Efficient RNA Interference Method During Caste Differentiation with Hormone Treatment in the Termite (Isoptera: Rhinotermitidae)

Overview

Journal Front Insect Sci

Date 2024 Mar 12

PMID 38469474

Authors

Ryutaro Suzuki

Yudai Masuoka

Ryohei H Suzuki

Kiyoto Maekawa

Affiliations

Soon will be listed here.

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 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 homolog () (2.0 µg/151.8 nL NFW) into workers with hormone treatments. The expression levels of were significantly reduced at 9 days after dsRNA injection. 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 .

Citing Articles

Candidate target genes of the male-specific expressed Doublesex in the termite Reticulitermes speratus.

Fujiwara K, Miyazaki S, Maekawa K PLoS One. 2024; 19(3):e0299900.

PMID: 38427681 PMC: 10906832. DOI: 10.1371/journal.pone.0299900.

References

Kucharski R, Maleszka J, Foret S, Maleszka R . Nutritional control of reproductive status in honeybees via DNA methylation. Science. 2008; 319(5871):1827-30. DOI: 10.1126/science.1153069. View

Saiki R, Hayashi Y, Toga K, Yaguchi H, Masuoka Y, Suzuki R . Comparison of gene expression profiles among caste differentiations in the termite Reticulitermes speratus. Sci Rep. 2022; 12(1):11947. PMC: 9279399. DOI: 10.1038/s41598-022-15984-z. View

Miyazaki S, Fujiwara K, Kai K, Masuoka Y, Gotoh H, Niimi T . Evolutionary transition of doublesex regulation from sex-specific splicing to male-specific transcription in termites. Sci Rep. 2021; 11(1):15992. PMC: 8346542. DOI: 10.1038/s41598-021-95423-7. View

Saiki R, Gotoh H, Toga K, Miura T, Maekawa K . High juvenile hormone titre and abdominal activation of JH signalling may induce reproduction of termite neotenics. Insect Mol Biol. 2015; 24(4):432-41. DOI: 10.1111/imb.12169. View

Terrapon N, Li C, Robertson H, Ji L, Meng X, Booth W . Molecular traces of alternative social organization in a termite genome. Nat Commun. 2014; 5:3636. DOI: 10.1038/ncomms4636. View

Masuoka Y, Toga K, Nalepa C, Maekawa K . A Crucial Caste Regulation Gene Detected by Comparing Termites and Sister Group Cockroaches. Genetics. 2018; 209(4):1225-1234. PMC: 6063233. DOI: 10.1534/genetics.118.301038. View

Miura T, Maekawa K . The making of the defensive caste: Physiology, development, and evolution of the soldier differentiation in termites. Evol Dev. 2020; 22(6):425-437. DOI: 10.1111/ede.12335. View

Abouheif E, Wray G . Evolution of the gene network underlying wing polyphenism in ants. Science. 2002; 297(5579):249-52. DOI: 10.1126/science.1071468. View

Zhou X, Oi F, Scharf M . Social exploitation of hexamerin: RNAi reveals a major caste-regulatory factor in termites. Proc Natl Acad Sci U S A. 2006; 103(12):4499-504. PMC: 1450200. DOI: 10.1073/pnas.0508866103. View

10.

Shigenobu S, Hayashi Y, Watanabe D, Tokuda G, Hojo M, Toga K . Genomic and transcriptomic analyses of the subterranean termite : Gene duplication facilitates social evolution. Proc Natl Acad Sci U S A. 2022; 119(3). PMC: 8785959. DOI: 10.1073/pnas.2110361119. View

11.

Toga K, Hojo M, Miura T, Maekawa K . Expression and function of a limb-patterning gene Distal-less in the soldier-specific morphogenesis in the nasute termite Nasutitermes takasagoensis. Evol Dev. 2012; 14(3):286-95. DOI: 10.1111/j.1525-142X.2012.00545.x. View

12.

Saleh M, van Rij R, Hekele A, Gillis A, Foley E, OFarrell P . The endocytic pathway mediates cell entry of dsRNA to induce RNAi silencing. Nat Cell Biol. 2006; 8(8):793-802. PMC: 2731564. DOI: 10.1038/ncb1439. View

13.

Masuoka Y, Yaguchi H, Suzuki R, Maekawa K . Knockdown of the juvenile hormone receptor gene inhibits soldier-specific morphogenesis in the damp-wood termite Zootermopsis nevadensis (Isoptera: Archotermopsidae). Insect Biochem Mol Biol. 2015; 64:25-31. DOI: 10.1016/j.ibmb.2015.07.013. View

14.

Watanabe D, Gotoh H, Miura T, Maekawa K . Social interactions affecting caste development through physiological actions in termites. Front Physiol. 2014; 5:127. PMC: 3988372. DOI: 10.3389/fphys.2014.00127. View

15.

Masuoka Y, Yaguchi H, Toga K, Shigenobu S, Maekawa K . TGFβ signaling related genes are involved in hormonal mediation during termite soldier differentiation. PLoS Genet. 2018; 14(4):e1007338. PMC: 5912798. DOI: 10.1371/journal.pgen.1007338. View

16.

Andersen C, Jensen J, Orntoft T . Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res. 2004; 64(15):5245-50. DOI: 10.1158/0008-5472.CAN-04-0496. View

17.

Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A . Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 2002; 3(7):RESEARCH0034. PMC: 126239. DOI: 10.1186/gb-2002-3-7-research0034. View

18.

Harrison M, Jongepier E, Robertson H, Arning N, Bitard-Feildel T, Chao H . Hemimetabolous genomes reveal molecular basis of termite eusociality. Nat Ecol Evol. 2018; 2(3):557-566. PMC: 6482461. DOI: 10.1038/s41559-017-0459-1. View

19.

Untergasser A, Nijveen H, Rao X, Bisseling T, Geurts R, Leunissen J . Primer3Plus, an enhanced web interface to Primer3. Nucleic Acids Res. 2007; 35(Web Server issue):W71-4. PMC: 1933133. DOI: 10.1093/nar/gkm306. View

20.

Maekawa K, Hayashi Y, Lo N . Termite sociogenomics: evolution and regulation of caste-specific expressed genes. Curr Opin Insect Sci. 2022; 50:100880. DOI: 10.1016/j.cois.2022.100880. View