Freshwater Shrimp Macrobrachium Olfersii Research Articles

Long-term exposure of the freshwater shrimp Macrobrachium olfersii to elevated salinity: Effects on gill (Na+,K+)-ATPase α-subunit expression and K+-phosphatase activity

The kinetic properties of a microsomal gill (Na+,K+)-ATPase from the freshwater shrimp, Macrobrachium olfersii, acclimated to 21 per thousand salinity for 10 days were investigated using the substrate p-nitrophenylphosphate. The enzyme hydrolyzed this substrate obeying cooperative kinetics at a rate of 123.6+/-4.9 U mg-1 and K0.5=1.31+/-0.05 mmol L-1. Stimulation of K+-phosphatase activity by magnesium (Vmax=125.3+/-7.5 U mg-1; K0.5=2.09+/-0.06 mmol L-1), potassium (Vmax=134.2+/-6.7 U mg-1; K0.5=1.33+/-0.06 mmol L-1) and ammonium ions (Vmax=130.1+/-5.9 U mg-1; K0.5=11.4+/-0.5 mmol L-1) was also cooperative. While orthovanadate abolished p-nitrophenylphosphatase activity, ouabain inhibition reached 80% (KI=304.9+/-18.3 micromol L-1). The kinetic parameters estimated differ significantly from those for freshwater-acclimated shrimps, suggesting expression of different isoenzymes during salinity adaptation. Despite the approximately 2-fold reduction in K+-phosphatase specific activity, Western blotting analysis revealed similar alpha-subunit expression in gill tissue from shrimps acclimated to 21 per thousand salinity or fresh water, although expression of phosphate-hydrolyzing enzymes other than (Na+,K+)-ATPase was stimulated by high salinity acclimation.

Free amino acid pools as effectors of osmostic adjustment in different tissues of the freshwater shrimp macrobrachium olfersii (crustacea, decapoda) during long-term salinity acclimation

To examine osmotic regulation during long-term acclimation to a hyperosmotic medium, hemolymph osmolality, [Na+] and total protein, tissue hydration, and free amino acid (FAA) pools in abdominal muscle, gills, central nervous tissue and hemolymph were quantified in the diadromous freshwater (FW) shrimp, Macrobrachium olfersii, during direct exposure to 21‰S seawater over a 20-day period. Hemolymph osmolality and [Na+] reach stable maxima within 24 h while total protein is unchanged. Muscle and nerve tissues rapidly lose water while gills hydrate; all tissues attain maximum hydration (+5%) by 5 days, declining to FW values except for gills. Total FAA are highest in muscle, reach a maximum by 2 days (+64%), declining to FW values. Gill FAA increase by 110% after 24 h, diminishing to FW values. Nerve FAA increase 187% within 24 h, and remain elevated. Hemolymph FAA decrease (−75%) after 24 h, stabilizing well below the FW concentration. During acclimation, muscle glycine (+247%), gill taurine (+253%) and proline (+150%), and nerve proline (+426%), glycine (+415%) and alanine (+139%) increase, while hemolymph leucine (−70%) decreases. Total FAA pools contribute 10–20% to intracellular (22–70 mmol/kg) and 0.5–2.4% to hemolymph (3–7 mOsm/kg) osmolalities during direct acclimation from FW. These data emphasize the modest participation of FAA pools in intracellular osmotic regulation during physiological adaptation by M. olfersii to osmotic challenge, accentuating the role of anisosmotic extracellular regulation, suggesting that, during the invasion of freshwater by the Crustacea, dependence on intracellular adjustment employing FAA as osmotic effectors, has become progressively reduced.

Nitrophenylphosphate as a tool to characterize gill Na +, K +-ATPase activity in hyperregulating Crustacea

The kinetic properties of a gill Na +, K +-ATPase from the freshwater shrimp Macrobrachium olfersii were studied using p-nitrophenylphosphate (PNPP) as a substrate. Sucrose gradient centrifugation of the microsomal fraction revealed a single protein fraction that hydrolyzed PNPP. The Na +, K +-ATPase hydrolyzed PNPP (K +-phosphatase activity) obeying Michaelis–Menten kinetics with K M=1.72±0.06 mmol l −1 and V max=259.1±11.6 U mg −1. ATP was a competitive inhibitor of K +-phosphatase activity with a K i=50.1±2.5 μmol l −1. A cooperative effect for the stimulation of the enzyme by potassium ( K 0.5=3.62±0.18 mmol l −1; n H=1.5) and magnesium ions ( K 0.5=0.61±0.02 mmol l −1, n H=1.3) was found. Sodium ions had no effect on K +-phosphatase activity up to 1.0 mmol l −1, but above 80 mmol l −1 inhibited the original activity by approximately 75%. In the range of 0–10 mmol l −1, sodium ions did not affect stimulation of the K +-phosphatase activity by potassium ions. Ouabain ( K i=762.4±26.7 μmol l −1) and orthovanadate ( K i=0.25±0.01 μmol l −1) completely inhibited the K +-phosphatase activity, while thapsigargin, oligomycin, sodium azide and bafilomycin were without effect. These data demonstrate that the activity measured corresponds to that of the K +-phosphatase activity of the Na +, K +-ATPase alone and suggest that the use of PNPP as a substrate to characterize K +-phosphatase activity may be a useful technique in comparative osmoregulatory studies of Na +, K +-ATPase activities in crustacean gill tissues, and for consistent comparisons with well known mechanistic properties of the vertebrate enzyme.

Identification and quantification of carotenoid pigments during the embryonic development of the freshwater shrimp Macrobrachium olfersii (crustacea, decapoda)

The principal carotenoids present during the 16‐day embryonic development period and in the first post‐embryonic stage (zoea I) were identified and quantified in the diadromous, neotropical, freshwater shrimp Macrobrachium olfersii, employing differential extraction, absorption spectra analysis and thin layer chromatography. The characteristic crustacean pigments α‐ and β‐carotene and astaxanthin, and a mixture of four unidentified yellow pigments were found. Total carotenoids increase significantly at the mid‐point of embryogenesis (day 8), declining to a minimum in the recently hatched first zoea. This decrease appears to reflect the establishment of carotenoid catabolism, or of carotenoid use in protein and membrane formation and stabilization towards the end of embryogenesis, as well as the depletion of yolk protein reserves by the non‐feeding first zoea.

Characterization of (Na +, K +)-ATPase in gill microsomes of the freshwater shrimp Macrobrachium olfersii

To better understand the adaptive strategies that led to freshwater invasion by hyper-regulating Crustacea, we prepared a microsomal (Na +, K +)-ATPase by differential centrifugation of a gill homogenate from the freshwater shrimp Macrobrachium olfersii. Sucrose gradient centrifugation revealed a light fraction containing most of the (Na +, K +)-ATPase activity, contaminated with other ATPases, and a heavy fraction containing negligible (Na +, K +)-ATPase activity. Western blotting showed that M. olfersii gill contains a single α-subunit isoform of about 110 kDa. The (Na +, K +)-ATPase hydrolyzed ATP with Michaelis–Menten kinetics with K 0.5=165±5 μM and V max=686.1±24.7 U mg −1. Stimulation by potassium ( K 0.5=2.4±0.1 mM) and magnesium ions ( K 0.5=0.76±0.03 mM) also obeyed Michaelis–Menten kinetics, while that by sodium ions ( K 0.5=6.0±0.2 mM) exhibited site–site interactions ( n=1.6). Ouabain ( K 0.5=61.6±2.8 μM) and vanadate ( K 0.5=3.2±0.1 μM) inhibited up to 70% of the total ATPase activity, while thapsigargin and ethacrynic acid did not affect activity. The remaining 30% activity was inhibited by oligomycin, sodium azide and bafilomycin A 1. These data suggest that the (Na +, K +)-ATPase corresponds to about 70% of the total ATPase activity; the remaining 30%, i.e. the ouabain-insensitive ATPase activity, apparently correspond to F 0F 1- and V-ATPases, but not Ca-stimulated and Na- or K-stimulated ATPases. The data confirm the recent invasion of the freshwater biotope by M. olfersii and suggest that (Na +, K +)-ATPase activity may be regulated by the Na + concentration of the external medium.

The calcium dependence of pigment translocation in freshwater shrimp red ovarian chromatophores.

The roles of calcium in cell signaling consequent to chromatophorotropin action and as an activator of mechanochemical transport proteins responsible for pigment granule translocation were investigated in the red ovarian chromatosomes of the freshwater shrimp Macrobrachium olfersii. Chromatosomes were perfused with known concentrations of free Ca++ (10(-3) to 10(-9) M) prepared in Mg(++)-EGTA-buffered physiological saline after selectively permeabilizing with 25 microM calcium ionophore A23187 or with 10(-8) M red pigment concentrating hormone (RPCH). The degree of pigment aggregation and the translocation velocity of the leading edges of the pigment mass were recorded in individual chromatosomes during aggregation induced by RPCH or A23187 and dispersion induced by low Ca++. Aggregation is Ca++ dependent, showing a dual extracellular and intracellular requirement. After perfusion with reduced Ca++ (10(-4) to 10(-9) M), RPCH triggers partial aggregation (approximately 65%), although the maximum translocation velocities (approximately 16.5 microns/min) and velocity profiles are unaffected. After aggregation induced at or below 10(-5) M Ca++, spontaneous pigment dispersion ensues, suggesting a Ca++ requirement for RPCH coupling to its receptor, or a concentration-dependent, Ca(++)-induced Ca(++)-release mechanism. The Ca(++)-channel blockers Mn++ (5 mM) and verapamil (50 microM) have no effect on RPCH-triggered aggregation. An intracellular Ca++ requirement for aggregation was demonstrated in chromatosomes in which the Ca++ gradient across the cell membrane was dissipated with A23187. At free [Ca++] above 10(-3) M, aggregation is complete; at 10(-4) M, aggregation is partial, followed by spontaneous dispersion; below 10(-5) M Ca++, pigments do not aggregate but disperse slightly. Aggregation velocities diminish from 11.6 +/- 1.2 microns/min at 5.5 mM Ca++ to 7.4 +/- 1.3 microns/min at 10(-4) M Ca++. Half-maximum aggregation occurs at 3.2 x 10(-5) M Ca++ and half-maximum translocation velocity at 4.8 x 10(-5) M Ca++. Pigment redispersion after 5.5 mM Ca(++)-A23187-induced aggregation is initiated by reducing extracellular Ca++: slight dispersion begins at 10(-7) M, complete dispersion being attained at 10(-9) M Ca++. Dispersion velocities increase from 0.6 +/- 0.2 to 3.1 +/- 0.5 microns/min. Half-maximum dispersion occurs at 7.6 x 10(-9) M Ca++ and half-maximum translocation velocity at 2.9 x 10(-9) M Ca++. These data reveal an extracellular and an intracellular Ca++ requirement for RPCH action, and demonstrate that the centripetal or centrifugal direction of pigment movement, the translocation velocity, and the degree of pigment aggregation or dispersion attained are calcium-dependent properties of the granule translocation apparatus.

Ultracytochemical location of Na(+)/K(+)-atpase activity and effect of high salinity acclimation in gill and renal epithelia of the freshwater shrimp Macrobrachium olfersii (Crustacea, Decapoda).

Accumulation sites of lead phosphate reaction product consequent to Na(+)/K(+)-ATPase activity in gill and renal epithelia of the freshwater shrimp Macrobrachium olfersii were located ultracytochemically by para-nitrophenyl-phosphate hydrolysis and lead precipitation, and quantified per unit membrane area and cytoplasmic volume. In shrimps in freshwater (<0.5 per thousand S, 20 mOsm/kg H(2)O, 0.7 mEq Na(+)/liter), numerous sites of electron-dense, Na(+)/K(+)-ATPase reaction product accumulation were demonstrated in the membrane invaginations of the mitochondria-rich, intralamellar septal cells (12.5 +/- 1.7 sites/microm(2) membrane, 179 +/- 22 sites/microm(3) cytoplasm, mean+/- SEM, N </= 7) and in the basal region of the medial renal tubules (19.8 +/- 1.8 sites/microm(2) membrane, 437 +/- 53 sites/microm(3) cytoplasm), but not in the pillar cells whose apical flanges form the primary interface with the external medium. A putative, ouabain-insensitive Na(+)- or H(+)-ATPase was found in the apical microvilli of the medial renal tubules (17.4 +/- 1.7 sites/microm(2) membrane, 629 +/- 101 sites/microm(3) cytoplasm). This restricted location of Na(+)/K(+)-ATPase activity within the gill epithelium suggests that during uptake, Na(+) moves across the apical pillar cell membrane, passes through specialized, basolateral coupling junctions into the septal cell cytoplasm and is pumped into the hemolymph via the Na(+)/K(+)-ATPase in the invagination membranes. In shrimps acclimated to a high-salinity medium (21 per thousand S, 630 mOsm/kg H(2)O, 280 mEq Na(+)/liter) for 2 and 5 days, the mean number of sites of para-nitrophenylphosphatase activity/microm(2) membrane and /microm(3) cytoplasm for both epithelia increases markedly by 83 and 163%, respectively. However, after 10 days acclimation, the number of sites declines dramatically, attaining values far below those for shrimps in freshwater. These acclimation-induced alterations in numerical density/microm(3) cytoplasm cannot be accounted for by corresponding changes in membrane surface density (microm(2) membrane/microm(3) cytoplasm) and reflect a real alteration in the number of Na(+)/K(+)-ATPase reaction product sites/unit membrane area. These data suggest that neither the gill nor the renal Na(+)/K(+)-ATPase systems function at maximal activity in shrimps in freshwater, possibly due to the low Na(+) concentration, and are initially stimulated by the increase in external ionic concentration. However, these powerful Na(+) transport systems respond to salt loading by a notable reduction in the number of hydrolysis sites, possibly through the incorporation of the Na(+)/K(+)-ATPase into isolated membrane vesicles in the basal invaginations of the medial renal tubules, together with ultrastructural alterations like the spatial isolation of the mitochondria by multiple membrane stacks in the intralamellar septal cells. J. Exp. Zool. 284:617-628, 1999.

Regulation of hemolymph osmolytes and gill Na +/K +-ATPase activities during acclimation to saline media in the freshwater shrimp Macrobrachium olfersii (Wiegmann, 1836) (Decapoda, Palaemonidae)

To evaluate the mechanisms of long-term osmotic adaptation to saline media, total hemolymph osmolytes, hemolymph sodium and chloride concentrations, and gill Na +/K +-ATPase activities were measured in the freshwater shrimp Macrobrachium olfersii (Wiegman) after acclimation for 10 or 20 days to media of <0.5, 21 and 28‰ salinity. Total hemolymph osmolytes are maintained strongly hyperosmotic to the external medium in low and moderate salinities, becoming slightly hyperosmotic at high salinity. In contrast, however, hemolymph [Na +] and [Cl −] are distinctly hyporegulated at the higher salinities. This difference in response pattern to acclimation apparently results from the presence of free amino acids in the hemolymph as a consequence of the synthesis of intracellular organic osmolytes. The resulting hyperosmotic regulation both avoids water loss from the animal in saline media and provides an osmotic gradient, allowing the uptake of water from the medium for excretion of the salt load. Gill Na +/K +-ATPase activities decrease by ≈35% after acclimation to saline media, suggesting that the rates of the cellular mechanisms responsible for salt uptake in freshwater become reduced, avoiding excessive salt loading. A Na +-ATPase activity is salinity independent. These data are examined with regard to the intrinsic coupling between the physiological alterations taking place in hemolymph osmolytes during acclimation to saline media and the concomitant ultrastructural rearrangements in the salt transporting tissues of the gills, and their possible neurosecretory control mechanisms.

The Route of Ion and Water Movements Across the Gill Epithelium of the Freshwater Shrimp Macrobrachium olfersii (Decapoda, Palaemonidae): Evidence From Ultrastructural Changes Induced by Acclimation to Saline Media.

The ultrastructure of the pillar cells in the gill lamellae of the freshwater shrimp Macrobrachium olfersii was examined to evaluate the routes of salt and water movement across the gill epithelium and into the hemolymph. Alterations were morphometrically quantified in shrimp maintained in fresh water (FW, <0.5{permill} salinity) and after acclimation to saline media (21{permill} or 28{permill} salinity). The tissue interface between the hemolymph and the external medium consists exclusively of the thin apical flange regions of the pillar cells, the upper membrane of which is highly amplified by dense microvilli and overlain by a thin cuticle. The lower flange membrane, bathed by the hemolymph, is smooth and not invaginated. Contiguous flanges are strongly bound by junctional structures including desmosomes and septate junctions. The basal surface of the pillar cell perikaryon is linked to the adjacent septal cells through many basolateral junctions. The septal cell plasmalemma is abundantly and deeply invaginated, each infolding enclosing numerous mitochondria; these characteristics are typical of salt-transporting machinery. After shrimps were acclimated to saline media for 10 days, the thickness of the pillar cell flanges was significantly reduced (from 1.3 to {approx}0.4 {mu}m), as was the height (from 0.8 to 0.3 {mu}m) and density (from 4.0 to {approx} 1.8 microvilli/{mu}m) of the apical microvilli. This reduction in the apical surface area of the pillar cells appears to lead to decreased ionic permeability, concomitant with a reduction in Na+/K+-ATPase activity, thus limiting Na+ uptake. In contrast to the brachyurans, in which the respiratory and ion-transporting mechanisms are differentially located in the anterior and posterior gills, in palaemonid shrimps the pillar cells apparently play a dual role: ions move preferentially through ion transporters in the microvilli above the pillar cell perikaryon, while respiratory gases are exchanged through the fine flange regions in contact with the hemolymph.

Neuroendocrine modulation of osmoregulatory parameters in the freshwater shrimp Macrobrachium olfersii (Wiegmann) (Crustacea, Decapoda)

Freshwater crustaceans typically exhibit considerable differences in the osmolality and ionic concentrations of their hemolymph compared to the surrounding medium. This steady state equilibrium is maintained by powerful mechanisms of ion uptake and reabsorption, and the excretion of a dilute urine. When acutely exposed to saline media, a new equilibrium is established through readjustment of ion capture and reabsorption rates, and ionic permeability, apparently regulated by neurosecretory mechanisms. This concept is investigated in the euryhaline, freshwater shrimp Macrobrachium olfersii (Wiegmann), using homogenates of the optic ganglia (OG) and ventral nerve cord (VNC). Homogenates prepared from hyperosmoregulating shrimps maintained in freshwater (FW) were injected into the abdominal musculature of test shrimps exposed to FW or sea-water (SW) for 0, 1, 3 or 6 h; changes in hemolymph osmolality and [Cl −], and in body water content and heart rate were measured. Control shrimps received saline alone. Both neurohomogenates reduced hemolymph osmolality and [Cl −], and increased heart rate in SW-exposed shrimps. Body water increased in OG homogenate-injected shrimps in FW while heart rate decreased; the converse occurred in VNC homogenate-injected shrimps in SW. These data show clear effects of putative neurosecretory factors on important osmoregulatory parameters and are interpreted to demonstrate the modulation of osmoregulatory capability by neurosecretory mechanisms.

Neuroendocrine Control of Osmotic Regulation in the Freshwater Shrimp Macrobrachium olfersii (Wiegmann) (Crustacea, Decapoda): Free Amino Acid Concentrations in the Hemolymph

The participation of neuroendocrine factors present within the central nervous system in the regulation of hemolymph free amino acid (FAA) concentrations was examined in the freshwater shrimp Macrobrachium olfersii. Test shrimps were injected intramuscularly with homogenates prepared from the eyestalks (ES), ventral nerve cord (VNC), supraesophageal (SEG), or thoracic ganglia (TG) of donor shrimps previously exposed for 6 hr to a high-salinity medium (HSM, 21% salinity). After injection of the homogenate, the shrimps were maintained for up to 6 hr in either freshwater (FW) or HSM. Hemolymph was sampled by cardiac puncture and prepared for reverse phase HPLC, derivatizing the FAA with phenylisothiocyanate. An FAA profile was determined and the [FAA]:[Cl-] ratios for the four FAA present in highest concentration (Gly, Arg, Ala, and Pro for ES and VNC experiments; Glu, Leu, Ala, and Val for SEG and TG experiments) were obtained. Nonparametric analyses revealed specific, notable effects resulting from homogenate injection, e.g., ES homogenate increased [Pro]/[Cl-] ratios in FW-exposed shrimps; SEG homogenate increased [Glu]/[Cl-] and [Val]/[Cl-] ratios in HSM exposed shrimps; and TG homogenate increased [FAA]/[Cl-] ratios for Glu, Leu, Ala, and Val in HSM-exposed shrimps. Total FAA concentrations decreased after exposure of the shrimps to HSM but were increased by the injection of ES homogenate in FW exposed shrimps and by TG homogenate in HSM exposed shrimps. The total [FAA]/[Cl-] ratio was also increased by TG homogenate in HSM-exposed animals. There were no clear effects on [Cl-] alone. These data are consistent with the notion that FAA titers in the hemolymph of M. olfersii reflect anisosmotic extracellular regulation and its modulation by endocrine factors which accumulate in neurosecretory cells of the ES, SEG, and TG, particularly the latter. The role of such modulation in the osmoregulatory process in vivo is discussed.

Fine Structure of the Gills of the Fresh-Water Shrimp Macrobrachium olfersii (Decapoda): Effect of Acclimation to High Salinity Medium and Evidence for Involvement of the Lamellar Septum in Ion Uptake

Fine Structure of the Gills of the Fresh-Water Shrimp Macrobrachium olfersii (Decapoda): Effect of Acclimation to High Salinity Medium and Evidence for Involvement of the Lamellar Septum in Ion Uptake

FINE STRUCTURE OF THE GILLS OF THE FRESH-WATER SHRIMP MACROBRACHIUM OLFERSII (DECAPODA): EFFECT OF ACCLIMATION TO HIGH SALINITY MEDIUM AND EVIDENCE FOR INVOLVEMENT OF THE LAMELLAR SEPTUM IN ION UPTAKE

ABSTRACT The microanatomy and ultrastructure of the sixth gill of the fresh-water shrimp Macrobrachium olfersii was examined in shrimps maintained either in fresh water or acclimated to a high salinity medium (HSM, sea water of 21‰) for 10 days. At its base in the gill shaft, each phyllobranchiate lamella possesses a central afferent vessel and 2 lateral efferent vessels. The lamella exhibits a nonfenestrated, medial septum over its full extension which divides the lamella into 2 compartments through which hemolymph flow is directed by pillar cells disposed perpendicularly above and below the septum. The pillar cells possess apical microvilli apposed to the cuticle, while their basal regions are strongly attached to the septal cells by areas of desmosomal contact. Wide apical flanges spread radially from the columnar pillar cells as sheets of thin cytoplasm of about 850 nm in thickness. The flanges also exhibit microvilli, and adjacent flanges are connected by areas of septate junctions; there is no elaboration of the basolateral membrane. The pillar cells and their flanges are overlaid by a thin cuticle (175 nm thick), resulting in a hemolymph-water diffusion barrier of about 1 μm in the region of the flanges. The septal cells are rich in mitochondria and exhibit numerous infoldings of their plasma membrane which individually envelop each mitochondrion, probably providing an energy source for active salt transport through the pillar cells. The gill can thus be considered a mixed function gill with a dual role in gas exchange and salt transport. The 10-day acclimation period to HSM induced changes in this basic arrangement, quantified by morphometric analysis. The surface density of the plasma membrane infoldings increased significantly from 2.01 ± 0.15 μm2 membrane/μm3 cytoplasm in shrimps maintained in fresh water to 2.91 ± 0.19 μm2 membrane/μm3 cytoplasm in those acclimated to HSM. The infoldings themselves became arranged in stacked layers separating the mitochondria (7.07 ± 1.51 cf. 13.73 ± 2.48 test segments intersecting more than one invagination). The mitochondrial volume fraction (12.56 ± 0.69 cf. 14.26 ± 0.71%) was unchanged as was the length to width ratio of the mitochondrial profiles (2.74 ± 0.17 cf. 3.23 ± 0.18). The data are discussed in terms of the significance of the physical coupling between membrane invaginations and mitochondria in the septal cells, and in terms of the possible evolution of the lamellar structure in the phyllobranchiate gills of the Palaemonidae.

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) 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‰, S) and were injected into shrimps subsequently maintained for 1, 3, or 6 hr in freshwater (FW, 0‰ S) 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 −] 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 [CI −] 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 2+] 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.

Neuroendocrine regulation of osmotic and ionic concentrations in the hemolymph of the freshwater shrimp Macrobrachium olfersii (Wiegmann) (crustacea, decapoda)

Putative neuroendocrine mediation of osmotic and ionic responses to acute exposure to high salinity medium was investigated in the freshwater shrimp Macrobrachium olfersii (Wiegmann). Homogenates of supra-esophageal or thoracic ganglia, prepared from shrimps exposed to seawater of 21‰ S for 6 hr, were injected into the abdominal musculature of shrimps previously exposed to freshwater and subsequently exposed to either freshwater or seawater (21‰ 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 filtered seawater, isosmotic to the hemolymph, and treated similarly. In control shrimps, the osmotic, Na +, Cl −, K +, Mg 2+, and Ca 2+ concentrations in the hemolymph increased ( P≤0.05) after 1-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 Mg 2+ concentrations decreased ( P ≤ 0.05) with time. In shrimps injected with homogenates of thoracic ganglion and exposed to seawater, hemolymph osmotic, K +, and Mg 2+ concentrations increased ( P ≤ 0.05); Na +, Cl −, and Ca 2+ concentrations remained unchanged. In injected shrimps exposed to freshwater, hemolymph osmotic concentration alone increased ( P ≤ 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. olfersii 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.

The effect of eyestalk ablation on haemolymph osmotic and ionic concentrations during acute salinity exposure in the freshwater shrimp Macrobrachium olfersii (Wiegmann) (Crustacea, Decapoda)

The effect of bilateral eyestalk ablation on haemolymph osmotic and Na+, Cl−, K+, Mg2+ and Ca2+ concentrations was investigated in the freshwater shrimp Macrobrachium olfersii during acute exposure (1, 3, 6, 12 and 24 h) to sea-water of 21% S. Analysis of variance treatment indicated that haemolymph osmotic and Na+, Cl−, K+, Mg2+ and Ca+ concentrations were affected by exposure time (p ≤ 0.01). However, only [Na+] was affected by eyestalk ablation (p < 0.01), destalked shrimps exhibiting haemolymph Na+ concentrations approximately 15 to 30% lower than control shrimps. These results are interpreted to indicate that less Na+ enters the haemolymph of eyestalkless shrimps on exposure to seawater of 21% S, presumably resulting from the absence of an eyestalk-located neurofactor. A factor causing the influx of Na+ into the haemolymph is thus proposed to be present in the eyestalk ganglionic system of intact Macrobrachium olfersii in freshwater. Reduction in secretion of the factor may act to reduce Na+ entry in high salt environments.

O 2 consumption and acute salinity exposure in the freshwater shrimp Macrobrachium olfersii (Wiegmann) (Crustacea:Decapoda): whole animal and tissue respiration

The time course of O 2 consumption after acute salinity exposure (1, 3, 6, 12, and 24 h to 0, 7, 14, 21, 28, and 35 S) was examined in isolated supraesophageal ganglia, gills, and intact Macrobrachium olfersii (Wiegmann), a hyperosmoregulating freshwater palaemonid shrimp, to establish patterns of metabolic adjustment during salinity adaptation. In whole shrimps, O 2 uptake rates decline with salinity increase to 21 S, subsequently increasing with further salinity increase. The rates increase to maxima after 6–12-h exposure in low salinities, decreasing steadily with time in high salinities. In gill preparations, O 2 consumption rates increase to a maximum in 14 S, then decline; they are maximal after 3–6-h exposure to low salinities and diminish with time in high salinities. In the supraesophageal ganglion, rates of O 2 uptake, always measured in seawater of 18 S, are also maximal when shrimps are exposed to 14 S, subsequently declining or levelling off. Rates decrease with time in shrimps exposed to very low salinities, and are stable in 21 S, reaching maxima after 3–6-h exposure of shrimps to all other media. Both tissues thus exhibit characteristic response patterns of O 2 consumption rate which appear to depend on their functional significance within the context of the whole organism. Such data are interpreted to indicate an interrelationship between O 2 consumption and osmoregulatory capability.

The time course of osmotic regulation in the freshwater shrimp Macrobrachium olfersii (Wiegmann) (Decapoda, Palaemonidae)

Alterations in hemolymph osmotic concentration (HOC) in the decapod shrimp Macrobrachium olfersii (Wiegmann) were examined as a function of salinity (0, 7, 14, 21, 28 and 35 S) and exposure time (1,3,6,12 and 24 h). This freshwater palaemonid maintains a high HOC in river water (362.9 ± 7.8 mOsm) which is virtually unaffected by salinity variation up to 14 S or exposure time. However, in 21 and 28 S, HOC increases rapidly until 6 h, remaining stable to 12 h, then increasing to 24 h; in 35 S, HOC increases continually with time. M. olfersii hyperregulates its HOC in salinities up to isosmotic (14 S), exhibiting various degrees of HOC response (strong hyporegulation to isosmoticity) in higher salinities, as a function of time. Hemolymph isosmotic points increase little during the first 6 h of exposure, rising rapidly after 12 and 24 h. Given its high HOC in freshwater, tolerance of high salinities and the dependence of its larval stages on saline water, M. olfersii is considered to be a recent, although efficient, colonizer of the freshwater biotope.

The effect of salinity on respiratory metabolism in selected ontogenetic stages of the freshwater shrimp Macrobrachium olfersii (Decapoda, palaemonidae)

1. 1. The effect of acute salinity exposure (0, 7, 14, 21, 28 and 35%.S) on the respiratory metabolism of selected ontogenetic stages (zoeae, postlarvae and adults) of the freshwater shrimp Macrobrachium olfersiiwas examined. 2. 2. Metabolic rates are salinity independent from 14 to 28%. S in zoeae 1–4, but tend to increase with increasing salinity in zoeae 5 and 8. Postlarvae exhibit maximal rates in midrange salinities while in adult shrimps, oxygen consumption rates decrease with salinity increase. 3. 3. Salinity has little effect on the metabolism-weight relationship, regression analysis indicating that b varies from 0.69 in 0%. S to 0.62 in 35%. S. 4. 4. Data are discussed as to whether larval responses reflect adaptation to the adult biotope and whether development of the larval neurosecretory system might affect metabolic response to salinity exposure.