Fig. 2
Role of GABA receptor-mediated GABA signaling during infection by mosquito-borne viruses. a–c dsRNA-mediated knockdown of the AaGABA A -R1 gene impaired DENV-2 infection in A. aegypti. a, b Silencing of AaGABA A -R1 enhanced DENV-2 infection as measured by qPCR a and a plaque assay b. The viral loads were assessed on 6 days post-infection via a plaque assay. c dsRNA-mediated knockdown of the AaGABA A -R1 gene in mosquitoes. Mosquitoes inoculated with GFP dsRNA served as negative controls. The AaGABA A -R1 abundance was assessed by SYBR Green qPCR on 6 days post dsRNA microinjection. Ten M.I.D.50 of DENV-2 were inoculated on 3 days post dsRNA inoculation. d, e Thoracic inoculation of GABA receptor-inhibitory insecticides decreased DENV-2 replication in A. aegypti. Either Fipronil d or Bilobalide e was premixed with 10 M.I.D.50 of DENV-2 and subsequently inoculated into mosquitoes. Mosquitoes inoculated with Deltamethrin and 10 M.I.D.50 of DENV-2 served as unrelated controls. Mosquitoes inoculated with PBS and DENV-2 served as mock controls. The number on the X-axis represent the amount of insecticides inoculated per mosquitoes. f Insecticide exposure reduced DENV-2 replication in A. aegypti. Mosquitoes infected by oral membrane feeding were exposed to bottles sprayed with Fipronil (50 μg/bottle) or Bilobalide (100 μg/bottle) for 2 h. Deltamethrin (50 ng/bottle) served as an unrelated control. Infected mosquitoes exposed to PBS served as mock controls. For mosquito oral infection, 6 × 105 p.f.u./ml of DENV-2 was used. Surviving mosquitoes were transferred to new culture containers for further rearing. The viral loads were assessed over time post-infection via TaqMan qPCR and were normalized to A. aegypti actin (AAEL011197). g Thoracic inoculation of GABA facilitated DENV-2 infection in A. aegypti. Serial concentrations of GABA with 10 M.I.D.50 of DENV-2 were microinjected into mosquito thoraxes. Mosquitoes inoculated with PBS and DENV-2 served as negative controls. The number on the X-axis represents the amount of GABA inoculated per mosquito. a, d, e, g The viral loads were assessed at 3 days post-infection via TaqMan qPCR and were normalized to A. aegypti actin (AAEL011197). a–g One dot represents one mosquito, and the horizontal line represents the median of the results. The data were analyzed statistically using the non-parametric Mann–Whitney test. The results were reproduced at least two times. The primers and probes used for PCR are presented in Supplementary Data 2
