The recent Zika crisis once again highlighted the cosmopolitan role the yellow fever mosquito Aedes aegypti plays as a prolific vector for emerging and resurgent arboviral diseases. To blood-feed and consequently transmit pathogens, Ae. aegypti and other anthropophilic disease vectors rely on their finely-tuned senses to track a variety of chemical and physical cues including body odor, carbon dioxide, moisture, heat, and visual contrast emanating from humans. Despite their direct role in mediating such epidemiologically important behaviors, the mosquito neurons and receptors that detect human-related sensory stimuli remain largely unknown. These cellular and molecular pathways represent key targets to disrupt mosquito behavior and develop new strategies to control mosquito-borne diseases. Here, we propose to develop a genetic method in Aedes aegypti that will facilitate the identification, isolation, and transcriptional profiling of mosquito sensory neurons activated by human-related cues. To accomplish this goal, we will use the QF2/QUAS system of binary expression to direct neuronal expression of the biosensor CaMPARI, a modified GFP molecule that changes fluorescence from green to red with the coincident presence of high calcium concentrations and violet light. Application of this technique will directly facilitate the identification of mosquito sensory neurons in peripheral sensory appendages (antennae, maxillary palp, proboscis, legs, wings) activated by human-related cues. This activity-dependent biosensor is particularly well suited to identify sensory neurons responding to target stimuli as: 1) neuronal activation of sensory neurons in response to cues includes a large increase in intracellular calcium, and 2) peripheral organs containing sensory neurons are experimentally easy to reach with violet light.
The specific aims are: 1) To develop a genetic method in Aedes aegypti mosquitoes for labeling sensory neurons activated by human-related cues, and 2) To identify candidate molecular receptors from activated mosquito sensory neurons that may detect human-related cues. We will examine appendages for neuronal CaMPARI labeling using a number of sensory stimuli: 1) human-derived olfactory cues (whole human odor, lactic acid, ammonia and CO2), 2) temperatures representing body warmth (37?C), and 3) moisture (relative humidity = 70%). To isolate the activated neurons, we will perform cryosections of appendages containing CaMPARI-labelled neurons, followed by laser capture microdissection. Finally, we will generate transcriptional profiles using RNA-seq analyses to identify candidate molecular receptors that may mediate detection of these target human-related cues in CaMPARI labeled neurons. This study will generate a new genetic tool to dissect mosquito sensory biology, identify populations of neurons in Aedes aegypti activated by key human-derived cues, and generate a candidate list of associated receptors that may be targeted for novel mosquito behavioral disruption strategies.
Hundreds of thousands are adversely affect every year from diseases spread by mosquitoes. In addition, mosquito populations that might transmit diseases continue to encroach on urban populations. Mosquitoes utilize a diverse array of senses to navigate and identify human hosts for biting, and a better understanding of these sensory systems could lead to new effective methods to control mosquito populations.
