Comprehensive Screening and Validation of Stable Internal Reference Genes for Accurate qRT-PCR Analysis in Holotrichia parallela under Diverse Biological Conditions and Environmental Stresses

<i>Holotrichia parallela</i> is among the world's most destructive pests. For accurate qPCR and gene expression studies, the selection of stable and appropriate reference genes is crucial. However, a thorough evaluation of potential reference genes for use in <i>H. parallela</i> research is lacking. In this study, 11 reference genes (<i>GAPDH</i>, <i>RPL32</i>, <i>RPL7A</i>, <i>RPS18</i>, <i>RPL13a</i>, <i>RPL18</i>, <i>Actin</i>, <i>RPS7</i>, <i>RPS3</i>, <i>VATB</i>,and <i>EF1A</i>) were evaluated under different biological conditions and environmental stresses. The stability of 11 potential reference gene transcripts was evaluated through various computational tools, including geNorm, BestKeeper, NormFinder, theΔCt method, and the RefFinder program. Under various developmental stages and RNAi conditions, <i>RPL18</i> and <i>RPL13a</i> exhibited the greatest stability. <i>RPL13a</i>, <i>RPL18</i>, and <i>RPL32</i> were the most stable genes in both male and female adults. Under differing tissue conditions, <i>RPL13a</i> and <i>RPS3</i> stood out as the most reliable. Moreover, under varying photoperiod conditions, <i>RPL13a</i>, <i>RPS3</i> and <i>RPL32</i> were the most stable genes. Lastly, <i>Actin</i> and <i>RPL13a</i> were the most stable genes across different temperatures. These findings offer essential criteria for selecting suitable reference genes across diverse experimental settings, thereby establishing a solid basis for accurate gene expression studies in <i>H. parallela</i> using RT-qPCR.