Carboxylic acids that drive mosquito attraction to humans activate ionotropic receptors.

The mosquito, Aedes aegypti, is highly anthropophilic and transmits debilitating arboviruses within human populations and between humans and non-human primates. Female mosquitoes are attracted to sources of blood by responding to odor plumes that are emitted by their preferred hosts. Acidic volatile compounds, including carboxylic acids, represent particularly salient odors driving this attraction. Importantly, carboxylic acids are major constituents of human sweat and volatiles generated by skin microbes. As such, they are likely to impact human host preference, a dominant factor in disease transmission cycles. A more complete understanding of mosquito host attraction will necessitate the elucidation of molecular mechanisms of volatile odor detection that function in peripheral sensory neurons. Recent studies have shown that members of the variant ionotropic glutamate receptor gene family are necessary for physiological and behavioral responses to acidic volatiles in Aedes. In this study, we have identified a subfamily of variant ionotropic receptors that share sequence homology across several important vector species and are likely to be activated by carboxylic acids. Moreover, we demonstrate that selected members of this subfamily are activated by short-chain carboxylic acids in a heterologous cell expression system. Our results are consistent with the hypothesis that members of this receptor class underlie acidic volatile sensitivity in vector mosquitoes and provide a frame of reference for future development of novel mosquito attractant and repellent technologies.

Quantifying Mosquito Attraction Behavior Using Olfactometry.

When blood feeding from human hosts, female mosquitoes can transmit life-threatening pathogens to humans, including dengue virus, chikungunya virus, and Zika virus. Olfaction is the primary sense mosquitoes use to locate and differentiate hosts and studying it can lead to new strategies to reduce the risk of disease. To effectively study host-seeking behavior in mosquitoes, a repeatable, quantitative assay that isolates olfaction from other cues is critical for interpreting mosquito behavior. Here, we contribute an overview of methods and best practices for the study of mosquito attraction (or lack thereof) by using olfactometry to quantify behavior. In the accompanying protocols, we present an olfactory-based behavioral assay using a uniport olfactometer that measures mosquito attraction rate to specific stimuli. We include construction details, setup of the uniport olfactometer, details of the behavioral assay, and data analysis guidelines, as well as how to prepare the mosquitoes before their introduction into the olfactometer. This uniport olfactometer behavioral assay is currently one of the most reliable methods to study mosquito attraction to a single olfactory stimulus.

Building a Uniport Olfactometer to Assess Mosquito Responses to Odors.

The uniport olfactometer behavioral assay is currently one of the most reliable single-choice methods to use to study mosquito attraction to olfactory stimuli. It allows for the reproducible calculation of mosquito attraction rate to human hosts or to other olfactory stimuli. Here, we present the design of our modified uniport olfactometer. Consistent carbon-filtered air flows through the assay, creating positive pressure that reduces odor contamination from the room. It includes a precision-milled white acrylic base to facilitate easy setup and consistent placement of the component parts. Our design can be made by a commercial acrylic fabricator or an academic machine shop. This olfactometer is designed to assess the responses of mosquitoes but could be applied to other insects that fly upwind toward an odor stimulus. In a companion protocol, we detail how to perform the experiments with mosquitoes by using the uniport olfactometer.

Quantifying Mosquito Attraction Using a Uniport Olfactometer.

Female mosquitoes respond to the world around them by using chemosensory organs, such as their antennae, to detect volatile compounds emitted from a vertebrate host. These chemosensory systems facilitate the interpretation of external stimuli from the periphery by connecting to the central nervous system and eliciting behaviors necessary for survival, such as obtaining a blood meal. This innate behavior leads to the transmission of pathogens, including dengue virus, chikungunya virus, and Zika virus. Olfaction is a primary sense mosquitoes use to differentiate between vertebrate hosts, and studying it can lead to novel strategies to reduce the risk of disease. In this protocol, we present an olfactory-driven behavioral assay using a uniport olfactometer that measures mosquito attraction rate to a specific stimulus. We include details of the behavioral assay and data analysis as well as how to prepare the mosquitoes before their introduction into the olfactometer. This uniport olfactometer behavioral assay is currently one of the most reliable methods to study mosquito attraction to a single stimulus.

Aedes aegypti Mosquitoes Detect Acidic Volatiles Found in Human Odor Using the IR8a Pathway.

Mosquitoes use olfaction as a primary means of detecting their hosts. Previously, the functional ablation of a family of Aedes aegypti olfactory receptors, the odorant receptors (ORs), was not sufficient to reduce host seeking in the presence of carbon dioxide (CO2). This suggests the olfactory receptors that remain, such as the ionotropic receptors (IRs), could play a significant role in host detection. To test this, we disrupted the Ir8a co-receptor in Ae. aegypti using CRISPR/Cas9. We found that Ir8a mutant female mosquitoes are not attracted to lactic acid, a behaviorally active component of human sweat, and they lack odor-evoked responses to acidic volatiles. The loss of Ir8a reduces mosquito attraction to humans and their odor. We show that the CO2-detection pathway is necessary but not sufficient for IR8a to detect human odor. Our study reveals that the IR8a pathway is crucial for an anthropophilic vector mosquito to effectively seek hosts. VIDEO ABSTRACT.