Organ-specific splice variants of aquaporin water channel AgAQP1 in the malaria vector Anopheles gambiae

Abstract

Background: Aquaporin (AQP) water channels are important for water homeostasis in all organisms. Malaria transmission is dependent on Anopheles mosquitoes. Water balance is a major factor influencing mosquito survival, which may indirectly affect pathogen transmission.

Methodology/principal findings: We obtained full-length mRNA sequences for Anopheles gambiae aquaporin 1 (AgAQP1) and identified two splice variants for the gene. In vitro expression analysis showed that both variants transported water and were inhibited by Hg(2+). One splice variant (AgAQP1A) was exclusively expressed in adult female ovaries indicating a function in mosquito reproduction. The other splice variant (AgAQP1B) was expressed in the midgut, malpighian tubules and the head in adult mosquitoes. Immunolabeling showed that in malpighian tubules, AgAQP1 is expressed in principal cells in the proximal portion and in stellate cells in the distal portion. Moreover, AgAQP1 is expressed in Johnston's organ (the "ear"), which is important for courtship behavior.

Conclusions and significance: These results suggest that AgAQP1 may play roles associated with mating (courtship) and reproduction in addition to water homeostasis in this important African malaria vector.

Figure 1. Gene and polypeptide structures of AgAQP1A and B.

A, genetic structures of the splice variants AgAQP1A and AgAQP1B on Chromosome 3R. The structures are identical from 5’ end to the 4th intron. AgAQP1A consists of 5 exons while AgAQP1B consists of 6 exons. Untranslated regions (UTRs) are shown in red. B, amino-acid sequence alignment by ClustalW2 of the two variants. Predicted transmembrane domains are boxed, while NPA motifs are shaded with grey. Divergent residues at C termini are in white letters in black background. Arrowheads indicate conserved R/ar residues (F53, H177 and R192). * indicates identical residue : indicates similar residue.

Figure 2. H₂O permeability of AgAQP1A and B expressed in Xenopus laevis oocytes.

Osmotic water permeability in 70 mOsm hypotonic solution (mean ± SD). Coefficient of osmotic water permeability (P _f in 10^-4 cm/s) is shown in the y-axis. HgCl₂: incubation in 0.5 mM HgCl₂ for 5 min; 2ME: 5 mM 2-mercaptoethanol for 10 min following HgCl₂ treatment. Statistical significance is indicated with asterisks (**: P < 0.01).

Figure 3. Expression profile of AgAQP1A and AgAQP1B by qRT-PCR (mean ± SD).

A, in developmental stages, AgAQP1B is expressed in larval to adult stages with dramatic upregulation in young adults. AgAQP1A is expressed only in older adult females. B, AgAQP1B is expressed throughout the mature adult body, while AgAQP1A is expressed only in female abdomen. C, Ovaries specifically express AgAQP1A. AgAQP1A (D) and AgAQP1B (E) expression in ovaries from unfed and blood-fed mosquitoes. AgAQP1A is highly upregulated in blood-fed ovaries during egg maturation (≤ 48 h post blood feeding: pbf) returning to baseline 72 h pbf, while AgAQP1B is transiently upregulated at 24 h pbf. Note expression of AgAQP1A is much higher than AgAQP1B (see scales on vertical axes).

Figure 4. Whole-mount confocal microscopy of AgAQP1 localization in the larval and adult female alimentary canals.

This figure shows two montages of maximum projection images of dissected alimentary canals from larval (A) and adult (B) Anopheles gambiae. Each panel shows TRITC immunofluorescence (red) for AgAQP1 localization (upper figure of each panel) and a merged image with DAPI staining of nuclear DNA (lower figure in each panel). Anterior is to the right. Prominent staining for AgAQP1 in the larval gut (panel A) is evident in the cells of the Cardia (labeled C), the Gastric Caecae (GC) the midgut Transitional Zone (TZ) the proximal Principal Cells of the Malpighian Tubules (PC) and the Stellate Cells (SC) of the Malpighian Tubules. In panel B, the adult alimentary canal shows areas of prominent staining in the Foregut (FG), Midgut (MG), proximal Principal Cells (PC) and Stellate Cells (SC) of the Malpighian tubules, Hindgut (HG) and the Rectal Pads (RP).

Figure 5. Confocal microscopy of AgAQP1 in the larval gastric caecae and Malpighian tubule area indicating basal localization.

This figure shows a single larval gut at the region of the Gastric Cacae (GC) (A) and midgut-hindgut junction with Malpighian tubules (B). TRITC (red) staining indicates AgAQP1, Cy5 (blue) indicates the basal membrane marker NaK-ATPase and GSL-1 (green) indicates a plant lectin that binds to various extracellular matrices including the caecal membrane which is internal to the caecal cavity. The top row of each panel shows a maximum projection of several z-planes. Note that in the merge panel at the right end, red and blue signals on the lobes of the cacae co-localize, indicating co-localization of AgAQP1 and NaK-ATPase on the basal membranes of the caecal cells. The lower row of panels showing GC are from the same z-stack but present a single z plane. Again, note that AgAQP1 and the NaK-ATPase colocalize. Note the intense labeling for AgAQP1 in the proximal Principal Cells and the Stellate cells of the Malpighian tubules. As in the GC, AgAQP1 labeling in the Malpighian tubules is on the basal surface of both Principal cells. Scale bar 120 µm.

Figure 6. Localization of AgAQP1 in adult and larval mosquitoes by immunofluorescence of sections.

Fluorescence micrograph images using anti-AgAQP1 (green). Blue: DAPI (cell nuclei). A, AgAQP1 is expressed on the basal side of the female midgut, (lumen indicated by red arrowheads). B, preimmune. C, AgAQP1 is expressed in oviduct (Od). Immunofluorescence appears to be in posterior portion of the oviduct (A: anus). In this figure, Malpighian tubule principal cells with large nuclei (yellow triangles) with immunolabeled stellate cells (yellow arrows) either traverse or cross section are included. Hindgut rectum with rectal pads is also seen (HG: hindgut; OV: ovary). D, preimmune. E and F, AgAQP1 is expressed in Johnston’s organ (JO). G, preimmune. Shown are the male JO. Scale bar A, B, F, G: 16 µm; C, D, E: 60 µm.