Liposcelis entomophila is a nuisance pest that seriously threatens the safety of stored grains owing in part of pesticide resistance. To facilitate better control of L. entomophila, future studies need to focus on gene function characterization. This requires the identification of suitable reference genes (RGs) for reverse-transcription quantitative PCR (RT-qPCR), which is crucial for the normalization of target gene expression. Here, we evaluated the expression stability of 12 candidate RGs in L. entomophila across six experimental conditions (developmental stage, population, body part, phosphine fumigation, hypoxia induction, and temperature treatment). Under developmental conditions, ACTB, HSP70, and RPL23 were the most stable RGs. Among the different populations and body parts, RPL23, EF1a, and ACTB were the most stable RGs. Comparison of gene expression among adults under different environmental conditions revealed that at least seven RGs were fit for normalizing gene expression data. Among them, RPL23, EF1a, and ACTB showed the best fit as RGs in RT-qPCR analysis. After validation, our results demonstrated that EF1a and ACTB, as well as their combination, were the ideal RGs for L. entomophila across various experimental conditions. This study provides a comprehensive RG selection suggestion for gene expression studies of L. entomophila and will facilitate future functional genomic research on the development and tolerance of environmental factors in psocids.

Liposcelis entomophila是一种令人讨厌的害虫，由于部分农药抗药性，严重威胁储存粮食的安全。为了更好地控制L. entomophila，未来的研究需要关注基因功能表征。这需要为逆转录定量 PCR (RT-qPCR) 确定合适的参考基因 (RG)，这对于目标基因表达的标准化至关重要。在这里，我们评估了 12 个候选 RGs 在6 个实验条件下（发育阶段、种群、身体部位、磷化氢熏蒸、缺氧诱导和温度处理）在L. entomophila 中的表达稳定性。在发育条件下，ACTB、HSP70和RPL23是最稳定的 RG。在不同的人群和身体部位中，RPL23、EF1a和ACTB是最稳定的 RG。不同环境条件下成人基因表达的比较表明，至少有 7 个 RGs 适合标准化基因表达数据。其中，RPL23、EF1a和ACTB在 RT-qPCR 分析中显示出最适合作为 RGs。经过验证，我们的结果表明EF1a和ACTB以及它们的组合是L. entomophila的理想 RGs在各种实验条件下。该研究为L. entomophila 的基因表达研究提供了全面的 RG 选择建议，并将促进未来对 psocids 环境因素的发展和耐受性的功能基因组研究。