<i>Minos</i> -mediated transgenesis in the pantry moth <i>Plodia interpunctella</i>

Transposon-mediated transgenesis has been widely used to study gene function in Lepidoptera, with <i>piggyBac</i> being the most commonly employed system. However, because the <i>piggyBac</i> transposase originates from a lepidopteran genome, it raises concerns about endogenous activation, remobilization, and silencing of transgenes, thus questioning its suitability as an optimal tool in Lepidoptera. As an alternative, we evaluated the dipteran-derived <i>Minos</i> transposase for stable germline transformation in the pantry moth, <i>Plodia interpunctella</i>. We injected syncytial embryos with transposase mRNA, along with donor plasmids encoding <i>3xP3::EGFP</i> and <i>3xP3::mCherry</i> markers of eye and glial tissues. Across multiple experiments, we found that G₀ injectees could transmit <i>Minos</i> transgenes through the germline even in the absence of visible marker expression in the soma, and that large mating pools of G₀ founders consistently produced transgenic offspring at efficiencies exceeding 10%. Using these methods, we generated transgenic lines with a dual expression plasmid, using <i>3xP3::mCherry</i> for driving red fluorescence in eyes and glial tissues, as well as the <i>Fibroin-L</i> promoter expressing the recently developed mBaoJin fluorescent protein in the silk glands. This demonstrated the feasibility of screening two pairs of promoter activity in tissues of interest. Collectively, these results-along with previous findings in the silkworm <i>Bombyx mori</i>-demonstrate that <i>Minos</i> achieves robust germline integration of transgenes in Lepidoptera, offering a valuable pathway to the genetic modification of species where the remobilization or suppression of <i>piggyBac</i> elements might be rampant.