title: Phenotypic screen for RNAi effects in the codling moth Cydia pomonella.
abstract: RNAi-based technologies have the potential to augment, or replace existing pest management strategies. However, some insect taxa are less susceptible to the induction of the post-transcriptional gene silencing effect than others, such as the Lepidoptera. Here we describe experiments to investigate the induction of RNAi in the codling moth, Cydia pomonella, a major lepidopteran pest of apple, pear, and walnut. Prior to a knockdown screen, fluorescently labeled small interfering RNA (siRNA) and double-stranded RNA (dsRNA) derived from green fluorescent protein (GFP) coding sequence were delivered to the surface of artificial diet to which neonate larvae were introduced and subsequently examined for the distribution of fluorescence in their tissues. Fluorescence was highly concentrated in the midgut but its presence in other tissues was equivocal. Next, dsRNAs were made for C. pomonella genes orthologous to those that have well defined deleterious phenotypes in Drosophila melanogaster. A screen was conducted using dsRNAs encoding cullin-1 (Cpcul1), maleless (Cpmle), musashi (Cpmsi), a homeobox gene (CpHbx), and pumilio (Cppum). The dsRNAs designed from these target genes were administered to neonate larvae by delivery to the surface of the growth medium. None of the dsRNA treatments affected larval viability, however Cpcul1-dsRNA had a significant effect on larval growth, with the average length of larvae about 3mm, compared to about 4mm in the control groups. Measurement of Cpcul1 transcript levels by quantitative real-time PCR (qRT-PCR) revealed a dose-dependent RNAi effect in response to increasing amount of Cpcul1-dsRNA. Despite their reduced size, Cpcul1-dsRNA-treated larvae molted normally and matured to adulthood in a manner similar to controls. In an additional experiment, Cpcul1-siRNA was found to induce similar stunting effect as that induced by Cpcul1-dsRNA.
authors.affiliation: Department of Entomology, Cornell University, New York State Agricultural Experiment Station, Geneva, NY 14456, USA; Department of Entomology, Nanjing Agricultural University, Nanjing, Jiangsu 210095, PR China.
authors.forename: Jinda
authors.initials: J
authors.lastname: Wang
authors.affiliation: Department of Entomology, Cornell University, New York State Agricultural Experiment Station, Geneva, NY 14456, USA.
authors.forename: Liuqi
authors.initials: L
authors.lastname: Gu
authors.affiliation: Department of Entomology, Cornell University, New York State Agricultural Experiment Station, Geneva, NY 14456, USA; Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI 48109, USA.
authors.forename: Stephen
authors.initials: S
authors.lastname: Ireland
authors.affiliation: USDA-ARS, Yakima Agricultural Research Laboratory, Wapato, WA 98951, USA.
authors.forename: Stephen F
authors.initials: SF
authors.lastname: Garczynski
authors.affiliation: Department of Entomology, Cornell University, New York State Agricultural Experiment Station, Geneva, NY 14456, USA. Electronic address: dck2@cornell.edu.
authors.forename: Douglas C
authors.initials: DC
authors.lastname: Knipple
mesh_terms: D000818:Animals; D003001:Cloning, Molecular; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D019476:Insect Proteins / Q000235:genetics* / Q000378:metabolism; D007814:Larva / Q000502:physiology; D009036:Moths / Q000235:genetics / Q000502:physiology*; D010802:Phylogeny; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000378:metabolism
chemical_list: D019476:Insect Proteins; D012330:RNA, Double-Stranded; D049452:Green Fluorescent Proteins
keywords: Cullin; Cydia pomonella; RNAi; siRNA