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

<i>Propsilocerus akamusi</i> (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 <i>P. akamusi</i> genome through sequence alignment with their <i>Drosophila melanogaster</i> 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. <i>RPL32</i> exhibited the highest expression stability across different body parts of adults under varying temperature conditions, and <i>RPS11</i> and <i>RPL8</i> showed the greatest stability across developmental stages and in larvae exposed to different temperatures. Furthermore, under nickel chloride and deltamethrin treatments, <i>RPS11</i> and <i>RPL8</i> 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, <i>RPL32</i> and <i>RPL4</i> exhibited the most stable expression. Across different developmental stages, <i>RPS11</i> and <i>RPL8</i> performed best in terms of expression stability. Under different temperature treatments, <i>RPL32</i> and <i>RPL4</i> remained stable in adults, while <i>RPS11</i> and <i>RPL8</i> showed the greatest stability in larvae. Similarly, under nickel chloride and deltamethrin treatments, <i>RPS11</i> and <i>RPL8</i> 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.