Limits...
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.

Show MeSH

Related in: MedlinePlus

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.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4216128&req=5

pone-0111858-g005: 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.

Mentions: In order to determine whether AgAmt and AgRh50 can form functional ammonium transporters, we used the Xenopus laevis heterologous expression system and two-electrode voltage clamp electrophysiology (TEVC). Oocytes injected with AgAmt cRNAs consistently evoked inward currents when voltage clamped at −80 mV in response to perfusions of ammonium chloride concentrations ranging from 200 nM to 200 µM (Figure 5A). As a negative control, buffer-injected oocytes were perfused with the same concentrations of ammonium chloride, yet no currents were observed (Figure 5A). A similar characterization of both splice forms of AgRh50 was attempted using the oocyte system; however, currents were not observed when AgRh50a or AgRh50b cRNA-injected oocytes were perfused with ammonium and methylammonium (data not shown). While we can reasonably conclude the observed inward currents from AgAmt-injected oocytes are likely due to the functional presence of AgAmt in the oocyte plasmid membrane, we cannot exclude the possibility that the observed currents are due to the interaction of the exogenously expressed transporter with endogenous proteins (Figure 5A). Previous studies have demonstrated that X. laevis oocytes produce endogenous inward currents in response to concentrations of ammonium at or above 1 mM [48], [49]. AgAmt-injected oocytes showed increasing responses to ammonium chloride at concentrations up to 500 mM that were always much larger in amplitude than endogenous currents recorded from water-injected controls (Figure 5B). These results suggest that the total whole cell current in AgAmt-injected oocytes is the summation of currents produced by the endogenously expressed ammonium transporters and the AgAmt transporter, which is responsible for the larger amplitudes compared to controls (Figure 5B).


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

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

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.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0111858-g005: 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.
Mentions: In order to determine whether AgAmt and AgRh50 can form functional ammonium transporters, we used the Xenopus laevis heterologous expression system and two-electrode voltage clamp electrophysiology (TEVC). Oocytes injected with AgAmt cRNAs consistently evoked inward currents when voltage clamped at −80 mV in response to perfusions of ammonium chloride concentrations ranging from 200 nM to 200 µM (Figure 5A). As a negative control, buffer-injected oocytes were perfused with the same concentrations of ammonium chloride, yet no currents were observed (Figure 5A). A similar characterization of both splice forms of AgRh50 was attempted using the oocyte system; however, currents were not observed when AgRh50a or AgRh50b cRNA-injected oocytes were perfused with ammonium and methylammonium (data not shown). While we can reasonably conclude the observed inward currents from AgAmt-injected oocytes are likely due to the functional presence of AgAmt in the oocyte plasmid membrane, we cannot exclude the possibility that the observed currents are due to the interaction of the exogenously expressed transporter with endogenous proteins (Figure 5A). Previous studies have demonstrated that X. laevis oocytes produce endogenous inward currents in response to concentrations of ammonium at or above 1 mM [48], [49]. AgAmt-injected oocytes showed increasing responses to ammonium chloride at concentrations up to 500 mM that were always much larger in amplitude than endogenous currents recorded from water-injected controls (Figure 5B). These results suggest that the total whole cell current in AgAmt-injected oocytes is the summation of currents produced by the endogenously expressed ammonium transporters and the AgAmt transporter, which is responsible for the larger amplitudes compared to controls (Figure 5B).

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