Refining the annotation of Rhodnius prolixus aspartic proteases A1 family genes through proteogenomics

Rhodnius prolixus Stål (Hemiptera: Reduviidae: Triatominae) a hematophagous model organism and vector of Chagas disease, relies on a complex repertoire of digestive enzymes to process its blood meals. Among these, aspartic proteases from the A1 peptidase family play a crucial role in nutrient breakdown. This study aims to refine the gene annotation of the A1 peptidase family in this organism through proteogenomics. A comprehensive analysis of aspartic protease gene sequences and protein isoforms, identified by proteomics, revealed discrepancies in existing gene annotations, including the identification of novel open reading frames and the consolidation of previously separated gene sequences. Our efforts led to the correction of seven gene annotations, reducing the total count of A1 peptidase genes from 19 to 15. Notably, 11 of these genes were confirmed at the protein level, while two were supported by transcriptomic data. Furthermore, our findings highlight instances of alternative splicing, as seen in RPRC015076, where proteoforms T1IFK7 and R4G5J6 are expressed through intron retention. This study not only provides a more accurate and comprehensive genomic framework for the A1 peptidase family but also offers new insights into the functional complexity and regulation of digestive enzymes in R. prolixus. These findings pave the way for future studies on insect digestive biology and their potential applications in vector control strategies.