Determination of suitable reference genes for RT-qPCR normalisation in Bombyx mori (Lepidoptera: Bombycidae) infected by the parasitoid Exorista sorbillans (Diptera, Tachinidae).

The silkworm Bombyx mori (Lepidoptera: Bombycidae) is a lepidopteran model insect of great economic importance. The parasitoid Exorista sorbillans (Diptera, Tachinidae) is the major pest of B. mori and also a promising candidate for biological control. However, the molecular interactions between hosts and dipteran parasitoids have only partially been studied. Gene expression analysis by reverse-transcription quantitative real-time polymerase chain reaction (RT-qPCR) is indispensable to characterise their interactions. Accurate normalisation of RT-qPCR-based gene expression requires the use of reference genes that are constantly expressed irrespective of experimental conditions. In this study, the expression stability of 13 traditionally used reference genes was estimated by five statistical algorithms (ΔCt, geNorm, Normfinder, BestKeeper, and RefFinder) to determine the best reference genes for gene expression studies in different tissues of B. mori under E. sorbillans parasitism. Specifically, TATA-box-binding protein was the best reference gene in epidermis and testis, while elongation factor 1α was the most stable gene in prothoracic gland and midgut. Elongation factor 1γ, ribosomal protein L3, actin A1, ribosomal protein L40, glyceraldehyde-3-phosphate dehydrogenase and eukaryotic translation initiation factor 4A were the most suitable genes in head, silk gland, fat body, haemolymph, Malpighian tubule and ovary, respectively. Our study offers a set of suitable reference genes for gene expression normalisation in B. mori under the parasitic stress of E. sorbillans, which will benefit the in-depth exploration of host-dipteran parasitoid interactions, and also provide insights for further improvements of B. mori resistance against parasitoids and biocontrol efficacy of dipteran parasitoids.