Blood meal acquisition enhances arbovirus replication in mosquitoes through activation of the GABAergic system

Mosquitoes are hematophagous insects that carry-on and transmit many human viruses. However, little information is available regarding the common mechanisms underlying the infection of mosquitoes by these viruses. In this study, we reveal that the hematophagous nature of mosquitoes contributes to arboviral infection after a blood meal, which suppresses antiviral innate immunity by activating the GABAergic pathway. dsRNA-mediated interruption of the GABA signaling and blockage of the GABAA receptor by the specific inhibitors both significantly impaired arbovirus replication. Consistently, inoculation of GABA enhanced arboviral infection, indicating that GABA signaling facilitates the arboviral infection of mosquitoes. The ingestion of blood by mosquitoes resulted in robust GABA production from glutamic acid derived from blood protein digestion. The oral introduction of glutamic acid increased virus acquisition by mosquitoes via activation of the GABAergic system. Our study reveals that blood meals enhance arbovirus replication in mosquitoes through activation of the GABAergic system.

replication in the AaGABA A -R1 silencing mosquitoes.
(A) Incubation of Fipronil and Bilobalide did not affect the DENV-2 infectivity. 6 × 10 5 pfu DENV-2 was premixed with either 50 µg/ml Fipronil or 100 µg/ml Bilobalide for 30 mins, respectively. The virus incubated with PBS served as negative control. After 30 mins post-incubation, the viral titer was determined by a plaque assay. The experiment was reproduced by 2 times. The data are presented as the mean ± s.e.m. B) Both GABA A -receptor targeting insecticides did not have additional effects on DENV-2 replication in the AaGABA A -R1-silenced mosquitoes. Either 15 ng of Fipronil or 30 ng of Bilobalide mixed with 10 M.I.D. 50 of DENV-2 were inoculated into the AaGABA A -R1 dsRNA-treated mosquitoes. The viral loads were assessed on 3 days post-infection via TaqMan qPCR and were normalized to A. aegypti actin (AAEL011197). The primers and probes used for PCR are presented in Supplementary Data 2. One dot represents 1 mosquito, and the horizontal line represents the median of the results. The data were analyzed statistically using the non-parametric Mann-Whitney test. The data from two independent experiments were combined. The number on the X-axis represent the amount of insecticides inoculated per mosquitoes. (C) Insecticide exposure reduced ZIKV replication in A. aegypti. Mosquitoes infected by oral membrane blood feeding were exposed to bottles sprayed with Fipronil (50 µg/ bottle), Bilobalide (100 µg/ bottle) or Deltamethrin (50 ng/ bottle) for 2 hours. For mosquito oral infection, 5 × 10 5 p.f.u. ml -1 of ZIKV was used. Infected mosquitoes exposed to PBS served as mock controls. The mosquitoes that survived were transferred to new culture containers for further rearing. The viral loads were assessed over time post-infection by SYBR Green qPCR and were normalized to A. aegypti actin (AAEL011197).

Supplementary
(A-C) The data were analyzed statistically using the non-parametric Mann-Whitney test. The data from 2 independent experiments were combined.

A. aegypti
Two independent dsRNAs against the AaGABA A -R1 gene were inoculated into mosquitoes.
Mosquitoes inoculated with GFP dsRNA served as negative controls. The abundance of AaGABA A -R1 was assessed by SYBR Green qPCR on 3 days post dsRNA inoculation. The data were analyzed statistically using the non-parametric Mann-Whitney test. The experiment was reproduced twice. The data are presented as the mean ± s.e.m. Figure 12. Regulation of the Imd-mediated gut immunity by feeding with GABA.

Supplementary
(A-D) Regulation of Imd signaling by feeding with GABA. Since proliferation of the microbiota could skew the Imd signaling, the gut commensal microbiome was removed by feeding antibiotics. The antibiotic-treated mosquitoes were fed by 120 ng of GABA with a sucrose meal. Antibiotic-treated mosquitoes fed a sucrose meal without GABA served as negative controls. The midguts were dissected at 18 hours after feeding to determine the expression of the AaImd (A), AaRel2 (B), AaDef-C (C) and AaCec-N (D) genes by SYBR Green qPCR. The experiment was reproduced twice. The data are presented as the mean ± s.e.m.
(E) Regulation of the gut microbiome by feeding with GABA. Normal mosquitoes were fed with a sucrose meal, with or without 120ng of GABA. The midguts were dissected at 24 hours after feeding. The burden of the gut bacteria was determined by qPCR. The qPCR primers for 16S rDNA from the gut bacteria are described in Supplementary Data 2. One dot represents one mosquito gut. The horizontal line represents the median value of the results.
(A-E) The data were analyzed statistically using the non-parametric Mann-Whitney test. The qPCR primers for the AaImd, AaRel2, AaDef-C, AaCec-N genes and gut bacterial 16S rDNA are described in Supplementary Data 2.

Supplementary Figure 13. Measurement of fully engorged mosquitoes immediately collected after either sucrose meal or blood feeding
The 5-7 days old female mosquitoes were fed by either 1% sucrose or the whole human blood, via feeders of the Hemotek feeding system. The fully engorged mosquitoes were immediately collected for the size (A) and weight (B) comparison. There was no significant difference in the size and weight of mosquitoes fed by sucrose and human blood. (B) One dot represents a mosquito. The data were statistically analyzed by the Student's t test.

Supplementary Figure 14. Tracking the acquisition of sucrose liquid in the mosquito midgut and crop
The previous studies showed that digestion of sugar in insects may take place in either the midgut 40-42 or the sack-like crop 42 . The acquisition and digestion location is influenced by the meal size consumed, sugar concentration and other factors 43 . We therefore investigated which tissue is exactly used for stocking the virus-spiked sucrose liquid after a meal. The A. aegypti mosquitoes were fed by 1% sucrose (50% v/v), the supernatant from DENV-2-infected Vero cells (50% v/v), with 0.1% Ponceau S (the final concentration). Both the midgut and the crop were dissected at 0, 2, 4, 8 hours after the sucrose meal for investigation. The result indicated that the sucrose liquid was immediately acquired into the midgut rather than the crops after a meal of the sucrose-medium mixture. The experiment was reproduced twice.

Supplementary Figure 15. Silencing AaGAD1 offset the increase in DENV-2 infectious ratios caused by blood meals
The mosquitoes were inoculated with dsRNAs against the AaGAD1 and GFP genes. Three days post-dsRNA inoculation, a mixture that containing either human blood or 1% sucrose (500 µl) and supernatant from DENV-2-infected Vero cells (cultured in serum-free medium) (500 µl) was used to feed A. aegypti via an in vitro blood feeding system. Mosquito infectivity was determined by TaqMan qPCR 8 days post-blood meal. The experiment was reproduced twice. The number of infected mosquitoes relative to the total number of mosquitoes is shown at the top of each column. Each dot represents a mosquito. The data upper mosquito number are represented as the percentage of mosquito infection. Differences in the mosquito infective ratios were compared using Fisher's exact test.