Electroporation-mediated functional analysis method of genes in the giant insect Trypoxylus dichotomus

The Japanese rhinoceros beetle, Trypoxylus dichotomus, possesses large horns on its head and thorax, features whose biological significance has been explored across various fields, including evolutionary developmental biology, behavioral ecology, and materials science. To investigate the molecular basis of these characteristics, systemic larval RNA interference (RNAi) has been employed as a primary loss-of-function genetic tool. However, gain-of-function analyses and region-specific gene function assessments remain underdeveloped, thereby limiting the comprehensive understanding of the molecular mechanisms involved. To address this limitation, we developed an in vivo electroporation technique to introduce exogenous DNA vectors directly into the somatic tissues of T. dichotomus larvae to express the genes of interest. Additionally, we utilized the piggyBac transposon system to insert the exogenous DNA vectors into the host genome for stable gene expression. Our findings indicate that the T. dichotomus actin A3 gene promoter exhibits sufficient transcriptional activity in the early postembryonic stage of T. dichotomus via electroporation. Furthermore, we observed that this promoter functions effectively across a diverse range of insect species, including the harlequin ladybug, Harmonia axyridis and the silkworm, Bombyx mori, suggesting the broad applicability of the T. dichotomus actin A3 promoter in various insects.