Journal of Mosquito Research 2024, Vol.14, No.2, 61-66 http://emtoscipublisher.com/index.php/jmr 63 Figure 3 reveals the integration of the driver-responder-marker (DRM) system into the olfactory gene orco in the African malaria mosquito, Anopheles gambiae. Part A displays a diagram of the DRM system integrated into the orco gene using CRISPR-Cas9 mediated homology-directed repair (HDR) technology, and the construction of a binary-use orcoQF2 driver line through excision of the responder gene by Cre-loxP technology. Part B shows the expression of orco+olfactory sensory neurons in the antennae, maxillary palps, and labial palps. Part C exhibits the heterologous expression of the Act5C-ECFP marker in different T2A-QF2 integration lines, highlighting the positional effects of specific gene loci on responder and marker transgene expression. It shows the expression of Act5C-ECFP marking T2A-QF2 driver integration in the midgut of larvae, and the expression of 3xP3-ECFP marking QUAS-GCaMP6f responder integration in the ventral nerve cord and optic lobes. These results not only show the locality dependence of gene expression but also emphasize the complexity of gene expression regulation that must be considered when implementing gene editing strategies in complex biological systems. Figure 3 A DRM integration into the Anopheles gambiae orco gene reveals positional effects modulating responder and marker transgene expression Figure 4 displays the response of Gr22+ olfactory sensory neurons on the antennae of Anopheles gambiae to carbon dioxide (CO2). Part A shows the activity of Gr22+neurons before and after exposure to 1% CO2 stimulus, indicated by changes in GCaMP6f fluorescence. Under a 1% CO2 environment, a marked increase in fluorescent response reveals the neurons' sensitivity to CO2. Part B tracks the activity changes of Gr22+neurons following stimulation with different concentrations of CO2, with mean ± standard error of the mean (SEM) illustrating an increase in neuronal response intensity as CO2 concentration rises. Part C quantifies the maximum amplitude of fluorescence change under a 1-second CO2 pulse, with mean ± SEM again confirming the neurons' activity dependence on CO2 concentration changes. Bilateral Wilcoxon signed-rank tests indicate that responses at higher CO2 concentrations are significantly stronger than at baseline levels. These data clearly indicate that Gr22+ olfactory neurons can specifically respond to changes in CO2 concentration, a finding crucial for understanding how mosquitoes utilize olfaction to locate hosts.
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