Efficienthyperactive piggyBactransgenesis inPlodiapantry moths

WhilepiggyBactransposon-based transgenesis is widely used in various emerging model organisms, its relatively low transposition rate in butterflies and moths has hindered its use for routine genetic transformation in Lepidoptera. Here, we tested the suitability of a codon-optimizedhyperactive piggyBactransposase (hyPBase) in mRNA form to deliver and integrate transgenic cassettes into the genome of the pantry mothPlodia interpunctella. Co-injection ofhyPBasemRNA with donor plasmids successfully integrated 1.5-4.4 kb expression cassettes driving the fluorescent markers EGFP, DsRed, or EYFP in eyes and glia with the3xP3promoter. Somatic integration and expression of the transgene in the G0injected generation was detectable from 72-hr embryos and onward in larvae, pupae and adults carrying a recessive white-eyed mutation. Overall, 2.5% of injected eggs survived into transgene-bearing adults with mosaic fluorescence. Subsequent outcrossing of fluorescent G0founders transmitted single-insertion copies of3xP3::EGFPand3xP3::EYFPand generated stable isogenic lines. Random in-crossing of a small cohort of G0founders expressing3xP3::DsRedyielded a stable transgenic line segregating for more than one transgene insertion site. We discuss howhyPBasecan be used to generate stable transgenic resources inPlodiaand other moths.