Diversity of culturable bacteria including Pantoea in wild mosquito Aedes albopictus

Abstract

Background: The microbiota has been shown to play an important role in the biology of insects. In recent decades, significant efforts have been made to better understand the diversity of symbiotic bacteria associated with mosquitoes and assess their influence on pathogen transmission. Here, we report the bacterial composition found in field-caught Aedes albopictus populations by using culture-dependent methods.

Results: A total of 104 mosquito imagos (56 males and 48 females) were caught from four contrasting biotopes of Madagascar and their bacterial contents were screened by plating whole body homogenates on three different culture media. From 281 bacterial colony types obtained, amplified ribosomal DNA restriction analysis (ARDRA) showed they had 40 distinct ribotypes. Sequencing and BLAST analysis of the 16S rDNA genes responsible for each representative profile made it possible to identify 27 genera distributed in three major phyla. In female mosquitoes, bacterial isolates were mostly Proteobacteria (51.3%) followed by Firmicutes (30.3%) and Actinobacteria (18.4%). Conversely, Actinobacteria was the most abundant phylum in male mosquitoes (48%) followed by Proteobacteria (30.6%) and Firmicutes (20.4%). The relative abundance and composition of isolates also varied between sampling sites, ranging from 3 distinct families in Ankazobe to 8 in Tsimbazaza Park, and Toamasina and Ambohidratrimo. Pantoea was the most common genus in both females and males from all sampling sites, except for Ambohidratrimo. No differences in genome size were found between Pantoea isolates from mosquitoes and reference strains in pulse field gel electrophoresis. However, according to the numbers and sizes of plasmids, mosquito isolates clustered into three different groups with other strains isolated from insects but distinct from isolates from the environment.

Conclusions: The recent upsurge in research into the functional role of the insect microbiota prompts the interest to better explore the role some bacteria detected here may have in the mosquito biology. Future studies of culturable bacteria might decipher whether they have a biological role in the invasiveness of Ae. albopictus. As a possible candidate for paratransgenesis, the predominant genus Pantoea will be characterized to better understand its genetic contents and any possible influence it may have on vector competence of Ae. albopictus.

Figure 1

Frequency of culturable isolates from field populations of Ae. albopictus according to sampling site and isolation medium.

Figure 2

PFGE of undigested genomic DNA of Pantoea mosquito isolates and their reference strains. Chromosomal DNA from Hansenula wingei was used as a reference (BioRad). Characteristics of the samples are indicated in Table 3.

Figure 3

Electrophoretic profiles of high-molecular-weight plasmids from Pantoea mosquito isolates obtained using a modified Eckhardt procedure. Plasmids from Azospirillum brazilense strains En-Ab79 and Sp245 were used as references [38,39]. Characteristics of the samples are indicated in Table 3.

Figure 4

Phylogenetic analysis based on partial 16S rRNA gene sequences of Pantoea obtained from this study and some of those available in GenBank. Identification and GenBank accession numbers are indicated for each sample. The phylogenetic tree was constructed using the Hasegawa, Kishino and Yano maximum likelihood method, with bootstrap analysis with 1000 replicates. Numbers on branches indicate support for each clade ≥ 50%. Only one representative sequence of Ae. albopictus Pantoea isolates is listed in the tree corresponding to the classification of the 45 Pantoea isolates according to their ARDRA profile and sequence composition.