Abstract
Anopheline mosquitoes transmit over 200 million cases of malaria and cause nearly half a million deaths annually. Infected female mosquitoes transmit disease through bites as they require nourishment from a blood meal to produce eggs. To locate hosts, female mosquitoes rely on several sensory cues including CO2, humidity, odors, and heat, but the molecular mechanisms of stimulus detection are largely unknown. The Garrity lab previously identified Ionotropic Receptor 21a (IR21a) as necessary for both warm and cold avoidance in Drosophila. I hypothesize that the Anopheles ortholog of this receptor may serve a similar function and be involved in heat seeking. An initial AgIr21a mutant generated by CRISPR/Cas9 and the nonhomologous end joining (NHEJ) pathway exhibited dramatic reductions in heat-seeking. To firmly establish a role for AgIr21a in this behavior, sought to generate a second mutant allele of AgIr21a and introduce genetic tools into the locus to enable subsequent cell-specific experimental manipulation. Achieving these goals required designing and building transgenesis vectors for disrupting the AgIr21a gene by homology directed repair. These vectors not only permit the generation of mutation, but also the insertion of a fluorophore for identifying the mutation, as well as a recombination targeting sequence that enables subsequent integration of genetic tools into the AgIr21a locus. The resulting second mutant allele, AgIr21aT2A-QF-eYFP, shows similar heat-seeking defects to the NHEJ mutant, further supporting a role for IR21a in this process. In addition, I generated a toolbox of adaptable parent vectors to facilitate future genetic manipulations of Anopheles gambiae.