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

Abstract

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 GABA_A 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.

Fig. 1

Genes regulated by Flavivirus, Alphavirus a Orthobunyavirus infections in A. aegypti. a Genes regulated by infections with various viruses in A. aegypti. For RNA-Seq analysis, total RNA was extracted with TRIzol from the mosquitoes infected with 100 M.I.D.₅₀ of viruses on 1 and 6 days post-viral inoculation. The log₂ ratio (read number in the virus-induced whole mosquito/read number in the control whole mosquito) was exploited to evaluate gene regulation. Genes with a log₂ ratio ≤−1.5 or ≥1.5 were selected for further analysis. b Venn diagram schematic representation of up-regulated or down-regulated genes by virus infection

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

Fig. 3

Role of the GABA production system during infection with mosquito-borne viruses. a Schematic representation of the GABAergic system. b, c GABA generation was induced by DENV-2 infection in A. aegypti. b The expression of AaGAD1 was enhanced by DENV-2 infection. The abundance of AaGAD1 was assessed by SYBR Green qPCR on 1 and 6 days post-infection. c GABA induction by DENV-2 infection in A. aegypti. Ten M.I.D.₅₀ of DENV-2 were inoculated into the thoracic region of mosquitoes. Mosquitoes that were microinjected with PBS served as mock controls. The data are presented as the mean ± s.e.m. The results were reproduced three times. d–f Role of the GABA production system during infection with mosquito-borne viruses. AaGAD1 was silenced by inoculation with AaGAD1 dsRNA. Mosquitoes inoculated with GFP dsRNA served as negative controls. Three days post dsRNA treatment, 10 M.I.D.₅₀ of DENV-2 d, SINV e or TAHV f, with or without 120 ng of GABA, were microinjected into A. aegypti mosquitoes. The viral loads were assessed at 3 days post-infection via TaqMan qPCR d or SYBR Green qPCR e, f 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. d–f The number on the X-axis represents the amount of GABA inoculated per mosquito. b–f The data were analyzed statistically using the non-parametric Mann–Whitney test. The results were reproduced three times

Fig. 4

The GABAergic system suppresses the gene expression of the Imd pathway. a Regulation of immune-related genes in the AaGABA _A -R1-silenced mosquitoes. Two independent dsRNAs were exploited to knockdown the AaGABA _A -R1 gene in A. aegypti, respectively. The GFP dsRNA-treated mosquitoes served as controls. The total RNA was isolated on 3 days post dsRNA inoculation for RNA-Seq analysis. Immune-related genes were clustered according to immune pathways and factors. b–g Regulation of Imd-related genes by AaGABA _A -R1 silencing in A. aegypti. AaGABA _A -R1 was silenced by two independent dsRNAs. GFP dsRNA-treated mosquitoes served as controls. Expression of AaImd b, AaRel2 c, AaDef-A d, AaDef-C e, AaDef-D f and AaCec-N g were determined by SYBR Green qPCR at 3 days post dsRNA microinjection. h–k Knockdown of AaImd enhanced virus infection in A. aegypti. h Inoculation of AaImd dsRNA significantly suppressed the AaImd expression. Mosquitoes inoculated with GFP dsRNA served as negative controls. AaImd abundance was assessed by SYBR Green qPCR at 6 days post dsRNA microinjection. Silencing AaImd enhanced infection of DENV-2 i, SINV j and TAHV k in A. aegypti. In total 10 M.I.D.₅₀ of each virus were inoculated at 3 days post dsRNA inoculation. l PGN-mediated activation of Imd signaling offset the increase in DENV replication in GABA-inoculated mosquitoes. Serial concentrations of PGN with 10 M.ID.₅₀ of DENV-2, with or without 120 ng of GABA, were co-inoculated into mosquitoes. Mosquitoes inoculated with PBS served as mock controls. m Silencing the Imd gene offset the reduction in DENV-2 infection in the AaGABA _A -R1-silenced mosquitoes. Mosquitoes inoculated with GFP dsRNA served as negative controls. Ten M.I.D.₅₀ of each DENV-2 were inoculated at 3 days post dsRNA inoculation. b–h The data are presented as the mean ± s.e.m. The results were reproduced twice. i–m The viral loads were assessed at 3 days post-infection via TaqMan qPCR. One dot represents one mosquito and the horizontal line represents the median of the results. b–m The genes were normalized to A. aegypti actin (AAEL011197). The data were analyzed statistically using the non-parametric Mann–Whitney test. The data from at least two independent experiments were combined

Fig. 5

Glutamate-mediated GABA induction by blood ingestion. a–c Feeding blood enhanced the infection of DENV-2 in A. aegypti a, JEV in C. pipiens pallens b and TAHV in C. pipiens pallens c. Either human blood or 1% sucrose (500 µl) was premixed with supernatant from 6 × 10⁵ p.f.u./ml of DENV-2-infected a, 5 × 10⁵ p.f.u./ml of JEV-infected b or 5 × 10⁶ p.f.u./ml of TAHV-infected c Vero cells (cultured in serum-free medium) (500 µl) to feed mosquitoes via an in vitro membrane blood meal. Mosquito infectivity was determined by TaqMan qPCR at 8 days post-blood meal. The number of infected mosquitoes relative to the total number of mosquitoes is shown at the top of each column. Each dot represents one 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. d and e Protein ingestion augmented the amount of glutamic acid d and GABA e in the mosquitoes over time after a meal. Either whole human blood or 70 mg/ml BSA was used for oral feeding of A. aegypti. Mosquitoes fed 1% sucrose served as negative controls. The fed mosquitoes were collected over time after feeding and were used to assess the amounts of glutamic acid and GABA present. The numbers on the Y-axis represents the amount of GABA inoculated per mosquito. The data are presented as the mean ± S.E.M. Differences were considered significant if P < 0.05. *P < 0.05; **P < 0.01; and ***P < 0.001. The data were analyzed statistically using the non-parametric Mann–Whitney test. The results were reproduced twice. a–e The data from at least two independent experiments were combined

Fig. 6

Glutamate-mediated GABA generation facilitates infection of mosquito vectors by mosquito-borne viruses. a The oral introduction of glutamic acid enhanced the prevalence of DENV-2 infection in A. aegypti. A mixture, which contained 1% sucrose (500 µl), supernatant from 6 × 10⁵ p.f.u./ml of DENV-2-infected Vero cells (cultured in serum-free medium) (500 µl), and 10 µg/ml or 100 µg/ml glutamic acid, was used to feed A. aegypti via an in vitro blood feeding system. Mosquitoes fed this mixture without glutamic acid served as a negative control. b, c Knockdown of the AaGAD1 gene suppressed glutamate-mediated GABA generation b, resulting in reduced DENV infection c with sucrose feeding. AaGAD1 was silenced by inoculation with AaGAD1 dsRNA. Mosquitoes inoculated with GFP dsRNA served as negative controls. Three days post dsRNA treatment, a mixture that containing 1% sucrose (500 µl), supernatant from DENV-2-infected Vero cells (cultured in serum-free medium) (500 µl), and 100 µg/ml glutamic acid, was used to feed A. aegypti via an in vitro blood feeding system. d–f Sucrose feeding with glutamic acid enhanced the prevalence of SINV infection in A. aegypti d, JEV infection in C. pipiens pallens e and TAHV infection in C. pipiens pallens f. A mixture, which contained 1% sucrose (500 µl), supernatant from either 2 × 10⁶ p.f.u. ml⁻¹ of SINV-infected d, 5 × 10⁵ p.f.u./ml of JEV-infected e and 5 × 10⁶ p.f.u./ml of TAHV-infected f Vero cells (cultured in serum-free medium) (500 µl), and 100 µg/ml glutamic acid, was used to feed mosquitoes via an in vitro blood feeding system. Mosquitoes fed on this mixture without glutamic acid served as negative controls. b The numbers on the Y-axis represents the amount of GABA inoculated per mosquito. The results were reproduced twice. a, c–f Mosquito infectivity was determined by TaqMan qPCR or SYBR Green at 8 days post-blood meal. The number of infected mosquitoes relative to the total numbers of mosquitoes is shown at the top of each column. Each dot represents one 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. a–f The data from at least two independent experiments were combined