Detection of endosymbiotic, environmental, and potential bacterial pathogens in diverse mosquito taxa from Colombian tropical forests using RNAseq

<h4>Introduction</h4>Mosquitoes of the subfamily <i>Culicinae</i> transmit pathogens of major medical and veterinary importance, particularly in tropical regions where urbanization and ecological change promote arbovirus circulation. In Colombia, rural <i>Culicinae</i> species are diverse and harbor microbiomes that may influence vector competence, yet their bacterial communities remain poorly characterized.<h4>Methods</h4>We characterized the bacterial microbiota of multiple <i>Culicinae</i> species and morphotypes collected from two rural localities in Antioquia, Colombia, using an integrated metagenomic approach. Ribosomal 16S rRNA sequences were extracted from total RNA-seq datasets to infer bacterial community composition and assess <i>α</i>- and <i>β</i>-diversity. Diversity metrics (Chao1 and Shannon indices), Discriminant Analysis of Principal Components (DAPC), and Bray-Curtis ordination were used to evaluate community structure. In parallel, <i>de novo</i> assembled contigs were taxonomically annotated against the NCBI NR bacterial database to obtain complementary taxonomic and functional insights.<h4>Results</h4>Culex morphotypes exhibited the highest richness and evenness, whereas <i>Aedes</i> and <i>Trichoprosopon</i> showed lower diversity. Ordination and DAPC analyses revealed partial clustering by species and tribe. Both the 16S and assembly-based analyses showed complex bacterial assemblages dominated by <i>Wolbachia</i> (up to 60% of reads in several <i>Aedes</i> and Culex morphotypes), followed by environmental genera such as <i>Pseudomonas</i> and <i>Acinetobacter</i> (10-20%). Lower-abundance taxa of medical and veterinary importance-including <i>Salmonella</i>, <i>Borrelia</i>, and <i>Clostridium</i> (<5%)-were also detected. Bacterial community structure differed among mosquito species; <i>Aedes albopictus</i> was enriched in lactic acid bacteria, while <i>Culex</i> morphotypes exhibited broader environmental and endosymbiotic profiles.<h4>Discussion</h4>This study provides the first comprehensive metagenomic description of bacterial communities associated with rural <i>Culicinae</i> mosquitoes in Colombia. The predominance of symbionts such as <i>Wolbachia</i> and <i>Spiroplasma</i>, coupled with distinct bacterial signatures among host species, highlights the ecological complexity of these microbiomes and their potential relevance for microbiome-based strategies in sustainable arboviral disease management.