Marjorie L. Patrick

P-type Na+/K+-ATPase and V-type H+-ATPase expression patterns in the osmoregulatory organs of larval and adult mosquito Aedes aegypti.

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.

Characterization of ion and acid‐base transport in the fresh water adapted mummichog (Fundulus heteroclitus)

We examined whether ionoregulatory mechanisms of fresh water Fundulus heteroclitus in vivo are similar to those of typical freshwater species (e.g., rainbow trout, goldfish, and catfish). Under control conditions ([NaCl]ext ~1 mmol/l), the mummichog exhibits very large Na+ influx and efflux rates but virtually no Cl– influx and a small Cl– efflux component. External NaCl levels were varied to reveal a saturable, low affinity (Km = 1,723 ± 223 μmol/l), high capacity (Jmax = 2,258 ± 288 nEq/g/h) Na+ uptake system that was independent of both Na+ efflux and ammonia excretion. A measurable Cl– influx did not occur until NaCl levels surpassed 2 mmol/l and did not saturate within the freshwater range, suggesting a completely different uptake mechanism. Cl– efflux was also independent of Cl– influx. A systemic acidosis (intraperitoneal HCl injection) was induced in order to investigate the connection between ionoregulation and acid-base balance. The acidosis did not affect influx rates but induced an elevated Cl– efflux and an attenuated Na+ efflux. This resulted in an excess of net Cl– loss over Na+ loss which effected a net acid excretion by strong ion difference theory. These results concur with the measured acid-base fluxes which indicate that over 50% of the acid load was excreted within 4 h by differential efflux modulation. Therefore an ion/acid-base link does exist in the mummichog but differs in nature from that of other freshwater fish. Indeed, virtually all of these findings differ from the current model for most other teleosts, indicating that alternate models of ionoregulation in fresh water exist. J. Exp. Zool. 279:208–219, 1997.

NaCl transport and ultrastructure of opercular epithelium from a freshwater-adapted euryhaline teleost, Fundulus heteroclitus

We adapted killifish to defined freshwater (FW: 1.0 mM Na, 1.0 mM Cl, + 0.1 mM Ca) and by fluorescence light microscopy and scanning and transmission electron microscopy found that the opercular epithelium retained mitochondria rich (MR) cells that were significantly larger but less numerous than in SW-acclimated tissues. Opercular epithelia mounted in vitro with FW bathing the mucosal surface take up Cl against a large negative inside transepithelial potential (Vt, grand mean –64.1 mV) and concentration gradient; the observed flux ratio was significantly different (P < 0.001) from that predicted for passive ion distribution but the net flux was consistently negative. The Na flux ratio suggested that Na was passively distributed. Vt was largely a Na diffusion potential, based on unilateral manipulations of [Na]. Cl unidirectional uptake was unaffected by mucosally added SITS (0.1 mM) but was inhibited by SCN (1.0 mM) and by anaerobiosis. Killifish transferred from SW to FW for 48 h had reduced Cl secretion by the opercular epithelium compared to SW controls but had not yet developed Cl uptake, indicating a slow adaptive process for development of Cl absorptive transport. Opercular epithelia of FW adapted fish, if bathed with isotonic saline on both sides, has a modest net Na and Cl uptake, unlike SW opercular epithelium that strongly secretes Cl under similar conditions. FW killifish opercular epithelium may provide a model to study ion regulation by euryhaline fish. J. Exp. Zool. 277:23–37, 1997.

Ion and acid–base regulation in the freshwater mummichog (Fundulus heteroclitus): a departure from the standard model for freshwater teleosts

Abstract Ion and acid–base balance were examined in the freshwater-adapted mummichog ( Fundulus heteroclitus ) using a series of treatments designed to perturb the coupling mechanisms. Unidirectional Cl − uptake ( J Cl in ) was extremely low whereas J Na in was substantial (three- to sixfold higher); comparable differences occurred in unidirectional efflux rates ( J Cl out , J Na out ). J Cl in was refractory to all treatments, suggesting that Cl − /base exchange was unimportant or absent. Indeed, no base excretion or modulation of ion fluxes occurred for acid–base balance for up to 8 h after NaHCO 3 − loading (injections of 1000 or 3000 nequiv.·g −1 ). Acute environmental low pH (4.5) and amiloride (10 −4 M) treatments caused concurrent inhibition of J Na in and net H + excretion ( J H+ net ), indicating the presence of Na + /H + exchange. J Na in was elevated and J H+ net restored during recovery from both treatments, but this exchange did not appear to be dynamically adjusted for acid–base homeostasis. High external ammonia exposure (1 mmol·l −1 ) initially blocked ammonia excretion ( J Amm net ) but had no effect on J Na in , whereas high pH (9.4) reduced both J Amm net and J Na in . Inhibition of J Na in by the low pH and amiloride treatments had no effect on J Amm net . These results indicate that ammonia excretion is entirely diffusive and independent of both Na + uptake and the protons that are transported via the Na + /H + coupling. In addition, ureagenesis served as a compensatory mechanism during high external ammonia exposure, as a marked elevation in urea excretion partially replaced the inhibited J Amm net . In all treatments, changes in the Na + –Cl − net flux differential were consistent with changes in J H+ net measured by traditional water titration techniques, indicating that the former can be used as an estimate of the acid–base status of the fish. Overall, the results demonstrate that the freshwater-adapted F. heteroclitus does not conform to the ion/acid–base relationships described in the standard model based on commonly studied species such as trout, goldfish, and catfish.

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 Jmax values, ranging from 1,150 to 1,750 nmol g−1 h−1, while Km values varied to a greater extent. Three species had Km values <33 μmol L−1, while the rest had Km \documentclass{aastex} \usepackage{amsbsy} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{bm} \usepackage{mathrsfs} \usepackage{pifont} \usepackage{stmaryrd} \usepackage{textcomp} \usepackage{portland,xspace} \usepackage{amsmath,amsxtra} \usepackage[OT2,OT1]{fontenc} \newcommand\cyr{ \renewcommand\rmdefault{wncyr} \renewcommand\sfdefault{wncyss} \renewcommand\encodingdefault{OT2} \normalfont \selectfont} \DeclareTextFontCommand{\textcyr}{\cyr} \pagestyle{empty} \DeclareMathSizes{10}{9}{7}{6} \begin{document} \landscape

Effects of Water pH and Calcium Concentration on Ion Balance in Fish of the Rio Negro, Amazon

\mathrm{values}\,\geq 110

Ion and Acid‐Base Balance in Three Species of Amazonian Fish during Gradual Acidification of Extremely Soft Water

\end{document} μmol L−1. Because of the extremely low Na+ concentration of Rio Negro water, the differences in Km 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 μmol L−1) by most species. Unlike other species with high Km values, the catfish Corydoras julii maintained high rates of Na+ uptake in dilute waters by having a Jmax 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, Jmax fell >50%, and Km dropped about 70%. The unusual acclimatory drop in Km 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.

Unusual physiology of scale-less carp, Gymnocypris przewalskii, in Lake Qinghai: a high altitude alkaline saline lake

Stenohaline freshwater stingrays (Potamotrygon spp.) are endemic to the very dilute (Na+, Cl−, \documentclass{aastex} \usepackage{amsbsy} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{bm} \usepackage{mathrsfs} \usepackage{pifont} \usepackage{stmaryrd} \usepackage{textcomp} \usepackage{portland,xspace} \usepackage{amsmath,amsxtra} \usepackage[OT2,OT1]{fontenc} \newcommand\cyr{ \renewcommand\rmdefault{wncyr} \renewcommand\sfdefault{wncyss} \renewcommand\encodingdefault{OT2} \normalfont \selectfont} \DeclareTextFontCommand{\textcyr}{\cyr} \pagestyle{empty} \DeclareMathSizes{10}{9}{7}{6} \begin{document} \landscape