely exposed to a high salinity medium or maintained in freshwater. MATERIAL
AND METHODS
Intermoult, female specimens of the freshwater shrimp M. olfersii were collected from the marginal vegetation of the Guaeca River (0% S, approximately 20”) in the State of Sao Paulo, Brazil. In the laboratory, shrimps were maintained unfed, in tanks containing about 10 liters river water, at approximately 25”, for 1 or 2 days prior to use. To obtain homogenates, groups of about 25 shrimps were exposed to aerated seawater of 21% S for 6 hr in a constant temperature chamber at 25”. The supraesophageal and thoracic ganglia were then exposed as described by McNamara and Moreira (1987); after the various connectives were cut, the ganglia were removed with iridectomy scissors and jeweler’s forceps and placed separately in filtered seawater, isosmotic to the hemolymph (300 mOsm), on ice. Twenty ganglia of each type were then triturated separately in 100 ul of filtered, isosmotic seawater with a glass pestle and a pinch of fine carborundum powder, in a I-ml polyethylene tube. The resulting homogenates were centrifuged at 10,OOOgfor 15 mitt, the supematants providing
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MCNAMARA,
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water-soluble extracts of each ganglion, containing 2 ganglion equivalents/IO ~1. Groups of 10 shrimp, previously exposed to aerated river water for 6 hr at 25”, each received 10 ul of either homogenate, injected ventrally into the abdominal musculature by means of a microsyringe. The injected animals were then exposed to aerated river water or to seawater of 21X0 S for 1, 3, or 6 hr in a constant temperature chamber at 25”. Controls for the injected animals exposed to high salinity medium consisted of shrimps injected with 10 ~1 filtered, isosmotic seawater and likewise exposed to seawater of 21%0. For shrimps injected with homogenate and exposed to freshwater alone, controls consisted of animals injected with isosmotic seawater and exposed to freshwater for up to 3 hr. After each exposure period, a single hemolymph sample of approximately 70-100 ~1 was removed from each shrimp by intracardiac puncture with a 25-8 needle and plastic insulin syringe, placed in a 1.5ml stoppered, polyethylene vial and frozen at *- 20”. At the end of the experiments, hemolymph samples were thawed at room temperature, centrifuged at 6,000 rpm for 15 min, and subsamples taken for osmotic and ionic analyses. Osmotic concentration was determined by the freezing point depression method (Salomao, 1980) and chloride concentration with a Beckman/ Spinco microtitrator, using mercuric nitrate as the titulant and diphenylcarbazone as the indicator. After appropriate dilution, cation concentrations were determined by flame spectrophotometry, Na+ (1:SOOO)and K+ (1:150) by emission, and Mg’+ (1:150) and Ca” (1:50) by absorption, using a Zeiss PMQII flame spectrophotometer. Data are presented as the mean * SEM and were analyzed statistically using one- or two-way analyses of variance to determine whether exposure time or homogenate injection or the interaction between these two factors influenced osmotic and ionic responses (P s 0.05). The Student-Newman-Keels procedure (Zar, 1984) and a t test were used to locate significant differences among means (P s 0.05). All statistical analyses were performed using the Statgraphics statistical package (Statistical Graphics Corporation).
RESULTS Summary of Analyses of Variance for Major Eficts on Hemolymph Parameters
The two-way analysis of variance treatment for control and injected M. olfersii exposed to a high salinity medium showed that both the application of homogenate of supra-esophageal ganglion and exposure
AND RIBEIRO
time affected the osmotic concentration and the concentrations of sodium, chloride, potassium, and magnesium in the hemolymph. The interaction factor was significant for magnesium concentration alone. In shrimps injected with homogenate of thoracic ganglion and subsequently exposed to high salinity medium, the concentrations of sodium, chloride, and potassium were affected by both exposure time and homogenate application. In this group, exposure time alone influenced the osmotic and magnesium concentrations while homogenate injection alone affected calcium concentrations. There was an interaction effect between exposure time and homogenate application for all ionic species. The one-way analysis of variance treatment for shrimps injected with homogenate of supra-esophageal ganglion and subsequently exposed to freshwater indicated that the concentrations of sodium, chloride, potassium, and magnesium in the hemolymph were altered during the postinjection interval. In shrimps injected with homogenates of thoracic ganglion, only the osmotic concentration of the hemolymph was influenced during the postinjection period of exposure to freshwater. Specific Effects on the Time Course of Changes in Hemolymph Parameters
Specific data on the effect of injection of homogenates of either supra-esophageal or thoracic ganglia on the osmotic and ionic concentrations of the hemolymph of M. olfersii subsequently exposed to either a high salinity gradient or freshwater are given in Figs. 1 to 6. Control shrimps. In control shrimps exposed to high salinity medium, the osmotic concentration (Fig. 1) and those of sodium (Fig. 2), chloride (Fig. 3), potassium (Fig. 4), and calcium (Fig. 6) in the hemolymph increased markedly after 1 hr, subsequent values not differing. Magnesium concentration (Fig. 5) likewise increased after 1 hr
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FIG. 1. Osmotic concentration (moan/kg water) of the hemolymph of the freshwater shrimp Macrobruchium olfersii in freshwater, after exposure to a high salinity medium (21%0 S, 608 mOsmkg water), or after the injection of 10 ul of homogenate of either supra-esophageal (SEC) or thoracic ganglion (TG) and subsequent exposure to either freshwater (0) or high salinity medium (21) for specific time periods. Data are given as the mean 2 SEM (8 < N > 17). ‘Significantly different from value for noninjected shrimps maintained in freshwater (time = 0), or immediately preceding value, at P < 0.05 (Student-Newman-Keuls test).
exposure, decreasing however from 3 to 6 hr exposure. The time course of alteration in hemolymph parameters in control shrimps maintained in freshwater was monitored only at time = 0 and 3 hr, the values not being significantly different. Values for the other intervals were assumed to be unchanged since no alteration in external osmotic or ionic gradients was applied, other experimental conditions being virtually constant. Shrimps injected with homogenate of supra-esophageal ganglion. In shrimps injected with homogenate of supra-esophageal ganglion and subsequently exposed to high salinity medium, the osmotic concentration of the hemolymph (Fig. 1) and those of sodium (Fig. 2), chloride (Fig. 3), potassium (Fig. 4), and calcium (Fig. 6) did not vary with exposure time; magnesium concentration increased after 3 hr exposure (Fig. 5). Ionic concentrations in the hemolymph of these shrimps were significantly lower than those for the control
shrimps in the case of sodium and chloride for all exposure intervals, for potassium after l-hr exposure and for magnesium up to 3-hr exposure. In shrimps injected with homogenate and exposed to freshwater, the concentrations of sodium (Fig. 2) and chloride (Fig. 3) in the hemolymph decreased after 3 hr while those of potassium (Fig. 4) and magnesium (Fig. 5) decreased after 6 hr. Osmotic and calcium concentrations remained constant. Shrimps injected with homogenate of thoracic ganglion. In shrimps injected with homogenate of thoracic ganglion and subsequently exposed to high salinity medium, the osmotic concentration of the hemolymph (Fig. 1) increased after 6 hr exposure. The concentrations of sodium (Fig. 2) and chloride (Fig. 3) in the hemolymph did not vary with exposure time. Potassium concentration (Fig. 4) increased from 3- to 6-hr exposure while magnesium concentration (Fig. 5) increased after I-hr exposure. Calcium concentration (Fig. 6) decreased
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6 Exposure Time, h FIG. 2. Concentration of sodium (meqil) in the hemolymph of the freshwater shrimp Macrobruchium olfersii in freshwater, after exposure to a high salinity medium (21X0 S, 280 meqkter Na+), or after the injection of 10 ul of homogenate of either supra-esophageal (SEG) or thoracic ganglion (TG) and subsequent exposure to either freshwater (0) or high salinity medium (21) for specific time periods. Data are given as the mean 2 SEM (8 < N > 16). ‘Significantly different from value for noninjected shrimps maintained in freshwater (time = 0), or immediately preceding value, at P s 0.05 (StudentNewman-Keuls test). *Significantly different from respective value for noninjected shrimps exposed to high salinity medium, at P < 0.05 (t test).
after I-hr exposure, subsequently increasing to initial values. The ionic concentrations in the hemolymph of these shrimps were significantly lower than those for the control shrimps in the case of sodium and calcium for all exposure periods, for chloride up to 3-hr exposure and for potassium after I-hr exposure. Magnesium concentrations were significantly higher than those in control shrimps for all exposure periods. In shrimps injected with homogenate and exposed to freshwater, the osmotic concentration of the hemolymph (Fig. 1) increased after l-hr exposure, decreasing after 3-hr exposure. No alteration was noted in the concentration of any ionic species. DISCUSSION The present study demonstrates that homogenates of supra-esophageal and thoracic ganglia taken from freshwater shrimps exposed to a strong salinity gradient induce
alterations in hemolymph osmotic and ionic concentrations when injected into other shrimps subsequently exposed either to freshwater or high salinity medium. Administration of homogenate of supraesophageal ganglion reduces the osmotic, sodium, chloride, potassium, magnesium, and calcium concentrations in the hemolymph of shrimps exposed to high salinity medium when compared to salineinjected controls exposed to the same high salinity. A decrease occurs in the sodium, chloride, potassium, and magnesium concentrations of the hemolymph of shrimps exposed to freshwater on comparison with saline-injected controls maintained in freshwater. These data do not coincide with those presented by Kamemoto and Tullis (1972) showing that injection of homogenates of supra-esophageal ganglion from Macrobrachium rosenbergii into the freshwater crayfish P. clarkii exposed to 0.1 M NaCl results in an increase in hemolymph chlo-
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6 Exposure Time, h FIG. 3. Concentration of chloride (meqil) in the hemolymph of the freshwater shrimp Mucrobrachium olfersii in freshwater, after exposure to a high salinity medium (21%0 S, 330 meqkter Cl-), or after the injection of 10 ~1 of homogenate of either supra-esophageal (SEG) or thoracic ganglion (TG) and subsequent exposure to either freshwater (0) or high salinity medium (21) for specific time periods. Data are given as the mean f SEM (8 < N > 16). ‘Significantly different from value for noninjected shrimps maintained in freshwater (time = 0), or immediately preceding value, at P 6 0.05 (StudentNewman-Keuls test). 2Signifcantly different from respective value for noninjected shrimps exposed to high salinity medium, at P s 0.05 (r test).
ride concentration and sodium influx but no change in water movements within 2.5 hr. The data are also in conflict with the demonstration by these authors that a brain homogenate from P. clarkii increases hemolymph chloride concentration and sodium influx similarly to that from M. rosenbergii when assayed on the crayfish itself. However, McNamara et al. (1990) have shown that eyestalkless M. olfersii, exposed to seawater for up to 24 hr, exhibit reduced concentrations of sodium in the hemolymph when compared to intact, control shrimps likewise exposed to seawater, suggesting the existence of a factor in the optic ganglia which stimulates sodium uptake in freshwater. This factor may be similar to that found in the supra-esophageal ganglion of M. rosenbergii by Kamemoto and Tullis (1972). The discrepancy noted in comparison with the data of Kamemoto and Tullis (1972) may reflect the fact that in the present experiments, the shrimps providing
the homogenates were previously exposed to seawater, thereby increasing the synthesis of granules in the neurosecretory cells of the medial protocerebrum (McNamara and Sesso, 1985). In contrast, the brain homogenates used by Kamemoto and Tullis (1972) were derived from M. rosenbergii and P. clarkii apparently maintained in freshwater and thus may not contain the same factor or the same proportion of factors as found in supra-esophageal homogenates from M. olfersii. McNamara and Moreira (1987) have demonstrated an increase in the rates of oxygen consumption by isolated supra-esophageal ganglia after removal from intact M. oifersii exposed to high salinity gradients, which also suggests augmented ganglionic neurosynthetic activity associated with exposure to high salinity. The oxygen consumption rate of M. elfersii maintained in freshwater increases differentially after the injection of homogenate of supra-esophageal ganglia derived from shrimps exposed to either freshwater or sa-
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MCNAMARA,
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Exposure Time, h FIG. 4. Concentration of potassium (meq/l) in the hemolymph of the freshwater shrimp Macrobruchhm olfersii in freshwater, after exposure to a high salinity medium (21%0 S, 6.2 meq/liter K+), or after the injection of 10 ~1 of homogenate of either supra-esophageal (SEG) or thoracic ganglion (TG) and subsequent exposure to either freshwater (0) or high salinity medium (21) for specific time periods. Data are given as the mean f SEM (6 < N > 14). ‘Significantly different from value for noninjected shrimps maintained in freshwater (time = 0), or immediately preceding value, at P < 0.05 (Student-Newman-Keuls test). 2Signiticantly different from respective value for noninjected shrimps exposed to high salinity medium, at P s 0.05 (t test).
line water (Souza and Moreira, 1987), reproducing the increases in rate of oxygen consumption demonstrated for this shrimp in freshwater and at high salinities (McNamara and Moreira, 1987)which possibly reflect the metabolic consequences of acute osmotic adaptation (McNamara, 1987). The data obtained in the present study are consistent, however, with those obtained by Kamemoto and Tullis (1972) on injecting homogenates of supra-esophageal ganglion derived from the marine crabs Metapograpsus messor (semi-terrestrial/ estuarine) and Thalamita crenata (marine/ estuarine) into the freshwater crayfish P. clarkii, provoking a decrease in hemolymph chloride concentration and sodium and water influxes. A supra-esophageal homogenate from the marine shrimp Spirontocaris marmoratus assayed on P. clarkii reduces water influx alone. Thus, on exposure to seawater, the freshwater shrimp M. oifersii apparently synthetizes a neurofactor in the supra-esophageal ganglion which induces
modifications in hemolymph ionic concentration similar to those provoked by supraesophageal homogenates from marine crabs, although not present in the marine shrimp S. marmoratus. Viewed then, in terms of the invasion and colonization of the freshwater biotope by the freshwater decapods, M. ofirsii appears to exhibit osmotic and ionic responses to neurofactor application more closely identifiable with those of marine/estuarine crustaceans rather than with those of a truly freshwater form such as the freshwater astacid crayfish, substantiating the notion that this species is indeed a recent invader of the freshwater habitat (McNamara, 1987). Similarly to the action of homogenate of supra-esophageal ganglion, the injection of thoracic ganglion homogenate in M. olfersii likewise produces a reduction in sodium, chloride, potassium, magnesium, and calcium concentrations in the hemolymph of shrimps exposed to high salinity medium, compared to saline-injected control
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Exposure Time, h FIG. 5. Concentration of magnesium (meq/l) in the hemolymph of the freshwater shrimp Macrobrachium olfersii in freshwater, after exposure to a high salinity medium (21X0 S, 33.4 meq/liter Mg2+), or after the injection of 10 ~1 of homogenate of either supra-esophageal (SEG) or thoracic ganglion (TG) and subsequent exposure to either freshwater (0) or high salinity medium (21) for specific time periods. Data are given as the mean 2 SEM (8 < IV > 13). ‘Significantly different from value for noninjected shrimps maintained in freshwater (time = 0), or immediately preceding value, at P s 0.05 (Student-Newman-Keuls test). ZSignificantly different from respective value for noninjected shrimps exposed to high salinity medium, at P s 0.05 (t test).
shrimps similarly exposed to high salinity; however, in shrimps exposed to freshwater, injection of thoracic ganglion homogenate, while causing an increase in hemolymph osmotic concentration, does not alter ionic concentrations. Few comparative data are available concerning the effects of homogenates of thoracic ganglia. Kamemoto and Tullis (1972) have shown that there is a decrease in chloride concentration in the hemolymph of the marine crab Grapsus sanguineus and in Metapograpsus messor after the injection of homogenates of thoracic ganglion compared to controls when both crabs are placed in 25% seawater. A decrease in water influx but no change in sodium influx occurs after injection of homogenate of thoracic ganglion from Thalamita crenafa into P. clarkii maintained in 0.1 M NaCl or into the crab itself on exposure to 50% seawater (Tullis and Kamemoto, 1974). This tendency does not appear to be consistent with that noted after the injection of thoracic
ganglion homogenate into M. olfersii exposed to high salinity medium where a decrease in ionic concentrations of the hemolymph is found. Clearly, alterations in water and ion permeabilities, in addition to specific osmolyte concentrations and hydration levels, must be considered when interpreting data on the effects of putative neurofactors present in the crustacean central nervous system. Although such data are still lacking for M. olfersii, a possible mechanism accounting for the decreased ionic concentrations of the hemolymph encountered in M. olfersii, injected with homogenates of either supraesophageal or thoracic ganglia, and subsequently exposed to high salinity medium, would be a decrease in apparent ionic permeability. It is unlikely that a net flux of water into the animal (hemolymph osmotic concentration = 387 mOsm after 6 hr) would occur against its concentration gradient (osmotic concentration of external medium = 608 mOsm); likewise, it is im-
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MCNAMARA,
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6 Exposure
Time, h
FIG. 6. Concentration of calcium (meq/l) in the hemolymph of the freshwater shrimp Mucrobruchium olfersii in freshwater, after exposure to a high salinity medium (2110 S, 6.2 meqkter Ca”), or after the injection of 10 pl of homogenate of either supra-esophageal (SEG) or thoracic ganglion (TG) and subsequent exposure to either freshwater (0) or high salinity medium (21) for specific time periods. Data are given as the mean f SEM (3 < N > 11). ‘Significantly different from value for noninjected shrimps maintained in freshwater (time = O), or immediately preceding value, at P s 0.05 (StudentNewman-Keuls test). ‘Significantly different from respective value for noninjected shrimps exposed to high salinity medium, at P s 0.05(t test).
probable that a net eftlux of the principal ions involved (Na+, Cl-) would take place against their concentration gradients, (i.e., hemolymph [Na+] = approximately 150 meq/liter after 6 hr vs external medium [Na+] = 280 meqfliter). Thus, the reduced ionic concentrations in the hemolymph most probably reflect a decrease in the apparent permeability to ion intlux rather than an increase in ion efflux. In the case of shrimps injected with homogenate of supra-esophageal ganglion and exposed to freshwater, clearly net water influx or net Na+ and Cl- efflux must be considered. The reduced ionic concentrations in the hemolymph can only have resulted from osmotic dilution, i.e., increased rates of water influx (see Tullis and Kamemoto, 1974) and/or ion efflux, either in the urine as the result of decreased reabsorption, or through increased apparent permeability of the body surfaces, possibly the gills. It is feasible that the homogenate may have reduced the efftciency of the inwardly directed, Na+ transporting system,
which subsequently becomes unable to compensate for diffusional effluxes, resulting in a net decrease in hemolymph Na+ concentration. The rapid increase in osmotic concentration of the hemolymph in M. olfersii injected with homogenate of thoracic ganglion and exposed to freshwater, which is not reflected in the concentrations of the principal ions measured, may result from an increase in the quantity of organic osmolytes in the hemolymph, possibly free amino acids. Tan and Choong (1981) have demonstrated that gradual acclimation (3day steps) of M. rosenbergii from freshwater to varying dilutions of seawater up to 30% S induces a decrease in hemolymph protein content and an increase in muscle ninhydrin positive substances and free amino acids. Neurofactors associated with the thoracic ganglion thus may be involved in the regulation of hemolymph protein hydrolysis and free amino acid concentration. Curiously, Kamemoto and Tullis (1972) have shown that the hemolymph chloride
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concentration of the freshwater crab Potamon dehaani maintained in freshwater increases after injection of thoracic ganglion homogenate from the crab itself. The present study thus has revealed the presence of neurofactors located in the ganglia of the central nervous system of the freshwater shrimp M. olfersii which are apparently involved in ionic regulation, under conditions frequently encountered in the normal biotope of the species. The existence of such capability substantiates the hypothesis that the establishment of successful limnetic species requires specific physiological mechanisms capable of compensating for the phenomena of osmotic dilution and ion loss typically encountered by such organisms. ACKNOWLEDGMENTS The authors thank Alice G. Lima and Alexandre C. Coutinho for skillful technical assistance. This study, partially conducted at the Centro de Biologia Marinha, Universidade de S&o Paul0 in Szlo Sebastiao, SP, was financed by research grants from the Conselho Nacional de Desenvolvimento Cientifico e TecnoMgico (303282/84,403353&t, 403635/84) to J. C. McNamara.
REFERENCES Ahearn, G. A. (1978). Allosteric cotransport of sodium, chloride, and calcium by the intestine of freshwater prawns. J. Membr. Biol. 42, 281-300. Ahearn, G. A., and Tomquist, A. (1977). Aliosteric cooperativity during intestinal cotransport of sodium and chloride in freshwater prawns. Biochim. Biophys. Acta 471, 273-279. Berlind, A., and Kamemoto, F. I. (1977). Rapid water permeability changes in eyestalkless euryhahne crabs and in isolated, perfused gills. Comp. Biothem. Physiol. 58A, 383-385. Cantelmo, A. (1977). Water permeability of isolated tissues from decapod crustaceans. 1. Effect of osmotic conditions. Comp. Biochem. Physiol. 58A, 343-348. Charmantier, G., Charmantier-Daures, M., and Aiken, D. E. (1984). Neuroendocrine control of hydromineral regulation in the American lobster Homarus americanus H. Mime-Edwards 1837 (Crustacea, Decapoda). 1. Juveniles. Gen. Comp. Endocrinol. 54, 8-19.
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