Report of Systematic Zoology Lab Practicum, Volume 4: e02; August, 2013

Partial sequence of the nuclear 28S rRNA gene of the sea snail Omphalius rusticus (Mollusca: Gastropoda: Trochidae) from the intertidal zone in Oshoro Bay on the Sea-of-Japan coast of Hokkaido, northern Japan

Yuriko Saheki and Kenta Tahara

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

Material and Methods
A sea snail was obtained in the intertidal zone of Oshoro Bay, Hokkaido, Japan, about 43°12′N, 140°51′E, on 20 May 2013, by Yuriko Saheki and Kenta Tahara. It was photographed by Takumi Onishi and fixed in 99% EtOH; the species was identified by Hiroshi Kajihara as Omphalius rusticus (Gmelin, 1791) by a reference to Sasaki (2000), DNA was extracted from a part of the foot 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 ICHU2110164 (contact: Dr. Hiroshi Kajihara, kazi@mail.sci.hokudai.ac.jp).
      An about 1.2Kb fragment of 28S rRNA gene was amplified by polymerase chain reaction (PCR) using LSU5 (5′-ACCCGCTGAAYTTAAGCA-3′) and LSU3 (5′-TCCTGAGGGAAACTTCGG-3′) (Littlewood et al. 1994). A hot start 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. Amplification by the primer pair LCO1490 and HCO2198 (Folmer et al. 1994) was unsuccesful.
      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 v5.1 software (Tamura et al. 2007).


Results
Phylum Molluscaa
Class Gastropoda
Order Vetigastropoda
Family Trochidae
Omphalius rusticus (Gmelin, 1791)
[Japanese name: koshidaka-gangara]
(Figs 1, 2)

A total of 1054 bp of 28S rRNA sequence was determined from Omphalius rusticus (see Appendix). A nucleotide BLAST search (Altschul et al. 1997) at the NCBI website (https://blast.ncbi.nlm.nih.gov/) resulted in that our sequence from Oshoro is completely identical (query coverage = 100%; E value = 0.0; identity = 100%) to EU530051, which is a sequence of Omphalius rusticus from Russia (Williams et al 2008).



Fig. 1. Omphalius rusticus (Gmelin, 1791), ICHU2110164, dorsal veiw of the shell.


Fig. 2. Omphalius rusticus (Gmelin, 1791), ICHU2110164, ventral veiw of the shell; the species can be distinguished from Chlorostoma lischkei and C. turbinatum in that the color around the umblicus is not greenish; also, spiral ribs are more developed on the base in C. lischkei and C. turbinatum than in O. rusticus



References

Altschul, S. F., Madden, T. L., Schaffer, 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.