Electroporation-mediated RNA interference reveals a role of the multicopper oxidase 2 gene in dragonfly cuticular pigmentation
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Dragonflies are colorful insects, and recent RNA sequencing studies have identified a number of candidate genes potentially involved in their color pattern formation and color vision. However, functional aspects of such genes have not been assessed due to the lack of molecular genetic tools applicable to dragonflies. We established an electroporation-mediated RNA interference (RNAi) procedure using the tiny dragonfly Nannophya pygmaea Rambur, 1842 (Odonata: Libellulidae) that targets the multicopper oxidase 2 gene (MCO2; also known as laccase2 gene) responsible for cuticular pigmentation in many insects. RNA sequencing of N. pygmaea and genomic survey of the dragonfly Ladona fulva identified four multicopper oxidase family genes: MCO1, MCO2, MCO3 and multicopper oxidase-related protein gene (MCORP). In N. pygmaea, MCO2 was specifically expressed around the cuticular pigmentation period, whereas MCO1 was constantly expressed. MCORP was expressed at adult stages, and MCO3 was scarcely expressed. When we applied in vivo electroporation, final instar larvae injected with MCO2 small interfering RNA became adults with patchy unpigmented regions. RNAi without in vivo electroporation did not affect cuticular pigmentation, suggesting that dragonflies do not show a systemic RNAi response. These results indicate that MCO2 is required for cuticular pigmentation across diverse insects, and highlight the usefulness of the electroporation-mediated RNAi method in dragonflies.
KeywordsNannophya pygmaea Laccase2 gene Gene knockdown Pigment Body color
We thank Hiroyuki Futahashi for collecting dragonfly samples. We would like to acknowledge i5K, Stephen Richards and Oliver Niehuis for allowing us access to the L. fulva genome data. This work was supported by the Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research 26660276 and 26711021 (to R. F.), and the Cooperative Research Grant of the Genome Research for BioResource, NODAI Genome Research Center, Tokyo University of Agriculture (to R. F., R. K.-M., and S. Y.). This research is partially supported by the Platform Project for Supporting Drug Discovery and Life Science Research (Platform for Drug Discovery, Informatics, and Structural Life Science) of the Japan Agency for Medical Research and Development.
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