Isolated anterior midguts of larval Aedes aegypti were bathed in aerated mosquito saline containing serotonin (0.2 micromol L(-1)) and perfused with NaCl (100 mmol L(-1)). The lumen negative transepithelial voltage (V(te)) was measured and luminal alkalinization was determined through the color change of luminal m-cresol purple from yellow to purple after luminal perfusion stops. Addition of 10 mmol L(-1) amino acids (arginine, glutamine, histidine or proline) or dicarboxylic acids (malate or succinate) to the luminal perfusate resulted in more negative V(te) values, whereas addition of glucose was without effect. In the presence of TRIS chloride as luminal perfusate, addition of nutrients did not change V(te). These results are consistent with Na(+)-dependent absorption of amino acids and dicarboxylic acids. Effects of serotonin withdrawal indicated that nutrient absorption is stimulated by this hormone. Strong luminal alkalinization was observed with mosquito saline containing serotonin on the hemolymph-side and 100 mmol L(-1) NaCl in the lumen, indicating that alkalinization does not depend on luminal nutrients. Omission of glucose or dicarboxylic acids from the hemolymph-side solution had no effect on luminal alkalinization, whereas omission of amino acids significantly decelerated it. Re-addition of amino acids restored alkalinization, suggesting the involvement of amino acid metabolism in luminal alkalinization.

Recently, Na + /K + -ATPase has been detected in the luminal membrane of the anterior midgut of larval yellow fever mosquitoes (Aedes aegypti) with immunohistochemical techniques. In this study, the possible involvement of this ATPase in strong alkalinization was investigated on the level of whole larvae, isolated and perfused midgut preparations and on the molecular level of the Na + /K + -ATPase protein. Ouabain (5 mM) did not inhibit the capability of intact larval mosquitoes to alkalinize their anterior midgut. Also in isolated and perfused midgut preparations the perfusion of the lumen with ouabain (5 mM) did not result in a significant change of the transepithelial voltage or the capacity of luminal alkalinization. Na + /K + -ATPase activity was completely abolished when KCl was substituted with choline chloride, suggesting that the enzyme cannot act as an ATP-driven Na + /H + -exchanger. Altogether the results of the present investigation indicate that apical Na + /K + -ATPase is not of direct importance for strong luminal alkalinization in the anterior midgut of larval yellow fever mosquitoes.Strong luminal alkalinization of up to pH values of 12 has been observed in midgut regions of many larvae of endopterygote insects, including members of the orders Coleoptera, Diptera, Trichoptera and Lepidoptera (for references see Clark, '99). Two tissues have been studied especially intensively with regard to their mechanisms and capacities of alkalinization during the past 20 years: the midgut of the tobacco hornworm (Manduca sexta) and the anterior midgut of larval yellow fever mosquitoes (Aedes aegypti). For the lepidopteran larvae, a transport model has been established in which luminal V-type H + -pumps (V-ATPases) energize luminal, electrogenic K + /2H + -exchangers, resulting in active K + secretion and strong luminal alkalinization (for a review see Wieczorek et al., '99). Unfortunately, this mechanism can so far not be tested with the isolated epithelium, because it loses the capacity for strong luminal alkalinization after isolation, whereas active K + secretion continues in vitro at a high rate (Clark et al., '98). Strong luminal alkalinization in the anterior midgut of larval yellow fever mosquitoes appears to significantly differ from lepidopterans, including the methodological aspect that it can readily be observed with isolated and perfused midgut preparations (Onken et al., 2008). In larval A. aegypti, the V-ATPase is not localized in the luminal membrane of the anterior midgut, but instead in the basolateral membrane (Zhuang et al., '99). The epithelium of larval yellow fever mosquitoes generates a lumen negative transepithelial voltage (Clark et

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