The characterization of ion regulation in Amazonian mosquito larvae: evidence of phenotypic plasticity, population-based disparity, and novel mechanisms of ion uptake

Abstract

This study is the first step in characterizing ion uptake mechanisms of mosquito larvae from the Amazon region of Brazil. Hemolymph NaCl levels and rates of unidirectional Na(+) and Cl(-) uptake were measured in larvae of Aedes aegypti and Culex quinquefasciatus in a series of environmental manipulations that are known to challenge ion regulation in other aquatic animals. Despite being reared for numerous generations in dilute media (20 micromol L(-1) NaCl), both species were able to maintain high hemolymph NaCl concentrations, a departure from previous studies. Exposure to distilled water or high-NaCl media did not affect hemolymph ion levels, but pH 3 caused significant decreases in hemolymph Na(+) and Cl(-) levels in both species. Exposure to water from Rio Negro (pH 5.5), an organically rich but ion-poor body of water, did not disturb hemolymph Na(+) and Cl(-) levels or the uptake of these ions. Acute exposure to control media or Rio Negro water titrated to pH 3.5 caused inhibition of Na(+) uptake and stimulation of Cl(-) uptake in C. quinquefasciatus, but A. aegypti larvae experienced only a significant reduction of Na(+) uptake in Rio Negro/pH 3.5 treatment. The stimulation of Cl(-) uptake at low pH has been documented only in aquatic insects and differs from all other invertebrate and vertebrate species. A similar pattern of Na(+) uptake inhibition and Cl(-) uptake stimulation was observed in A. aegypti larvae exposed to bafilomycin A(1), a blocker of V-type H(+) ATPase. Culex quinquefasciatus larvae were unaffected by this drug. Both Na(+) and Cl(-) uptake were reduced when C. quinquefasciatus larvae were exposed to acetazolamide, indicating that H(+) and HCO(3)(-), derived from hydration of CO(2), are involved with Na(+) and Cl(-) uptake. Kinetic analysis of Na(+) and Cl(-) uptake in C. quinquefasciatus, A. aegypti, and Anopheles nuneztovari larvae indicate that these Amazonian species share similar high-capacity and high-affinity mechanisms. Comparison of the Amazonian C. quinquefasciatus with a Californian population provided evidence of both phenotypic plasticity and population disparity in Na(+) and Cl(-) uptake, respectively. When the California population of C. quinquefasciatus was reared in a medium similar to that of the Amazonian group (60 micromol L(-1) NaCl) instead of 4,000 micromol L(-1) NaCl, larvae increased both Na(+) uptake capacity (J(max)) and affinity (i.e., reduced K(m)), yet Cl(-) uptake did not change from its nonsaturating, low-capacity pattern. In the reverse experiment, Amazonian C. quinquefasciatus demonstrated plasticity in both Na(+) and Cl(-) uptake by significantly reducing rates when held in 4,000 micromol L(-1) NaCl for 3 d.