In press. Metatranscriptomic profiling of the bacterial and fungal microbiota of field-collected Anopheles darlingi from Colombia: Community composition and functional insights

Mosquitoes harbor diverse microorganisms that can influence host biology. While microbiota research in Anopheles has primarily focused on the metabarcoding approach, knowledge regarding the transcriptionally active microbiota and its functional roles in the host remains limited. This study aimed to characterize the transcriptionally active bacterial and fungal microbiota in field-collected Anopheles darlingi, a primary malaria vector in the Neotropics, and in addition, to explore its functional profile. Mosquitoes were collected in malaria-endemic regions of Colombia. Total RNA was extracted from pooled specimens to prepare cDNA libraries and sequenced by Illumina NovaSeq 6000. Results on microbiota composition showed predominance of bacterial (65 %) over fungal reads (35 %). The most abundant bacterial families included Enterobacteriaceae, Pseudomonadaceae, Acetobacteraceae and Moraxellaceae; the dominant genera were Halopseudomonas, Acinetobacter, Klebsiella, Escherichia and Asaia. In the fungal microbiota composition predominated Aspergillaceae and Hypoxylaceae, primarily represented by Aspergillus and Hypoxylon. Functional analysis showed transcripts encoding an MFS family transporter - an arabinose efflux permease, an ABC-type branched-chain amino acid transport system, diaminopimelate decarboxylase and O-succinylbenzoic acid-CoA ligase (MenE). These transcripts were associated with functional categories related to carbohydrate transport and metabolism, as well as the transport and biosynthesis of essential amino acids and vitamins. These findings offer valuable insights into the microbiota-mosquito dynamic and establish a foundation for further research into the role of microbiota in mosquito biology.