Sialotranscriptome of Simulium pertinax and functional characterization of SpVaP: a novel recombinant simuliid-specific vasodilator.

The salivary glands of hematophagous arthropods possess a remarkable molecular diversity, reflecting evolutionary adaptations to blood feeding lifestyle and vector-host interactions. In this study, we characterized the salivary-gland transcriptome of adult female Simulium pertinax, a medically important black fly species widely distributed in South America. Through RNA sequencing and bioinformatics analyses, we identified 15,708 high-quality transcripts, of which 1947 clusters were related to secreted proteins, distributed across 23 distinct functional families, including digestive enzymes, protease inhibitors, antimicrobial peptides, and hemostasis modulators. Phylogenetic analyses of selected hematophagy-associated families, including Kunitz-domain inhibitors, Apyrases, Kazal-type inhibitors, Serpins, and SVEP homologs, revealed a combination of conserved evolutionary relationships and lineage-specific diversification among simuliids. Notably, the SVEP (Simulium Vasodilator Erythema Protein) family was highly represented and appears to be restricted to black flies. Given that its biological role remains largely uncharacterized, we selected a novel SVEP variant for heterologous expression and functional characterization, which was designated as recombinant Simulium pertinax Vasoactive Protein (rSpVaP). This recombinant protein elicited vasodilator activity in the arterioles of the mouse cremaster muscle, as well as significant paw edema. These findings support rSpVaP as a biologically active SVEP-related salivary vasoactive protein in simuliids, underscoring its contribution to facilitating blood-feeding. Taken together, these results highlight the value of combining transcriptomic profiling with functional characterization to uncover the molecular adaptations underlying hematophagy in black flies, enhancing our understanding of vector-host interactions and the evolutionary pressures shaping the sialotranscriptome.