Fig. 3. Immunohistochemical detection of Na⁺/K⁺-ATPase (red) and CA9 (green) in An. albimanus larvae reared in freshwater and transferred to 25% ASW during the 2nd-instar stage for 24 h (A), during the 3rd-instar stage for 24 h (B) and during the 4th-instar stage for 24 h (C) and 48 h (D). Also shown are larvae reared in 25% ASW and transferred to freshwater during the 2nd-instar stage for 24 h (E), during the 3rd-instar stage for 72 h (F) and during the 4th-instar stage for 48 h (G). Distribution of Na⁺/K⁺-ATPase in each sample is indicted as a ratio of Na⁺/K⁺-ATPase peak pixel intensity in the DAR cells vs the non-DAR cells (H). Lowercase bar labels in H correspond to the uppercase letters in Fig. 3 panels (i.e. Fig. 3A corresponds to Fig. 3H bar `a'). Localization of CA9 is indicative of DAR cells. Na⁺/K⁺-ATPase and CA9 co-localization is indicated in yellow. In each set of images, (i) shows both Na⁺/K⁺-ATPase and CA9 localization whereas (ii) is the same picture showing only Na⁺/K⁺-ATPase localization. If reared in freshwater and exposed to 25% ASW during the 2nd- (A,H bar a) or 3rd- (B,H bar b) instar stages, a change in Na⁺/K⁺-ATPase localization from the non-DAR cells to DAR cells was evident within 24 h. Fourth-instar larvae exposed only for 24 h expressed Na⁺/K⁺-ATPase in both DAR and non-DAR cells, as if in an intermediate stage (C,H bar c). However, a change in Na⁺/K⁺-ATPase localization from the non-DAR cells to DAR cells was evident after 48 h (D,H bar d). When larvae were reared in 25% ASW and transferred to freshwater slightly different results were found. Whereas 2nd-instar larvae shifted Na⁺/K⁺-ATPase localization from DAR to non-DAR cells within 24 h (E,H bar e), 3rd- (F,H bar f) and 4th- (G,H bar g) instar larvae did not fully shift Na⁺/K⁺-ATPase localization after 72 h or 48 h, respectively, and expressed the protein in both DAR and non-DAR cells. ASW, artificial seawater; L, lumen. Scale bars: A,B,C,E, 75 µm; D, G, 150 µm; and F, 65.17 µm. Error bars indicate means ± s.e.m.