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First published online October 19, 2007
Journal of Experimental Biology 210, 3848-3861 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.007872
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Molecular cloning, phylogeny and localization of AgNHA1: the first Na+/H+ antiporter (NHA) from a metazoan, Anopheles gambiae

Mark R. Rheault1,*, Bernard A. Okech1, Stephen B. W. Keen2, Melissa M. Miller1, Ella A. Meleshkevitch3, Paul J. Linser1, Dmitri Y. Boudko3 and William R. Harvey1,{dagger}

1 The Whitney Laboratory for Marine Bioscience, University of Florida, 9505 Ocean Shore Boulevard, St Augustine, FL 32080, USA
2 Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA
3 Rosalind Franklin University of Medicine and Science, North Chicago, IL 60064, USA


Figure 1
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  		Fig. 1. Comparison of insect NHA1 amino acid sequences. (A) Alignment of AgNHA1 with other predicted insect NHA1 amino acid sequences. Predicted transmembrane spanning domains for AgNHA1 are numbered and indicated by a red bar above the alignment. (B) A comparison of the hydropathy plots of the insect NHA1 amino acid sequences (TopPred II parameters were: GES-hydrophobicity scales; upper cut-off=1.0; lower cut-off=0.6; core window size=10; wedge window size=5; Critical loop length=60; and critical transmembrane spacer=2). Sequences were aligned relative to amino acid position (horizontal scale). Transmembrane-spanning regions predicted for AgNHA1 are indicated by the vertical red bars. Ae, Aedes aegypti; Ag, An. gambiae; Am, Apis mellifera; Ds, D. pseudoobscura; Dm, D. melanogaster; Tc, Tribolium castaneum.

 

Figure 2
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  		Fig. 2. Cartoon of the predicted secondary structure of AgNHA1. Color code for amino acids: positively charged, blue; negatively charged, red; non-polar or zwitterionic, white. Putative phosphorylation sites, green, N-glycosylation sites, orange; antigenic sequences used for antibody production, purple.

 

Figure 3
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  		Fig. 3. Phylogenetic analysis of the cation proton antiporter (CPA) superfamily. The phylogenetic tree was constructed using maximum likelihood (ML) analysis and is shown as a ML-distance tree. It was deduced from an alignment of 65 sequences of prokaryotic and eukaryotic CPA genes and is arbitrarily rooted with the E. coli NhaA sequence. ML support values are shown at branch nodes. Background highlighting: CPA1 genes, purple; CPA2 genes, yellow; further CPA2 gene highlighting: bacteria, red; vertebrate, green; nematode, orange; insect, blue. The scale bar indicates the estimated number of amino acid substitutions per site. Abbreviations as in Table 1.

 

Figure 4
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  		Fig. 4. qPCR analyses of AgNHA1 transcript expression in fourth instar larval tissues of An. gambiae. Data are presented as the mean + s.e.m. of three averaged replicates of three independent experiments. An. gambiae 18S ribosomal RNA was used as a reference gene. Results were normalized to values for carcass at one. VNC, ventral nerve cord; SG/cardia, salivary glands and cardia; GC, gastric caeca; AMG, anterior midgut; PMG, posterior midgut; MT, Malpighian tubules.

 

Figure 5
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  		Fig. 5. The transcription patterns of AgNHA1 in tissues of fourth instar larval An. gambiae. AgNHA1 was detected by in situ hybridization using in vitro transcribed DIG-labeled antisense RNA of AgNHA1. Specific labeling appears purple or dark blue in color. The DIG-labeled sense control showed no specific staining (A). Expression of AgNHA1 transcript was observed in the gastric caeca, GC (B,C), posterior midgut (B,D) and the rectum (B,D). AgNHA1 transcript expression was also observed in the various ganglia of the ventral nerve cord (VNC). AgNHA1 was also expressed in the caudal ganglion (E), the abdominal ganglia (F) and the trilobed thoracic a.k.a. subesophageal ganglia (G). Scale bars, 500 µm (A–D); 75 µm (E–G).

 

Figure 6
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  		Fig. 6. Western blot of An. gambiae membrane proteins under reducing conditions. 25 µg of proteins were loaded in all lanes and subsequently probed with pre-immune serum in Lane 1, AgNHA1 antibody in Lane 2 and AgNHA1 antibody plus synthetic peptide (blocked antibody) in Lane 3. No protein was detected in the blot that was probed with pre-immune serum (Lane 1). Labeling of a single band in Lane 2 at the predicted molecular mass of 71 kDa shows that the antibody is specific for AgNHA1. Decreased signal in Lane 3 shows that the antibody is blocked by AgNHA1 synthetic peptide.

 

Figure 7
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  		Fig. 7. Immunolocalization of AgNHA1 (green; FITC) and P-type Na+/K+ ATPase (red; TRITC) counterstained with a nuclear DNA marker (pseudo-blue; DRAQ-5) in isolated tissues of fourth instar An. gambiae larvae. (A) A composite whole mount, showing AgNHA1 expressed in the gastric caeca (GC), anterior midgut (AMG), selected cells of the posterior midgut (PMG), proximal Malpighian tubule (MT) and in a subset of cells in the rectum. (B) AgNHA1 was expressed in two bands of highly immunoreactive cells in the cardia. (C) AgNHA1 was highly expressed in the caudal tip of the gastric caeca. Scale in C is the same as B. (D) In the anterior midgut, AgNHA1 expression appeared to have a vesicular pattern of staining. (E) AgNHA1 stained cells (arrows) in the PMG and intensely stained cells in the proximal portion of the MTs (see arrow). Scale in E is the same as D. (F) AgNHA1 is expressed in the subset of cells in the rectum. AgNHA1 staining did not occur in the dorsal anterior rectal (DAR) cells. Similarly, Na+/K+-ATPase stained much of the rectum but did not stain its DAR cells (G). (H) An overlay of the AgNHA1 and Na+/K+-ATPase expression in the rectum. Colocalization of AgNHA1 and Na+/K+-ATPase in the same cells is indicated by the orange staining pattern. Scale in G,H is the same as F. In addition, AgNHA1 was expressed in the neuropil of the sub-esophageal ganglion (SOG) (I) and the abdominal ganglion (AG) (J). The scale in J is the same as in I.

 

Figure 8
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  		Fig. 8. Immunolocalization of AgNHA1 in mosquito midgut paraffin sections. (A) Vesicular staining pattern in the anterior midgut cells (AMG). In numerous sections the staining pattern appeared to be in line with the nucleus of the cell or just apical of the nuclei. (B) Apical localization of AgNHA1 in the posterior midgut cells. (C,D) Sections of AMG and PMG showing the lack of immunostaining by pre-immunization serum. (E,F) Sections in which the antibody was blocked by the AgNHA1 peptide; there was no specific staining other than background.

 

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© The Company of Biologists Ltd 2007