Report of Systematic Zoology Lab Practicum, August, 2011


Cytochrome c oxidase subunit I partial sequence of Acanthochitona achates (Mollusca: Polyplacophora)


Masumi Abe

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



Material and Methods
A chiton was obtained subtidally at Oshoro Bay, Hokkaido, Japan, about 43°12′N, 140°51′E, on 13 June 2011 by Masumi Abe, photographed and identified by Hiroshi Kajihara as Acanthochitona achates (Gould, 1859) based on Saito (2000: 23, fig. 9a, b), and fixed in 99% EtOH. DNA was extracted from soft parts (hard parts remained as morphological voucher specimen) 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 ICHU2209056 (contact: Dr. Hiroshi Kajihara, kazi@mail.sci.hokudai.ac.jp).
      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, iCycler (Bio-Rad), 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 v5.05 software (Tamura et al. 2011).

Results
A total of 679 bp of COI sequence was determined from Acanthochitona achates (see Appendix).

Taxonomy
Phylum Mollusca
Class Polyplacophora
Order Neoloricata
Family Acanthochitonidae
Genus Acanthochitona Gray, 1821
Acanthochitona achates (Gould, 1859)
(Figs 1–2)



Fig. 1. Acanthochitona achates (ICHU22090056), dorsal view.



Fig. 2. Acanthochitona achates (ICHU22090056), dorsal view with a ruler.



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.

Folmer, O., Black, M., Hoeh, W., Lutz, R. and Vrijenhoek, R. 1994. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Molecular Marine Biology and Biotechnology 3: 294–299.

Saito, H. 2000. Polyplacophora. Pp. 5–23. In: Okutani, T. (Ed.) Marine Mollusks in Japan. Tokai University Press, Tokyo.

Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. and Kumar, S. 2011. MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Phylogenetics and Evolution 28: 2731–2739.

Appendix
COI sequence from ICHU22090056 identidied as Acanthochitona achates (Gould, 1859).

ATCTGGTTTGGTTGGAACGGCTTTAAGTTTATTGATTCGAGCGGAGTTAGGTCAGCCTGGGACTCTTTTAGGGGACGATCAGTTATATAATGTGATTGTAACTGCTCATGCTTTTGTTATAATTTTTTTTTTGGTTATACCAATGATAATTGGGGGGTTTGGTAATTGACTTGTTCCATTAATATTAGGGGCTCCTGACATGGCTTTTCCTCGCTTAAATAATATGAGTTTCTGACTGTTACCCCCTTCTTTGTGTCTTTTATTAGCTTCTGGGGCAGTTGAAAGCGGAGTAGGTACGGGGTGGACTGTTTATCCTCCTTTAGCTAGAAATGTAGCTCATGCTGGAGGGTCTGTAGATCTTGCTATTTTTTCTCTCCATTTGGCTGGTGTTTCTTCAATTTTAGGGGCTGTAAATTTTATTACTACTGTGTTGAATATACGGTGAAGTGGGATACAATTGGAGCGTTTACCTTTATTTGTTTGGTCGGTTAAGATTACTGCTGTACTTCTTTTATTGTCTTTACCTGTCCTTGCTGGGGGGATTACTATACTTTTAACTGATCGTAATTTTAATACAGCTTTTTT