Introducing Exogenous DNA Vectors Directly into <i>Trypoxylus dichotomus</i> Larvae Via In Vivo Electroporation.

Abstract

In the Japanese rhinoceros beetle <i>Trypoxylus dichotomus</i>, gene function studies have relied mainly on systemic larval RNA interference (RNAi), as gain-of-function techniques remain underdeveloped and germline transgenesis is impractical given the species' approximately one-year generation time. In addition, because larval RNAi is systemic, it has been difficult to analyze the function of lethal genes. Here, we present a simple and efficient protocol for the direct introduction of exogenous DNA into <i>T. dichotomus</i> larvae via in vivo electroporation. This protocol includes optimized procedures for adult breeding and egg collection, as well as a rigorously parameterized electroporation technique that delivers a <i>piggyBac</i> transposon vector into region-specific larval tissues. Within one day after electroporation, treated larvae exhibit mosaic expression of a reporter gene, enabling rapid tissue-specific functional analysis without the need to establish stable germline transgenic lines. Moreover, the key promoter used in this system (<i>T. dichotomus actinA3</i> promoter) is effective across diverse insect species, indicating that the method can be readily adapted to other non-model insects. Overall, this electroporation-based approach provides a valuable gain-of-function tool for <i>T. dichotomus</i> and potentially many other insect species. Key features • A simple and efficient method for in vivo electroporation of <i>T. dichotomus</i> larvae. • Applicable to other insect species. • The <i>T. dichotomus actinA3</i> promoter functions effectively across a diverse range of insect species. • An optimized method for egg collection in <i>T. dichotomus.</i>