The Selection and Validation of Reference Genes for RT-qPCR Analysis of the Predatory Natural Enemy Orius nagaii (Hemiptera: Anthocoridae)

<i>Orius nagaii</i> is a highly effective natural enemy for controlling thrips, tetranychids, aphids, and various Lepidoptera pests. Nevertheless, the molecular mechanisms underlying its interactions with host pests remain unclear. Screening for optimal reference genes is a prerequisite for using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to investigate the interrelationship. Here, ten commonly used reference genes (<i>Act</i>, <i>GAPDH</i>, <i>β-Tub</i>, <i>EF1-α</i>, <i>RPS10</i>, <i>RPS15</i>, <i>RPL6</i>, <i>RPL13</i>, <i>RPL32</i>, and <i>HSP90</i>) were selected, and their expression stability across developmental stages, tissues, temperatures, and host conditions were evaluated using <i>RefFinder</i>, which uses multiple analytical approaches (<i>NormFinder</i>, <i>geNorm</i>, the <i>ΔCt</i> method, and <i>BestKeeper</i>). The findings suggested that the most reliable normalization can be achieved by selecting the two reference genes for all conditions, with the optimal pairs being <i>RPS10</i> and <i>RPL32</i> for the developmental stage, <i>RPS10</i> and <i>RPS15</i> for tissue, <i>RPS10</i> and <i>RPS15</i> for the host, and <i>EF1-α</i> and <i>RPL13</i> for temperature. Also, the best and least stable reference genes were chosen to compare the relative transcript levels of the <i>TBX1</i> in various tissues, which exhibited considerable variation. Our findings will significantly enhance the reliability of RT-qPCR and provide a foundation for further research on the expression patterns of crucial genes that are implicated in the interaction between <i>O. nagaii</i> and its host pests.