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

Cristian Robayo-Cuevas¹Howard Junca²Sandra Uribe³Andres Mauricio Gomez-Palacio^1*

• ¹Universidad Pedagógica y Tecnológica de Colombia, Tunja, Colombia
• ²Microbiomas Foundation, Chía, Colombia
• ³Universidad Nacional de Colombia - Sede Medellin, Medellín, Colombia

Mosquitoes of the subfamily Culicinae transmit pathogens of major medical and veterinary importance, particularly in tropical regions where urbanization and ecological change promote arbovirus (mosquito-borne viruses) circulation. In Colombia, rural Culicinae species are diverse and harbor microbiomes that may influence vector competence, yet their bacterial communities remain poorly studied. Here, we characterized the bacterial microbiota of several Culicinae species and morphotypes collected from two rural localities in Antioquia, Colombia, using an integrated metagenomic framework. Ribosomal 16S rRNA sequences were extracted from total RNA-seq data to infer bacterial community composition and assess α-and β-diversity across mosquito species. Culex morphotypes exhibited the highest richness and evenness (Chao1, Shannon indices), whereas Aedes and Trichoprosopon showed the lowest diversity. Discriminant Analysis of Principal Components (DAPC) and Bray–Curtis ordination revealed partial clustering by species and tribe. Concurrently, de novo assembled contigs were taxonomically annotated against the NCBI NR bacterial database to provide complementary taxonomic and functional insights. Both approaches revealed complex bacterial assemblages dominated by Wolbachia (up to 60% of reads in several Aedes and Culex morphotypes), followed by environmental genera such as Pseudomonas and Acinetobacter (10–20%), and lower-abundance taxa of medical and veterinary importance including Salmonella, Borrelia, and Clostridium (<5%). Bacterial community structure differed among mosquito species, with Aedes albopictus enriched in lactic acid bacteria, whereas Culex morphotypes exhibited broader environmental and endosymbiotic profiles. Notably, Wolbachia and Spiroplasma—both recognized for their roles in microbiome modulation and pathogen interference—were detected across multiple samples. Altogether, our findings provide the first comprehensive metagenomic description of bacterial communities associated with rural Culicinae mosquitoes in Colombia, highlighting their ecological diversity and potential for microbiome modulation strategies in sustainable arboviral disease management.