Report of Systematic Zoology Lab Practicum, Volume 5: e12; August, 2014


Cytochrome c oxidase subunit I gene partial sequence of Quasitetrastemma nigrifrons (Nemertea: Hoplonemerta: Monostilifera) from Oshoro Bay, Hokkaido, Japan


Chie Itoh and Kaho Sekiguchi

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



Material and Methods
A ribbonworm was obtained among laminarian holdfasts in Oshoro Bay, Hokkaido, Japan, about 43°12′N, 140°51′E, on 28 May 2014 by Chie Itoh and Kaho Sekiguchi, identified as Quasitetrastemma nigrifrons (Coe, 1904) by Hiroshi Kajihara, then photographed and fixed in 99% EtOH by Takumi Onishi. DNA was extracted from the posterior half of the body 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.
      PCR amplification of the mitochondrial cytochrome c oxidase subunit I gene (COI) was performed using LCO1490 (5′-GGTCAACAAATCATAAAGATATTGG-3′) and HCO2198 (5′-TAAACTTCAGGGTGACCAAAAAATCA-3′) (Folmer et al. 1994) with 2720 Thermal Cycler (Applied Bopsystems) 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 sec; 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 v.5 software (Tamura et al. 2011).

Results
Phylum Nemertea
Class Hoplonemertea
Subclass Hoplonemertea
Order Monostilifera
Family Tetrastemmatidae
Genus Quasitetrastemma Chernyshev, 2004
Quasitetrastemma nigrifrons (Coe, 1904)
[Japanese name: menoko-himomushi]
(Figs 1–3)

A total of 619 bp of partial sequence of cytochrome c oxidase subunit I gene was determined from Quasitetrastemma nigrifrons (Coe, 1904) (see Appendix).



Fig. 1. Quasitetrastemma nigrifrons (Coe, 1904), ICHU02120952, entire animal; the animal was a female with nearly mature oocytes contained in the ovaries along the intestine.




Fig. 2. Quasitetrastemma nigrifrons (Coe, 1904), ICHU02120952, enlargement of head; the animal belongs to the variety spadix.




Fig. 3. Quasitetrastemma nigrifrons (Coe, 1904), ICHU02120952, enlargement of head, slightly oblique view to show the two pairs of ocelli (indicated by arrowheads).






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 (MEGA) software version 5.0. Molecular Phylogenetics and Evolution 28: 2731–2739.




Appendix
COI gene sequence (619 bases) from ICHU02120952 identidied as Quasitetrastemma nigrifrons (Coe, 1904).

ACTTTTAATTTTTTATTTGGAGTATGATCTGGGTTAGTTGGAACTGCATTGAGTTTATTAATTCGTGCTGAGTTAGGACAGCCTGGTGCTTTGTTAGGTGATGACCAGTTATATAATGTAATTGTAACTGCTCATGCATTTGTTATAATTTTTTTTTTAGTAATACCAGTAATAATTGGNGGATTTGGGAATTGATTGGTTCCGTTAATGTTAGGTGCGCCGGATATGGCTTTTCCTCGTATAAATAATATGAGATTTTGGTTATTGCCTCCTGCATTAACTCTTCTTCTTTCTTCTGGAGCGGTTGAGAGAGGTGCTGGAACTGGATGGACTGTTTACCCTCCTTTATCTAGAAATATTGCTCATGCTGGTGGATCAGTAGATTTGGCAATTTTTTCTTTACATTTAGCTGGTGTATCTTCGATTTTAGGTGCTATTAACTTTATTACTACTATTATTAATATGCGATGATACGGAATACAGTTTGAGCGGCTTTCTTTATTTGTTTGATCTGTAAAAATTACAGCTGTTCTATTGCTTTTATCTTTGCCAGTGTTGGCTGGGGCTATTACTATGTTGTTAACTGATCGTAATTTTAATACTTCTTTTTTTGATCCTG