Evaluation of suitable reference genes for expression analysis using quantitative real-time polymerase chain reaction in the parasitoid Exorista sorbillans (Diptera: Tachinidae)

The parasitoid Exorista sorbillans (Diptera: Tachinidae) is a larval endoparasitoid of the silkworm Bombyx mori, causing severe damage to silkworm cocoon industry. It is also an important natural enemy resource of insect pests in agriculture and forestry. Despite their roles in biocontrol and pest status on sericulture, there has been limited research on the functional studies of dipteran parasitoids. Quantitative real-time polymerase chain reaction (qRT-PCR) is the most commonly used to address gene functions. Using qRT-PCR, stably expressed reference genes under different experimental conditions are required to normalize the expression of target genes. However, no information regarding suitable qRT-PCR reference genes in dipteran parasitoids has been reported. In this study, we evaluate the expression stability of nine commonly used reference genes in insects including eukaryotic translation elongation factor 1 delta (eEF1 delta), elongation factor 2, 18S ribosomal RNA (18S rRNA), tubulin 3, actin87, ribosomal protein 49 (RP49), ribosomal protein S15, glyceraldehyde-3-phosphate dehydrogenase, and TATA-box binding protein (TBP) in E. sorbillans under different treatments, including tissues, developmental stages, genders, feeding density and pesticide stress, using increment C-t, BestKeeper, geNorm, Normfinder and RefFinder, respectively. The results showed that the genes RP49, eEF1 delta and 18S rRNA were recommended as the most suitable reference genes in E. sorbillans across all experimental conditions. This finding provides the necessary foundation for future functional studies in E. sorbillans and its effective use in both sericulture and pest control.

Automated summary

This study evaluated the expression stability of nine commonly used reference genes in the dipteran parasitoid, Exorista sorbillans, and identified RP49, eEF1 delta, and 18S rRNA as the most suitable reference genes. This finding provides the necessary foundation for future functional studies in E. sorbillans and its effective use in both sericulture and pest control.