Report of Systematic Zoology Lab Practicum, Volume 5: e03; August, 2014

28S large subunit ribosomal RNA gene partial sequence of Arabella iricolor (Annelida: Oenonidae) from Oshoro Bay, Hokkaido, Japan

Yu Yoshida and Shota Murakami

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

Material and Methods
A polychaete was obtained among laminarian holdfasts in Oshoro Bay, Hokkaido, Japan, about 43°22′N, 140°85′E, on 23 May 2014 by Yu Yoshida and Shota Murakami, identified by Hiroshi Kajihara as Arabella iricolor (Montagu, 1804), and then photographed and fixed in 99% EtOH by Takumi Onishi. Total DNA was extracted from the posterior 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 ICHU2110564 (contact: Dr. Hiroshi Kajihara, kazi@mail.sci.hokudai.ac.jp).
      Hot start PCRs were 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). Amplification of the mitochondrial cytochrome c oxidase subunit I (COI) gene using LCO1490 (5′-GGTCAACAAATCATAAAGATATTGG-3′) and HCO2198 (5′-TAAACTTCAGGGTGACCAAAAAATCA-3′) (Folmer et al. 1994) was unsuccessful. An about 1.2K-bp fragment of the 28S rRNA gene was amplified by using the primer pair LSU5 (ACCCGCTGAAYTTAAGCA) and LSU3 (TCCTGAGGGAAACTTCGG) (Littlewood. 1994). The 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 (COI) or 3 min (28S); and a final elongation at 72°C for 7 min.
      The PCR products were 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 ver. 5 software (Tamura et al. 2011).


Results
Phylum Annelida
Family Oenonidae Kinberg, 1865
Genus Arabella Grube, 1850
Arabella iricolor (Montagu, 1804)
[Japanese name: seguro-isome]

(Figs 1, 2)

Only 341 bases near the 5′ end of the PCR products were reliably determined (see Appendix), because the chromatogram in the 3′ region was more or less disturbed. A nucleotide BLAST search (Altschul et al. 1997) at the NCBI website (https://blast.ncbi.nlm.nih.gov/) showed that our sequence from Oshoro was most similar to AY838857 (100% in query coverage; E value = 6e-109; 89% identity), a sequence of the congener Arabella semimaculata (Moore, 1911) (Struck et al. 2006). There was no 28S sequence of A. iricolor as of writing.



Fig. 1. Arabella iricolor (Montagu, 1804), ICHU2110564, head, dorsolateral view.


Fig. 2. Arabella iricolor (Montagu, 1804), ICHU2110564, tail.




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.