Diverse strategies for ion regulation in fish collected from the ion-poor, acidic Rio Negro

We measured unidirectional ion fluxes of fish collected directly from the Rio Negro, an extremely dilute, acidic blackwater tributary of the Amazon. Kinetic analysis of Na(+) uptake revealed that most species had fairly similar J(max) values, ranging from 1,150 to 1,750 nmol g(-1) h(-1), while K(m) values varied to a greater extent. Three species had K(m) values <33 micromol L(-1), while the rest had K(m) values >or=110 micromol L(-1). Because of the extremely low Na(+) concentration of Rio Negro water, the differences in K(m) values yield very different rates of Na(+) uptake. However, regardless of the rate of Na(+) uptake, measurements of Na(+) efflux show that Na(+) balance was maintained at very low Na(+) levels (<50 micromol L(-1)) by most species. Unlike other species with high K(m) values, the catfish Corydoras julii maintained high rates of Na(+) uptake in dilute waters by having a J(max) value at least 100% higher than the other species. Corydoras julii also demonstrated the ability to modulate kinetic parameters in response to changes in water chemistry. After 2 wk in 2 mmol L(-1) NaCl, J(max) fell >50%, and K(m) dropped about 70%. The unusual acclimatory drop in K(m) may represent a mechanism to ensure high rates of Na(+) uptake on return to dilute water. As well as being tolerant of extremely dilute waters, Rio Negro fish generally were fairly tolerant of low pH. Still, there were significant differences in sensitivity to pH among the species on the basis of degree of stimulation of Na(+) efflux at low pH. There were also differences in sensitivity to low pH of Na(+) uptake, and two species maintained significant rates of uptake even at pH 3.5. When fish were exposed to low pH in Rio Negro water instead of deionized water (with the same concentrations of major ions), the effects of low pH were reduced. This suggests that high concentrations of dissolved organic molecules in the water, which give it its dark tea color, may interact with the branchial epithelium in some protective manner.     

Ion regulatory patterns of mosquito larvae collected from breeding sites in the Amazon rain forest

We examined the ion composition of mosquito breeding sites located in the Amazon rain forest and the ion regulatory patterns of larvae from these habitats. We found larvae of Toxorhynchites haemorroidalis, Limatus durhamii, Culex (Carrollia) bonnei, and Culex (Culex) sp. residing in fallen palm bracts, leaves, and tree holes that were filled with water. These breeding sites had micromolar levels of Na(+) (1.6-99 micromol L(-1)), but K(+) and Cl(-) concentrations were higher and varied over a large range (231-17,615 micromol L(-1) K(+); 355-2,700 micromol L(-1) Cl(-)). Despite the variability in environmental ion levels and ratios, all four species maintain high hemolymph NaCl levels (80-120 mmol L(-1) Na(+); 60-80 mmol L(-1) Cl(-)). However, the species differed in the means by which they maintain hemolymph ion balance, as indicated by the range of unidirectional Na(+) and Cl(-) uptake rates. Toxorhynchites haemorroidalis had extremely low rates of Na(+) uptake and undetectable Cl(-) uptake, whereas L. durhamii had high rates of uptake for both ions. This variability in rates of uptake may reflect species differences in rates of diffusive ion loss (i.e., permeability). We observed the same curious pattern of Na(+) inhibition and Cl(-) stimulation by low-pH exposure in all four species of mosquitoes, as has been documented in other mosquitoes and aquatic insects. Kinetic analyses of Na(+) and Cl(-) uptake in C. bonnei larvae revealed an unusual pattern of Na(+) uptake that increases linearly (nonsaturable) to extremely high rates, while Cl(-) uptake is a low-affinity, low-capacity system. This pattern contrasts with L. durhamii and Culex (Culex) sp. larvae, which had large increases in both Na(+) and Cl(-) uptake when external NaCl levels were increased. Our results suggest that although these rain forest mosquito larvae are residing in habitats with similar low Na(+), high Cl(-) composition and maintain similar hemolymph NaCl levels, the underlying mechanisms of ion regulation differ among the species.     

Protection by natural blackwater against disturbances in ion fluxes caused by low pH exposure in freshwater stingrays endemic to the Rio Negro

Stenohaline freshwater stingrays (Potamotrygon spp.) are endemic to the very dilute (Na(+), Cl(-), Ca2(+) 300 micromol L(-1) in reference water (low DOC) to about 100 micromol L(-1) in blackwater (high DOC). In reference water, both JNain and JClin were inhibited >90%, both JNaout and JClout more than doubled, and J(Amm) did not change at pH 4.0. In blackwater, the inhibition of influxes was attenuated, the increases in outflux did not occur, and J(Amm) increased by 60% at pH 4.0. Addition of 100 micromol L(-1) Ca(2+) to reference water prevented the increases in JNaout and JClout and allowed J(Amm) to increase at pH 4.0, which demonstrates that the gills are sensitive to Ca(2+). However, addition of Ca(2+) to blackwater had no effect on the responses to pH 4.0. Addition of commercial humic acid to reference water did not duplicate the effects of natural Rio Negro blackwater at the same DOC level; instead, it greatly exacerbated the increases in JNaout and JClout at low pH and prevented any protective influence of added Ca(2+). Thus, blackwater DOC appears to be very different from commercial humic acid. Biogeochemical modeling indicated that blackwater DOC prevents Ca(2+) binding, but not H(+) binding, to the gills and that the protective effects of blackwater cannot be attributed to its higher buffer capacity or its elevated Al or Fe levels. Natural DOC may act directly at the gills at low pH to exert a protective effect and, when doing so, may override any protective action of Ca(2+) that might otherwise occur.     

A pharmacological examination of Na+ and Cl- transport in two species of freshwater fish

We examined branchial Na(+) and Cl(-) uptake in two species of stenohaline, freshwater fish (goldfish and the Amazonian neon tetra). Kinetic analysis revealed that the two species had similar uptake capacities and affinities for Na(+) and Cl(-). However, while uptakes of Na(+) and Cl(-) (JNain and JClin, respectively) by goldfish were completely inhibited at pH 4.5 and below, uptake in tetras was unaffected by pH down to 3.25. Examination of Cl(-) transport with blockers indicated that goldfish and neon tetras utilize Cl(-)/HCO-3 exchange; SITS and SCN(-) inhibited Cl(-) uptake in both species. In contrast, large differences in Na(+) transport were indicated between the species. In goldfish, exposure to four Na(+)/H(+) exchange blockers, as well as the Na(+) channel blocker phenamil, strongly inhibited JNain. Further, Na(+) and Cl(-) uptake were strongly inhibited by the Na(+)/K(+)/Cl(-) cotransport inhibitor furosemide, as was JNain in "Cl(-)-free" water and JClin in "Na(+)-free" water. This suggests the presence of multiple transporters and possibly even a direct linkage between the transport of Na(+) and Cl(-) in goldfish. In contrast, none of these drugs strongly reduced Na(+) transport in neon tetras, which raises the possibility of a significantly different Na(+) transport mechanism in this acid-tolerant species.     

Proton pump-driven cutaneous chloride uptake in anuran amphibia

Krogh introduced the concept of active ion uptake across surface epithelia of freshwater animals, and proved independent transports of Na(+) and Cl(-) in anuran skin and fish gill. He suggested that the fluxes of Na(+) and Cl(-) involve exchanges with ions of similar charge. In the so-called Krogh model, Cl(-)/HCO(3)(-) and Na(+)/H(+) antiporters are located in the apical membrane of the osmoregulatory epithelium. More recent studies have shown that H(+) excretion in anuran skin is due to a V-ATPase in mitochondria-rich (MR) cells. The pump has been localized by immunostaining and H(+) fluxes estimated by pH-stat titration and mathematical modelling of pH-profiles in the unstirred layer on the external side of the epithelium. H(+) secretion is voltage-dependent, sensitive to carbonic-anhydrase inhibitors, and rheogenic with a charge/ion-flux ratio of unity. Cl(-) uptake from freshwater is saturating, voltage independent, and sensitive to DIDS and carbonic-anhydrase inhibitors. Depending on anuran species and probably on acid/base balance of the animal, apical exit of protons is coupled to an exchange of Cl(-) with base (HCO(3)(-)) either in the apical membrane (gamma-type of MR cell) or in the basolateral membrane (alpha-type MR cell). The gamma-cell model accounts for the rheogenic active uptake of Cl(-) observed in several anuran species. There is indirect evidence also for non-rheogenic active uptake accomplished by a beta-type MR cell with apical base secretion and basolateral proton pumping. Several studies have indicated that the transport modes of MR cells are regulated via ion- and acid/base balance of the animal, but the signalling mechanisms have not been investigated. Estimates of energy consumption by the H(+)-ATPase and the Na(+)/K(+)-ATPase indicate that the gamma-cell accomplishes uptake of NaCl in normal and diluted freshwater. Under common freshwater conditions with serosa-positive or zero V(t), the K(+) conductance of the basolateral membrane would have to maintain the inward driving force for Na(+) uptake across the apical membrane. With the K(+) equilibrium potential across the basolateral membrane estimated to -105 mV, this would apply to external Na(+) concentrations down to 40-120 micromol/l. NaCl uptake from concentrations down to 10 micromol/l, as observed by Krogh, presupposes that the H(+) pump hyperpolarizes the apical membrane, which would then have to be associated with serosa-negative V(t). In diluted freshwater, exchange of cellular HCO(3)(-) with external Cl(-) seems to be possible only if the proton pump has the additional function of keeping the external concentration of HCO(3)(-) low. Quantitative considerations also lead to the conclusion that with the above extreme demand, at physiological intracellular pH of 7.2, the influx of Cl(-) via the apical antiporter and the passive exit of Cl(-) via basolateral channels would be possible within a common range of intracellular Cl(-) concentrations.     

Ingestion, dissolution, and proteolysis of the Bacillus sphaericus toxin by mosquito larvae

Larvae of Culex quinquefasciatus are much more susceptible to the toxin of Bacillus sphaericus than are larvae of Aedes aegypti. In the present study, the rate of ingestion, dissolution, and the cleavage by midgut proteases of the B. sphaericus toxin were compared in larvae of these species to determine whether these factors account for the differences in susceptibility. During filter feeding, larvae of both species removed significant quantities of B. sphaericus toxin from suspensions. Filtration rates for 1 hr, the time at which C. quinquefasciatus exhibited marked intoxication, were higher for A. aegypti (576-713 microliters/larva/hr) than for C. quinquefasciatus (446-544 microliters/larva/hr). Within 24 hr of exposure, A. aegypti larvae ingested 97-99% of the toxin particulates and suffered not more than 10% mortality in suspensions which induced complete mortality in C. quinquefasciatus within 2 hr of exposure. Quantification of the particulate toxin present in larvae after exposure to B. sphaericus suspensions revealed that larvae of both species contained only minor amounts of the toxin, suggesting the larvae had been able to solubilize the toxin after ingestion. Proteases recovered from the feces of larvae cleaved at 43-kDa protein isolated from B. sphaericus toxin extract to 40 kDa in both species. Thus, differences in susceptibility to the B. sphaericus toxin between A. aegypti and C. quinquefasciatus are not due to differences in rates of ingestion, dissolution, or the specificity of proteases.     

Patterns of ion regulation in acidophilic fish native to the ion-poor, acidic Rio Negro

The Na⁺ uptake mechanism of cardinal tetras Paracheirodon axelrodi displayed specialization for operation in dilute waters of the amazonian Rio Negro. Kinetic analysis revealed low K_m and high J_max values which ensure high rates of uptake even in very dilute waters. In contrast, Na⁺ uptake of angelfish Pterophyllum scalare did not appear to be specialized for dilute waters at all, with much higher K_m and lower J_max values. Na⁺ uptake in cardinal tetras was high and completely unaffected down to pH 3·5, while uptake in angelfish was much lower and progressively inhibited by dropping pH; it was completely shut down at pH 3·5. During chronic exposure to pH 4·0 and 3·5, angelfish showed no ability to restore Na⁺ uptake and at pH 3·5 all individuals died between day 3 and 6 presumably due to small, but steady loss of Na⁺. At low pH, both species displayed a strong ability to prevent stimulation of diffusive Na+ losses. In angelfish, the ability to control diffusive ion losses at low pH was related to a high branchial affinity for Ca²⁺. For cardinal tetras, the rate of Na⁺ loss appeared to be independent of water Ca²⁺ concentration. The specializations of cardinal tetras are similar to other characid fishes tested, but the results for angelfish reveal a new pattern of ion regulation in acidophilic species from the Rio Negro. The differences between the species may be the result of their different distributions, cardinal tetras are found in the central Rio Negro region while angelfish are more peripheral, or they may reflect phylogenetic differences.     

Lotus tenuis tolerates the interactive effects of salinity and waterlogging by 'excluding' Na+ and Cl- from the xylem

Salinity and waterlogging interact to reduce growth of poorly adapted species by, amongst other processes, increasing the rate of Na(+) and Cl(-) transport to shoots. Xylem concentrations of these ions were measured in sap collected using xylem-feeding spittlebugs (Philaenus spumarius) from Lotus tenuis and Lotus corniculatus in saline (NaCl) and anoxic (stagnant) treatments. In aerated NaCl solution (200 mM), L. corniculatus had 50% higher Cl(-) concentrations in the xylem and shoot compared with L. tenuis, whereas concentrations of Na(+) and K(+) did not differ between the species. In stagnant-plus-NaCl solution, xylem Cl(-) and Na(+) concentrations of L. corniculatus increased to twice those of L. tenuis. These differences in xylem ion concentrations, which were not caused by variation in transpiration between the two species, contributed to lower net accumulation of Na(+) and Cl(-) in shoots of L. tenuis, indicating that ion transport mechanisms in roots of L. tenuis were contributing to better 'exclusion' of Cl(-) and Na(+) from shoots, compared with L. corniculatus. Root porosity was also higher in L. tenuis, due to constitutive aerenchyma, than in L. corniculatus, suggesting that enhanced root aeration contributed to the maintenance of Na(+) and Cl(-) 'exclusion' in L. tenuis exposed to stagnant-plus-NaCl treatment. Lotus tenuis also had greater dry mass than L. corniculatus after 56 d in NaCl or stagnant-plus-NaCl treatment. Thus, Cl(-) 'exclusion' is a key trait contributing to salt tolerance of L. tenuis, and 'exclusion' of both Cl(-) and Na(+) from the xylem enables L. tenuis to tolerate, better than L. corniculatus, the interactive stresses of salinity and waterlogging.     

Effects of temperature, pH and salinity on the infection of Leptolegnia chapmanii Seymour (Peronosporomycetes) in mosquito larvae

The effects of temperature, pH, and NaCl concentrations on the infectivity of zoospores of Leptolegnia chapmanii (Argentine isolate) were determined for Aedes aegypti and Culex pipiens under laboratory conditions. Zoospores of L. chapmanii were infectious at temperatures between 10 and 35 degrees C but not at 5 or 40 degrees C. At the permissive temperatures, mortality rates in young instars were much higher than in older instars and larvae of Ae. aegypti were more susceptible to L. chapmanii than larvae of Cx. pipiens. At 25 degrees C, Ae. aegypti larvae challenged with L. chapmanii zoospores resulted in 100% infection at pH levels ranging from 4 to 10. Larvae of Cx. pipiens exposed to similar pH and zoospore concentrations resulted in increasing mortality rates from 62% to 99% at pH 4 to 7, respectively, and then decreased to 71% at pH 10. Aedes aegypti larvae exposed to L. chapmanii zoospores in NaCl concentrations ranging from 0 to 7 parts per thousand (ppt) at 25 degrees C resulted in 100% mortality while mortality rates for Cx. pipiens decreases from 96% in distilled water to 31.5% in water with 6 ppt NaCl. Control Cx. pipiens larvae died when exposed at a NaCl concentration of 7 ppt. Vegetative growth of L. chapmanii was negatively affected by NaCl concentrations. These results have demonstrated that the Argentinean isolate of L. chapmanii tolerated a wide range of temperatures, pH, and salinity, suggesting that it has the potential to adapt to a wide variety of mosquito habitats.     

Salt tolerance in a Hordeum marinum-Triticum aestivum amphiploid, and its parents

Growth, grain production, and physiological traits were evaluated for Hordeum marinum, Triticum aestivum (cv. Chinese Spring), and a H. marinum-T. aestivum amphiploid, when exposed to NaCl treatments in a nutrient solution. H. marinum was more salt tolerant than T. aestivum and the amphiploid was intermediate, both for vegetative growth and relative grain production. H. marinum was best able to 'exclude' Na(+) and Cl(-), particularly at high external NaCl. At 300 mM NaCl, concentrations of Na(+) (153 micromol g(-1) dry mass) and Cl(-) (75 micromol g(-1) dry mass) in the youngest fully-expanded leaf blade of H. marinum were, respectively, only 7% and 4% of those in T. aestivum; and in the amphiploid the Na(+) and Cl(-) concentrations were 39% and 36% of those in T. aestivum. Glycinebetaine and proline concentrations in the youngest fully-expanded leaf blade of plants exposed to 200 mM NaCl were highest in H. marinum (128 and 60 micromol g(-1) dry mass, respectively), lowest in T. aestivum (85 and 37 micromol g(-1) dry mass), and intermediate in the amphiploid (108 and 54 micromol g(-1) dry mass). Thus, salt tolerance of H. marinum was expressed in the H. marinum-T. aestivum amphiploid.     

Ionoregulatory specializations for exceptional tolerance of ion-poor, acidic waters in the neon tetra (Paracheirodon innesi)

To better understand how fish are able to inhabit dilute waters of low pH, we examined ionoregulation in exceptionally acid-tolerant neon tetras (Paracheirodon innesi), which are native to the ion-poor, acidic Rio Negro, Amazon. Overall ion balance was unaffected by 2-wk exposure to pH 4.0 and 3.5. Measurements of unidirectional Na+ fluxes during exposure to pH 3.5 showed that tetras experienced only a mild, ionic disturbance of short duration (相似文献   

Effect of divalent cations on ion fluxes and leaf photochemistry in salinized barley leaves

Photosynthetic characteristics, leaf ionic content, and net fluxes of Na(+), K(+), and Cl(-) were studied in barley (Hordeum vulgare L) plants grown hydroponically at various Na/Ca ratios. Five weeks of moderate (50 mM) or high (100 mM) NaCl stress caused a significant decline in chlorophyll content, chlorophyll fluorescence characteristics, and stomatal conductance (g(s)) in plant leaves grown at low calcium level. Supplemental Ca(2+) enabled normal photochemical efficiency of PSII (F(v)/F(m) around 0.83), restored chlorophyll content to 80-90% of control, but had a much smaller (50% of control) effect on g(s). In experiments on excised leaves, not only Ca(2+), but also other divalent cations (in particular, Ba(2+) and Mg(2+)), significantly ameliorated the otherwise toxic effect of NaCl on leaf photochemistry, thus attributing potential targets for such amelioration to leaf tissues. To study the underlying ionic mechanisms of this process, the MIFE technique was used to measure the kinetics of net Na(+), K(+), and Cl(-) fluxes from salinized barley leaf mesophyll in response to physiological concentrations of Ca(2+), Ba(2+), Mg(2+), and Zn(2+). Addition of 20 mM Na(+) as NaCl or Na(2)SO(4) to the bath caused significant uptake of Na(+) and efflux of K(+). These effects were reversed by adding 1 mM divalent cations to the bath solution, with the relative efficiency Ba(2+)>Zn(2+)=Ca(2+)>Mg(2+). Effect of divalent cations on Na(+) efflux was transient, while their application caused a prolonged shift towards K(+) uptake. This suggests that, in addition to their known ability to block non-selective cation channels (NSCC) responsible for Na(+) entry, divalent cations also control the activity or gating properties of K(+) transporters at the mesophyll cell plasma membrane, thereby assisting in maintaining the high K/Na ratio required for optimal leaf photosynthesis.     

Insecticidal, repellent and oviposition-deterrent activity of selected essential oils against Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus

Essential oils extracted from 10 medicinal plants were evaluated for larvicidal, adulticidal, ovicidal, oviposition-deterrent and repellent activities towards three mosquito species; Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus. The essential oils of Juniperus macropoda and Pimpinella anisum were highly effective as both larvicidal and ovicidal. The essential oil of P. anisum showed toxicity against 4th instar larvae of A. stephensi and A. aegypti with equivalent LD95 values of 115.7 microg/ml, whereas it was 149.7 microg/ml against C. quinquefasciatus larvae. Essential oils of Zingiber officinale and Rosmarinus officinalis were found to be ovicidal and repellent, respectively towards the three mosquito species. The essential oil of Cinnamomum zeylanicum resulted into highest repellent (RD95) values of 49.6, 53.9 and 44.2 mg/mat against A. stephensi, A. aegypti and C. quinquefasciatus, respectively apart from oviposition-deterrent potential.     

Regulation of paracellular ion conductances by NaCl gradients in renal epithelial cells

In the present study, we clarified how the NaCl gradient across the epithelial cells regulates the paracellular ion conductance. Under isotonic conditions, the absorption-directed NaCl gradient elevated the paracellular conductances of Na(+) (G(Na)) and Cl(-) (G(Cl)), while the secretion-directed NaCl gradient diminished the G(Na) and G(Cl). We further investigated the paracellular ionic conductances of NMDG (G(NMDG)) and gluconate (G(gluconate)) by replacing Na(+) with NMDG or Cl(-) with gluconate. The G(NMDG) was lower than the G(Na) and the replacement of Na(+) with NMDG decreased the G(Cl). The G(gluconate) was lower than the G(Cl) and the replacement of Cl(-) with gluconate also decreased the G(Na). These observations suggest the interaction of cations and anions on paracellular ionic conductances; i.e., cations affect paracellular anion conductances and anions affect paracellular cation conductances.     

Co-expression of Bacillus thuringiensis Cry4Ba and Cyt2Aa2 in Escherichia coli revealed high synergism against Aedes aegypti and Culex quinquefasciatus larvae

Cry4Ba is a delta-endotoxin produced by Bacillus thuringiensis subsp. israelensis and Cyt2Aa2 is a cytolytic delta-endotoxin produced by B. thuringiensis subsp. darmstadiensis. Cry4Ba produced in Escherichia coli was toxic to Aedes aegypti larvae (LC(50)=140 ng ml(-1)) but virtually inactive to Culex quinquefasciatus larvae. Cyt2Aa2 expressed in E. coli exhibited moderate activity against A. aegypti and C. quinquefasciatus larvae with LC(50) values of 350 and 250 ng ml(-1), respectively. Co-expression of both toxins in E. coli dramatically increased toxicity to both A. aegypti andC. quinquefasciatus larvae (LC(50)=7 and 20 ng ml(-1), respectively). This is the first report to demonstrate that Cry4Ba and Cyt2Aa2 have high synergistic activity against C. quinquefasciatus larvae.     

Comparative study of hemocytes and associated cells of some medically important dipterans

The aim of this work is to study, characterize, and compare different morphological types of hemocytes of Glossina austeni, G. morsitans, Calliphora erythrocephala, Stomoxys calcitrans, Lucilia sericata, Aedes aegypti, and Culex quinquefasciatus. This information is intended to provide a basis for future studies of the cellular defense mechanisms of these dipterans. Seven morphological types of hemocytes were identified by phase-contrast optics: prohemocytes, plasmatocytes, thrombocytoids, granulocytes, adipohemocytes, oenocytoids, and spindle cells of various sizes. Adipohemocytes are difficult to distinguish from both fat body cells and granulocytes. All seven cell types are not present in every species. For example, thrombocytoids and spindle cells were not found in A. aegypti or C. quinquefasciatus, and oenocytoids were observed only in A. aegypti, C. quinquefasciatus, and in C. erythrocephala. In addition to the hemocytes, fat body cells and nephrocytes are also freely present in the hemolymph of some species but may have gained access to the blood during the bleeding process. With electron microscopy and with thick plastic sections of G. austeni hemolymph, only nephrocytes, plasmatocytes, granulocytes, and spindle cells were identified with any certainty, and the large spindle cells are morphologically very different from those found in the other dipterans. They are rigid cells supported by microtubules running throughout their entire length. These hemocytes are present in large numbers only in newly emerged flies, they are absent in larvae and young pupae, and are rare in old adults. Their disappearance from the hemolymph of newly emerged Glossina appears to be a result of phagocytosis by the plasmatocytes.     

Rubidium uptake is increased in shore crabs, Carcinus maenas, acclimated to dilute seawater

In dilute seawater, Carcinus maenas hyperosmoregulates by actively absorbing Na, K, and Cl. Here we characterize K uptake using a novel technique. Rb was used as a tracer for K transport, and hemolymph Rb levels were measured using cation chromatography. Hemolymph Rb was detectable at 0.1 mmol L(-1), which enabled determination of initial rate of Rb uptake. Crabs maintained for 3 wk in dilute artificial seawater (35% ASW crabs) maintained Na and K above the level of the external media and had elevated Na-K-ATPase activity in the posterior gills. In assay conditions matched to 100% ASW, Rb uptake was the same in 35% ASW crabs (0.45+/-0.04 micromol g(-1) h(-1)) and in crabs acclimated to normal seawater (100% ASW crabs, 0.49+/-0.05 micromol g(-1) h(-1)). In assay conditions matched to 35% ASW, Rb uptake was greater in 35% ASW crabs (0.28+/-0.03 micromol g(-1) h(-1)) compared with 100% ASW crabs (0.10+/-0.04 micromol g(-1) h(-1)). Low external [Rb] or reduced salinity were found to contribute independently to the difference between 100% ASW and 35% ASW crabs. Thus, whole-body Rb uptake in crabs can be measured by cation chromatography, and Rb uptake is greater in 35% ASW crabs than in 100% ASW crabs.     

Water chloride provides partial protection during chronic exposure to waterborne silver in rainbow trout (Oncorhynchus mykiss) embryos and larvae

Rainbow trout embryos and larvae were continuously exposed (at 12.5 degrees C) to waterborne silver in a flow-through setup, from fertilization to swim-up, at nominal silver concentrations of 0, 0.1, or 1.0 microg/L total silver (as AgNO(3)) at three different water Cl(-) levels (30, 300, and 3,000 microM, added as KCl). Exposures were conducted in synthetic soft water (hardness 20 mg CaCO(3)/L generated from reconstituted reverse osmosis freshwater). Continuous exposure to 1.0 microg/L total silver for 58 d at 30 microM water Cl(-) resulted in a pronounced ionoregulatory disturbance (as indicated by a reduction in whole body Na(+),K(+)-ATPase activity, unidirectional Na(+) uptake [Jin Na(+)], and whole body Na(+) and Cl(-) levels) and a reduction in extractable protein and wet weight. Thus, the mechanism of chronic silver toxicity appears to be similar to that observed during acute silver exposure in juvenile and adult fish, specifically an ionoregulatory disturbance. Higher water Cl(-) levels (300 and 3,000 microM Cl(-)) offered some degree of protection from the ionoregulatory disturbance, with only minor protective effects in terms of mortality. The protective effects of water Cl(-) on the toxicity of silver (as AgNO(3)) appear to be far less during chronic than during acute exposure. Mortality and larval Na(+) concentration, Jin Na(+), and Na(+),K(+)-ATPase activity all appear to be correlated with silver body burden and calculated water Ag(+) during chronic silver exposure. Thus, there appears to be potential to model chronic toxicity but not simply by recalibration of an acute model. A chronic model must be based on real chronic data because the protective effects of various ligands appear to be quantitatively very different from those in the acute situation.     

Na+ versus Cl- transport in the intact killifish after rapid salinity transfer

Much of the early research elucidating the general mechanisms of euryhalinity was performed on the common killifish. More recently, its opercular epithelium with abundant mitochondria-rich cells has proven to be a powerful model for analyzing the mechanisms of active NaCl transport under Ussing conditions in vitro (i.e., with isotonic saline on both surfaces, at short-circuit). However, it is unclear whether this preparation duplicates the gill under real world conditions-i.e., at open-circuit, with real seawater (SW) or freshwater (FW) on the mucosal surface. There have been only limited studies, mostly about 35 years ago, on ion transport in the intact killifish. Therefore, using radioisotopes (22Na, 36Cl), we developed and evaluated methods for the independent measurement of unidirectional Na(+) and Cl(-) influx and efflux rates and internal pools in intact killifish acclimated to 10% SW and abruptly transferred to either 100% SW or FW. Internal Na(+) pools were disturbed less than internal Cl(-) pools by transfer, and were corrected after 3 days in 100% SW or 7 days in FW. Influx and efflux rates in 10% SW were about 3000 micromol kg(-1) h(-1) and increased to 15,000-18,000 micromol kg(-1) h(-1) after transfer to 100% SW, remaining approximately equal and equimolar for Na(+) and Cl(-), and stable from 0.5 to 7 days post-transfer. After transfer to FW, Na(+) influx and efflux rates dropped to 1000-1500 micromol kg(-1) h(-1), with efflux slightly exceeding influx, and remained approximately stable from 0.5 to 7 days. However, while Cl(-) efflux responded similarly, Cl(-) influx rate dropped immediately to negligible values (20-50 micromol kg(-1) h(-1)) without recovery through 7 days. These results differ from early ion transport data in 100% SW, and demonstrate that fluxes stabilize quickly after salinity transfer. They also show that the intact animal responds more quickly than the epithelium, provide qualitative but not quantitative support for the opercular epithelium as a model for the gill under real world SW conditions, and no support for its use as a gill model under real world FW conditions, where branchial Cl(-) uptake is negligible.     

Non-catalytic role of carbonic anhydrase in rat intestinal absorption

Carbonic anhydrase (CA) inhibition reduces NaCl absorption in rat distal ileum, a pH-sensitive, low CA activity tissue, and in distal colon, a CO(2)-sensitive, high CA activity tissue. We hypothesized that CA plays a non-catalytic role in NaCl absorption in these segments. Unidirectional fluxes of Na(+) and Cl(-), and total HCO(3)(-) generation (estimated as the sum of radiolabeled HCO(3)(-) and CO(2) produced from glucose) were measured in Ussing chambers in nominally CO(2), HCO(3)(-)-free HEPES Ringer. Measurements were made in the presence and absence of 0.1 mM methazolamide, a membrane-permeant CA inhibitor. Ringer pH reduction from 7.6 to 7.1 stimulated ileal but not colonic Na(+) and Cl(-) absorption. In the ileum, methazolamide reduced J(ms)(Na) and J(ms)(Cl) and caused net Cl(-) secretion at pH 7.6, and prevented the stimulatory effect of lowering pH. In the colon, methazolamide reduced Na(+) and Cl(-) absorption at pH 7.6. Total HCO(3)(-) generation was minimal in HEPES at pH 7.6 and 7.1 in both segments, was minimally affected by methazolamide, and did not account for the changes in Cl(-) absorption caused by pH or methazolamide. We conclude that CA plays a role in ileal and colonic NaCl absorption independent of its catalytic function.