Pyrosequencind of 16S rRNA genes to assess the bacterial microbiota associated with wild tiger mosquito Aedes albopictus

The Asian tiger mosquito Aedes (Stegomya) albopictus is an invasive species that has spread across the world in the past couple of decades, showing a considerable adaptation capability in contrasted climates and environment. As it is now admitted that microbial symbionts often mediate or constrain adaptation of many insects, the contribution of bacterial symbionts in Aedes ecology is a challenging aspect that need to be investigated. Although some bacterial species have already been identified from Aedes albopictus using classical methods, an accurate appreciation of mosquito associated bacterial diversity is needed to obtain a comprehensive picture of the bacterial diversity before envisaging to decipher their potential biological function. Using next generation pyrosequencing of 16S rRNA amplicons, we surveyed the bacteria associated with field populations of Ae. albopictus from Madagascar, Indian Ocean. Different aspects of amplicon preparation and depth of sequencing were tested to determine the best modalities to retrieve the deepest bacterial diversity. The results revealed that all mosquito populations sampled in different regions has a bacterial microbiota largely dominated by a single bacterial taxon, Wolbachia pipientis, which accounts for about 96% to 99% of the sequences. As Aedes albopictus is known to harbour two Wolbachia strains, quantitative PCR estimated the relative density of wAlbA and wAlbB from 0.0625 to 0.547 and from 0.203 to 1.4 (bacteria-to-host ratio), respectively. Diversity indices showed low values due to the high abundance of Wolbachia sequences that outnumber and underestimate other taxa. These results show the limits of high-throughput sequencing to reveal the most complete picture of the bacterial microbiota in the presence of a dominant endosymbiotic bacterium. In such situation, further studies should implement alternative strategies to specifically remove sequences of a particular abundant endosymbiont.