Unraveling the miRNA Landscape in the Salivary glands and Saliva of the Tsetse Fly, Vector of African Trypanosomes

MicroRNAs (miRNAs) are emerging as potential mediators of cross-species gene regulation in vector-borne diseases. In this study, we investigate miRNA presence in the tsetse fly (Glossina morsitans)-trypanosome system, focusing on salivary glands and saliva during Trypanosoma brucei infections. Using small RNA sequencing and a consensus approach combining two bioinformatics tools, miRDeep2 and sRNAtoolbox, we identified 54 unique miRNAs in tsetse saliva and salivary glands, with none originating from T. brucei. To ensure the accuracy of miRNA annotations, a robust pipeline was implemented, including de novo prediction, comparative analyses across tools, and manual curation to minimize false positives. Among the identified miRNAs, 12 were novel to tsetse flies, and 5 represented putative novel miRNAs. Salivary gland samples contained the majority of detected miRNAs, with a subset also present in saliva, with some of those saliva miRNAs exhibiting notably high relative abundance. This study demonstrates the power of integrated bioinformatics pipelines to investigate miRNAs in non-model organisms and unconventional matrices like insect saliva. Our findings contribute to the growing understanding of miRNA roles in host-vector-pathogen interactions and lay the groundwork for future functional validation studies to elucidate their biological significance. Overall design: miRNA-seq profiling of Glossina morsitans salivary glands and saliva, either infected or non-infected with Trypanosoma brucei