Report of Systematic Zoology Lab Practicum, Volume 4: e05; August, 2013

Cytochrome c oxidase subunit I gene partial sequence of Aeolidia papillosa (Linnaeus, 1761) (Mollusca: Gastropoda: Nudibranchia)

Madoka Asano and Kohei Oguchi

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

Material and Methods
A sea slug was obtained intertidally at Oshoro Bay, Hokkaido, Japan, about 43°12′N, 140°51′E, on 20 May 2013 by Kohei Oguchi, photographed and identified by Hiroshi Kajihara as Aeolidia papillosa (Linnaeus, 1761), and fixed in 99% EtOH. DNA was extracted from foots 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 ICHU2110427 (contact: Dr. Hiroshi Kajihara, kazi@mail.sci.hokudai.ac.jp).
      An about 600-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 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 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 5 software (Tamura et al. 2011).

Results
A total of 611 bp of COI sequence was determined from Aeolidia papillosa (see Appendix). A BLAST search (Altschul et al. 1997), as implemented in the NCBI website (http://www.ncbi.nlm.nih.gov/), resulted in 98% maximum identity (with 0.0 E value) of the present sequence witw JX087536, a 658-bp strech of COI sequence from A. papillosa from Washington, USA. (Carmona et al., unpublished data).

Taxonomy
Phylum Mollusca
Class Gastropoda
Subclass Opisthobranchia
Order Nudibranchia
Suborder Aeolidina
Family Aeolidiae
Genus Aeolidia Cuvier, 1798
Aeolidia papillosa (Linnaeus, 1761)
[Japanese name: oomino-umiushi]
(Figs 1, 2)



Fig. 1. Aeolidia papillosa (Linnaeus, 1761), ICHU2110427, dorsal veiw, taken in life.



Fig. 2. Aeolidia papillosa (Linnaeus, 1761), ICHU2110427, ventral view, taken in life.



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.