Report of Systematic Zoology Lab Practicum, August, 2012

28S rDNA partial sequence of Oerstedia phoresiae (Nemertea: Hoplonemerta: Monostilifera)

Toshihiro Arae and Mayu Horie

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

Material and Methods
A nemertean was obtained among laminarian holdfasts in Oshoro Bay, Hokkaido, Japan, about 43°12′N, 140°51′E, on 28 May 2012 by Toshihiro Arae and Mayu Horie, photographed and identified as Oerstedia phoresiae (Kulikova, 1987) by Hiroshi Kajihara, and fixed in 99% EtOH. DNA was extracted from the entire animal 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.
      Amplification of mitochondrial cytochrome c oxidase subunit I gene (COI) using LCO1490 (5′-GGTCAACAAATCATAAAGATATTGG-3′) and HCO2198 (5′-TAAACTTCAGGGTGACCAAAAAATCA-3′) (Folmer et al. 1994) was unsuccessful.
      An about 1000-bp fragment of 28S rRNA gene was amplified by polymerase chain reaction (PCR) using LSU5 (5′-ACCCGCTGAAYTTAAGCA-3′) andLSU3(5′-TCCTGAGGGAAACTTCGG-3′) (Littlewood et al. 1994). A hot start PCR was performed by a thermal cycler, DNA Engine(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 v.5 software (Tamura et al. 2011).

Results
A total of 1077 bp of partial 28S rDNA sequence was determined from Oerstedia phoresiae (Kurikova, 1987) (see Appendix). This is the first record of the species from Japanese waters.

Taxonomy
Phylum Nemertea
Class Enopla
Subclass Hoplonemertea
Order Monostilifera
Family Oerstediidae
Genus Oerstedia Quatrefages, 1846
Oerstedia phoresiae (Kurikova, 1987)
(Figs 1, 2)



Fig. 1. Oerstedia phoresiae (Kurikova, 1987), ICHU22100102, entire animal.




Fig. 2. Oerstedia phoresiae (Kurikova, 1987), ICHU22100102, enlargement of head.



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.