Report of Systematic Zoology Lab Practicum, Volume 5: e06; August, 2014

Cytochrome c oxidase subunit I gene partial sequence of an unidentified polynoid polychaete (Annelida: Polynoidae) from Oshoro Bay on the Sea-of-Japan coast of Hokkaido, northern Japan

Yuuki Ohya and Masakazu Hoshino

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

Material and Methods
A scale worm was obtained from Oshoro Bay, Hokkaido, Japan, about 43°12′N, 140°51′E, on 19 May 2014 by Yuuki Ohya and Masakazu Hoshino, photographed and fixed in 99% EtOH by Takumi Onishi. Total DNA was extracted from the anterior one tenth 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 ICHU2120527 (contact: Hiroshi Kajihara, kazi@mail.sci.hokudai.ac.jp).
      PCR amplification of the mitochondrial cytochrome c oxidase subunit I gene (COI) was performed using LCO1490 (5′-GGTCAACAAATCATAAAGATATTGG-3′) and HCO2198 (5′-TAAACTTCAGGGTGACCAAAAAATCA-3′) (Folmer et al. 1994) with 2720 Thermal Cycler (Applied Bopsystems) in a 20-µl reaction volume containing 1 µl of template total DNA (approximately 10–100 ng) and 19 µl of premix 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 Annelida
Family Polynoidae Kinberg, 1856
Polynoidae gen. et sp. indet.

(Figs 1, 2)

A total of 658 bp of partial sequence of COI was determined from Polynoidae sp. (see Appendix). A nucleotide BLAST search (Altschul et al. 1997) at the NCBI website (https://blast.ncbi.nlm.nih.gov/) resulted in that our sequence from Oshoro was most similar to two polynoid sequences with the same scores (83% similarity; E value = 1e-169; 100% in query coverage): KJ676625 from Austrolaenilla antarctica Bergström, 1916 (Neal et al. 2014) and HQ024272 from Bylgides groenlandicus (Malmgren, 1867) (Carr et al. 2011).


Fig. 1. ICHU2120527, an unidentified polynoid, head, dorsal view.




Fig. 2. ICHU2120527, an unidentified polynoid, tail, dorsal view.



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.