Evolutionary trade-offs in osmotic and ionic regulation and expression of gill ion transporter genes in high latitude, cold clime Neotropical crabs from the 'end of the world'.

Osmoregulatory findings on crabs from high Neotropical latitudes are entirely lacking. Seeking to identify the consequences of evolution at low temperature, we examined hyperosmotic/hypo-osmotic and ionic regulation and gill ion transporter gene expression in two sub-Antarctic Eubrachyura from the Beagle Channel, Tierra del Fuego. Despite sharing the same osmotic niche, Acanthocyclus albatrossis tolerates a wider salinity range (2-65‰ S) than Halicarcinus planatus (5-60‰ S); their respective lower and upper critical salinities are 4‰ and 12‰ S, and 63‰ and 50‰ S. Acanthocyclus albatrossis is a weak hyperosmotic regulator, while H. planatus hyperosmoconforms; isosmotic points are 1380 and ∼1340 mOsm kg-1 H2O, respectively. Both crabs hyper/hypo-regulate [Cl-] well with iso-chloride points at 452 and 316 mmol l-1 Cl-, respectively. [Na+] is hyper-regulated at all salinities. mRNA expression of gill Na+/K+-ATPase is salinity sensitive in A. albatrossis, increasing ∼1.9-fold at 5‰ compared with 30‰ S, decreasing at 40-60‰ S. Expression in H. planatus is very low salinity sensitive, increasing ∼4.7-fold over 30‰ S, but decreasing at 50‰ S. V-ATPase expression decreases in A. albatrossis at low and high salinities as in H. planatus. Na+/K+/2Cl- symporter expression in A. albatrossis increases 2.6-fold at 5‰ S, but decreases at 60‰ S versus 30‰ S. Chloride uptake may be mediated by increased Na+/K+/2Cl- expression but Cl- secretion is independent of symporter expression. These unrelated eubrachyurans exhibit similar systemic osmoregulatory characteristics and are better adapted to dilute media; however, the expression of genes underlying ion uptake and secretion shows marked interspecific divergence. Cold clime crabs may limit osmoregulatory energy expenditure by hyper/hypo-regulating hemolymph [Cl-] alone, apportioning resources for other energy-demanding processes.

Strategies of Invertebrate Osmoregulation: An Evolutionary Blueprint for Transmuting into Fresh Water from the Sea.

Early marine invertebrates like the Branchiopoda began their sojourn into dilute media some 500 million years ago in the Middle Cambrian. Others like the Mollusca, Annelida, and many crustacean taxa have followed, accompanying major marine transgressions and regressions, shifting landmasses, orogenies, and glaciations. In adapting to these events and new habitats, such invertebrates acquired novel physiological abilities that attenuate the ion loss and water gain that constitute severe challenges to life in dilute media. Among these taxon-specific adaptations, selected from the subcellular to organismal levels of organization, and constituting a feasible evolutionary blueprint for invading freshwater, are reduced body permeability and surface (S) to volume (V) ratios, lowered osmotic concentrations, increased osmotic gradients, increased surface areas of interface epithelia, relocation of membrane proteins in ion-transporting cells, and augmented transport enzyme abundance, activity, and affinity. We examine these adaptations in taxa that have penetrated into freshwater, revealing diversified modifications, a consequence of distinct body plans, morpho-physiological resources, and occupation routes. Contingent on life history and reproductive strategy, numerous patterns of osmotic regulation have emerged, including intracellular isosmotic regulation in weak hyper-regulators and well-developed anisosmotic extracellular regulation in strong hyper-regulators, likely reflecting inertial adaptations to early life in an estuarine environment. In this review, we address osmoregulation in those freshwater invertebrate lineages that have successfully invaded this biotope. Our analyses show that across 66 freshwater invertebrate species from six phyla/classes that have transmuted into freshwater from the sea, hemolymph osmolalities decrease logarithmically with increasing S:V ratios. The arthropods have the highest osmolalities, from 300 to 650 mOsmoles/kg H2O in the Decapoda with 220-320 mOsmoles/kg H2O in the Insecta; osmolalities in the Annelida range from 150 to 200 mOsmoles/kg H2O, and the Mollusca showing the lowest osmolalities at 40-120 mOsmoles/kg H2O. Overall, osmolalities reach a cut-off at ∼200 mOsmoles/kg H2O, independently of increasing S:V ratio. The ability of species with small S:V ratios to maintain large osmotic gradients is mirrored in their putatively higher Na+/K+-ATPase activities that drive ion uptake processes. Selection pressures on these morpho-physiological characteristics have led to differential osmoregulatory abilities, rendering possible the conquest of freshwater while retaining some tolerance of the ancestral medium.

Salt transport by the gill Na+-K+-2Cl- symporter in palaemonid shrimps: exploring physiological, molecular and evolutionary landscapes.

Palaemonid shrimps inhabit osmotic niches from marine to continental waters. They hyper-regulate hemolymph osmolality and ionic concentrations in dilute media, hypo-regulating in concentrated media. Their gill epithelia express ion transporters like the Na+-K+-2Cl- symporter (NKCC) thought to play a role in salt secretion. To examine Cl- hypo-regulatory capability and phylogenetic correlations between gill NKCC mRNA levels and protein expression, we used palaemonids ranging from marine tide pools through estuaries (Palaemon) to coastal and continental fresh waters (Macrobrachium). We established the species' upper critical salinity limits (UL50) and short- (24 h) and long-term (120h) hypo-regulatory abilities at salinities of 80% of their UL50's (80%UL50). The Palaemon species exhibited the highest UL50's and greatest hypo-regulatory capabilities; among the Macrobrachium species, UL50's were higher in the diadromous than in the hololimnetic species. While basal transcript levels of gill NKCC mRNA were highest in P. pandaliformis, levels were unaffected by salinity or exposure time in all species. However, gill NKCC protein abundance increased after 120-h exposure at the 80%UL50 in all Macrobrachium species, except M. potiuna. Unexpectedly, hemolymph hyper-osmoregulatory capability in acclimatization media correlated with gill NKCC protein synthesis, while gill NKCC mRNA expression correlated with hemolymph hyper-Cl- regulation in Macrobrachium. These findings, together with the evolutionary history of osmoregulation in this shrimp clade, suggest a role for the gill NKCC symporter in both salt uptake and secretion. The evolution of NKCC protein expression responsiveness, unlike hemolymph hypo-regulation and NKCC mRNA expression, may have been driven by environmental salinity during niche radiation. SUMMARY STATEMENT: While mRNA expression of the gill Na+-K+-2Cl- symporter is unchanged during acclimation of palaemonid shrimps to saline media, protein expression is up regulated, revealing a role in chloride secretion.

Contrasting strategies of osmotic and ionic regulation in freshwater crabs and shrimps: gene expression of gill ion transporters.

Owing to their extraordinary niche diversity, the Crustacea are ideal for comprehending the evolution of osmoregulation. The processes that effect systemic hydro-electrolytic homeostasis maintain hemolymph ionic composition via membrane transporters located in highly specialized gill ionocytes. We evaluated physiological and molecular hyper- and hypo-osmoregulatory mechanisms in two phylogenetically distant, freshwater crustaceans, the crab Dilocarcinus pagei and the shrimp Macrobrachium jelskii, when osmotically challenged for up to 10 days. When in distilled water, D. pagei survived without mortality, hemolymph osmolality and [Cl-] increased briefly, stabilizing at initial values, while [Na+] decreased continually. Expression of gill V-type H+-ATPase (V-ATPase), Na+/K+-ATPase and Na+/K+/2Cl- symporter genes was unchanged. In M. jelskii, hemolymph osmolality, [Cl-] and [Na+] decreased continually for 12 h, the shrimps surviving only around 15-24 h exposure. Gill transporter gene expression increased 2- to 5-fold. After 10 days exposure to brackish water (25‰S), D. pagei was isosmotic, iso-chloremic and iso-natriuremic. Gill V-ATPase expression decreased while Na+/K+-ATPase and Na+/K+/2Cl- symporter expression was unchanged. In M. jelskii (20‰S), hemolymph was hypo-regulated, particularly [Cl-]. Transporter expression initially increased 3- to 12-fold, declining to control values. Gill V-ATPase expression underlies the ability of D. pagei to survive in fresh water while V-ATPase, Na+/K+-ATPase and Na+/K+/2Cl- symporter expression enables M. jelskii to confront hyper/hypo-osmotic challenges. These findings reveal divergent responses in two unrelated crustaceans inhabiting a similar osmotic niche. While D. pagei does not secrete salt, tolerating elevated cellular isosmoticity, M. jelskii exhibits clear hypo-osmoregulatory ability. Each species has evolved distinct strategies at the transcriptional and systemic levels during its adaptation to fresh water.

Transcriptional, translational and systemic alterations during the time course of osmoregulatory acclimation in two palaemonid shrimps from distinct osmotic niches.

Palaemonid shrimps exhibit numerous adaptive strategies, both in their life cycles and in biochemical, physiological, morphological and behavioral characteristics that reflect the wide variety of habitats in which they occur, including species that are of particular interest when analyzing adaptive osmoregulatory strategies. The present investigation evaluates the short- (hours) and long-term (days) time courses of responses of two palaemonid shrimps from separate yet overlapping osmotic niches, Palaemon northropi (marine) and Macrobrachium acanthurus (diadromous, fresh water), to differential salinity challenges at distinct levels of structural organization: (i) transcriptional, analyzing quantitative expression of gill mRNAs that encode for subunits of the Na+/K+-ATPase and V(H+)-ATPase ion transporters; (ii) translational, examining the kinetic behavior of gill Na+/K+-ATPase specific activity; and (iii) systemic, accompanying consequent adjustment of hemolymph osmolality. Palaemon northropi is an excellent hyper-hypo-osmoregulator in dilute and concentrated seawater, respectively. Macrobrachium acanthurus is a strong hyper-regulator in fresh water and hypo-regulates hemolymph osmolality and particularly [Cl-] in brackish water. Hemolymph hyper-regulation in fresh water (Macrobrachium acanthurus) and dilute seawater (Palaemon northropi) is underlain by augmented expression of both the gill Na+/K+-ATPase and V(H+)-ATPase. In contrast, in neither species is hypo-regulation sustained by changes in Na+/K+-ATPase mRNA expression levels, but rather by regulating enzyme specific activity. The integrated time course of Na+/K+- and V(H+)-ATPase expression and Na+/K+-ATPase activity in the gills of these palaemonid shrimps during acclimation to different salinities reveals versatility in their levels of regulation, and in the roles of these ion transporting pumps in sustaining processes of hyper- and hypo-osmotic and chloride regulation.

Polyamines regulate phosphorylation-dephosphorylation kinetics in a crustacean gill (Na+, K+)-ATPase.

Aiming to clarify the mechanism of inhibition of (Na+, K+)-ATPase activity by polyamines, we examined the effects of exogenous putrescine, spermidine, and spermine on the kinetic behavior of phosphoenzyme-linked partial reactions using a microsomal gill (Na+, K+)-ATPase from juvenile and adult M. amazonicum, a freshwater palaemonid shrimp. The time course of phosphointermediate formation is greater (0.089 ± 0.006 s-1) in adults than in juveniles (0.053 ± 0.003 s-1) for spermidine, but similar to juveniles (0.059 ± 0.004 s-1) for putrescine. Maximum phosphointermediate formation for the (Na+, K+)-ATPase from juveniles decreased by 46% and 32% with spermidine and putrescine, respectively. In adults, maximum phosphointermediate levels decreased by 50% and 8%, respectively. For both spermidine and putrescine, dephosphorylation rates were higher for adults than for juveniles, and were higher than in controls without polyamines. Spermine had a negligible effect (<10%) on phosphorylation/dephosphorylation rates of both juvenile and adult enzymes. This is the first report on the effects of polyamines on phosphoenzyme-linked partial reactions in juvenile and adult M. amazonicum gill (Na+, K+)-ATPases. Our findings suggest that the phosphorylation/dephosphorylation steps of this gill enzyme may be regulated by polyamines during ontogenetic development.

Low salinity-induced alterations in epithelial ultrastructure, Na+/K+-ATPase immunolocalization and enzyme kinetic characteristics in the gills of the thinstripe hermit crab, Clibanarius vittatus (Anomura, Diogenidae).

Fresh caught Clibanarius vittatus [SW, 31‰ salinity (S)] were acclimated to a dilute medium (15‰ S) for 10 days, employing silver staining to locate gill ion transporting tissue, immunofluorescence to localize the Na+/K+-ATPase α-subunit in the lamellae, and electron microscopy to portray ultrastructural changes in the gill epithelia. Na+/K+-ATPase activity was characterized kinetically in a gill microsomal fraction, including synergistic stimulation by NH4+ plus K+. Silver staining revealed that all 26 phyllobranchiate arthro- and pleurobranchiae participate in ion transport. Na+/K+-ATPase α-subunit staining was weak in SW crabs and distributed exclusively and irregularly within the intralamellar septal cells, particularly at the septal-pillar cell body junctions, and septal cell cytoplasm facing the hemolymph space. In 15‰ S crabs, α-subunit localization was intense, occupying the entire thickened septum. Pillar cells and flanges did not stain. Mitochondria and membrane foldings increased in the pillar cell flanges and intralamellar septal cells, greatly amplifying surface area. Only a single ATP binding site (VM  =  130.8 ± 10.5 nmol min-1 mg protein-1; K0.5  =  55.3 ± 1.7 µmol l-1) obeying Michaelis-Menten kinetics was disclosed. Na+/K+-ATPase activity was modulated by Mg2+, Na+, and NH4+, exhibiting site-site interactions; K+ modulation showed Michaelis-Menten kinetics. K+ plus NH4+ synergistically stimulated activity ≈ 1.7-fold. Ouabain inhibited total ATPase activity by ≈ 70% (KI  =  220-300 µmol l-1), revealing phosphohydrolytic activities other than the Na+/K+-ATPase. Despite ample phylogenetic separation, the phyllobranchiate lamellae of the Anomura and Caridea share many ultrastructural features, that is, an intralamellar septum and opposed abutting pillar cells, similar Na+/K+-ATPase distribution, and comparable kinetic characteristics. These findings suggest either convergent evolution at the structural and biochemical levels, or preservation of traits present in a remote common ancestor.

A kinetic characterization of (Na+, K+)-ATPase activity in the gills of the pelagic seabob shrimp Xiphopenaeus kroyeri (Decapoda, Penaeidae).

We characterize the kinetic properties of a gill (Na(+), K(+))-ATPase from the pelagic marine seabob Xiphopenaeus kroyeri. Sucrose density gradient centrifugation revealed membrane fractions distributed mainly into a heavy fraction showing considerable (Na(+), K(+))-ATPase activity, but also containing mitochondrial F0F1- and Na(+)- and V-ATPases. Western blot analysis identified a single immunoreactive band against the (Na(+), K(+))-ATPase α-subunit with an Mr of ≈ 110 kDa. The α-subunit was immunolocalized to the intralamellar septum of the gill lamellae. The (Na(+), K(+))-ATPase hydrolyzed ATP obeying Michaelis-Menten kinetics with VM = 109.5 ± 3.2 nmol Pi min(-1) mg(-1) and KM = 0.03 ± 0.003 mmol L(-1). Mg(2+) (VM = 109.8 ± 2.1 nmol Pi min(-1 )mg(-1), K0.5 = 0.60 ± 0.03 mmol L(-1)), Na(+) (VM = 117.6 ± 3.5 nmol Pi min(-1 ) mg(-1), K0.5 = 5.36 ± 0.14 mmol L(-1)), K(+) (VM = 112.9 ± 1.4 nmol Pi min(-1 )mg(-1), K0.5 = 1.32 ± 0.08 mmol L(-1)), and NH4 (+) (VM = 200.8 ± 7.1 nmol Pi min(-1 )mg(-1), K0.5 = 2.70 ± 0.04 mmol L(-1)) stimulated (Na(+), K(+))-ATPase activity following site-site interactions. K(+) plus NH4 (+) does not synergistically stimulate (Na(+), K(+))-ATPase activity, although each ion modulates affinity of the other. The enzyme exhibits a single site for K(+) binding that can be occupied by NH4 (+), stimulating the enzyme. Ouabain (KI = 84.0 ± 2.1 µmol L(-1)) and orthovanadate (KI = 0.157 ± 0.001 µmol L(-1)) inhibited total ATPase activity by ≈ 50 and ≈ 44 %, respectively. Ouabain inhibition increases ≈ 80 % in the presence of NH4 (+) with a threefold lower KI, suggesting that NH4 (+) is likely transported as a K(+) congener.

Pigment granule translocation in red ovarian chromatophores from the palaemonid shrimp Macrobrachium olfersi (Weigmann, 1836): functional roles for the cytoskeleton and its molecular motors.

The binding of red pigment concentrating hormone (RPCH) to membrane receptors in crustacean chromatophores triggers Ca²âº/cGMP signaling cascades that activate cytoskeletal motors, driving pigment granule translocation. We investigate the distributions of microfilaments and microtubules and their associated molecular motors, myosin and dynein, by confocal and transmission electron microscopy, evaluating a functional role for the cytoskeleton in pigment translocation using inhibitors of polymer turnover and motor activity in vitro. Microtubules occupy the chromatophore cell extensions whether the pigment granules are aggregated or dispersed. The inhibition of microtubule turnover by taxol induces pigment aggregation and inhibits re-dispersion. Phalloidin-FITC actin labeling, together with tannic acid fixation and ultrastructural analysis, reveals that microfilaments form networks associated with the pigment granules. Actin polymerization induced by jasplaquinolide strongly inhibits RPCH-induced aggregation, causes spontaneous pigment dispersion, and inhibits pigment re-dispersion. Inhibition of actin polymerization by latrunculin-A completely impedes pigment aggregation and re-dispersion. Confocal immunocytochemistry shows that non-muscle myosin II (NMMII) co-localizes mainly with pigment granules while blebbistatin inhibition of NMMII strongly reduces the RPCH response, also inducing spontaneous pigment dispersion. Myosin II and dynein also co-localize with the pigment granules. Inhibition of dynein ATPase by erythro-9-(2-hydroxy-3-nonyl) adenine induces aggregation, inhibits RPCH-triggered aggregation, and inhibits re-dispersion. Granule aggregation and dispersion depend mainly on microfilament integrity although microtubules may be involved. Both cytoskeletal polymers are functional only when subunit turnover is active. Myosin and dynein may be the molecular motors that drive pigment aggregation. These mechanisms of granule translocation in crustacean chromatophores share various features with those of vertebrate pigment cells.

Kinetic analysis of gill (Na⁺,K⁺)-ATPase activity in selected ontogenetic stages of the Amazon River shrimp, Macrobrachium amazonicum (Decapoda, Palaemonidae): interactions at ATP- and cation-binding sites.

We investigated modulation by ATP, Mg²âº, Na⁺, K⁺ and NH4⁺ and inhibition by ouabain of (Na⁺,K⁺)-ATPase activity in microsomal homogenates of whole zoeae I and decapodid III (formerly zoea IX) and whole-body and gill homogenates of juvenile and adult Amazon River shrimps, Macrobrachium amazonicum. (Na⁺,K⁺)-ATPase-specific activity was increased twofold in decapodid III compared to zoea I, juveniles and adults, suggesting an important role in this ontogenetic stage. The apparent affinity for ATP (K(M) = 0.09 ± 0.01 mmol L⁻¹) of the decapodid III (Na⁺,K⁺)-ATPase, about twofold greater than the other stages, further highlights this relevance. Modulation of (Na⁺,K⁺-ATPase activity by K⁺ also revealed a threefold greater affinity for K⁺ (K0.5 = 0.91 ± 0.04 mmol L⁻¹) in decapodid III than in other stages; NH4⁺ had no modulatory effect. The affinity for Na⁺ (K0.5 = 13.2 ± 0.6 mmol L⁻¹) of zoea I (Na⁺,K⁺)-ATPase was fourfold less than other stages. Modulation by Na⁺, Mg²âº and NH4⁺ obeyed cooperative kinetics, while K⁺ modulation exhibited Michaelis-Menten behavior. Rates of maximal Mg²âº stimulation of ouabain-insensitive ATPase activity differed in each ontogenetic stage, suggesting that Mg²âº-stimulated ATPases other than (Na⁺,K⁺)-ATPase are present. Ouabain inhibition suggests that, among the various ATPase activities present in the different stages, Na⁺-ATPase may be involved in the ontogeny of osmoregulation in larval M. amazonicum. The NH4⁺-stimulated, ouabain-insensitive ATPase activity seen in zoea I and decapodid III may reflect a stage-specific means of ammonia excretion since functional gills are absent in the early larval stages.

Differential adjustment in gill Na+/K+- and V-ATPase activities and transporter mRNA expression during osmoregulatory acclimation in the cinnamon shrimp Macrobrachium amazonicum (Decapoda, Palaemonidae).

We evaluate osmotic and chloride (Cl(-)) regulatory capability in the diadromous shrimp Macrobrachium amazonicum, and the accompanying alterations in hemolymph osmolality and [Cl(-)], gill Na(+)/K(+)-ATPase activity, and expression of gill Na(+)/K(+)-ATPase α-subunit and V-ATPase B subunit mRNA during salinity (S) acclimation. We also characterize V-ATPase kinetics and the organization of transport-related membrane systems in the gill epithelium. Macrobrachium amazonicum strongly hyper-regulates hemolymph osmolality and [Cl(-)] in freshwater and in salinities up to 25‰ S. During a 10-day acclimation period to 25‰ S, hemolymph became isosmotic and hypo-chloremic after 5 days, [Cl(-)] alone remaining hyporegulated thereafter. Gill Na(+)/K(+)-ATPase α-subunit mRNA expression increased 6.5 times initial values after 1 h, then decreased to 3 to 4 times initial values by 24 h and to 1.5 times initial values after 10 days at 25‰ S. This increased expression was accompanied by a sharp decrease at 5 h then recovery of initial Na(+)/K(+)-ATPase activity within 24 h, declining again after 5 days, which suggests transient Cl(-) secretion. V-ATPase B-subunit mRNA expression increased 1.5-fold within 1 h, then reduced sharply to 0.3 times initial values by 5 h, and remained unchanged for the remainder of the 10-day period. V-ATPase activity dropped sharply and was negligible after a 10-day acclimation period to 21‰ S, revealing a marked downregulation of ion uptake mechanisms. The gill epithelium consists of thick, apical pillar cell flanges, the perikarya of which are coupled to an intralamellar septum. These two cell types respectively exhibit extensive apical evaginations and deep membrane invaginations, both of which are associated with numerous mitochondria, characterizing an ion transporting epithelium. These changes in Na(+)/K(+)- and V-ATPase activities and in mRNA expression during salinity acclimation appear to underpin ion uptake and Cl(-) secretion by the palaemonid shrimp gill.

Structural and biochemical correlates of Na+,K+-ATPase driven ion uptake across the posterior gill epithelium of the true freshwater crab, Dilocarcinus pagei (Brachyura, Trichodactylidae).

To better comprehend the structural and biochemical underpinnings of ion uptake across the gills of true freshwater crabs, we performed an ultrastructural, ultracytochemical and morphometric investigation, and kinetically characterized the Na(+),K(+)-ATPase, in posterior gill lamellae of Dilocarcinus pagei. Ultrastructurally, the lamellar epithelia are markedly asymmetrical: the thick, mushroom-shaped, proximal ionocytes contain elongate mitochondria (41% cell volume) associated with numerous (≈14 µm² membrane per µm³cytoplasm), deep invaginations that house the Na(+),K(+)-ATPase, revealed ultracytochemically. Their apical surface is amplified (7.5 µm² µm⁻²)) by stubby evaginations whose bases adjoin mitochondria below the subcuticular space. The apical membrane of the thin, distal ionocytes shows few evaginations (1.6 µm² µm⁻²), each surrounding a mitochondrion, abundant in the cytoplasm below the subcuticular space; basolateral invaginations and mitochondria are few. Fine basal cytoplasmic bridges project across the hemolymph space, penetrating into the thick ionocytes, suggesting ion movement between the epithelia. Microsomal Na(+),K(+)-ATPase specific activity resembles marine crabs but is ≈5-fold less than in species from fluctuating salinities, and freshwater shrimps, suggesting ion loss compensation by strategies other than Na(+) uptake. Enzyme apparent K(+) affinity attains 14-fold that of marine crabs, emphasizing the relevance of elevated K(+) affinity to the conquest of fresh water. Western blotting and biphasic ouabain inhibition disclose two α-subunit isoforms comprising distinct functional isoenzymes. While enzyme activity is not synergistically stimulated by NH(4) (+) and K(+), each increases affinity for the other, possibly assuring appropriate intracellular K(+) concentrations. These findings reveal specific structural and biochemical adaptations that may have allowed the establishment of the Brachyura in fresh water.

Association of kinesin and myosin with pigment granules in crustacean chromatophores.

Chromatic adaptation in crustaceans results from the differential distribution of colored pigment granules within their chromatophores consequent to cell signaling by neurosecretory peptides. However, the force transducing, mechanochemical protein motors responsible for granule translocation, and their molecular mechanisms of action, are not well understood. The present study uses immunocytochemical techniques and a motility assay in vitro to demonstrate that protein motors from the kinesin and myosin superfamilies are stably associated with membrane-bounded pigment granules in the red, ovarian chromatophores of the freshwater, palaemonid shrimp, Macrobrachium olfersii. Monoclonal antibodies against conventional kinesin heavy chain, and an anti-myosin whole serum, labeled pigment-containing fragments prepared from homogenates of chromatophores with fully dispersed or aggregated pigments: this finding infers a permanent association between the protein motors and the pigment granules, and suggests that such motors may be regulated while bound to their cargos. The pigment aggregator appears to be a myosin since the anti-myosin whole serum attenuated hormonally triggered pigment aggregation in the motility assay in vitro, and induced pigment hyper-dispersion in some chromatophores. Western blots of the chromatophore-containing, ovarian tissue homogenate demonstrated protein bands consistent with myosin II and myosin XII, either of which may be the pigment aggregator. This study provides the first direct evidence for myosin and kinesin protein motors directly and stably associated with pigment granules in crustacean chromatophores, and may represent the first successful isolation of myosin class XII.

Ion-motive ATPases and active, transbranchial NaCl uptake in the red freshwater crab, Dilocarcinus pagei (Decapoda, Trichodactylidae).

The present investigation examined the microanatomy and mRNA expression and activity of ion-motive ATPases, in anterior and posterior gills of a South American, true freshwater crab, Dilocarcinus pagei. Like diadromous crabs, the anterior gills of this hololimnetic trichodactylid exhibit a highly attenuated (2-5 microm), symmetrical epithelium on both lamellar surfaces. In sharp contrast, the posterior gill lamellar epithelia are markedly asymmetrical. Their proximal side consists of thick (18-20 microm) cells, displaying features typical of a transporting epithelium, while the distal epithelium is thin (3-10 microm) and formed entirely by apical pillar cell flanges. Both anterior and posterior gills express Na+/K+- and V-ATPases. Phylogenetic analysis of partial cDNA sequences for the Na+/K+-ATPase alpha-subunit and V-ATPase B-subunit among various crab species confirmed the previous classification and grouping of D. pagei based on morphological criteria. Semi-quantitative RT-PCR clearly showed that mRNA for both ion pump subunits is more intensely expressed in posterior gills. Na+/K+-ATPase activity in the posterior gills was nearly fourfold that of anterior gills, while V-ATPase and F-ATPase activities did not differ. A negative short-circuit current (Isc) was measured using the distal side of split, posterior gill lamellae, mounted in a modified Ussing chamber and perfused symmetrically with identical hemolymph-like salines. Although hemolymph-side ouabain did not affect this current, concanamycin significantly reduced Isc without altering preparation conductance, suggesting V-ATPase-driven Cl- absorption on the distal side of the posterior gill lamellae, as known to occur in diadromous crabs adapted to freshwater. These findings suggest that active Na+ uptake predominates across the thick proximal epithelium, and Cl- uptake across the thin, distal epithelium of the posterior gill lamellae.

Modulation of gill Na+,K+-ATPase activity by ammonium ions: Putative coupling of nitrogen excretion and ion uptake in the freshwater shrimp Macrobrachium olfersii.

The effect of NH4+ ions on (Na+,K+)-ATPase hydrolytic activity was examined in a gill microsomal fraction from M. olfersii. In the absence of NH4+ ions, K+ ions stimulated ATP hydrolysis, exhibiting cooperative kinetics (nH=0.8), to a maximal specific activity of V=556.1+/-22.2 nmol.min(-1).mg(-1) with K(0.5)=2.4+/-0.1 mmol.L(-1). No further stimulation by K+ ions was observed in the presence of 50 mmol.L(-1) NH4+ ions. ATP hydrolysis was also stimulated by NH4+ ions obeying Michaelian kinetics to a maximal specific activity of V=744.8+/-22.3 nmol.min(-1).mg(-1) and KM=8.4+/-0.2 mmol.L(-1). In the presence of 10 mmol.L(-1) K+ ions, ATP hydrolysis was synergistically stimulated by NH4+ ions to V=689.8+/-13.8 nmol.min(-1).mg(-1) and K(0.5)=6.6+/-0.1 mmol.L(-1), suggesting that NH4+ ions bind to different sites than K+ ions. PNPP hydrolysis was also stimulated cooperatively by K+ or NH4+ ions to maximal values of V= 235.5+/-11.8 nmol.min(-1).mg(-1) and V=234.8+/-7.0 nmol.min(-1).mg(-1), respectively. In contrast to ATP hydrolysis, K(+)-phosphatase activity was not synergistically stimulated by NH4+ and K+ ions. These data suggest that at high NH4+ ion concentrations, the (Na+, K+)-ATPase exposes a new site; the subsequent binding of NH4+ ions stimulates ATP hydrolysis to rates higher than those for K+ ions alone. This is the first demonstration that (Na+, K+)-ATPase activity in a freshwater shrimp gill is modulated by ammonium ions, independently of K+ ions, an effect that may constitute a fine-tuning mechanism of physiological relevance to osmoregulatory and excretory processes in palaemonid shrimps.