Background

The Japanese rhinoceros beetle Trypoxylus dichotomus is useful from different perspectives, such as in developmental biology, morphology, ethology, ecology, biomimetics, aerodynamics, and drug discovery. For instance, Siva-Jothy (1987) revealed the feeding time of T. dichotomus from ecological research, Miyanoshita et al. (1996) isolated a new antibacterial peptide from larvae of T. dichotomus, Hongo (2007) clarified the relationship between horn length and body size in T. dichotomus, Emlen et al. (2012) showed that signaling through the insulin receptor is involved in T. dichotomus horn development, Chen et al. (2017) clarified that the elytron plate of T. dichotomus has a mechanism of high energy absorption, and Ohde et al. (2018) identified several horn formation genes using RNA-sequencing analysis and RNAi techniques.

Recently, the genome of T. dichotomus was clarified (Ogata, 2021; Morita et al., 2022), and various candidate genes required for a specific phenotype of interest were listed by RNA-sequencing analysis (Ohde et al., 2018; Zinna et al., 2018). Furthermore, the functions of the candidate genes were investigated using RNA interference (RNAi) method, the only gene function analysis tool for T. dichotomus developed so far (Emlen et al., 2012; Ito et al., 2013; Gotoh et al., 2015; Adachi et al., 2018, 2020; Ohde et al., 2018; Morita et al., 2019).

RNAi method for small insects generally requires specialized setups for microinjection, such as those for preparing glass capillaries, loading double-stranded RNA (dsRNA) into microcapillaries, injecting dsRNA into insects using high pressure, and manipulating the needle for injection under a stereomicroscope (e.g., Kim et al., 2004; Niimi et al., 2005; Masumoto et al., 2009; Posnien et al., 2009). The RNAi method in T. dichotomus does not require most of the above setups, it only requires a 1-mL syringe for dsRNA injection, since the beetle’s larval body size is as large as a mouse. This study provides a detailed protocol for the RNAi method in the third instar larvae of T. dichotomus (Figure 1). This method can also be applied to the first and second instar larvae and pupae of T. dichotomus.

Figure 1. Overview of RNA interference method procedure in Trypoxylus dichotomus.