Efficient RNA interference method during caste differentiation with hormone treatment in the termite Reticulitermes speratus (Isoptera: Rhinotermitidae)

Ryutaro Suzuki; Yudai Masuoka; Ryohei H Suzuki; Kiyoto Maekawa

doi:10.3389/finsc.2023.1188343

Efficient RNA interference method during caste differentiation with hormone treatment in the termite Reticulitermes speratus (Isoptera: Rhinotermitidae)

Front Insect Sci. 2023 May 9:3:1188343. doi: 10.3389/finsc.2023.1188343. eCollection 2023.

Authors

Ryutaro Suzuki^{1

2}, Yudai Masuoka^{1

3}, Ryohei H Suzuki^{1

4}, Kiyoto Maekawa⁵

Affiliations

¹ Graduate School of Science and Engineering, University of Toyama, Toyama, Japan.
² Ishikawa Insect Museum, Hakusan, Japan.
³ Institute of Agrobiological Sciences, National Agriculture and Food Research Organization, Tsukuba, Japan.
⁴ Research and Development Headquarters, Earth Corporation, Ltd. Ako, Japan.
⁵ Academic Assembly, University of Toyama, Toyama, Japan.

Abstract

Unveiling the proximate mechanism of caste differentiation is crucial for understanding insect social evolution, and gene function analysis is an important tool in this endeavor. The RNA interference (RNAi) technique is useful in termites, but its knockdown effects may differ among species. One of the most important model species in the field of termite sociogenomics is Reticulitermes speratus Kolbe (Isoptera: Rhinotermitidae). Presoldier and worker differentiation of this species can be artificially induced by juvenile hormone and 20-hydroxyecdysone application, respectively. However, appropriate RNAi technique of genes expressed during caste differentiation has never been considered. To clarify this issue, first, we injected nine different volumes of nuclease-free water (NFW, 0-404.8 nL) into workers and found that survival and caste differentiation rates were strongly reduced by the application of the top three largest volumes. Second, we injected double-stranded (ds) RNA of ecdysone receptor homolog (RsEcR) (2.0 µg/151.8 nL NFW) into workers with hormone treatments. The expression levels of RsEcR were significantly reduced at 9 days after dsRNA injection. RsEcR RNAi strongly affected both molting events during presoldier and worker differentiation induced by hormone treatments. The present results highlight the need for caution regarding injection volumes for RNAi experiments using hormone treatments. We suggest that the injection of dsRNA solution (2 µg; approximately 100-200 nL) is suitable for RNAi experiments during caste differentiation induced by hormone application in R. speratus.

Keywords: 20-hydroxyecdysone; RNAi; caste differentiation; ecdysone receptor gene; juvenile hormone.

Grants and funding

This study was supported in part by Grants-in-Aid for JSPS Fellows (No. 18J15134 to RS) and Scientific Research (No. 21K19293 and 22H02672 to KM) from the Japan Society for the Promotion of Science.