Report of Systematic Zoology Lab Practicum, August, 2010

Cytochrome c oxidase subunit I partial sequence of Zygonemertes shintai (Nemertea: Hoplonemertea: Monostilifera: Amphiporidae)

Takumi Onishi

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

Material and Methods
A ribon worm specimen was obtained among mussels at Oshoro Bay, Hokkaido, Japan, about 43°12′N, 140°51′E, on 31 May 2010 by Takumi Onishi, photographed and identified by Hiroshi Kajihara as Zygonemertes shintai based on Kajihara (2002), and fixed in 99% EtOH. DNA was extracted from the anterior half 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 ICHU22080267 (contact: Dr. Hiroshi Kajihara, kazi@mail.sci.hokudai.ac.jp).
      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).

Results
A total of 589 bp of COI sequence was determined from Zygonemertes shintai Kajihara, 2002 (see Appendix).

Taxonomy
Order Monostilifera
Family Amphiporidae McIntosh, 1874
Genus Zygonemertes Montgomery, 1897
Zygonemertes shintai Kajihara, 2002
(Figs 1–3)



Fig. 1. Zygonemertes shintai Kajihara, 2002 (ICHU22080171), contracted animal.



Fig. 2. Zygonemertes shintai Kajihara, 2002 (ICHU22080171), ventral surface.





References

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.