Larval anopheline mosquito recta exhibit a dramatic change in localization patterns of ion transport proteins in response to shifting salinity: a comparison between anopheline and culicine larvae

doi:10.1242/jeb.019299

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First published online September 19, 2008
Journal of Experimental Biology 211, 3067-3076 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.019299

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Larval anopheline mosquito recta exhibit a dramatic change in localization patterns of ion transport proteins in response to shifting salinity: a comparison between anopheline and culicine larvae

Kristin E. Smith, Leslie A. VanEkeris, Bernard A. Okech, William R. Harvey and Paul J. Linser^*

The Whitney Laboratory for Marine Bioscience, University of Florida, 9505 Ocean Shore Boulevard, St Augustine, FL 32080, USA

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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.

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Fig. 2. Immunohistochemical detection of carbonic anhydrase (CA9) (blue) protein localization in longitudinal sections of the recta of freshwater-reared Ae. aegypti (A), An. gambiae (B), Oc. taeniorhynchus (C) and An. albimanus (D). Na⁺/K⁺-ATPase (red) was used as a counterstain. CA localization did not change in larvae reared in saline water; therefore, only images of freshwater-reared larvae are shown. CA localized to the dorsal anterior rectum (DAR) cells in An. gambiae and An. albimanus and to the anterior rectum (AR) of Oc. taeniorhynchus. CA was not detected in Ae. aegypti rectum. Arrowheads indicate the junction between DAR and non-DAR cells. Arrows indicate junction between anterior and posterior recta. L, lumen; PR, posterior rectum. Scale bars: A, 150 µm; B, 75 µm; C, 149.36 µm; and D, 99.32 µm.

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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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Fig. 4. Comparison of the rectal structure of the freshwater culicine Ae. aegypti (A), the saline-tolerant culicine Oc. taeniorhynchus (B), and the freshwater anopheline, An. gambiae (C) using confocal microscopy of whole mount immunohistochemical preparations. Similar data have previously been reported (Patrick and Gill, 2003

; Smith et al., 2007

; Okech et al., 2008

). Protein localizations are shown for visual distinction of rectal segments in each species. Freshwater culicine larvae possess a structurally uniform rectum as shown by Na⁺/K⁺-ATPase (red) localization (A) whereas saline-tolerant culicine larvae possess a rectum structurally divided into distinct anterior (AR) and posterior (PR) regions as shown by the localization of Na⁺/K⁺-ATPase (red) to the AR and V-ATPase (green) to the PR (B). By contrast, both freshwater and saline-tolerant anopheline larvae possess a unique type of segmented rectum, which differs from culicines and consists of a patch of cells on the dorsal anterior region of the rectum (DAR cells), which are distinguishable from the rest of the rectum (non-DAR cells) as shown by the localization of Na⁺/K⁺-ATPase (red) to the non-DAR cells and CA9 (blue) to the DAR cells (C). AR, anterior rectum; DAR, dorsal anterior rectum; PR, posterior rectum. Scale bars: A,C, 150 µm; and B, 186 µm.

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Fig. 5. A comparison of the major protein localization patterns found in the recta of freshwater-reared culicines and anophelines with those in saline water-reared culicines and anophelines. This figure allows an easy comparison of the proteins in the anopheline and culicine recta. In all cases CA9 localizes to the cytoplasm, Na⁺K⁺-ATPase (NaK) localizes to the basal membrane and V-ATPase localizes to the apical membrane. The non-DAR (dorsal anterior rectum) cells of freshwater-reared anophelines resembled the recta of freshwater culicines in protein localization as both expressed Na⁺/K⁺-ATPase and V-ATPase. When reared in saline water, the anopheline rectum shifted Na⁺/K⁺-ATPase localization and resembled the saline-water culicine rectum in protein localization; anopheline DAR cells and culicine AR possessed CA9 and Na⁺/K⁺-ATPase whereas anopheline non-DAR cells and culicine PR possessed V-ATPase.

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