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Abstract The European corn borer (Ostrinia nubilalis) is an agricultural pest and burgeoning model for research on speciation, seasonal adaptation and insect resistance management. Although previous work inO. nubilalishas identified genes associated with differences in life cycle, reproduction, and resistance toBttoxins, the general lack of a robust gene‐editing protocol forO. nubilalishas been a barrier to functional validation of candidate genes. Here, we demonstrate an efficient and practical methodology for heritable gene mutagenesis inO. nubilalisusing the CRISPR/Cas9 genome editing system. Precise loss‐of‐function (LOF) mutations were generated at two circadian clock genes,period(per) andpigment‐dispersing factor receptor(pdfr), and a developmental gene,prothoracicotropic hormone(ptth). Precluding the need for a visible genetic marker, gene‐editing efficiency remained high across different single guide RNAs (sgRNA) and germline transmission of mutations to F₁offspring approached 100%. When single or dual sgRNAs were injected at a high concentration, gene‐specific phenotypic differences in behaviour and development were identified in F₀mutants. Specifically, F₀gene mutants demonstrated that PER, but not PDFR, is essential for normal timing of eclosion. PTTH F₀mutants were significantly heavier and exhibited a higher incidence of diapause. This work will accelerate future studies of gene function inO. nubilalisand facilitate the development of similar screens in other Lepidopteran and non‐model insects.