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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.
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The period gene alters daily and seasonal timing in Ostrinia nubilalis
Summary The timing of insects’ daily (feeding, movement) and seasonal (diapause, migration) rhythms affects their population dynamics and distribution. Yet, despite their implications for insect conservation and pest management, the genetic mechanisms underlying variation in timing are poorly understood. Prior research in the European corn borer moth (Ostrinia nubilalis) associated ecotype differences in seasonal diapause and daily activity with genetic variation at the circadian clock geneperiod(per). Here, we demonstrate that populations with divergent allele frequencies atperexhibit differences in daily behavior, seasonal development, and the expression of circadian clock genes. Specifically, later daily activity and shortened diapause were associated with a reduction and delay in the abundance of cyclingpermRNA. CRISPR/Cas9-mediated mutagenesis revealed thatperand/or an intact circadian clock network were essential for the appropriate timing of daily behavior and seasonal responsiveness. Furthermore, a reduction ofpergene dosage inperheterozygous mutants (per-/+) pleiotropically decreased the diapause incidence, shortened post-diapause development, and delayed the timing of daily behavior, in a manner phenotypically reminiscent of wild-type individuals. Altogether, this combination of observational and experimental research strongly suggests thatperis a master regulator of biological rhythms and may contribute to the observed life cycle differences between bivoltine (two generation) and univoltine (one generation)O. nubilalis. HighlightsNatural ecotypes with divergentperiod(per) genotypes differ in their daily and seasonal responses to photoperiodLater daily activity, reduced diapause incidence, and shorter post-diapause development is associated with reducedpermRNA abundanceperis essential for short-day recognition and daily timingReducedpergene dosage shortened post-diapause development and delayed locomotor activity
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- Award ID(s):
- 2416175
- PAR ID:
- 10588442
- Publisher / Repository:
- bioRxiv
- Date Published:
- Format(s):
- Medium: X
- Institution:
- bioRxiv
- Sponsoring Org:
- National Science Foundation
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