Microbiota activates IMD pathway and limits Sindbis infection in Aedes aegypti

Abstract

Background: Aedes aegypti is the main vector of important arboviruses such as dengue, Zika and chikungunya. During infections mosquitoes can activate the immune pathways Toll, IMD and JAK/STAT to limit pathogen replication.

Results: Here, we evaluate the immune response profile of Ae. aegypti against Sindbis virus (SINV). We analyzed gene expression of components of Toll, IMD and JAK/STAT pathways and showed that a blood meal and virus infection upregulated aaREL2 in a microbiota-dependent fashion, since this induction was prevented by antibiotic. The presence of the microbiota activates IMD and impaired the replication of SINV in the midgut. Constitutive activation of the IMD pathway, by Caspar depletion, leads to a decrease in microbiota levels and an increase in SINV loads.

Conclusion: Together, these results suggest that a blood meal is able to activate innate immune pathways, through a nutrient induced growth of microbiota, leading to upregulation of aaREL2 and IMD activation. Microbiota levels seemed to have a reciprocal interaction, where the proliferation of the microbiota activates IMD pathway that in turn controls bacterial levels, allowing SINV replication in Ae. aegypti mosquitoes. The activation of the IMD pathway seems to have an indirect effect in SINV levels that is induced by the microbiota.

Keywords: Aedes aegypti; IMD; Immune response; Microbiota; Sindbis virus.

Fig. 1

Whole body gene expression of Toll and IMD pathway genes are induced upon blood-feeding. Gene expression of the three main mosquito immune pathways was evaluated by qPCR using whole body samples 24 h post blood-feeding. a Toll pathway components. b IMD pathway components. c Jak/STAT pathway components. The blood-fed samples were compared with sugar fed samples. Relative expression was calculated using the ΔΔCt method using sugar fed expression as reference. RP49 gene was used as a reference gene. We performed Student’s t-test for statistical analysis between sugar and blood-fed conditions for each gene analyzed

Fig. 2

Transcription factor aaREL2 is upregulated in response to blood-feeding in the midgut of mosquitoes. Gene expression of the three main mosquito immune pathways was evaluated by qPCR in multiple tissues 24 h post blood-feeding. a aaREL1. b aaCactus. c aaMYD88. d aaSerpin. e aaREL2. f aaIMD. g aaDefensin. h aaSTAT. i TEP. Relative expression was calculated using the ddCt method using the lower tissue expression as reference. RP49 gene was used as a reference gene. We performed One-way ANOVA Tukey multiple comparison test for statistical analyses. Abbreviations: Hd, Head; Cc, carcass; Mg, Midgut; Ov, ovary; Tx, thorax

Fig. 3

SINV infection induces expression of molecules from Toll, IMD and Jak/STAT pathways. Gene expression of the Toll, IMD and Jak/STAT pathways were evaluated by qPCR in three different tissues: carcass, midgut and ovaries. Tissues were dissected for RNA extraction 4 days post-infection. a Carcass. b Midgut. c Ovary. Relative expression was calculated using the ΔΔCt method comparing each infected tissue with the respective non-infected one, represented as 1 (blank bars). We performed Student’s t-test for statistical analysis between tissues infected and non-infected. RP49 was used as a reference gene

Fig. 4

Microbiota depletion impacts aaREL2 expression in response to SINV infection. Relative gene expression of the transcription factors of Toll, IMD and Jak/STAT pathways was evaluated in response to microbiota depletion and SINV infection. Mosquitoes were either fed with blood, pretreated for 3 days with a sugar solution containing antibiotic (penicillin and streptomycin) and after that fed with blood or infected blood containing SINV particles. a Relative gene expression of aaREL1 4 days post-infection. b Relative gene expression of aaREL2 4 days post-infection. c Relative gene expression of aaSTAT 4 days post-infection. Relative expression was calculated using the ΔΔCt method and setting the blood condition as reference for comparison. RP49 was used as a reference gene. We performed One-way ANOVA Tukey multiple comparison test for statistical analyses

Fig. 5

Microbiota depletion, by antibiotic treatment or IMD activation, leads to SINV particles increase in Ae. aegypti. After 4 days post-infection the amount of viral RNA and mature particles were measured using mosquito whole body samples. Mosquitoes were maintained in normal sugar or pretreated with antibiotic solution before feeding an infectious blood meal. a Viral RNA amounts relative to the reference gene (RP49). Mosquitoes maintained in normal sugar and then infected with SINV were set as 1. b Plaque assay of whole body samples from mosquitoes infected with SINV 7 days after infection. Mosquitoes injected with dsLacZ-control and dsCaspar were infected with SINV and the levels of bacteria and virus were measured by qPCR. c Four days post-infection the 16S expression was measured in pools of five mosquitoes. d After 7 days post-infection, SINV RNA was measured in whole body of individual mosquitoes. We calculated the relative expression of both bacteria and virus using the LacZ-control condition as a reference. Statistical analysis were conducted using Student’s t-test (unpaired)

Fig. 6

Tripartite model of interaction between the microbiota, virus and the insect immune system