Ovarian development in swimming crabs: Comparative histochemistry and ultrastructure of Callinectes ornatus and Arenaeus cribrarius (Brachyura, Portunidae).

The ovarian development of Callinectes ornatus and Arenaeus cribrarius was described using histochemistry and ultrastructure. Both species shows the same ovarian stages, which are the juvenile (JUV), adult rudimentary (RUD), developing (DEV), intermediary (INT), mature (MAT), and spent (OV) stages. The JUV and RUD stages showed similar characteristics, and previtellogenesis is characterized by meiotic prophase chromosomes. In the primary vitellogenesis, the oocyte cytoplasm shows many small and large cytoplasmic glycoprotein vesicles. These vesicles correspond to the dilated cisternae of the rough endoplasmic reticulum (RER), which produces the immature (endogenous) yolk. Secondary vitellogenesis (exogenous phase) begins at the DEV stage with the fusion of pinocytic vesicles and vesicles with immature yolks to form mature yolk granules. At the INT stage, the formation of the chorion begins, and the mature yolks increase in size and number, while the RER diminishes. In the MAT stage, the oocytes are completely formed, and the cytoplasm is filled with mature yolk, lipid droplets, and glycogen. There are no significant variations between the gonadosomatic and hepatosomatic indices, which allows us to infer that the transfer of reserves from the hepatopancreas is nearly constant during ovarian development, since we observed primiparous and multiparous females in the same sampled population.

Shell occupation by the endemic western Atlantic hermit crab Isocheles sawayai (Diogenidae) from Caraguatatuba, Brazil / Ocupação de conchas pelo ermitão endêmico do Atlântico ocidental Isocheles sawayai (Diogenidae) em Caraguatatuba, Brasil

The aim of this study was to characterize the pattern of shell occupation by the hermit crab Isocheles sawayai Forest and Saint-Laurent, 1968, from the Caraguatatuba region. The percentage of shell types that were occupied and the morphometric relationships between hermit crabs and occupied shells were analyzed from systematized collections that were conducted monthly from July 2001 to June 2003. A total of 373 individuals were captured (297 males, 41 non-ovigerous females, 25 ovigerous females and 10 intersexes), occupying 17 species of gastropod shells. Stramonita haemastoma (Linnaeus, 1767) (49.87 percent) was the significantly most occupied species (χ2 = 89.30; P < 0.05) followed, with no significant difference, by Phalium granulatum (Born, 1778) (11.53 percent), Polinices hepaticus (Roding, 1798) (8.31 percent) and Cymatium parthenopeum (von Salis, 1793) (6.97 percent). All the morphometric relationships between hermit crabs and occupied shells showed high (r > 0.68) and significant (P < 0.05) correlation values, which is an important indication that in this I. sawayai population the animals occupied adequate shells. The high number of occupied shell species and relative plasticity indicated that, for the studied population, occupation is influenced by the shell availability.

O presente estudo caracterizou o padrão de ocupação de conchas de Isocheles sawayai Forest and Saint-Laurent, 1968, na região de Caraguatatuba. A porcentagem dos diferentes tipos de conchas ocupadas e as relações morfométricas entre os ermitões e as conchas ocupadas foram analisadas a partir de material de amostragem sistematizada, mensalmente, de julho/2001 a junho/2003. Foram capturados 373 indivíduos (297 machos, 41 fêmeas não-ovígeras, 25 fêmeas ovígeras e 10 indivíduos intersexo), ocupando 17 espécies de conchas de moluscos gastrópodes. Stramonita haemastoma (Linnaeus, 1767) (49,87 por cento) foi a espécie significativamente mais ocupada (χ2 = 89,30; p < 0,05) seguida, porém sem diferença significativa, por Phalium granulatum (Born, 1778) (11,53 por cento), Polinices hepaticus (Roding, 1798) (8,31 por cento) e Cymatium parthenopeum (von Salis, 1793) (6,97 por cento). Todas as relações morfométricas entre os ermitões e as conchas ocupadas apresentaram valores de coeficientes de correlação elevados (r > 0,68) e significativos (p < 0,05), o que é um importante indício de que, nessa população de I. sawayai, os animais ocupavam conchas adequadas. O elevado número de espécies de conchas ocupadas e a relativa plasticidade indicaram que, para a população estudada, a ocupação é influenciada pela disponibilidade de conchas.

The crustacean gill (Na+,K+)-ATPase: allosteric modulation of high- and low-affinity ATP-binding sites by sodium and potassium.

The blue crab, Callinectes danae, tolerates exposure to a wide salinity range employing mechanisms of compensatory ion uptake when in dilute media. Although the gill (Na+,K+)-ATPase is vital to hyperosmoregulatory ability, the interactions occurring at the sites of ATP binding on the molecule itself are unknown. Here, we investigate the modulation by Na+ and K+ of homotropic interactions between the ATP-binding sites, and of phosphoenzyme formation of the (Na+,K+)-ATPase from the posterior gills of this euryhaline crab. The contribution of the high- and low-affinity ATP-binding sites to maximum velocity was similar for both Na+ and K+. However, in contrast to Na+, a threshold K+ concentration triggers the appearance of the high-affinity binding sites, displacing the saturation curve to lower ATP concentrations.Further, a low-affinity site for phosphorylation is present on the enzyme. These findings reveal notable differences in the catalytic mechanism of the crustacean (Na+,K+)-ATPase compared to the vertebrate enzyme.

Regulation by the exogenous polyamine spermidine of Na,K-ATPase activity from the gills of the euryhaline swimming crab Callinectes danae (Brachyura, Portunidae).

Euryhaline crustaceans rarely hyporegulates and employ the driving force of the Na,K-ATPase, located at the basal surface of the gill epithelium, to maintain their hemolymph osmolality within a range compatible with cell function during hyper-regulation. Since polyamine levels increase during the adaptation of crustaceans to hyperosmotic media, we investigate the effect of exogenous polyamines on Na,K-ATPase activity in the posterior gills of Callinectes danae, a euryhaline swimming crab. Polyamine inhibition was dependent on cation concentration, charge and size in the following order: spermine>spermidine>putrescine. Spermidine affected K(0.5) values for Na(+) with minor alterations in K(0.5) values for K(+) and NH(4)(+), causing a decrease in maximal velocities under saturating Na(+), K(+) and NH(4)(+) concentrations. Phosphorylation measurements in the presence of 20 microM ATP revealed that the Na,K-ATPase possesses a high affinity site for this substrate. In the presence of 10 mM Na(+), both spermidine and spermine inhibited formation of the phosphoenzyme; however, in the presence of 100 mM Na(+), the addition of these polyamines allowed accumulation of the phosphoenzyme. The polyamines inhibited pumping activity, both by competing with Na(+) at the Na(+)-binding site, and by inhibiting enzyme dephosphorylation. These findings suggest that polyamine-induced inhibition of Na,K-ATPase activity may be physiologically relevant during migration to fully marine environments.

Shell occupation by the endemic western Atlantic hermit crab Isocheles sawayai (Diogenidae) from Caraguatatuba, Brazil.

The aim of this study was to characterize the pattern of shell occupation by the hermit crab Isocheles sawayai Forest and Saint-Laurent, 1968, from the Caraguatatuba region. The percentage of shell types that were occupied and the morphometric relationships between hermit crabs and occupied shells were analyzed from systematized collections that were conducted monthly from July 2001 to June 2003. A total of 373 individuals were captured (297 males, 41 non-ovigerous females, 25 ovigerous females and 10 intersexes), occupying 17 species of gastropod shells. Stramonita haemastoma (Linnaeus, 1767) (49.87%) was the significantly most occupied species (chi2 = 89.30; P < 0.05) followed, with no significant difference, by Phalium granulatum (Born, 1778) (11.53%), Polinices hepaticus (Roding, 1798) (8.31%) and Cymatium parthenopeum (von Salis, 1793) (6.97%). All the morphometric relationships between hermit crabs and occupied shells showed high (r > 0.68) and significant (P < 0.05) correlation values, which is an important indication that in this I. sawayai population the animals occupied adequate shells. The high number of occupied shell species and relative plasticity indicated that, for the studied population, occupation is influenced by the shell availability.

K+ and NH4(+) modulate gill (Na+, K+)-ATPase activity in the blue crab, Callinectes ornatus: fine tuning of ammonia excretion.

To better comprehend the mechanisms of ionic regulation, we investigate the modulation by Na+, K+, NH4(+) and ATP of the (Na+, K+)-ATPase in a microsomal fraction from Callinectes ornatus gills. ATP hydrolysis obeyed Michaelis-Menten kinetics with KM=0.61+/-0.03 mmol L(-1) and maximal rate of V=116.3+/-5.4 U mg(-1). Stimulation by Na+ (V=110.6+/-6.1 U mg(-1); K0.5=6.3+/-0.2 mmol L(-1)), Mg2+ (V=111.0+/-4.7 U mg(-1); K0.5=0.53+/-0.03 mmol L(-1)), NH4(+) (V=173.3+/-6.9 U mg(-1); K0.5=5.4+/-0.2 mmol L(-1)) and K+ (V=116.0+/-4.9 U mg(-1); K0.5=1.5+/-0.1 mmol L(-1)) followed a single saturation curve, although revealing site-site interactions. In the absence of NH4(+), ouabain (K(I)=74.5+/-1.2 micromol L(-1)) and orthovanadate inhibited ATPase activity by up to 87%; the inhibition patterns suggest the presence of F0F1 and K+-ATPases but not Na+-, V- or Ca2+-ATPase as contaminants. (Na+, K+)-ATPase activity was synergistically modulated by K+ and NH4(+). At 10 mmol L(-1) K+, increasing NH4(+) concentrations stimulated maximum activity to V=185.9+/-7.4 U mg(-1). However, at saturating NH4(+) (50 mmol L(-1)), increasing K+ concentrations did not stimulate activity further. Our findings provide evidence that the C. ornatus gill (Na+, K+)-ATPase may be particularly well suited for extremely efficient active NH4(+) excretion. At elevated NH4(+) concentrations, the enzyme is fully active, regardless of hemolymph K+ concentration, and K+ cannot displace NH4(+) from its exclusive binding sites. Further, the binding of NH4(+) to its specific sites induces an increase in enzyme apparent affinity for K+, which may contribute to maintaining K+ transport, assuring that exposure to elevated ammonia concentrations does not lead to a decrease in intracellular potassium levels. This is the first report of modulation by ammonium ions of C. ornatus gill (Na+, K+)-ATPase, and should further our understanding of NH4(+) excretion in benthic crabs.

Gill microsomal (Na+,K+)-ATPase from the blue crab Callinectes danae: Interactions at cationic sites.

Euryhaline crustaceans tolerate exposure to a wide range of dilute media, using compensatory, ion regulatory mechanisms. However, data on molecular interactions occurring at cationic sites on the crustacean gill (Na+,K+)-ATPase, a key enzyme in this hyperosmoregulatory process, are unavailable. We report that Na+ binding at the activating site leads to cooperative, heterotropic interactions that are insensitive to K+. The binding of K+ ions to their high affinity sites displaces Na+ ions from their sites. The increase in Na+ ion concentrations increases heterotropic interactions with the K+ ions, with no changes in K0.5 for K+ ion activation at the extracellular sites. Differently from mammalian (Na+,K+)-ATPases, that from C. danae exhibits additional NH4+ ion binding sites that synergistically activate the enzyme at saturating concentrations of Na+ and K+ ions. NH4+ binding is cooperative, and heterotropic NH4+ ion interactions are insensitive to Na+ ions, but Na+ ions displace NH4+ ions from their sites. NH4+ ions also displace Na+ ions from their sites. Mg2+ ions modulate enzyme stimulation by NH4+ ions, displacing NH4+ ion from its sites. These interactions may modulate NH4+ ion excretion and Na+ ion uptake by the gill epithelium in euryhaline crustaceans that confront hyposmotic media.

K+-Phosphatase activity of gill (Na+, K+)-ATPase from the blue crab, Callinectes danae: low-salinity acclimation and expression of the alpha-subunit.

The kinetic properties of a microsomal gill (Na(+), K(+)) ATPase from the blue crab, Callinectes danae, acclimated to 15 per thousand salinity for 10 days, were analyzed using the substrate p-nitrophenylphosphate. The (Na(+), K(+))-ATPase hydrolyzed the substrate obeying Michaelian kinetics at a rate of V=102.9+/-4.3 U.mg(-1) with K(0.5)=1.7+/-0.1 mmol.L(-1), while stimulation by magnesium (V=93.7+/-2.3 U.mg(-1); K(0.5)=1.40+/-0.03 mmol.L(-1)) and potassium ions (V=94.9+/-3.5 U.mg(-1); K(0.5)=2.9+/-0.1 mmol.L(-1)) was cooperative. K(+)-phosphatase activity was also stimulated by ammonium ions to a rate of V=106.2+/-2.2 U. mg(-1) with K(0.5)=9.8+/-0.2 mmol.L(-1), following cooperative kinetics (n(H)=2.9). However, K(+)-phosphatase activity was not stimulated further by K(+) plus NH(4) (+) ions. Sodium ions (K(I)=22.7+/-1.7 mmol.L(-1)), and orthovanadate (K(I)=28.1+/-1.4 nmol.L(-1)) completely inhibited PNPPase activity while ouabain inhibition reached almost 75% (K(I)=142.0+/-7.1 micromol.L(-1)). Western blotting analysis revealed increased expression of the (Na(+), K(+))-ATPase alpha-subunit in crabs acclimated to 15 per thousand salinity compared to those acclimated to 33 per thousand salinity. The increase in (Na(+), K(+))-ATPase activity in C. danae gill tissue in response to low-salinity acclimation apparently derives from the increased expression of the (Na(+), K( (+) ))-ATPase alpha-subunit; phosphate-hydrolyzing enzymes other than (Na(+), K(+))-ATPase are also expressed. These findings allow a better understanding of the kinetic behavior of the enzymes that underlie the osmoregulatory mechanisms of euryhaline crustaceans.

Gill (Na+,K+)-ATPase from the blue crab Callinectes danae: modulation of K+-phosphatase activity by potassium and ammonium ions.

The kinetic properties of a microsomal gill (Na(+),K(+))-ATPase from the blue crab Callinectes danae were analyzed using the substrate p-nitrophenylphosphate. The (Na(+),K(+))-ATPase hydrolyzed PNPP obeying cooperative kinetics (n=1.5) at a rate of V=125.4+/-7.5 U mg(-1) with K(0.5)=1.2+/-0.1 mmol l(-1); stimulation by potassium (V=121.0+/-6.1 U mg(-1); K(0.5)=2.1+/-0.1 mmol l(-1)) and magnesium ions (V=125.3+/-6.3 U mg(-1); K(0.5)=1.0+/-0.1 mmol l(-1)) was cooperative. Ammonium ions also stimulated the enzyme through site-site interactions (n(H)=2.7) to a rate of V=126.1+/-4.8 U mg(-1) with K(0.5)=13.7+/-0.5 mmol l(-1). However, K(+)-phosphatase activity was not stimulated further by K(+) plus NH(4)(+) ions. Sodium ions (K(I)=36.7+/-1.7 mmol l(-1)), ouabain (K(I)=830.3+/-42.5 micromol l(-1)) and orthovanadate (K(I)=34.0+/-1.4 nmol l(-1)) completely inhibited K(+)-phosphatase activity. The competitive inhibition by ATP (K(I)=57.2+/-2.6 micromol l(-1)) of PNPPase activity suggests that both substrates are hydrolyzed at the same site on the enzyme. These data reveal that the K(+)-phosphatase activity corresponds strictly to a (Na(+),K(+))-ATPase in C. danae gill tissue. This is the first known kinetic characterization of K(+)-phosphatase activity in the portunid crab C. danae and should provide a useful tool for comparative studies.

ESR dating of a subfossil shell from Couve Island, Ubatuba, Brazil.

The occurrence of subfossil material in bottom sediments of Couve Island, Ubatuba region, São Paulo State, is confirmed. The analyzed material consists of a bivalve shell that was dated by the electron spin resonance technique. By the additive irradiation method an archaeological absorbed dose of (25 +/- 5) Gy was obtained and calibration with other dating works in the same area allows the inference of an age of (25 +/- 5) x 10(2) years. The importance of this finding and its paleoecological implications could contribute to elucidating the nature of short-term environmental changes in the Brazilian coastline during the Holocene, as well as becoming an important tool to the understanding of the distribution and biological aspects of the bivalve fauna.

Modulation by ammonium ions of gill microsomal (Na+,K+)-ATPase in the swimming crab Callinectes danae: a possible mechanism for regulation of ammonia excretion.

The modulation by Na(+), K(+), NH(4)(+) and ATP of the (Na(+),K(+))-ATPase in a microsomal fraction from Callinectes danae gills was analyzed. ATP was hydrolyzed at high-affinity binding sites at a maximal rate of V=35.4+/-2.1 Umg(-1) and K(0.5)=54.0+/-3.6 nM, obeying cooperative kinetics (n(H)=3.6). At low-affinity sites, the enzyme hydrolyzed ATP obeying Michaelis-Menten kinetics with K(M)=55.0+/-3.0 microM and V=271.5+/-17.2 Umg(-1). This is the first demonstration of a crustacean (Na(+),K(+))-ATPase with two ATP hydrolyzing sites. Stimulation by sodium (K(0.5)=5.80+/-0.30 mM), magnesium (K(0.5)=0.48+/-0.02 mM) and potassium ions (K(0.5)=1.61+/-0.06 mM) exhibited site-site interactions, while that by ammonium ions obeyed Michaelis-Menten kinetics (K(M)=4.61+/-0.27 mM). Ouabain (K(I)=147.2+/-7.microM) and orthovanadate (K(I)=11.2+/-0.6 microM) completely inhibited ATPase activity, indicating the absence of contaminating ATPase and/or neutral phosphatase activities. Ammonium and potassium ions synergistically stimulated the enzyme, increasing specific activities up to 90%, suggesting that these ions bind to different sites on the molecule. The presence of each ion modulates enzyme stimulation by the other. The modulation of (Na(+),K(+))-ATPase activity by ammonium ions, and the excretion of NH(4)(+) in benthic crabs are discussed.