Report of Systematic Zoology Lab Practicum, Volume 6: e13; August, 2015

A partial sequence of the mitochondrion-encoded 16S rRNA gene from the polychaete Eulalia viridis (Annelida: Phyllodocidae) collected in Oshoro Bay, Hokkaido, northern Japan

Yuto Nozaki and Ryo Suzuki

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

Material and Methods
A polychaeta was collected from under stone in the intertidal zone of Oshoro Bay, Hokkaido, Japan, about 43°12&min;N, 140°51&min;E, on 1 June 2015 by Yuto Nozaki and Ryo Suzuki. It was identified as Eulalia viridis (Linnaeus, 1767) by Hiroshi Kajihara, then photographed alive and fixed in 99% by Shinri Tomioka. Total DNA was extracted from the part of legs 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 ICHU2130775 (contact: Dr. Hiroshi Kajihara, kazi@mail.sci.hokudai.ac.jp).
      PCR amplification was attempted with the primer pairs LoboF1 (5′-KBTCHACAAAYCAYAARGAYATHGG-3′) and LoboR1 (5′-TGTTTYTTYGGWCAYCCWGARGTTTA-3′) (Lobo et al. 2013) for the mitochondrial cytochrome c oxidase subunit I gene (COI) and 16S ar-L (5′-CGCCTGTTTATCAAAAACAT-3′) and br-H (5′-CCGGTCTGAACTCAGATCACGT-3′) (Palumbi et al. 1991) for the 16S rRNA gene. PCR products were visualized by electrophoresis in 1% agarose gel. Of the two gene markers that were attempted, only 16S rRNA gene was confirmed to be successfully amplified. The PCR was performed by a thermal cycler, 2720 Thermal Cycler (Applied Biosystems), 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 products of the 16S rRNA gene 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 v5.2 software (Tamura et al. 2011).


Results
Phylum Annelida
Class Polychaeta
Order Phyllodocida
Family Phyllodocidae Örsted, 1843
Genus Eulalia Savigny, 1822
Eulalia viridis (Linnaeus, 1767)
[Japanese name: samidori-sashiba]
(Figs 1, 2)
A 500-bp partial sequence of the mitochondrial 16S rRNA gene was determined from ICHU2130775, identified as Eulalia viridis (see Appendix). A nucleotide BLAST search (Altschul et al. 1997) at the NCBI website (https://blast.ncbi.nlm.nih.gov/) showed that our sequence was most similar to AY340455 (93% similarity; E value = 0.0; 100% in query coverage), a 16S rRNA partial sequence of the same species from Europe (Rousset et al. 2007).



Fig. 1. Eulalia viridis (Linnaeus, 1767) (ICHU2130775) collected in Oshoro Bay, Hokkaido, Japan, photographed by Shinri Tomioka.


Fig. 2. Eulalia viridis (Linnaeus, 1767) (ICHU2130775), magnification of head, photographed by Shinri Tomioka.



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.