Report of Systematic Zoology Lab Practicum, Volume 6: e14; August, 2015

Partial sequence of the mitochondrion-encoded 16S rRNA gene determined from the polychaete cf. Nereis heterocirrata (Annelida: Nereididae) collected in Oshoro Bay, Hokkaido, northern Japan

Ryotaro Nii and Tadasuke Okubo

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

Material and Methods
A polychaete was collected among byssi of the bivalves Mytilus trossulus Gould, 1850 and Septifer virgatus (Wiegmann, 1837) in the intertidal zone of Oshoro Bay, Hokkaido, Japan, about 43°12′N, 140°51′E, on 25 May 2015 by Ryotaro Nii, photographed and fixed in 99% EtOH by Shinri Tomioka; the specimen was tentatively identified by Hiroshi Kajihara as Nereis heterocirrata Treadwell, 1931 by a reference to Imajima (1996: 152). Total DNA was extracted from anterior portion 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 ICHU2130879.
      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 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 v5.2 software (Tamura et al. 2011).


Results
A total of 462 bp of 16S rRNA gene partial sequence was determined from ICHU2130879, identified as Nereis heterocirrata (see Appendix).


Taxonomy
Phylum Annelida
Family Nereididae Blainville, 1818
Genus Nereis Linnaeus, 1758
Nereis heterocirrata Treadwell, 1931
[Japanese name: higebuto-gokai]
(Fig. 1)


Fig. 1. ICHU2130879 tentatively identified as Nereis heterocirrata Treadwell, 1931, photographed by Shinri Tomioka; arrow indicates the ventral tentacular cirrus, which is characteristically thick in this species.

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.