GENERAL

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COMPARATIVE

ENDOCRINOLOGY

88, -+16-327 (1992)

Involvement of the Centrai Nervous System in Neuroendocrine Mediation of Osmotic and Ionic Regulation in the Freshwater Shrimp Macrobrachium olfersii (Crustacea, Decapoda) CAROLINAARRUDA Departamento de Fisiologia Feral, and *Departamento de Biologia,

FREIRE ANDJOHN CAMPBELL MCNAMARA"." lnstituto FFCL.

de BiociEncias, Universidade Universidade de Srio Paula,

de Srio Pa&, Ribeircio Preto.

Sdo Pa&o, Brasil; 14049 SP, Brasil

Accepted April 13, 1992 The presence of putative neurofactors within the central nervous system, i.e., the eyestalks (ES), ventral nerve cord (VNC). and supra-esophageal (SEG) and thoracic ganglia (TG), which are involved in osmotic and ionic regulation, was investigated in the euryhaline. freshwater shrimp, Macrobrachium olfersii. Homogenates were prepared from shrimps exposed for 6 hr to a high salinity medium (HSM, 21%0 S) and were injected into shrimps subsequently maintained for 1,3, or 6 hr in freshwater (FW, O%0S) or HSM. Osmolality and sodium, chloride, and calcium concentrations were determined in single hemolymph samples removed at each time interval. Heart rates and wet weights were measured before and after experimental treatments. Exposure to HSM increased [Na’] and [Cl -1 and heart rate. Injection of ES homogenate increased osmolality, [Na’] and [Cl-], and wet weight in shrimps maintained in FW; VNC homogenate also increased hemolymph [Cl-] in shrimps maintained in FW after injection, but reduced heart rate in shrimps subsequently exposed to HSM. Injection of TG homogenate reduced heart rate to a lesser extent in shrimps maintained in FW. Hemolymph {Ca’+] was not altered by homogenate injection. The exposure period of 6 hr to HSM appears to result in the accumulation of factors within the central nervous system that regulate the osmotic and ionic concentrations of the hemolymph, in addition to exerting antidiuretic and cardio-depressor actions. The coordinated action of these factors is intimately involved in the hyporegulatory processes that permit the survival of M. olfersii in media of elevated salinity. ii: 1992 Academic Pres\. Inc

Among the hyper-regulating aquatic Crustacea, the maintenance of hemolymph osmolality above that of the external medium is achieved through the coordinated action of several regulatory mechanisms. Such adaptive processes appear to have attained their greatest efficiency in the freshwater decapods which can absorb salt from the medium at a high rate by means of a high afTinity transport system while minimizing salt loss and water entry through the permeable body surfaces. In such hyperregulators, the principal sites of ion exchange with the environment are the gills, although cardiac, excretory, and intestinal functions play a role in determining the in’ To whom correspondence

should be addressed.

tegrated responses in normal media, or when subjected to artificial osmotic gradients (see reviews in Kirschner, 1979; Gilles and Pequeux, 1983; Mantel and Farmer, 1983). A number of investigations have substantiated the neuroendocrine control of integrated osmoregulatory responses in various crustacean species (see for example, Tullis and Kamemoto, 1974; Kamemoto, 1976; Berlind and Kamemoto, 1977; Davis and Hagadorn, 1982; Kamemoto and Oyama, 1985). Such studies suggest that neurofactors present within the central nervous system of hyper-regulators are responsible for the high active uptake of ions (Kamemoto and Tullis, 1972; IZhrenfeki and Isaia, 1974; Savage and Robinson, 1983; McNamara et 316

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Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

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al., 1990), the low osmotic and ionic permeabilities (Kato and Kamemoto, 1969), and the high rate of urine production (Norfolk and Craik, 1980). The control mechanisms of hypo-osmotic regulation are less well known, however. McNamara ef al. (1991) have shown that homogenates of the supra-esophageal and thoracic ganglia of M. olfersii exposed acutely to high salinity medium reduce the osmotic and ionic concentrations in the hemolymph of injected shrimps also exposed to high salinity medium, implicating factors associated with these structures in the processes of hypo-regulation. Corroborating this evidence, McNamara and Sesso (1985) have shown that neurosecretory granules accumulate within the antero-medial, protocerebral cells of the supra-esophageal ganglion, after acute exposure of M. olfersii to high salinity medium. The genus Macrobrachium consists of species exhibiting varying degrees of adaptation to the freshwater habitat. M. olfersii, for example, inhabits freshwater reaches of the fluvial system of the southern Brazilian coast (Holthuis, 1952), from estuaries to regions devoid of tidal influence. The larvae are entirely dependent on seawater for sustained development and return to river water after metamorphosis to postlarvae (McNamara et al., 1986). Adult M. olfersii strongly hyper-osmoregulate in freshwater and exhibit some hypo-osmoregulatory capability in media of high salinity (McNamara, 1987). The species thus represents an appropriate model on which to investigate neuroendocrine involvement in osmotic and ionic regulation. The present experiments attempt to demonstrate the participation of neuroendocrine factors located within the principal components of the central nervous system of M. olfersii, namely the eyestalks, ventral nerve cord, and supra-esophageal and thoracic ganglia, in the processes of osmotic and ionic regulation, by monitoring hemolymph osmotic and ionic concentrations, and changes in heart rate and water

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content, in animals injected with homogenates of these elements. MATERIAL

AND METHODS

Intermolt females of Mucrobrachium olfersii were collected from the marginal vegetation of the Patiba River (23”, 47’ S, 45” 33’ W) (O%OS, approximately 23”) in the State of S&o Paulo, Brazil. Shrimps were maintained unfed in the laboratory, in tanks containing aerated river water, at approximately 25”, and were used within 3 days of collection. Homogenates were obtained from the eyestalks (ES), ventral nerve cord (VNC), supra-esophageal ganglion (SEC+), or thoracic ganglion (TG), removed from groups of about 20 shrimps exposed for 6 hr to an aerated, high salinity medium (HSM) of 21%0 S (608 mOsm/kgH,O, 279 mEq Na+/Iiter, 330 mEq Cl-/liter, 6.2 mEq Ca*+/liter), in a constant temperature chamber at 25”. Experiments were conducted in two series: the first comprised experiments with ES and VNC homogenates, performed in March and April 1989 (fall), and the second consisted of experiments with SEG and TG homogenates, in October and November 1989 (spring). To eliminate possible seasonal differences, separate control experiments were performed for each series. To obtain components of the nervous system containing the putative neurofactors, after exposure to HSM, the ES were removed from each shrimp and the VNC dissected out between the junction of the cephalothorax and abdomen and the last abdominal segment. For the second series, the dissection procedures were as described by McNamara et al. (1991), the SEG and TG being obtained from the same individuals. To prepare each homogenate, the structures were placed in physiological saline of composition (in mM): Na+ 140, Ca*+ 10, Mg*+ 2, K’ 5 as chlorides (Cl169) according to McNamara et al. (1990). They were then triturated with finely ground glass and centrifuged at 30,OOOgand 12” for 20 min. The supematants were transferred to polyethylene vials and the volume was adjusted with physiological saline to provide a dose of two ganglion equivalents00 ~1. Pairs of homogenates were prepared simultaneously (ES and VNC, or SEC and TG) and were used on the same day. The experimental procedure consisted of the following steps. Heart rate was determined by making three consecutive measurements of the time necessary for 50 heart beats. Beats were counted visually in the cardiac area, using a stereoscopic dissecting microscope, in shrimps immobilized in moist cotton wool. The wet weight of each shrimp was then determined prior to the injection of 10 ul of either physiological saline or homogenate with a 50-ul microsyringe, ventrally into the abdominal musculature. Approximately 10 shrimps were used in all groups.

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The injected shrimps were then exposed individually to aerated river water (freshwater, FW, 0%~ S) or to HSM (21%0 S) for 1, 3, or 6 hr in a constant temperature chamber at 25”. After each exposure period, the heart rate and wet weight of each shrimp were again determined. Thus, initial and final values for each parameter were obtained from each shrimp. allowing calculation of the percentage variation in relative heart rate and relative wet weight, i.e., the difference between the final and initial values divided by the initial value, multiplied by 100, for each animal. A single hemolymph sample of approximately 70100 ~1 was then removed from each shrimp by intracardiac puncture with a 25-8 needle coupled to a I-ml plastic insulin syringe. Hemolymph samples were placed in 1S-ml stoppered polyethylene vials and frozen at - 20”. Each animal was then sacrificed by rapid immersion in boiling water, dried in an oven at 80” for 16 hr, and weighed after cooling under vacuum. After thawing, the osmolality of each hemolymph sample was measured by the freezing point depression method, using a microcryoscope (Salomao, 1980). Sodium concentration was obtained by emission after dilution of 10 t.~lof hemolymph in 50 ml distilled water (1:5000) using a Zeiss PMQII flame spectrophotometer. Chloride and calcium concentrations were determined by titration on a Beckman/Spinco Model 150 microtitrator, using 10 and 20 ~1 of hemolymph. respectively. Mercuric nitrate was the titrant and s-diphenylcarbazone the indicator for chloride, and EDTA the titrant and calcein the indicator for calcium. Control shrimps, injected with 10 u1 of physiological saline, were treated identically to those injected with homogenate. However, the group of control shrimps in FW at time = 0 hr consisted of approximately 10 animals in which heart rate and wet weight were determined and hemolymph was sampled but which were not injected with physiological saline. The shrimps exposed to FW represent controls both for those shrimps exposed to HSM alone and for those injected with homogenate and maintained in FW. The shrimps exposed to HSM alone represent controls for the groups injected with homogenate and exposed to HSM. The data are presented as the mean 2 SEM. After verifying normality of distribution (KolmogorovSmimov test), a two-way analysis of variance was used to determine the effect of exposure time (1. 3, 6 hr) and treatment (injection with physiological saline or homogenate), or their interaction, on each experimental parameter measured. When a significant treatment effect was found, the Student t test was used to compare the experimental groups with their respective controls, for each exposure period. When the effect of exposure time was indicated as significant. the Student-Newman-Keuls procedure was used to compare the means for each exposure period within either the injected or control groups. Differences were considered to be significant at P = 0.05 in all statistical procedures.

RESULTS

Control Parameters for Experiments with Homogenates of Eyestalks and Ventral Nerve Cord Neither hemolymph osmolality (Fig. 1A) nor [Na’] (Fig. 2A) was affected by exposure to HSM. Hemolymph [Cl-] (Fig. 3A) decreased with exposure time in FW control shrimps but was higher in shrimps exposed to HSM. Hemolymph [Ca2+ ] (Fig. 4A) was unchanged in shrimps exposed to HSM, compared to control shrimps in FW, and although there was a significant increase with exposure time in both groups,

A1

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tame,

Exposure

hme. h

h

FIG. 1. Osmolality (mOsm/kg IX&I) of the hemoiymph of ~~crobruckium ot#ZZssiiin&eshwa&r @%o S), after exposure to a hi& sidiity medinm (21%0 St 608 mOsmikg H,O), or after iqjection of homogenate of (A) either eyestalks (ES-) or ventral nerve cord (VNC) and (B) either supra-esophageal (SE@ or thoracic (TG) ganglia, and subsequent e%posure either to freshwater (@& S) or to high salinity n&inm (21%~ S) for specific time intervals. Data are the mean ‘-t SEM; (A) 10 6 N 6 12, (B) 7 G N 6 14. y different from respective control at same time intMvat (t test, P < 0.05). 2Signi&antly different from vahres for time intervals indicated as subscripts (SNK, P 4 0.05).

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time, h

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Exposure

tune, h

Exposure

1

t,me. h

3 time, h

6

FIG. 2. Sodium concentration (mEq/liter) in the hemolymph of Mucrobrachium olfersii in freshwater (O%,, S), after exposure to a high salinity medium (21%0 S, 279 mEq Na+iliter), or after injection of homogenate of (A) either eyestalks (ES) or ventral nerve cord (VNC) and (B) either supra-esophageal (SEC+) or thoracic (TG) ganglia, and subsequent exposure either to freshwater (O%OS) or to high salinity medium (21%0 S) for specific time intervals. Data are the mean of:SEM; (A) 9 < N s 12, (B), 7 < N =Z 15. ‘Significantly different from respective control at same time interval (t test, P < 0.05). 2Significantly different from values for time intervals indicated as subscripts (SNK, P c 0.05).

FIG. 3. Chloride concentration (mEq/liter) in the hemolymph of Mucrobrachium olfersii in freshwater (O%O S), after exposure to a high salinity medium (21%0 S, 330 mEq Cl-iliter), or after injection of homogenate of (A) either eyestalks (ES) or ventral nerve cord (VNC) and (B) either supra-esophageal (SEG) or thoracic (TG) ganglia, and subsequent exposure either to freshwater (O%OS) or to high salinity medium (21%0 S) for specific time intervals. Data are the mean 2 SEM; (A) 7 c N s 12, (B), 6 s N s 12. ‘Significantly different from respective control at same time interval (t test, P s 0.05). 2Significantly different from values for time intervals indicated as subscripts (SNK, P < 0.05).

specific increases could not be located statistically. The heart rate of control shrimps in FW at time = 0 hr was 357 f 3 1 beats/min (N = 12). Isolation in the constant temperature chamber in individual recipients reduced heart rate with exposure time (Fig. 5A). In shrimps exposed to HSM, heart rate increased with respect to FW control shrimps, and over the duration of the exposure period. The mean initial wet weight for all shrimps in this series was 1.393 -+ 0.028 g (N = 222). Tissue water represented 74.0 + 0.14% of wet weight, ranging from 69.6 to

81.2%. Shrimps in the FW control group at time = 0 hr had a mean wet weight of 1.687 + 0.108 g (iV = 12). There was little variation in relative wet weight (Fig. 6A) in control shrimps exposed to FW. Exposure to HSM did not affect relative wet weight compared to control shrimps in FW, and although the decrease with exposure time was significant in both the FW and HSM control groups, specific differences could not be located statistically. Effects of Eyestalk Homogenate Hemolymph

osmolality (Fig. IA) was not

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MCNAMARA

/

Itme, h

3 Exposure

Exposure

6 hme. h

lime, h

FIG. 4. Calcium concentration (mEq/liter) in the hemolymph of Macrobrachium olfersii in freshwater (O%O S), after exposure to a high salinity medium (21%~ S, 6.2 mEq Ca’+/liter), or after injection of homogenate of (A) either eyestalks (ES) or ventral nerve cord (VNC) and (B) either supra-esophageal (SEG) or thoracic (TG) ganglia, and subsequent exposure either to freshwater (O%OS) or to high salinity medium (21%0 S) for specific time intervals. Data are the mean t SEM; (A) 6 6 N < 11, (B), 3 < N d 9. ‘Significantly different from values for time intervals indicated as subscripts (SNK, P c 0.05).

affected by injection of ES homogenate and subsequent exposure to HSM, compared to control shrimps exposed to HSM. However, shrimps injected with ES homogenate and maintained in FW had a higher hemolymph osmolality than did control shrimps in FW. Injection of ES homogenate and subsequent exposure to HSM did not affect hemolymph [Na+] (Fig. 2A); however, in injected shrimps maintained in FW, hemolymph [Na+l was increased compared to control shrimps. Hemolymph [Cl-] decreased after 1 hr exposure in control shrimps maintained in FW (Fig. 3A). [Cl-] in the hemolymph in-

FIG. 5. Variation

in relative heart rate (%) of Macin freshwater (0%~ S), after exposure to a high salinity medium (21% S), or after injection of homogenate of (A) either eye&&s (ES) or ventral nerve cord (VNC) and (B) either supra-esophageal (SEG) or thoracic (TG) ganglia, and subsequent exposure either to freshwater (W& S) or to high sahnity medium (21o/ooS) for specific time intervals. Data are the mean t SEM; (A) 9 6 N 6 12, (B), 6 s N < 12. ‘Significantly different from respective control~at same time interval (t test, P C 0.05). 2Sign&icantly different from values for time intervals indicated as subscripts (SNK, P c 0.05). robrachium

olfemii

creased with exposure time in shrimps injected with ES homogenate and subsequently exposed to~HSM, as also occurred in control shrimps exposed to HSM, compared to control shrimps in FW; however, specific differences between the different exposure times could not be located statistically. In shrimps injected with ES homogenate and kept in FW, hemolymph [Cl-] was higher than in control shrimps exposed to FW, for all exposure intervals (Fig. 3A). Injection of ES ho~g~ in shrimps subsequently exposed-to HSM did not alter

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I 3

1 Exposure

-2

1

I 6

Effects of Homogenate Nerve Cord

of the Ventral

hme, h

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There was a reduction in relative wet weight with exposure time in shrimps injected with ES homogenate and exposed to HSM (Fig. 6A). Injection of ES homogenate in shrimps subsequently kept in FW caused an increase in relative wet weight compared to FW control shrimps, and a reduction in relative wet weight with exposure time (Fig. 6A).

A

I

OF

6 time, h

FIG. 6. Variation in relative wet weight (%) of Mnco/&xii in freshwater (0%~ S), after exposure to a high salinity medium (21%0 S), or after injection of homogenate of (A) either eyestalks (ES) or ventral nerve cord (VNC) and (B) either supra-esophageal (SEC+) or thoracic (TG) ganglia, and subsequent exposure either to freshwater (O%o S) or to high salinity medium (21%0 S) for specific time intervals. Data are the mean 2 SEM; (A) 8 G N G 12, (B), 7 G N 6 12. Significantly different from respective control at same time interval (t test, P G 0.05). *Significantly different from values for time intervals indicated as subscripts (SNK, P ==G0.05). robrachium

hemolymph [Ca”] (Fig. 4A) compared to control shrimps, although [Cazf] increased with exposure time in the shrimps injected with homogenate. There were no changes in [Ca*+l in shrimps injected with ES homogenate when kept in FW. Injection of ES homogenate and subsequent exposure of the shrimps to either HSM or FW had no effect on relative heart rate (Fig. 5A).

In shrimps injected with homogenate of VNC and subsequently exposed to HSM, hemolymph osmolality increased with exposure time (Fig. 1A). Homogenateinjected shrimps maintained in FW showed no change in osmolality. Injection of VNC homogenate had no effect on hemolymph [Na+] (Fig. 2A) in shrimps maintained in either FW or exposed to HSM. An increase in hemolymph [Cl-] occurred with exposure time in shrimps injected with homogenate of VNC and exposed to HSM, and in their controls (Fig. 3A), although specific differences could not be statistically located. Injection of VNC homogenate in shrimps maintained in FW caused an increase in [Cl-]. There was also an increase in hemolymph [Ca*‘] (Fig. 4A) in shrimps injected with VNC homogenate and exposed to HSM. No effect was seen in shrimps injected with VNC homogenate and maintained in FW. In shrimps injected with VNC homogenate and subsequently exposed to HSM, relative heart rate was decreased (Fig. 5A) compared to control shrimps exposed to HSM. Relative heart rate also increased with exposure time in both homogenateinjected and control groups. There was no effect on heart rate in shrimps injected with VNC homogenate and maintained in FW. Injection of VNC homogenate had no effect on relative wet weight in shrimps either

322 exposed to HSM or maintained 6A).

FREIRE

AND

in FW (Fig.

Control Parameters for Experiments Homogenates of Supra-esophageal Thoracic Ganglia

with and

Exposure to HSM had no effect on hemolymph osmolality (Fig. lB), although there was an interaction effect between exposure to HSM and time. An anomalous decrease in hemolymph osmolality, [Nail and [Cl-] occurred after 6 hr exposure in control shrimps maintained in FW. Both hemolymph [Na+] (Fig. 2B) and [Cl-] (Fig. 3B) increased after exposure to HSM, and there was an interaction effect between exposure to HSM and time for both ions. Hemolymph [Ca2’l (Fig. 4B) was very stable and unaltered by exposure to HSM. Heart rate in FW control shrimps at time = 0 hr was 272 + 25 beats/min (N = 15). Heart rate increased with exposure time (Fig. 5B) both in control shrimps maintained in FW and in those exposed to HSM. The initial wet weight of all shrimps used in this series was 1.483 2 0.033 g (N = 192). Tissue water represented 72.5 -t 0.15% of wet weight, ranging from 67.9 to 79.8%. The control shrimps in FW at time = 0 hr had a mean wet weight of 1.457 4 0.106 g (N = 15). Relative wet weight (Fig. 6B) was unaltered after exposure to HSM although there was a decrease with exposure time in control shrimps maintained in FW. Effects of Homogenate of the Supra-esophageal Ganglion

Hemolymph osmolality (Fig. 1B) was not affected by the injection of SEG homogenate and subsequent exposure to HSM, although osmolality decreased with exposure time in both the control and homogenateinjected shrimps maintained in FW. Injection of SEG homogenate did not affect hemolymph INa+] (Fig. 2B) in shrimps exposed to HSM but {Na+] decreased with

MCNAMARA

exposure time in injected shrimps subsequently maintained in FW. Injection of SEG homogenate with subsequent exposure to HSM caused an increase in hemolymph [Cl ] (Fig. 3B) with respect to control shrimps; values also increased with exposure time. There was no effect on [Cll] in shrimps injected with SEG homogenate and maintained in FW. Injection of SEG homogenate did not alter hemolymph [Ca”] (Fig. 4B) in shrimps posteriorly exposed to either HSM or FW. There was an interaction effect between homogenate injection and exposure time on relative heart rate (Fig. 5B) in shrimps injected with SEG homogenate and exposed either to HSM or to FW; heart rate tended to decrease in injected shrimps in FW and increase slightly in injected shrimps in HSM. Relative wet weight was unaffected in shrimps injected with SEG homogenate and exposed to HSM (Fig. 6B). There was a decrease in relative wet weight in shrimps injected with SEG homogenate and maintained in FW (Fig. 6B). Effects of Homogenate Thoracic Ganglion

of the

Hemolymph osmolality was unaffected by the injection of TG homogenate and exposure to HSM or to FW (Fig. 1B). An interaction effect occurred between homogenate injection and exposure time to HSM for hemolymph [Na+] (Fig. 2B). In shrimps injected with TG homogenate and maintained in FW, hemolymph [Na+] decreased with exposure time, accompanying FW control shrimps (Fig. 2B). There was also an interaction effect between homogenate injection and exposure time to HSM for hemolymph [Cl-l (Fig. 3B). There was no effect on hemolymph [Cl-] in shrimps injected with honkgenate and maintained in FW. Hemolymph ICa* + I (Fig. 4B) was not altered by injection of TG homogenate in

NEUROENDOCRINE

CONTROL

shrimps either exposed to HSM or maintained in FW. Relative heart rate (Fig. 5B) was unaffected in shrimps injected with TG homogenate and exposed to HSM. However, in shrimps injected with TG homogenate and maintained in FW, relative heart rate decreased compared to FW control shrimps (Fig. 5B). Relative wet weight was unaffected in shrimps injected with TG homogenate and exposed to HSM (Fig. 6B). An interaction effect between homogenate injection and exposure time occurred in shrimps injected with homogenate and maintained in FW (Fig. 6B). DISCUSSION The data obtained in the present study strongly support the involvement of neuroendocrine factors in osmotic and ionic regulation in the freshwater shrimp Macrobruchium olfersii when acutely exposed to high salinity medium. Briefly, exposure to HSM resulted in increased concentrations of sodium and chloride in the hemolymph while injection of ES homogenate caused an increase in hemolymph osmolality and in sodium and chloride concentrations in shrimps maintained in FW after injection. Homogenate of VNC also increased the chloride concentration of the hemolymph in shrimps maintained in FW, as did injection of SEG homogenate in shrimps exposed to HSM. Hemolymph calcium concentrations were very stable and unaltered by homogenate application. Heart rate was elevated in response to exposure to HSM but was reduced by injection of VNC homogenate in shrimps exposed to this medium, and to a lesser extent in shrimps injected with TG homogenate when maintained in FW. Wet weight increased after injection of ES homogenate in shrimps maintained in FW, indicating an increase in body water content.

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The increase in the osmotic and ionic concentrations of the hemolymph occurred after 6 hr exposure to HSM, a response fairly similar to that observed by McNamara (1987) and McNamara et al. (1990, 1991) for M. olfersii, where hemolymph concentrations increased immediately after exposure, attaining maxima around 3 hr and remaining stable until 6 hr, indicating capability to compensate rapidly for inc&ses in the osmolality of the extracellular fluid. Despite the difference in the time course of the response, the present results also demonstrate a distinct hyporegulatory capability by M. olfersii. While the maximum hemolymph concentrations were attained after a 6-hr exposure period to HSM, the osmolality and sodium and chloride concentrations in the extracellular fluid reach approximately only half those of the external medium. The application of ES homogenate resulted in an increase in hemolymph concentrations when the injected shrimps were subsequently maintained in FW. This suggests that a factor accumulates in the optic ganglia of shrimps acutely exposed to high salinity medium that either stimulates the active uptake rate of ions and/or reduces apparent ionic permeability, possibly resulting in a reduction in passive ion loss from the antenna1 gland and gills, thus increasing the concentration of the hemolymph, and leading to greater retention of ions in the extracellular fluid. This finding is in accordance with the reduction in hemolymph sodium concentration in M. olfersii reported by McNamara et al. (1990), after ES ablation in shrimps posteriorly exposed to HSM, also indicating the presence of a factor which stimulates Na+ uptake in the eyestalks of intact shrimps. That shrimps injected with ES homogenate and subsequently exposed to HSM do not exhibit this increase in hemolymph sodium concentration, may result from the interaction between the effect of

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the exogenous, injected neurofactor and the simultaneous stimulus for osmoregulation induced by exposure to HSM. There also may be a compensatory decrease in the activity of the ion absorption system induced by the high external salt concentration, as suggested by Siebers et al. (1982) and Winkler et al. (1988) for Carcinus maenas.

The effects of homogenates of VNC and SEG specifically on the chloride concentration of the hemolymph alone may indicate independence between the systems responsible for the transport and homeostasis of sodium and those for chloride, as suggested by Shaw (1960) for Astacus pallipes. This idea is corroborated by Kamemoto and Tullis’ (1972) study showing an increase in hemolymph chloride after eyestalk ligation, or injection of homogenate of thoracic ganglion, in the freshwater crab Potamon dehaani, maintained in freshwater. Considering the present results as consistent with the phenomena which occur during hyporegulation in crustaceans, the putative neurofactors disclosed might be expected to result in effects converse to those demonstrated, i.e., induce a decrease in the rate of salt uptake, resulting in lower hemolymph concentrations. Using an experimental protocol similar to that employed here, McNamara et al. (1991) have demonstrated active factors present in both the SEG and TG of M. olfersii exposed to HSM. The SEG factor in fact causes a reduction in osmotic and ionic concentrations in the hemolymph while the TG factor impedes the increase in hemolymph ionic concentrations seen after exposure to HSM. This latter factor also causes an increase in hemolymph osmolality in shrimps maintained in FW. The discrepancy with the results reported herein for the effects of these two homogenates may be ascribed perhaps to seasonal variations in the physiology of M. olfersii given that the experiments described by McNamara et al. (1991) were

MCNAMARA

conducted during summer (Jan-Feb). The differences seen in the response of’ the two control groups for each experimental series in the present study do suggest the existence of a seasonal effect on osmotic behavior. Osmotic and ionic concentrations of the hemolymph alone do not adequately describe the osmoregulatory capability of an organism; information on the movement of water is also necessary. Shrimps injected with ES homogenate and maintained in FW showed an increase in hemolymph concentrations, despite net water entry. This effect may result from increased salt reabsorption by the antenna1 gland, generating an increase in water retention, causing injected shrimps exposed to FW to retain water. In the case of shrimps injected with ES homogenate and subsequently exposed to HSM, the osmotic gradient would not favor water retention. This factor present in the eyestalks of shrimps exposed to HSM may thus be antagonistic to those postulated as responsible for the maintenance of a low water permeability in FW crustaceans, and in euryhaline, marine crustaceans exposed to diluted seawater (Kamemoto and Ono, 1969; Kato and Kamemoto, 1969; Ehrenfeld and Isaia, 1974). Since apparent epithelial permeabilities are strongly influenced by the flow of fluids through and over permeable surfaces, mainly the gills in the present case, heart activity was also analyzed. Exposure of M. olfersii to HSM induced an increase in heart rate, in the control shrimps for the first series of experiments. Heart rate also increases after exposure to salinities above or below normal in the shrimps Lysmata seticaudata, Palaemon serratus, and Crangon crangon (Spaargaren, 1973), and in Carcinus maenas exposed to dilute seawater (Hume and Berlind, 1976). Salinity apparently has no effect on heart rate in Palaemon longirostris (Campbell and Jones, 1990) while bradycardia and cardiac

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arrest occur, together with a reduction in rate of scaphognathite beat, after exposure of the freshwater crayfish Procambarus simulans to sodium chloride solution (Larimer, 1964). The cardio-depressor factors from the VNC and TG may be involved in compensatory control of heart rate in M. olfersii, acting antagonistically to the cardioactivating hormones released from the pericardial organs, and secreted by perikarya located within the TG of crustaceans (Berlind and Cooke, 1970; Kamemoto and Oyama, 1985; Stangier et al., 1987; Dircksen and Keller, 1988). It should be emphasized that to our knowledge, there has been no previous report of cardio-depressor factors in the decapod Crustacea. Contrary to expectations, homogenate of the TG did not increase heart rate in M. olfersii but rather caused a decrease. Possibly, only homogenates of the pericardial organs themselves possess the cardio-activating peptides or, alternatively, in M. olfersii, the effect is very rapid and not readily detectable within the initial hour of the experiments performed. Heart rate in M. olfersii appears to be inversely related to relative wet weight, i.e., the water content of the animal. There is a low and negative correlation (R = -0.299) between the two parameters, indicating a tendency toward water loss associated with increase in heart rate and water retention associated with reduction in heart rate. Such coupling may occur through the initial process of urine formation, i.e., ultra-filtration in the celomic sac (see Peterson and Loizzi, 1974; Robinson, 1982; Taylor and Harris, 1986). Increased heart rate may signify increased cardiac output (Hume and Berlind, 1976) and, consequently, more rapid dispersion of the neurofactors involved in osmoregulation. The present data suggest that this may be the case for M. olfersii. This idea is also consistent with the release of an antidiuretic factor from the ES of M. olfer-

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sii, after exposure to HSM. The factor may

act to prevent water loss through the urine in shrimps exposed to HSM and would be responsible for the increase in wet weight in animals injected with ES homogenate and subsequently kept in FW. Thus, the increase in heart rate that occurs on exposure to HSM may result in a higher rate of water loss through increased urine production. The anti-diuretic factor would act to reduce this loss and, together with the cardiodepressor factors from the VNC and TG, reduce heart rate and urine production. Despite the considerable variability noted in and the likely seasonal influence on the responses obtained, it has been possible to integrate the data on hemolymph concentrations, wet weight, and heart rate into a single, plausible model of neuroendocrine involvement in a hypo-regulating crustacean, acutely exposed to a strong salinity gradient. ACKNOWLEDGMENTS This work, partially conducted at the Centro de Biologia Marinha, Universidade de Sao Paul0 in Sao Sebastilo, SP, was financed by postgraduate scholarships to C.A.F. (CAPES, FAPESP 8811222-3) and a research grant to J.C.M. (FAPESP 89/0114-5), and represents part of a dissertation by C.A.F in fulfillment of the requirements for the Degree of Master of Science in Physiology at the Departamento de Fisiologia Get-al, Instituto de Biociencias, Universidade de S&o Paulo. The authors are most grateful to Dr. Luiz Carlos Salomlo for kindly providing laboratory space and equipment, and thank Marcos Ribeiro de Souza for the line drawings.

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Involvement of the central nervous system in neuroendocrine mediation of osmotic and ionic regulation in the freshwater shrimp Macrobrachium olfersii (Crustacea, Decapoda).

The presence of putative neurofactors within the central nervous system, i.e., the eyestalks (ES), ventral nerve cord (VNC), and supra-esophageal (SEG...
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