This work analysed the midgut microbiota composition of mosquitoes reared in the laboratory and collected in the wild and compared this midgut microbiota diversity with their respective breeding sites, using an original strategy based on a special culture, the culturomics technique.

Furthermore, for the definitive identification of mosquitoes collected in the field, a new innovative method based on the analysis of mosquito leg protein spectra obtained by MALDI-TOF MS was used [19, 28].

This approach has been applied here for the first time to mosquitoes collected in the field from Africa. This is further evidence that use of MALDI-TOF MS to identify mosquitoes is a rapid, accurate analysis technique, at low cost in terms of consumables [19, 28].

Several previous studies have already analysed mosquito microbiota and the water of their respective breeding sites [10]. Most of these studies used molecular approaches, mainly based on analysing sequences of the 16S ribosomal RNA gene and cultures of mosquito midgut microbiota [10, 29].

The culturomics approach used in this work revealed a wide diversity of the midgut microbiota of An. gambiae (wild and laboratory strains), Ae. albopictus (wild and laboratory strains) and C. quinquefasciatus (wild strains).

The majority of the bacteria detected in the microbiota of mosquitoes were gram-negative and belong to the phylum Proteobacteria, similar to other studies [10, 29]. However, 17 new bacterial species not previously identified in An gambiae midgut microbiota have been isolated here: Serratia ureilytica, Enterobacter kobei, Enterococcus faecium, Enterococcus avium, Enterococcus raffinosus, Elizabethkingia miricola, Acinetobacter baylyi, Cedecea neteri, Enterobacter asburiae, Pseudomonas gessardii, Streptococcus sanguinis, Streptococcus mitis, Staphylococcus epidermidis, Clostridium perfringens, Microbacterium maritypicum, Pseudomonas massiliensis and Rhodococcus erythropolis.

Interestingly, among the bacterial colonies submitted to MALDI-TOF MS identification, one isolated in breeding water (Mali) was not identified, and corresponded to Lactococcus chungangensis according to 16S sequencing. This bacterial species was then implemented in the MALDI-TOF MS.

Moreover, a bacterial species, Pseudomonas massiliensis, recently isolated in the Timone laboratory, which is under description (D. Raoult, personal communication), was isolated in the An. gambiae and C. quinquefasciatus midgut and their breeding water collected from Mali.

Six bacterial species were commonly found in the midgut of An. gambiae laboratory colonies from Marseille and its respective breeding water. Moreover, 12 and seven bacterial species were found only in the midgut of An. gambiae laboratory colonies from Marseille and its breeding water, respectively. Three bacterial species (Enterococcus faecium, Enterobacter cloacae and Staphylococcus epidermidis) were commonly found both in the midgut of An. gambiae wild strains and laboratory strains.

The gut microbiota of An. gambiae in the wild (Mali) was composed of seven genera: Enterobacter, Pasteurella, Pseudomonas, Bacillus, Enterococcus, Staphylococcus and Kocuria. The gut microbiota of An. gambiae from field collection (Cameroon) was found to be dominated by Comamonas, Serratia, Pseudomonas, Burkholderia and Brevundimonas bacteria by pyrosequencing analysis [30].

We found that some bacterial species were common in the midgut of An. gambiae and Ae. albopictus laboratory strains.

Comparing the Ae. albopictus laboratory strain with those of the Ae. albopictus wild strain, we observed that Serratia marcescens was the only bacterial species found in common. Eight bacterial species were only found in the midgut of Ae. albopictus in the laboratory. Conversely, four were specific for the midguts of the Ae. albopictus wild strain. A difference was observed between the midgut microbiota of Ae. albopictus laboratory and wild strains. In addition to the contribution to the knowledge of bacterial species associated with the microbiota of mosquito vectors, these results suggest that the environment plays a major role in variations of the midgut microbiota diversity of mosquitoes. All the bacteria isolated from the laboratory and wild mosquito microbiota and breeding water are ubiquitous in the environment and are found in water and soil, as well in association with plants, insects, humans and other animals [3133]. These results correlate with other studies; namely that the environmental conditions of the vectors are key determinants in shaping the midgut microbiota [24].

The main limitation of our growth conditions is that culturomics does not allow the growth of some strictly anaerobic bacteria [14]. Strategies are currently under development by the team culturomics to enable the growth of these bacteria considered uncultured by culturomics [34].

The sample size of laboratory mosquitoes used in this study is higher than the number of wild mosquitoes collected; this may explain the increase in the number of bacteria isolated in the laboratory mosquitoes.

Despite the previous studies of the mosquito midgut microbiota, it is still necessary to extend our knowledge in this domain by using new tools for exploration, such as culturomics. This culturomics approach allowed the isolation of bacterial species not previously associated with these vectors, and will aid the development of new control strategies for mosquito-borne diseases.