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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.
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