Pyrosequencing 16S rRNA genes of bacteria associated with wild tiger mosquito Aedes albopictus: a pilot study

Abstract

The Asian tiger mosquito Aedes (Stegomya) albopictus is an invasive species that has spread across the world in the last two decades, showing a great capacity to adapt to contrasting climates and environments. While demonstrated in many insects, the contribution of bacterial symbionts in Aedes ecology is a challenging aspect that needs to be investigated. Also some bacterial species have already been identified in Ae. albopictus using classical methods, but a more accurate survey of mosquito-associated bacterial diversity is needed to decipher the potential biological functions of bacterial symbionts in mediating or constraining insect adaptation. We surveyed the bacteria associated with field populations of Ae. albopictus from Madagascar by pyrosequencing 16S rRNA gene amplicons. Different aspects of amplicon preparation and sequencing depth were tested to optimize the breadth of bacterial diversity identified. The results revealed that all mosquitoes collected from different sites have a bacterial microbiota dominated by a single taxon, Wolbachia pipientis, which accounted for about 99% of all 92,615 sequences obtained. As Ae. albopictus is known to harbor two Wolbachia strains (wAlbA and wAlbB), a quantitative PCR was used to estimate the relative densities, (i.e., the bacteria-to-host gene ratios) of each strains in individual mosquitoes. Relative densities were between 6.25 × 100.01 and 5.47 × 100.1 for wAlbA and between 2.03 × 100.1 and 1.4 × 101 for wAlbB. Apart from Wolbachia, a total of 31 bacterial taxa were identified at the genus level using different method variations. Diversity index values were low and probably underestimated the true diversity due to the high abundance of Wolbachia sequences vastly outnumbering sequences from other taxa. Further studies should implement alternative strategies to specifically discard from analysis any sequences from Wolbachia, the dominant endosymbiotic bacterium in Ae. albopictus from this area.

Mosquito vector‐associated microbiota: Metabarcoding bacteria and eukaryotic symbionts across habitat types in Thailand endemic for dengue and other arthropod‐borne diseases

“…Those that applied the culture‐independent deep sequencing approach were mainly restricted to gut bacterial communities (Minard et al., 2012; Wang, Gilbreath, Kukutla, Yan, & Xu, 2011) or other isolated adult arthropod parts (Damiani et al., 2010; Sharma et al., 2014), although one study investigated bacteria diversity from whole Ae. albopictus (Minard et al., 2014). Studies using shotgun metagenomic sequencing (which is agnostic to the genetic material sequenced, as opposed to the gene‐specific amplicon‐based method used here) have identified both bacterial and eukaryotic microbes associated with mosquitoes, however with insufficient depth to perform diversity analyses (Chandler, Liu, & Bennett, 2015).…”

“…quinquefasciatus (Werren, Baldo, & Clark, 2008). Other bacterial taxa, such as Providencia and Erwinia (all Family Enterobacteriaceae), are commonly identified with Aedes, Culex , and other mosquitoes (Demaio, Pumpuni, Kent, & Beier, 1996; Minard et al., 2012, 2014; Rani, Sharma, Rajagopal, Adak, & Bhatnagar, 2009; Zouache, Michelland, Failloux, Grundmann, & Mavingui, 2012). Likewise, the most common eukaryotic microbes identified in this study have also been previously found associated with mosquitoes.…”

“…aegypti , respectively (Lantova & Volf, 2014), although cross‐infections can occur between larvae (Lantova & Volf, 2014). The observed differences between diversity indices of microbiota between vector species could be influenced by the ability of vector‐specific symbionts such as Wolbachia and Ascogregarina to colonize their hosts in high number, as suggested in another study (Minard et al., 2014). However, we expect that richness indices, rather than diversity indices that measure relative abundance, are more robust to the “saturation” effect of Wolbachia or Ascogregarina read numbers.…”

“…Recent improvements in the exploration of microscopic organisms through the use of metagenomic PCR (Hugenholtz, Goebel, & Pace, 1998; Liles, Manske, Bintrim, Handelsman, & Goodman, 2003) and next‐generation sequencing (Logares et al., 2012) have allowed more accurate and sensitive documentation of these organisms. Several studies have characterized and compared mosquito bacterial microbiota in natural habitats (Akorli et al., 2016; Buck et al., 2016; Gimonneau et al., 2014; Minard et al., 2012, 2014, 2015; Tchioffo et al., 2015; Yadav et al., 2015; Zouache et al., 2010) but few have characterized eukaryotes and in such cases have targeted fungi (Muturi, Bara, Rooney, & Hansen, 2016; Muturi, Ramirez, Rooney, & Dunlap, 2016; Steyn, Roets, & Botha, 2016). We document mosquito microbiota composition and diversity for vector species Aedes aegypti , Aedes albopictus , and Culex quinquefasciatus across ecologically distinct, yet geographically proximate, habitats in Thailand where many mosquito‐borne diseases circulate.…”

The microbiome modulates arbovirus transmission in mosquitoes

“…Studies exploiting high throughput sequencing have revealed that the bacterial microbiome of mosquito vectors has low diversity but is highly variable. Most studies thus far have focused on Anopheles mosquitoes [4 • ,5,6 • ,7 •• ,8 •• ,9], and while the number of studies examining the bacterial microbiomes from Aedes and Culex mosquitoes is growing [6 • ,8 •• ,10], this is an area that needs further attention. Although not as comprehensive, culture dependent approaches have been used for characterization of the microbiome of important Aedes and Culex arboviral vectors [11,12].…”