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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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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.
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pone-0111858-g007: 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.

Mentions: Given the lack of function of AgRh50 transcripts in Xenopus oocytes, we attempted to complement a S. cerevisiae ammonium transporter triple mutant with plasmids expressing the An. gambiae ammonium transporters, AgAmt, AgRh50a and AgRh50b under the control of the galactose promoter. In these studies the mep1-3Δ mutant lacks all 3 endogenous ammonium transporters and accordingly grows very poorly in media where ammonium salts are the only source of free nitrogen (Figure 7). When the An. gambiae ammonium transporters were expressed in the mutant background, partial complementation of the mep1-3Δ mutant was observed for AgRh50a and AgRh50b both in liquid and solid media (Figure 7). Although not restored to wild-type levels, clear improvement in growth of the mutant strain was consistently observed after 4–6 days of growth in liquid culture (Figure 7 B,C) and after 2–4 days on solid plates (Figure 7D). In addition to improved growth based on optical density measurements of liquid cultures, colony size was similarly increased on solid media, being approximately 2–3 times larger for AgRh50b and AhRh50a, respectively (Figure 7D). The average optical density of AgRh50a-expressing strains compared with the triple mutant was significant (p = 0.08) at a reduced constraint P<0.1 in a Mann-Whitney U test (Figure 7C). We did not observed complementation of the mutant phenotype in AgAmt transformants.


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

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

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.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0111858-g007: 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.
Mentions: Given the lack of function of AgRh50 transcripts in Xenopus oocytes, we attempted to complement a S. cerevisiae ammonium transporter triple mutant with plasmids expressing the An. gambiae ammonium transporters, AgAmt, AgRh50a and AgRh50b under the control of the galactose promoter. In these studies the mep1-3Δ mutant lacks all 3 endogenous ammonium transporters and accordingly grows very poorly in media where ammonium salts are the only source of free nitrogen (Figure 7). When the An. gambiae ammonium transporters were expressed in the mutant background, partial complementation of the mep1-3Δ mutant was observed for AgRh50a and AgRh50b both in liquid and solid media (Figure 7). Although not restored to wild-type levels, clear improvement in growth of the mutant strain was consistently observed after 4–6 days of growth in liquid culture (Figure 7 B,C) and after 2–4 days on solid plates (Figure 7D). In addition to improved growth based on optical density measurements of liquid cultures, colony size was similarly increased on solid media, being approximately 2–3 times larger for AgRh50b and AhRh50a, respectively (Figure 7D). The average optical density of AgRh50a-expressing strains compared with the triple mutant was significant (p = 0.08) at a reduced constraint P<0.1 in a Mann-Whitney U test (Figure 7C). We did not observed complementation of the mutant phenotype in AgAmt transformants.

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