Molecular characterisation and diversity analysis of cultivable gut microbiota of selected mosquito larvae from thrissur district, Kerala

Abstract

Mosquito holds microorganisms in their digestive tract to form gut microbiota. This colonization and acquisition begins from their immature larval stage. Recent research is now peeping into mosquito microbiomes to analyse their role in mosquito biology. This knowledge may provide more information regarding its potential for developing bio-control strategies. Meanwhile, little research is focussed on the fungal entities of mosquito larval gut, as more research has been concentrated mainly on gut bacteria. Therefore the rationale of the present study intended to characterise and analyse the diversity of bacterial and fungal gut content of Aedes aegypti, Culex quinquefasciatus, and Anopheles stephensi of three sites of Thrissur districts across seasons pre-monsoon, monsoon and post-monsoon from 2018-2020, to analyse the phylogeny of bacterial and fungal gut entities as well as physicochemical parameters of larval breeding water during these stipulated time period (2018-2020) from aforementioned sites and seasons. The study proposed to identify bacterial and fungal gut content from these mosquito larvae by 16S rRNA and ITS sequencing, respectively. A total of 1026 bacterial isolates grouped into 63 species and 239 fungal isolates fitting into 64 species were identified from these mosquito larval species by culture-dependent methods. The bacterial isolates belonging to phylum Pseudomonadota and fungal isolates belonging to division Ascomycota were predominant in these mosquito species. This study provides insight into the predominance, abundance, and coexistence of bacteria and fungi within and between the larval gut of mosquito species. These bacterial and fungal communities identified from the gut of mosquito larval species of Aedes aegypti, Culex quinquefasciatus, and Anopheles stephensi were used for phylogenetic analysis. The organisms were primarily classified based on their morphological characteristics and biochemical assays. However, the limitations and accuracy of these methods were rectifiedby molecular characterisation. For the present study, the 16S rRNA gene and ITS were used for molecular characterization and phylogenetic assessment of bacteria and fungi, respectively. Nuclear 16S rRNA gene sequences are beneficial for the differentiation between organisms at the genus level across all major phyla of bacteria, while ITS is a well-recognised marker for systematics and phylogeny. During the study, a total of 63 bacterial isolates and 64 fungal isolates were obtained. The reference sequences from the NCBI database were also used for the study. For phylogenetic analysis, bacteria were clustered into distinct families, including Pseudomonadaceae, Bacillaceae, Enterobacteriacea, Yersiniaceae, Moraxellales, and Paenibacillaceae, using their 16S rRNA sequences. On the other hand, a phylogenetic assessment of fungi was carried out after grouping them into various families, such as Davidiellaceae, Debaryomycetaceae, Pleosporaceae, Trichocomaceae, Aspergillaceae, Cordycipitaceae and Syncephalstracea based on their ITS sequences. The results of the study provide valuable insight into intra and interspecific genetic divergences between members of specific classes. Another finding of the study was the close genetic similarity observed between morphologically dissimilar and geographically distant species. The abiotic and biotic factors of breeding water influence the survival and proliferation of mosquito larvae. Mosquitoes prefer diverse aquatic bodies for egg-laying and larval survival. During the present study physico-chemical properties of breeding water, across seasons and their influence on the larval abundance of three mosquito larval species, such as Aedes aegypti, Anopheles stephensi, and Culex quinquefasciatus were assessed. Dissolved oxygen, pH, conductivity, total dissolved solids, and temperature were found to have a significant influence on seasonal variations and played a substantial role in determining the prevalence of these three mosquito larval species. The findings of this study provide valuable insight into important physicochemical parameters that affect the presence of mosquito larval species. The study provides an interconnectedness or idea of the physicochemical parameters of breeding waterand the microbial composition of that water, which in turn are linked to larval abundance and their role in shaping the gut microbiota of mosquito species. These results offer valuable information regarding the role of physicochemical parameters and their influence on larval abundance. This information can be used to create new vector control strategies.