Optimized genetic tools for neuroanatomical and functional mapping of the Aedes aegypti olfactory system.

The mosquito Aedes aegypti is an emerging model insect for invertebrate neurobiology. We detail the application of a dual transgenesis marker system that reports the nature of transgene integration with circular donor template for CRISPR-Cas9-mediated homology-directed repair at target mosquito chemoreceptor genes. Employing this approach, we demonstrate the establishment of cell-type-specific T2A-QF2 driver lines for the A. aegypti olfactory co-receptor genes Ir8a and orco via canonical homology-directed repair and the CO2 receptor complex gene Gr1 via noncanonical homology-directed repair involving duplication of the intended T2A-QF2 integration cassette separated by intervening donor plasmid sequence. Using Gr1+ olfactory sensory neurons as an example, we show that introgression of such T2A-QF2 driver and QUAS responder transgenes into a yellow cuticular pigmentation mutant strain facilitates transcuticular calcium imaging of CO2-evoked neural activity on the maxillary palps with enhanced sensitivity relative to wild-type mosquitoes enveloped by dark melanized cuticle. We further apply Cre-loxP excision to derive marker-free T2A-QF2 in-frame fusions to clearly map axonal projection patterns from olfactory sensory neurons expressing these 3 chemoreceptors into the A. aegypti antennal lobe devoid of background interference from 3xP3-based fluorescent transgenesis markers. The marker-free Gr1 T2A-QF2 driver facilitates clear recording of CO2-evoked responses in this central brain region using the genetically encoded calcium indicators GCaMP6s and CaMPARI2. Systematic application of these optimized methods to different chemoreceptors stands to enable mapping A. aegypti olfactory circuits at peripheral and central levels of olfactory coding at high resolution.