Abstract
To compensate for the limited number of morphological characteristics of fish eggs and larvae, we established a convenient and robust method of species identification for eggs of the Japanese eel (Anguilla japonica) using a real-time polymerase chain reaction (PCR) that can be performed onboard research ships at sea. A total of about 1.2 kbp of the mitochondrial 16S ribosomal RNA gene sequences from all species of Anguilla and 3 other anguilliform species were compared to design specific primer pairs and a probe for A. japonica. This real-time PCR amplification was conducted for a total of 44 specimens including A. japonica, A. marmorata, A. bicolor pacifica, and 6 other anguilliform species. Immediate PCR amplification was only observed in A. japonica. We then tested this method under onboard conditions and obtained the same result as had been produced in the laboratory. These results suggest that real-time PCR can be a powerful tool for detecting Japanese eel eggs and newly hatched larvae immediately after onboard sampling during research cruises and will allow targeted sampling efforts to occur rapidly in response to any positive onboard identification of the eggs and larvae of this species.
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Acknowledgements
We sincerely thank the following colleagues for assistance in sampling: S. Sasai, J. G. Inoue, A. Shinoda, and Y. Kimura. We are grateful to captain S. Suzuki and the crew of the RV Tansei-Maru, Ocean Research Institute, University of Tokyo, for their help in the onboard testing of the new method. Thanks are due to M. Otsuka, T. Saito, K. Takeishi, and H. Shiraga of Applied Biosystems Japan for their technical support and for providing us reagents and equipment for our molecular experiments. The paper greatly benefited from critical comments and suggestions on an early draft by M. J. Miller. We thank M. Oya for her assistance in many aspects of the project.
S.W. was partly supported by the Ito Foundation for Promotion of Ichthyology and the Sasakawa Scientific Research Grant from the Japan Science Society. S.W. and T. Y. were supported by Research Fellowship of the Japan Society for the Promotion of Science for Young Scientists. This study was supported partly by Grants-in-Aid (08456094, 10460081, 08041139, 11691177, and 12NP0201) from the Ministry of Education, Culture, Sports, Science and Technology of Japan; “Research for the Future” Program JSPS-RFTF 97L00901 from the Japan Society for the Promotion of Science; Touwa Shokuhin Shinkoukai; and the Eel Research Foundation of Nobori-kai.
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Watanabe, S., Minegishi, Y., Yoshinaga, T. et al. A Quick Method for Species Identification of Japanese Eel (Anguilla japonica) Using Real-Time PCR: An Onboard Application for Use During Sampling Surveys. Mar Biotechnol 6, 566–574 (2004). https://doi.org/10.1007/s10126-004-1000-5
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DOI: https://doi.org/10.1007/s10126-004-1000-5