SUMMARY This study describes the expression patterns of P-type Na+/K+-ATPase and V-type H+-ATPase in the larval and adult forms of the mosquito Aedes aegypti and provides insight into their relative importance in ion transport function of key osmoregulatory organs. RT-PCR assays indicate that, at the level of the gene,both ATPases are expressed in all of the osmoregulatory tissues of larvae(midgut, Malpighian tubules, rectum and anal papillae) and adults (stomach,Malpighian tubules, anterior hindgut and rectum). Immunohistochemical studies determined that both ATPases are present in high levels in all the relevant organs, with the exception of the larval rectum (P-type Na+/K+-ATPase only). In larval gastric caeca, ATPase location corresponds to the secretory (basal P-type Na+/K+-ATPase, apical V-type H+-ATPase) and ion-transporting (V-type H+-ATPase on both membranes) regions as previously described. The two ATPases switch membrane location along the length of the larval midgut, indicating three possible regionalizations,whereas the adult stomach has uniform expression of basolateral P-type Na+/K+-ATPase and apical V-type H+-ATPase in each cell. In both larval and adult Malpighian tubules, the distal principal cells exhibit high expression levels of V-type H+-ATPase (apically and cytoplasmically) whereas P-type Na+/K+-ATPase is highly expressed in stellate cells found only in the distal two-thirds of each tubule. By contrast, the proximal principal cells express both P-type Na+/K+-ATPase (basal) and V-type H+-ATPase(apical). These results suggest a functional segregation along the length of the Malpighian tubules based on cell type and region. P-type Na+/K+-ATPase is the only pump apparent in the larval rectum whereas in the larval anal papillae and the adult hindgut (including the anterior hindgut and rectum with rectal pads), P-type Na+/K+-ATPase and V-type H+-ATPase localize to the basal and apical membranes, respectively. We discuss our findings in light of previous physiological and morphological studies and re-examine our current models of ion transport in these two developmental stages of mosquitoes that cope with disparate osmoregulatory challenges.

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.

The transcripts of three putative ammonia (NH 3 /NH 4 + ) transporters, Rhesus-like glycoproteins AeRh50-1, AeRh50-2 and Amt/Mep-like AeAmt1 were detected in the anal papillae of larval Aedes aegypti. Quantitative PCR studies revealed 12-fold higher transcript levels of AeAmt1 in anal papillae relative to AeRh50-1, and levels of AeRh50-2 were even lower. Immunoblotting revealed AeAmt1 in anal papillae as a pre-protein with putative monomeric and trimeric forms. AeAmt1 was immunolocalized to the basal side of the anal papillae epithelium where it co-localized with Na +

We examined the metabolic and ionoregulatory responses of the Amazonian cichlid, Astronotus ocellatus, to 20 h exposure to severe hypoxia (0.37 +/- 0.19 mg O(2)/l; 4.6% air saturation) or 8 h severe hypoxia followed by 12 h recovery in normoxic water. During 20 h exposure to hypoxia, white muscle [ATP] was maintained at normoxic levels primarily through a 20% decrease in [creatine phosphate] (CrP) and an activation of glycolysis yielding lactate accumulation. Muscle lactate accumulation maintained cytoplasmic redox state ([NAD(+)]/[NADH]) and was associated with an inactivation of the mitochondrial enzyme pyruvate dehydrogenase (PDH). The inactivation of PDH was not associated with significant changes in cytoplasmic allosteric modulators ([ADP(free)], redox state, or [pyruvate]). Hypoxia exposure caused an approximately 65% decrease in gill Na(+)/K(+) ATPase activity, which was not matched by changes in Na(+)/K(+) ATPase alpha-subunit protein abundance indicating post-translational modification of Na(+)/K(+) ATPase was responsible for the decrease in activity. Despite decreases in gill Na(+)/K(+) ATPase activity, plasma [Na(+)] increased, but this increase was possibly due to a significant hemoconcentration and fluid shift out of the extracellular space. Hypoxia caused an increase in Na(+)/K(+) ATPase alpha-subunit mRNA abundance pointing to either reduced mRNA degradation during exposure to hypoxia or enhanced expression of Na(+)/K(+) ATPase alpha-subunit relative to other genes.

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.