Cross-Species Evaluation of qPCR Reference Genes in Diabrotica speciosa (Coleoptera: Chrysomelidae) using Diabrotica virgifera virgifera sequences.

The cucurbit beetle Diabrotica speciosa is one of the most important corn pests in Brazil, seen as responsible for about 10-13% losses in maze crops, especially in the southern region of Brazil. The development of sustainable technologies for specific pest control, such as RNAi, depends on the identification of target genes and on the characterization of the gene expression profile of these genes in different developmental stages. Gene expression analyses, on the other hand, depend on reliable reference genes for the normalization of RT-qPCR data. In this study, we performed a cross-species evaluation of qPCR reference genes already validated in Diabrotica virgiferavirgifera, due to its relevance as an important corn pest in North America. The expression of the reference genes EF1α, β-actin, β-tubulin, and GAPDH was evaluated in different developmental stages of the Diabrotica speciosa: egg, neonate, 3rd instar larva, pupa, and adult. The stability of the reference genes was analyzed using the ΔCt method, BestKeeper, GeNorm, and NormFinder algorithms available at RefFinder web-based tool. From all four genes, β-actin and EF1α were the most suitable for normalizing genes, with both expression and stability ranking position in accordance with the results reported for Diabrotica virgifera virgifera. Our findings provide valuable information for researchers studying gene expression during different developmental stages of this species and highlight the potential of validating cross-species reference genes.