Mosquito gut microbial ecosystem: Impact on host physiology and malaria competence

Microbes are the integral part of many insects that affect their host phenotypes. Mosquito associated microbes were shown to influence mosquito physiology, development, nutritional status, survival and immunity against pathogens. Host associated microbes can be modified to devise anti-effector molecules that act against mosquito or malaria parasite. In an effort to identify such candidate microbial species, there is a need to study the microbial diversity associated with the mosquito host. In this view, we have characterized the metagenomic profiling of bacterial communities across the life stages of Anopheles gambiae mosquito using 16S based pyrosequencing. Moreover, whole genome shotgun sequencing (WGS) was conducted in parallel to gain structure and functional insights of the microbial community. Our results indicate that mosquito gut harbors diverse bacterial communities across the life stages of mosquitoes. The adult diet mainly shifted the taxonomic composition and relative abundance of the communities. Flavobacteriaceae members dominated the sugar-fed gut, while, Enteric bacteria (especially Enterobacter , Serratia) proliferated extensively after a blood meal. Additionally, metagenomic study informed about the functional capacity of the gut bacteria, suggesting that blood thrived bacteria possess large antioxidant and metabolic capability. The results provide understanding of how bacteria live in harmony in a metagenomic environment; disruption of this balance could affect host life traits.;Next, metagenomic RNA-Seq was performed to analyze the transcriptome of host and microbial communities. The challenge of accessing the bacterial mRNA from a metagenomic sample was addressed, by developing a ribosomal RNA subtraction based protocol. Using community specific rRNA probes, we have successfully demonstrated that the microbial mRNA enrichment for RNA-Sequencing. The microbial transcriptome in the sugar and blood gut was analyzed using community-based and taxon-based approaches. The dual RNA-Seq analysis of host and microbial antioxidant genes informed how the oxidative stress was managed during blood digestion. Condition dependent differentially expressed genes in the gut were identified.;This proof-of-concept RNA-Seq workflow has demonstrated to be an efficient tool to interrogate metatranscriptomes of the host-associated microbiome. The developed pipeline of analysis could be applied to study additional microbial networks in the mosquito gut ecosystem. Finally, in the perspective of symbiosis, we evaluated the contributions of bacteria to the mosquito host. Understanding the impact of gut bacteria on the host fecundity and immunity can advance our knowledge of mosquito control strategies by targeting the key bacterial members of the microbial community. Oral feeding of bacterial isolates affected host phenotypes. Pseudomonas, Leucobacter, and Elizabethkingia effectively suppressed the malaria infection. While, Pseudomonas and Leucobacter appear to boost immunity, while decreasing the mosquito fecundity. In general, significant tradeoffs between infection and fecundity were observed. These findings indicate the oral introduction of modified bacteria via sugar meals can be a way to introduce the paratransgenic candidate bacteria to the mosquitoes. Further, this knowledge can be utilized for the development of bacteria-based malaria control strategies.