Report of Systematic Zoology Lab Practicum, August, 2010

Cytochrome c oxidase subunit I partial sequence of Harmothoe imbricata (Polychaeta: Phyllodocida: Polynoidae)

Yutaro Saiki

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

Material and Methods
A ragworm specimen was obtained subtidally at Oshoro Bay, Hokkaido, Japan, about 43°12′N, 140°51′E, on 10 May 2010 by Yutaro Saiki, photographed and identified by Hiroshi Kajihara as Harmothoe imbricata based on Uchida (1992: 334, pl. 66, fig. 3), and fixed in 99% EtOH. DNA was extracted from the part of the posterior half of 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 ICHU22080020 (contact: Dr. Hiroshi Kajihara,
      An about 600-bp fragment of mitochondrial cytochrome c oxidase subunit I gene (COI) 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, iCycler (Bio-Rad), 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°C 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 v4 software (Tamura et al. 2007).

A total of 601 bp of COI sequence was determined from Harmothoe imbricata (see Appendix).

Phylum Annelida
Class Polychaeta
Order Phyllodocida
Family Polynoidae Malmgren, 1867
Genus Harmothoe Kinberg, 1856
Harmothoe imbricata (Linnaeus, 1767)
(Figs 1–3)

Fig. 1. Harmothoe imbricata (ICHU22080020), dorsal view.

Fig. 2. Harmothoe imbricata (ICHU22080020), magnification of head.

Fig. 3. Harmothoe imbricata (ICHU22080020), further mangnification of the tip of the head.


Boom, R., Sol, C. J. A., Salimans, M. M. M., Jansen, C. L., Wertheim-van Dillen, P. M. E., and van der Noordaa, J. 1990. Rapid and simple method for purification of nucleic acids. Journal of Clinical Microbiology 28: 495–503.

Folmer, O., Black, M., Hoeh, W., Lutz, R. and Vrijenhoek, R. 1994. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Molecular Marine Biology and Biotechnology 3: 294–299.

Tamura, K., Dudley, J., Nei, M. and Kumar, S. 2007. MEGA4: Molecullar Evolutionary Genetics Analysis (MEGA) software version 4.0. Molecular Phylogenetics and Evolution 24: 1596–1599.

Uchida, H. 1992. Annelida. Pp. 310–373. In: Nishimura, S. (Ed.) Guide to Seashore Animals of Japan with Color Pictures and Keys. Vol.I Hoikusha, Osaka.

COI sequence from ICHU22080020 identidied as Harmothoe imbricata.