Reference Genes for Expression Analyses by qRT-PCR in Propsilocerus akamusi (Diptera: Chironomidae)

Propsilocerus akamusi (Tokunaga, 1938) larvae serve as key bioindicators for water quality assessment. This study identifies optimal reference genes for RT-qPCR under diverse experimental conditions. Fifteen candidate genes commonly employed in other insect species were selected, candidate genes commonly used in other insect species. Homologous genes were identified in the P. akamusi genome through sequence alignment with their Drosophila melanogaster counterparts. Expression stability across developmental stages, body parts, temperature variations, and deltamethrin/nickel chloride exposures was systematically evaluated using geNorm, NormFinder, BestKeeper, and ΔCt methods. RPL32 exhibited the highest expression stability across different body parts of adults under varying temperature conditions, and RPS11 and RPL8 showed the greatest stability across developmental stages and in larvae exposed to different temperatures. Furthermore, under nickel chloride and deltamethrin treatments, RPS11 and RPL8 maintained the highest expression stability. The results indicated that the expression stability of reference genes varied under different conditions. Among different body parts of adults, RPL32 and RPL4 exhibited the most stable expression. Across different developmental stages, RPS11 and RPL8 performed best in terms of expression stability. Under different temperature treatments, RPL32 and RPL4 remained stable in adults, while RPS11 and RPL8 showed the greatest stability in larvae. Similarly, under nickel chloride and deltamethrin treatments, RPS11 and RPL8 demonstrated the most stable expression in larvae. Through the above research, we can advance ecosystem-impact insights and bolster environmental protection and water quality monitoring.