Report of Systematic Zoology Lab Practicum, July, 2012


Cytochrome c oxidase subunit I partial sequence of Phascolosoma scolops (Sipuncula)


Yuri Tanaka and Akari Toyoda

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


Material and Methods
A peanut-worm was collected intertidally among mussel bed in Oshoro Bay, Hokkaido, Japan, about 43°12′N, 140°51′E, on 28 May 2012 by Yuri Tanaka, photographed and identified by Hiroshi Kajihara as Phascolosoma scolops (Selenka and De Man, 1883), and fixed in 99% EtOH. DNA was extracted from tail assembly 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 ICHU22100080 (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, DNA Engine, 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 v5 software (Tamura et al. 2011).


Results
A total of 646 bp of COI sequence was determined from ICHU22100080, identified as Phascolosoma scolops (Selenka and De Man, 1883) (see Appendix).


Taxonomy
Phylum Sipuncula
Class Phascolosomatidea
Order Phascolosomatiformes
Family Phascolosomatidae Stephen and Edmonds, 1972
Genus Phascolosoma Leukart, 1828
Phascolosoma scolops (Selenka and De Man, 1883)
(Figs 1, 2)


Fig. 1. Phascolosoma scolops (Selenka and De Man, 1883), ICHU22100080, photographed by Hiroshi Kajihara.


Fig. 2. Phascolosoma scolops (Keferstein, 1866), ICHU22100080.

(Selenka & de Man, 1883), ICHU22100080, photomicrograph of hooks, showing the characteristic triangular transparent area (indicated by asterisks).



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.

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

Appendix
COI sequence from ICHU22100080 identidied as Phascolosoma scolops (Selenka and De Man, 1883).

CAAAAAATCAAAATAGATGTCTGAAAAGAATAGGGTCTCCGCCCCCTCCTGGGTCAAAAAAGGCAGTATTTAAATTTCGATCTGTTAGTAATATTGTAATAGCTCCCGCTAATACAGGGAGAGCTAGAAGTAAAAGGATAACTGTAATAAAGGCTGCCCATACAAATAAAGGTACTCGTTCCCAAGAAAATATTCTAGGTCGTATATTTGTTACTGTAGAAATAAAATTTAATGCACCTAAAATTGATCTTACTCCGGCTAAATGAAGAGAGAAGATAGCTAAGTCTACTGATGCGCCTGCATGAGCTAGAGCACCAGATAAAGGAGGATAAACTGTTCAGCCAGTACCAACTCCTTTTTCTACGGCCCTAGATGCTAGTAGGAGACATAGAGCAGGGGGTAATAATCAAAAACTAAGATTATTTAAACGAGGAAAAGCTATATCTGGAGCTCCAATTATTAGTGGAATTAACCAGTTTCCAAATCCTCCAATTAGAACAGGTATTACTAAAAAGAAAATTATTAAAAATGCATGAGCTGTAACAATAACATTATAGAGCTGGTCTCTACCTAATAAAGATCCAGGCTGGCCAAGTTCCGCTCGAATAAGAAGTCTTATTGAAGTTCCCATGAGGCCTGATCAGAT