Unraveling the miRNA landscape in tsetse fly saliva and salivary glands during Trypanosoma brucei infections.

MicroRNAs (miRNAs) are emerging as potential mediators of cross-species gene regulation in vector-borne diseases. Using small RNA-sequencing, this study investigated the presence of miRNAs in the tsetse fly (Glossina morsitans)-trypanosome parasite system, focusing on the fly's salivary glands and saliva during Trypanosoma brucei infections. To ensure annotation accuracy, we implemented a robust pipeline incorporating de novo prediction, comparative analyses and manual curation to minimize false positives. This resulted in the detection of 54 unique miRNAs in tsetse saliva and salivary glands, among which 12 were novel to tsetse flies, 5 were putative novel miRNAs and none were originating from T. brucei. Salivary gland samples contained most detected miRNAs, with a large subset also present in saliva, some exhibiting high relative abundance. Interestingly, two miRNAs appeared more represented in saliva as compared to salivary glands (let-7 and miR-8) and one miRNA (miR-184) was only detected in uninfected saliva. The presence of a T. brucei infection in the salivary glands significantly reduced miRNA diversity and abundance in saliva. Target analysis of tsetse salivary miRNAs identified numerous potential host targets, but functional enrichment was limited. In conclusion, this is the first description of miRNAs in the salivary glands and saliva of a tsetse fly species, a blood-feeding vector of African trypanosomes. Our study emphasizes the importance of a robust bioinformatics pipeline in de novo detection of miRNAs in non-model organisms and unconventional matrices like insect saliva. Our work highlights the challenges and limitations of miRNA target prediction, underscoring the need for rigorous experimental validation of miRNAs in host-vector-pathogen interactions.