Report of Systematic Zoology Lab Practicum, Volume 5: e07; August, 2014

Mitochondrial cytichrome c oxidase subunit I gene partal sequence of an unidentified gammarid amphipod cf. Ampithoe sp. (Crustacea: Amphipoda) from Oshoro Bay, Hokkaido, Japan

Ayano Hata and Emi Kinoshita

Division of Biology, Department of Biological Sciences, School of Science, Hokkaido University, Sapporo 060-0810, Japan

Material and Methods
A gammarid amphipod was obtained from among laminarian holdfasts in Oshoro Bay, Hokkaido, Japan, about 43°22′N, 140°85′E, on 26 May 2014 by Ayano Hata and Emi Kinoshita, then photographed and fixed in 99% EtOH by Takumi Onishi. Total DNA was extracted from the abdomen of the body using the silica method (Boom et al. 1990) with some modifications. Extracted DNA was dissolved in 30 µl of deionized water and has been preserved at –20°C. Remaining morphological voucher specimen has been deposited at the Hokkaido University Museum under the catalogue number ICHU2120569 (contact: Dr. Hiroshi Kajihara, kazi@mail.sci.hokudai.ac.jp).
      An about 650-bp fragment of the mitochondrial cytochrome c oxidase subunit I (COI) gene was amplified by polymerase chain reaction (PCR) using LCO1490 (5′-GGTCAACAAATCATAAAGATATTGG-3′) and HCO2198 (5′-TAAACTTCAGGGTGACCAAAAAATCA-3′) (Folmer et al. 1994). A hot start PCR was performed by a thermal cycler in a 20-µl reaction volume containing 1 µl of template total DNA (approximately 10–100 ng) and 19 µl of a premixture made with 632-µl deionized water, 80-µl Ex Taq Buffer (TaKara Bio), 64-µl dNTP (each 25 mM), 8-µl each primer (each 10 µM), and 0.1-µl TaKara Ex Taq (5 U/µl, TaKaRa Bio). Thermal cycling condition comprised an initial denaturation at 95°C for 30 sec; 30 cycles of denaturation at 95°C for 30 sec, annealing at 45°C for 30 sec, and elongation at 72°C for 45 sec; and a final elongation at 72°C for 7 min.
      The PCR product was purified with the silica method (Boom et al. 1990). Both strands were sequenced with a BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) following the manufacturer's protocol, using the same primer set as the initial PCR amplification. Sequencing was performed with ABI Prism 3730 DNA Analyzer (Applied Biosystems). Chromatogram and sequence data were operated with MEGA v.5 software (Tamura et al. 2011).


Results Phylum Arthropoda
Subphylum Crustacea
Class Malacostraca
Order Amphipoda
cf. Ampithoe sp.
(Fig. 1)

We obtained a 643-bp partial sequence of the mitochondrion-encoded COI gene from our gammarid specimen ICHU2120569 (see Appendix). A nucleotide BLAST search (Altschul et al. 1997) at the NCBI website (https://blast.ncbi.nlm.nih.gov/) showed that our sequence was 95% identical with JX502959 (99% in query coverage; E value = 0.0) (Kim et al. unpublishe) from South Korea and 89% with GU048166 (99% in query coverage; E value = 0.0) (Pilgrim and Darling 2010) from the Pacific coast of the USA, both identified as Ampithoe lacertosa. Our material is closely related to them, if not conspecific. We tentatively labeled our material Ampithoe sp., although we did not attempt morphology-based identification.


Fig. 1. Cf. Ampithoe sp. (ICHU2120569) from Oshoro Bay, Hokkaido, Japan, photograph taken in life.




References

Altschul, S. F., Madden, T. L., Schäffer, A. A., Zhang, J., Zhang, Z., Miller, W., and Lipman, D. J. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research 25: 3389–3402.