Report of Systematic Zoology Lab Practicum, Volume 4: e04; August 2013

An attempt to sequence the cytochrome c oxidase subunit I gene from Vayssierea elegans (Mollusca: Gastropoda: Nudibranchia)

Yui Kato

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

Material and Methods
A sea slug was obtained subtidally at Oshoro Bay, Hokkaido, Japan, about 43°12′N, 140°51′E, on 20 May 2013 by Yui Kato, photographed and identified by Hiroshi Kajihara as Vayssierea elegans (Baba, 1930) by a reference to Hamatani (1992), 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.
      An about 650-bp fragment of mitochondrial cytochrome c oxidase subunit I gene (COI) was amplified by polymerase chain reaction (PCR) using LCO1490 (5′-GGTCAACAAATCATAAAGATATTGG-3′) and HCO2198 (5′-TAAACTTCAGGGTGACCAAAAAATCA-3′) (Folmer et al. 1994). A hot start PCR was performed by a thermal cycler, 2720 Thermal Cycler (Applied Biosystem), 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), 0.2-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 v5.2 software (Tamura et al. 2011).

Results and Discussion
A total of 626 bp of COI sequence was determined from Vaysseierea elegans (see Appendix). However, a BLAST search (Altschul et al. 1997), implemented at the NCBI website (http://www.ncbi.nlm.nih.gov/), indicated that the sequence represents either a bacterial symbiont/contamination or a pseudogene. The latter possibility is likely because the nucleotide sequence was not translatable into any significant amino acid sequence.

Taxonomy
Phylum Mollusca
Class Gastropoda
Order Nudibranchia
Family Vayssiereidae
Genus Vayssierea Risbec, 1928
Vayssierea elegans (Baba, 1930)
[Japanese name: Okada-umiushi]
(Fig. 1)



Fig. 1. Vayssierea elegnas (ICHU02110219), dorsal view.




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.