Fig. 1. Immunohistochemical detection of Na⁺/K⁺-ATPase (red) and V-ATPase (green) protein localization in longitudinal sections of the recta of Ae. aegypti (A), An. gambiae (B,C), Oc. taeniorhynchus (E,F,G) and An. albimanus (I,J,K) reared in either freshwater or saline water. Distribution of Na⁺/K⁺-ATPase is indicted as a ratio of Na⁺/K⁺-ATPase peak pixel intensity in the DAR cells (or AR) vs the non-DAR cells (or PR) (D,H,L). Arrowheads demark the junction between DAR and non-DAR cells in anophelines and arrows demark the junction between AR and PR in culicines. The inset images in panels J and K are identical to the corresponding panel but lack V-ATPase staining signal thereby giving a clearer view of Na⁺/K⁺-ATPase localization. Ae. aegypti protein localization did not change between larvae reared in freshwater or saline water: Na⁺/K⁺-ATPase localized to the basal infoldings and V-ATPase to the apical lamellae (A). In freshwater-reared An. gambiae and An. albimanus Na⁺/K⁺-ATPase localized to the basal infoldings of the non-DAR cells and V-ATPase to the apical lamellae of the non-DAR cells and cytoplasm of the DAR cells (B,I,J). The apparent cytoplasmic localization of V-ATPase in An. albimanus is shown in higher magnification (I). When acclimated to 60% ASW, An. gambiae showed Na⁺/K⁺-ATPase on the basal infoldings of both DAR and non-DAR cells, and V-ATPase appeared cytoplasmic in all cells as well as apical in the non-DAR cells (C). The change in Na⁺/K⁺-ATPase distribution can be quantified as a change in Na⁺/K⁺-ATPase peak pixel intensity from being significantly greater in the non-DAR cells (in larvae reared in freshwater) to being approximately the same in the DAR and non-DAR cells (in larvae acclimated to 60% ASW) (D). In saline-reared An. albimanus, Na⁺/K⁺-ATPase exhibited a drastic shift in localization to the DAR cells (J inset vs K inset). This shift can be quantified as a change in Na⁺/K⁺-ATPase peak pixel intensity from being significantly greater in the non-DAR cells (in larvae reared in freshwater) to being significantly greater in the DAR cells (in larvae reared in 50% ASW) (L). V-ATPase remained the same (K). In freshwater reared Oc. taeniorhynchus, Na⁺/K⁺-ATPase localized to the basal infoldings of the AR whereas V-ATPase localized to the apical lamellae of the PR (F). However, in most larvae, a weak V-ATPase signal was evident on the apical lamellae of the AR (E, asterisks). When reared in 100% ASW, protein localization did not change drastically, although AR apical V-ATPase signal was not evident (G). The absence of a change in Na⁺/K⁺-ATPase localization is apparent in (H) as larvae reared in both freshwater and 100% ASW have significantly more Na⁺/K⁺-ATPase pixel intensity in the AR compared with the PR. AR, anterior rectum; ASW, artificial seawater; DAR, dorsal anterior rectum; L, lumen; PR, posterior rectum. Scale bars: A, 150 µm; B,J,J inset, 75 µm; C, 86.13 µm; E,F, 149.36 µm; G,K,K inset, 99.32 µm; and I, 12 µm. Error bars indicate means ± s.e.m.