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Chilo partellus  is an invasive polyphagous pest that has not been effectively managed with chemical pesticides. To select potential dsRNAs for use in an alternate control strategy, it is crucial to identify and evaluate stable reference genes for knockdown expression studies. This study evaluates the expression stability of seven candidate reference genes in  C. partellus  larvae fed on crude bacterially-expressed dsRNAs and purified dsRNAs at different time intervals, as well as the developmental stages and sexes. The expression stabilities of the reference genes were evaluated with different software programmes, such as BestKeeper, NormFinder, deltaCt, geNorm, and RefFinder. The overall results rank  ELF  as the most stably expressed reference gene when larvae were fed with crude bacteria-induced dsRNAs and purified dsRNA. However,  Tubulin  and  HSP70  were more stable under different developmental stages and sexes. The expression levels of larvae that were fed crude bacteria-induced dsRNAs of  Chitinase  and  Acetylcholinesterase  were normalized with the four most stable reference genes ( ELF ,  HSP70 ,  V-ATPase  and  Tubulin ) and the least stable reference gene ( 18S  and  HSP70 ) based on the geNorm algorithm. The least stable reference gene showed inconsistent knockdown expression, thereby confirming that the validation of a suitable reference gene is crucial to improve assay accuracy for dsRNA-targeted gene selection in  C. partellus .

Identification and validation of potential reference gene for effective dsRNA knockdown analysis in Chilo partellus