title: Germline transformation of the western corn rootworm, Diabrotica virgifera virgifera.
abstract: The western corn rootworm (WCR), a major pest of maize, is notorious for rapidly adapting biochemically, behaviourally and developmentally to a variety of control methods. Despite much effort, the genetic basis of WCR adaptation remains a mystery. Since transformation-based applications such as transposon tagging and enhancer trapping have facilitated genetic dissection of model species such as Drosophila melanogaster, we developed a germline-transformation system for WCR in an effort to gain a greater understanding of the basic biology of this economically important insect. Here we report the use of a fluorescent-marked Minos element to create transgenic WCR. We demonstrate that the transgenic strains express both an eye-specific fluorescent marker and piggyBac transposase. We identified insertion-site junction sequences via inverse PCR and assessed insertion copy number using digital droplet PCR (ddPCR). Interestingly, most WCR identified as transgenic via visual screening for DsRed fluorescence proved to carry multiple Minos insertions when tested via ddPCR. A total of eight unique insertion strains were created by outcrossing the initial transgenic strains to nontransgenic WCR mates. Establishing transgenic technologies for this beetle is the first step towards bringing a wide range of transformation-based tools to bear on understanding WCR biology.
authors.affiliation: Department of Entomology, North Carolina State University, Raleigh, NC, USA.
authors.forename: F
authors.initials: F
authors.lastname: Chu
authors.affiliation: Department of Entomology, North Carolina State University, Raleigh, NC, USA.
authors.forename: W
authors.initials: W
authors.lastname: Klobasa
authors.affiliation: Department of Entomology, North Carolina State University, Raleigh, NC, USA.
authors.forename: P
authors.initials: P
authors.lastname: Wu
authors.affiliation: Department of Entomology, North Carolina State University, Raleigh, NC, USA.
authors.forename: S
authors.initials: S
authors.lastname: Pinzi
authors.affiliation: Department of Entomology, North Carolina State University, Raleigh, NC, USA.
authors.forename: N
authors.initials: N
authors.lastname: Grubbs
authors.affiliation: Department of Entomology, North Carolina State University, Raleigh, NC, USA.
authors.forename: S
authors.initials: S
authors.lastname: Gorski
authors.affiliation: Department of Entomology, North Carolina State University, Raleigh, NC, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Cardoza
authors.affiliation: Department of Entomology, North Carolina State University, Raleigh, NC, USA.
authors.forename: M D
authors.initials: MD
authors.lastname: Lorenzen
mesh_terms: D000818:Animals; D001517:Coleoptera / Q000235:genetics*; D005260:Female; D018014:Gene Transfer Techniques; D008164:Luminescent Proteins; D008297:Male; D019895:Transposases; D000097573:Red Fluorescent Protein
chemical_list: D008164:Luminescent Proteins; D019895:Transposases; D000097573:Red Fluorescent Protein; C473499:Minos transposase
keywords: Minos; embryonic microinjection; piggyBac; small-scale rearing