Pitts RJ et al. (2014), Antennal-expressed ammonium transporters in the...

XB-ART-49631

PLoS One 2014 Jan 01;910:e111858. doi: 10.1371/journal.pone.0111858.

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Antennal-expressed ammonium transporters in the malaria vector mosquito Anopheles gambiae.

Pitts RJ , Derryberry SL , Pulous FE , Zwiebel LJ .

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The principal Afrotropical malaria vector mosquito, Anopheles gambiae remains a significant threat to human health. In this anthropophagic species, females detect and respond to a range of human-derived volatile kairomones such as ammonia, lactic acid, and other carboxylic acids in their quest for blood meals. While the molecular underpinnings of mosquito olfaction and host seeking are becoming better understood, many questions remain unanswered. In this study, we have identified and characterized two candidate ammonium transporter genes, AgAmt and AgRh50 that are expressed in the mosquito antenna and may contribute to physiological and behavioral responses to ammonia, which is an important host kairomone for vector mosquitoes. AgAmt transcripts are highly enhanced in female antennae while a splice variant of AgRh50 appears to be antennal-specific. Functional expression of AgAmt in Xenopus laevis oocytes facilitates inward currents in response to both ammonium and methylammonium, while AgRh50 is able to partially complement a yeast ammonium transporter mutant strain, validating their conserved roles as ammonium transporters. We present evidence to suggest that both AgAmt and AgRh50 are in vivo ammonium transporters that are important for ammonia sensitivity in An. gambiae antennae, either by clearing ammonia from the sensillar lymph or by facilitating sensory neuron responses to environmental exposure. Accordingly, AgAmt and AgRh50 represent new and potentially important targets for the development of novel vector control strategies.

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Species referenced: Xenopus laevis
Genes referenced: pnma2

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	Figure 2. Amino acid plots of AgAmt (A) and AgRh50 (B) depicting the 11 transmembrane domains.AgRh50a and AgRh50b differ only in their C-terminal regions.
	Figure 3. Phylogenetic tree of ammonium transporter families.Neighbor-joining tree comparing relationships among ammonium transporter proteins from prokaryotes and eukaryotes. Rhesus (blue shaded region) and Amt (green shaded region) proteins segregate into distinct families, with insects having representatives in both groups. Bootstrap support for branches is indicated. Scale bar is 1% corrected distance. Sequences and species names are found in Table S1.
	Figure 4. An. gambiae ammonium transporter expression in adult tissues.Left panels: RT-PCR amplification of AgAmt, AgRh50a, AgRh50b using cDNAs derived from adult female bodies, heads, antennae, and maxillary palps. +/− indicates presence or absence of reverse transcriptase in cDNA synthesis reactions. 100 bp marker shown for each gel image. Amplicons are of the expected sizes for each transcript: AgAmt (266 bp), AgRh50a (223 bp), AgRh50b (213 bp). Right panels: qRT-PCR amplification of AgAmt, AgRh50a, and AgRh50b relative to body (x:body) expression using AgLap as a normalizing transcript in each of 3 biological replicates (Samples 1–3). AgAmt (black bars) and AgRh50b (white bars) transcripts are dramatically enhanced in antennae (a), while AgRh50a (gray bars) transcript is enhanced in heads (h), minus antennae (a) and palps (p).
	Figure 5. Function of AgAmt in Xenopus oocytes.Representative traces showing an AgAmt cRNA injected oocyte and water injected oocyte to concentrations of ammonium chloride. Black bars indicate stimulus length. Histogram showing normalized magnitude of inward current measured at steady state for AgAmt injected oocytes (green) and water injected oocytes (blue) to concentrations of ammonium chloride. Currents were normalized with respect to the average steady state current that resulted from AgAmt oocytes when stimulated with 200 µM ammonium chloride. Higher Concentrations: Representative traces showing an AgAmt cRNA injected oocyte and water injected oocyte to concentrations of ammonium chloride. Black bars indicate stimulus length. Histogram showing normalized magnitude of inward current measured at steady state for AgAmt injected oocytes (green) and water injected oocytes (blue) to concentrations of ammonium chloride. Currents were normalized with respect to the average steady state current that resulted from AgAmt oocytes when stimulated with 500 mM ammonium chloride. Different Compounds: Representative traces showing an AgAmt cRNA injected oocyte and water injected oocyte to different chloride salts at 200 µM. Black bars indicate stimulus length. Histogram showing normalized magnitude of inward current measured at steady state for AgAmt injected oocytes (green) and water injected oocytes (blue) to 200 µM chloride salt. Currents were normalized with respect to the average steady state current that resulted from AgAmt oocytes when stimulated with 200 µM ammonium chloride.
	Figure 6. I–V Plot of whole cell conductances in oocytes expressing AgAmt.Current-voltage relationship for AgAmt cRNA injected oocytes to ammonium chloride (orange) and methylammonium chloride (purple). The x-axis shows voltage measured in millivolts and the y-axis shows current measured in microamps.
	Figure 7. AgRh50 Complements a Yeast Ammonium Transporter Mutant.Representative growth curve of wild type yeast and mep1-3Δ triple mutant transformants in minimal medium supplemented with 1 mM Arginine (A) or 1 mM ammonium sulfate (B) as the sole nitrogen source. (C) Histogram plot of mean optical densities for yeast transformants grown in minimal medium supplemented with 1 mM Arginine (white bards) or 1 mM ammonium sulfate (black bars). Error bars are SEM; n = 4. (D) Growth of yeast transformants spotted onto solid minimal medium with indicated supplements as above. Circles are 10 ul spots of ∼1000–2000 cells each, showing confluent growth on 1 mM Arginine (top panels) and reduced growth on 1 mM ammonium sulfate (bottom panels). Mean colony area (mm2) indicated in lower corner of bottom panes.
	Figure 1. An. gambiae ammonium transporter gene structures.Schematic representation of the exon (boxes) and intron (lines) structure of AgAmt and AgRh50 genomic loci. Alternative splicing at 3′ end of AgRh50 indicated by white boxes (AgRh50a) and black box and shaded region of final exon (AgRh50b) and TAG and TGA stop codons, respectively. Scale bar is for exons only; introns lengths in base pairs are indicated above or below splice sites.

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Abuin, Functional architecture of olfactory ionotropic glutamate receptors. 2011, Pubmed