The microbiome composition of Aedes aegypti is not critical for Wolbachia-mediated inhibition of dengue virus

Abstract

Background: Dengue virus (DENV) is primarily vectored by the mosquito Aedes aegypti, and is estimated to cause 390 million human infections annually. A novel method for DENV control involves stable transinfection of Ae. aegypti with the common insect endosymbiont Wolbachia, which mediates an antiviral effect. However, the mechanism by which Wolbachia reduces the susceptibility of Ae. aegypti to DENV is not fully understood. In this study we assessed the potential of resident microbiota, which can play important roles in insect physiology and immune responses, to affect Wolbachia-mediated DENV blocking.

Methodology/findings: The microbiome of Ae. aegypti stably infected with Wolbachia strain wMel was compared to that of Ae. aegypti without Wolbachia, using 16s rDNA profiling. Our results indicate that although Wolbachia affected the relative abundance of several genera, the microbiome of both the Wolbachia-infected and uninfected mosquitoes was dominated by Elizabethkingia and unclassified Enterobacteriaceae. To assess the potential of the resident microbiota to affect the Wolbachia-mediated antiviral effect, we used antibiotic treatment before infection with DENV by blood-meal. In spite of a significant shift in the microbiome composition in response to the antibiotics, we detected no effect of antibiotic treatment on DENV infection rates, or on the DENV load of infected mosquitoes.

Conclusions/significance: Our findings indicate that stable infection with Wolbachia strain wMel produces few effects on the microbiome of laboratory-reared Ae. aegypti. Moreover, our findings suggest that the microbiome can be significantly altered without affecting the fundamental DENV blocking phenotype in these mosquitoes. Since Ae. aegypti are likely to encounter diverse microbiota in the field, this is a particularly important result in the context of using Wolbachia as a method for DENV control.

Fig 1. Microbiome composition of wt and wMel Ae. aegypti mosquitoes.

Relative abundance of microbiota present in wt (A) or wMel (B) Ae. aegypti lines as determined by sequencing of 16s rDNA following a DENV-infectious blood-meal. Where indicated (+ antibiotics) mosquitoes were treated with a combination of penicillin-streptomycin-kanamycin for 3 generations before profiling. Each bar represents a single mosquito. OTU are grouped by genus; ‘unclassified’ indicates OTU that were not classified at the genus level.

Fig 2. Relative abundance of bacterial genera corrected for Wolbachia.

Relative abundance of genera from 19 (- antibiotics) or 20 (+ antibiotics) wMel mosquitoes after OTU assigned to Wolbachia were removed; ‘unclassified’ indicates OTU that were not classified at the genus level.

Fig 3. Effects of Wolbachia and antibiotic treatment on relative abundance of taxa.

Mean relative abundance of Elizabethkingia (A), unclassified taxa (B), Serratia (C) and Chryseobacterium (D) calculated from the Wolbachia-corrected microbiome profiles based on 16s sequencing data (S2 Table) and expressed as a percentage of the total.

Fig 4. Infection rate and DENV genome copy number are unaffected by manipulation of the microbiome using antibiotic treatment.

(A) Percent of infected mosquitoes as determined by qRT-PCR; total numbers of mosquitoes tested per group are listed below each chart. (B) Total number of DENV genome copies per body for all mosquitoes identified as DENV-positive in (A). Statistical analysis was performed using a one-way ANOVA, only the effect of Wolbachia was significant. Data shows median and interquartile range.