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Amplification and Sequencing of the Complete mtDNA of the Endangered Bitterling, Acheilognathus longipinnis (Cyprinidae), using Environmental DNA from Aquarium Water

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Abstract

The Itasenpara bitterling, Acheilognathus longipinnis Regan, 1905, in the family Cyprinidae, is an endangered freshwater fish endemic to Japan. Designated a Japanese natural monument, research on A. longipinnis specimens is strictly restricted by law. In this study, we supplement existing genetic information and provide a resource for future studies that lack access to the biomaterials of this protected species. Using environmental DNA (eDNA) sourced from aquarium water, we amplified overlapping mitochondrial DNA (mtDNA) fragments of 4.9 to >8.0 kbps to obtain the complete mitochondrial genome of A. longipinnis. The 16 772 bp circular genome contains the 37 genes and two non-coding regions typically found in vertebrate mtDNAs. The mtDNA obtained has high nucleotide similarity with congeneric species. Moreover, the 12S rRNA and nd2 gene sequences of the mtDNA were identical to those previously reported for A. longipinnis, indicating that the mtDNA amplified from eDNA corresponds to that of A. longipinnis. This study represents a novel utilisation of museum and aquarium resources to obtain mtDNA data from a protected species. Our experimental procedure can potentially broaden the opportunities for obtaining long-sequence genetic information on very rare and/or legally sensitive organisms for which the development of a non-invasive eDNA survey procedure is desirable.

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ACKNOWLEDGMENTS

We would like to express our gratitude to Prof. Dr. Keiichi Takahashi, Director of the Lake Biwa Museum, who gave us permission to collect water from the rearing water of the Itasenpara bitterling.

Funding

This research was financially supported by the Grant for Collaborative Research, Nagahama Institute of Bio-Science and Technology (FY2020) to AK.

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Authors and Affiliations

  1. Nagahama Institute of Bio-Science and Technology, Shiga, Japan

    R. Kakehashi, S. Ito, K. Yasui, Ch. Kambayashi & A. Kurabayashi

  2. Lake Biwa Museum, Kusatsu, Shiga, Japan

    Sh. Kanao

  3. North-West University, Potchefstroom, South Africa

    A. Kurabayashi

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  1. R. Kakehashi
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Correspondence to A. Kurabayashi.

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Kakehashi, R., Ito, S., Yasui, K. et al. Amplification and Sequencing of the Complete mtDNA of the Endangered Bitterling, Acheilognathus longipinnis (Cyprinidae), using Environmental DNA from Aquarium Water. J. Ichthyol. 62, 280–288 (2022). https://doi.org/10.1134/S0032945222020072

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