Tuta absoluta is one of the most significant invasive pests affecting tomato plants worldwide. RT-qPCR has emerged as one of the most sensitive and accurate methods for detecting gene expression data. The screening of stable internal reference genes is the most critical step for studying the molecular mechanisms of environmental adaptability. The stable reference genes expressed in T. absoluta under specific experimental conditions have not yet been clarified. In this study, seven candidate reference genes (RPL27, RPS13, RPS15, EF1-α, TUB, TBP, and β-actin) and their optimal numbers were evaluated under biotic (developmental stages and adult tissues) and abiotic (insecticide, temperature, and plant VOC) conditions using four software programs. Our results identified the following reference genes and numbers as optimal: three genes (EF1-α, RPS13, and RPL27) for different developmental stages (egg, larva, pupa, unmated adult), two genes (RPS13 and TBP) for adult tissues (antenna, head, thorax, abdomen, leg), two genes (TBP and RPS13) for insecticides (Bacillus thuringiensis, chlorpyrifos, abamectin-aminomethyl, and chlorantraniliprole), two genes (RPL27 and TUB) for temperature-induced stresses (0, 25, and 40 °C), and two genes (RPS13 and TUB) for VOC-induced stresses (nonanal, α-phellandrene, and tomato leaves). Our results provide a reference for selecting appropriate reference genes for further study of the functional genes of T. absoluta under different experimental conditions.
