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.