Abstract
Herbivorous land crabs such as Chiromantes haematocheir and C. dehaani show biomass-degrading activities. In this study, we performed RNA-seq analysis to detect biomass-degrading enzymes. A de novo transcriptome assembly in the midgut glands of molting and non-molting C. haematocheir crabs was constructed using RNA sequencing. Illumina sequencing generated 44,937,002 and 44,394,310 reads from the two midgut glands. In total, 178,710 contigs with an average length of 750 bp and an N50 value of 1,235 bp were assembled, of which 37,890 contigs were annotated using BLASTx search against the NCBI database. We identified 22 contigs (11 genes) belonging to the laccase family and 44 contigs (22 genes) belonging to the peroxidase family. Sixteen contigs (three genes) belonging to the GH9 cellulase family were also detected. We selected the gene accounting for the majority of expressed laccase and analyzed its properties. The 24131-laccase transcript (2465 bp) had one complete open reading frame, nt 149–1987, encoding a protein of 613 amino acids with a deduced molecular mass of 67.708 kDa. The enzyme was shown to belong to the multicopper oxidase family. The 24131-laccase protein was confirmed to have oxidation activity against 2,6-dimethoxyphenol by ectopic expression in Escherichia coli. Laccase activity was significantly enhanced by feeding land crabs with plant diets. These data suggest that the enzyme plays an important role in the digestion of lignin in the guts of land crabs.
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Acknowledgements
This study was supported by JSPS KAKENHI Grant number JP19K05173, the Noda Institute for Scientific Research (NISR) research grant, and the IWATANI NAOJI FOUNDATION.
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Funding was provided by Meijo University, JSPS KAKENHI Grant number JP19K05173, and the Noda Institute for Scientific Research (NISR) research grant.
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All the authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by KM and YB. The first draft of the manuscript was written by KM, and all the authors commented on previous versions of the manuscript. All the authors read and approved the final manuscript.
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No approval of research ethics committees was required to accomplish the goals of this study because experimental work was conducted with unregulated invertebrate species in Japan. However, as land crabs are generally accepted to be higher invertebrates, this work was performed according to the guideline of the Meijo University Animal Ethics committee. The authors also referred to “The Basel Declaration” and “Guidelines for the treatment of animals in behavioral research and teaching”.
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Miyake, K., Baba, Y. De novo transcriptome assembly of the midgut glands of herbivorous land crabs, Chiromantes haematocheir, and identification of laccase genes involved in lignin degradation. J Comp Physiol B 192, 247–261 (2022). https://doi.org/10.1007/s00360-021-01424-3
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DOI: https://doi.org/10.1007/s00360-021-01424-3