Validation of reliable reference genes for comparison of gene expression across species in the Anopheles Hyrcanus Group

The Anopheles Hyrcanus Group comprises 25 recognized mosquito species that exhibit significant variations in critical vector characteristics despite having similar morphology and ecological niches. Cross-species transcriptional profiling with appropriate reference genes is essential to evaluate the physiological differences among these closely related species. This study aimed to identify reliable pan-species reference genes among six Anopheles Hyrcanus Group species found in South Korea. We assessed the expression stability of eight candidate genes across five developmental stages in each of the six species. Utilizing qPCR and analysis programs, our analysis revealed that the transcription of ribosomal protein L8 (RPL8) and ribosomal protein L13a was the most stable at the larval stage. In contrast, ribosomal protein L32 and ribosomal protein S17 (RPS17) exhibited stability across all tested adult stages. In comparisons across different developmental stages within individual species, RPS17 emerged as a reliable reference gene for Anopheles belenrae, Anopheles pullus, Anopheles sinensis, and Anopheles sineroides, whereas ribosomal protein S7 and RPL8 were suitable for Anopheles kleini and Anopheles lesteri. These selected reference genes may facilitate comparative gene expression profiling within the Anopheles Hyrcanus Group, thereby providing insights into the molecular mechanisms underlying their differences, such as distinct vector competence and baseline insecticide susceptibility.