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Summary: Anopheles mosquitoes, as vectors for the malaria parasite, are a global threat to human health. To find and bite a human, they utilize neurons within their sensory appendages. However, the identity and quantification of sensory appendage neurons are lacking. Here we use a neurogenetic approach to label all neurons in Anopheles coluzzii mosquitoes. We utilize the homology assisted CRISPR knock-in (HACK) approach to generate a T2A-QF2w knock-in of the synaptic gene bruchpilot. We use a membrane-targeted GFP reporter to visualize the neurons in the brain and to quantify neurons in all major chemosensory appendages (antenna, maxillary palp, labella, tarsi, and ovipositor). By comparing labeling of brp>GFP and Orco>GFP mosquitoes, we predict the extent of neurons expressing ionotropic receptors (IRs) or other chemosensory receptors. This work introduces a valuable genetic tool for the functional analysis of Anopheles mosquito neurobiology and initiates characterization of the sensory neurons that guide mosquito behavior.

Abstract Background Live imaging of whole invertebrates can be accomplished with X-ray micro-computed tomography (micro-CT) at 10-100 μm spatial resolution. However, image quality could be compromised by the movement of live subjects, producing image artefacts. We tested the feasibility of using CO2 gas to induce temporary full-immobilization of sufficient duration to image live insects based on their ability to tolerate hypoxic conditions. Additionally, we investigated the effects of these prolonged hypoxic conditions on several life history traits of a lepidopteran species. Methods Live Colorado potato beetle (CPB) and true armyworm (TAW) adults were immobilized under a constant CO2 gas flow (0.5 L/min), and scanned using micro-CT (80 kVp; 450 μA). An L8 (24) orthogonal array (OA) was used to evaluate the effects of prolonged CO2-induced anesthesia on the recovery, longevity, and incidence of mating of TAW adults. The variable factors were age (immature and mature), sex (female and male), exposure time (3 and 7 h), and exposure regime (single and repeated). Results With this method, successful 3D reconstruction and visualizations of CPB and TAW adults were produced at 20 micron voxel spacing at an acceptable radiation dose and image noise level. From the inverse-square relationship found between the radiation doses and image noise levels, the optimal scanning protocol produced an entrance dose of 6.2 ± 0.04 Gy with images of 129.6 ± 5.1 HU noise level during a 2.7 h scan. Independent OA experiments indicated that CO2 gas did not result in death of exposed TAW adults, except when older males were exposed for longer durations. Exposure time and sex were more influential factors affecting recovery, longevity, and mating success than age and exposure regime following CO2 exposure. Conclusion We have demonstrated that using CO2 gas during micro-CT imaging effectively induces safe, repeatable, whole-body, and temporary immobilization of live insects for 3D visualizations without motion artefacts. Moreover, we have shown that exposed TAW individuals made a full recovery with very little impact on subsequent longevity, and mating success post hypoxia. This method is applicable to other imaging modalities and could be used for routine exploratory and time-course studies, for repeated scanning of live and intact individuals.