Efficient transgenic system for the firebrat Thermobia domestica utilizing hyPBase and G0 founder prescreening

The firebrat, Thermobia domestica, is an apterygote model insect with favorable traits, including rapid generation turnover, high fecundity, and ease of laboratory rearing. We previously developed a method for embryo injection and CRISPR/Cas9-based genome editing in Thermobia. In the present study, we established a highly efficient transgenesis system using the hyperactive piggyBac transposase (hyPBase) to expand genetic manipulation techniques in Thermobia. By injecting embryos with a mixture of hyPBase mRNA and a donor plasmid expressing GFP under the control of an eye enhancer, we achieved the first successful transgenesis in Thermobia. Eye-specific GFP expression was observed in 5.7 % of G₀ individuals hatched from injected eggs. Notably, these GFP-positive G₀ founders exhibited significantly elevated germline transmission rates (53.3 %) compared with GFP-negative G₀ founders (19.0 %). Additionally, a significant difference in the proportion of G₁ transgenic progeny emerged between the GFP-positive and GFP-negative G₀ groups (20.0 % vs. 2.7 %), highlighting the utility of GFP expression as a predictor of transgenic G₁ offspring from injected G₀ founders. Furthermore, multiple transgene insertions mediated by hyPBase contributed to the increased transformation efficiency observed in G₀ founders with high transmission rates. Our findings offer valuable genetic toolkits for Thermobia that will facilitate advanced research on fundamental biological processes, such as the evolution of wings and metamorphosis.