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

Pitts RJ, Derryberry SL, Pulous FE, Zwiebel LJ - PLoS ONE (2014)

Bottom Line: While the molecular underpinnings of mosquito olfaction and host seeking are becoming better understood, many questions remain unanswered.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.Accordingly, AgAmt and AgRh50 represent new and potentially important targets for the development of novel vector control strategies.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee, United States of America; Vanderbilt Institute for Global Health, Nashville, Tennessee, United States of America.

ABSTRACT
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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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).
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pone-0111858-g004: 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).

Mentions: RNA sequencing and RT-PCR confirmed the presence of AgAmt and AgRh50 transcripts in several adult tissues including antennae, maxillary palps, heads and whole bodies of An. gambiae (Table 1, Figure 4). Strikingly, transcripts for AgAmt and the AgRh50b splice variant were highly enriched in antennae compared with other tissues, while the AgRh50a transcript was much more evenly distributed in abundance (Table 1, Figure 4). Quantitative PCRs (qPCRs) confirmed the enhanced antennal specificity of AgAmt and AgRh50b, which were >100 fold overabundant in antennae relative to bodies (Figure 4). This analysis also revealed an enhancement (>4.5–24 fold) of AgRh50a transcripts in heads devoid of chemosensory appendages relative to bodies (Figure 4). While AgAmt and AgRh50b protein levels have yet to be determined in these tissues, enhanced levels of AgAmt and AgRh50 transcripts in antennae are consistent with important roles for ammonium transporters in antennae function.


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

Pitts RJ, Derryberry SL, Pulous FE, Zwiebel LJ - PLoS ONE (2014)

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).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4216128&req=5

pone-0111858-g004: 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).
Mentions: RNA sequencing and RT-PCR confirmed the presence of AgAmt and AgRh50 transcripts in several adult tissues including antennae, maxillary palps, heads and whole bodies of An. gambiae (Table 1, Figure 4). Strikingly, transcripts for AgAmt and the AgRh50b splice variant were highly enriched in antennae compared with other tissues, while the AgRh50a transcript was much more evenly distributed in abundance (Table 1, Figure 4). Quantitative PCRs (qPCRs) confirmed the enhanced antennal specificity of AgAmt and AgRh50b, which were >100 fold overabundant in antennae relative to bodies (Figure 4). This analysis also revealed an enhancement (>4.5–24 fold) of AgRh50a transcripts in heads devoid of chemosensory appendages relative to bodies (Figure 4). While AgAmt and AgRh50b protein levels have yet to be determined in these tissues, enhanced levels of AgAmt and AgRh50 transcripts in antennae are consistent with important roles for ammonium transporters in antennae function.

Bottom Line: While the molecular underpinnings of mosquito olfaction and host seeking are becoming better understood, many questions remain unanswered.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.Accordingly, AgAmt and AgRh50 represent new and potentially important targets for the development of novel vector control strategies.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee, United States of America; Vanderbilt Institute for Global Health, Nashville, Tennessee, United States of America.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus