Report of Systematic Zoology Lab Practicum, August, 2010


28S ribosomal RNA gene partial sequence of Idotea ochotensis (Crustacea: Isopoda: Idoteidae)


Yoriko Kajita

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



Material and Methods
An ovigerous female valviferan was obtained among laminarian holdfasts at Oshoro Bay, Hokkaido, Japan, about 43°12′N, 140°51′E, on 31 May 2010 by Yoriko Kajita, photographed and identified by Hiroshi Kajihara as Idotea ochotensis Brandt, 1851 based on Nunomura (1992: 216, pl. 81-12), and fixed in 99% EtOH. DNA was extracted from the pleonites 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 ICHU22080063 (contact: Dr. Hiroshi Kajihara, kazi@mail.sci.hokudai.ac.jp).
      Amplification of mitochondrial cytochrome c oxidase subunit I gene (COI) by polymerase chain reaction (PCR) using LCO1490 (5′-GGTCAACAAATCATAAAGATATTGG-3′) and HCO2198 (5′-TAAACTTCAGGGTGACCAAAAAATCA-3′) (Folmer et al. 1994) was unsuccessful.
      An about 1.5K-bp fragment of 28S ribosomal RNA gene was amplified by PCR using LSU5 (5′-ACCCGCTGAAYTTAAGCA-3′) and LSU3 (5′-TCCTGAGGGAAACTTCGG-3′) (Littlewood 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), 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). Single strand was sequenced with a BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) following the manufacturer's protocol, using LSU3. Sequencing was performed with ABI Prism 3730 DNA Analyzer (Applied Biosystems). Chromatogram and sequence data were operated with MEGA v4 software (Tamura et al. 2007).

Results
A total of 653 bp of 28S rDNA sequence was determined from Idotea ochotensis (see Appendix).

Taxonomy
Order Isopoda
Family Idoteidae Samouelle, 1819
Genus Idotea Fabricius, 1798
Idotea ochotensis Brandt, 1851
(Figs 1, 2)



Fig. 1. Idotea ochotensis (ICHU22080063), dorsal view.


Fig. 2. Idotea ochotensis (ICHU22080063), ventral 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.

Littlewood, D, T. 1994. Molecular phylogenetics of cupped oysters based on partial 28S rRNA gene sequence Molecular Phylogenetics and Evolution 3: 221–229.

Nunomura, N. 1992. Isopoda. Pp. 205–233. In: Nishimura, S. (Ed.) Guide to Seashore Animals of Japan with Color Pictures and Keys. Vol. II. Hoikusha, Osaka.

Tamura, K., Dudley, J., Nei, M. andvKumar, S. 2007. MEGA4: Molecullar Evolutionary Genetics Analysisv(MEGA) software version 4.0. Molecular Phylogenetics and Evolution 24: 1596–1599.


Appendix
28S rDNA sequence from ICHU22080063 identidied as Idotea ochotensis.

CGATAGGGTCTTTCGCCCCTATACCCAGCTCCGTACGAATCGATTTGGCACGTCAGAAACCGCTACGGACCTCCACCAGAGTTTCCTCTGGCTTCATCCTGGCCAGGCATAGATCACCATCTTTCGGGTACTAGCATGTACGCTCTTGGAACCCGCCTATCTTCTTTCCCTAACCGGCCCCTCGAAAGGGGTCGGAACTAGGGAAGAGAGGATAATATGGCGGGCCCCGGACCTGCGCCCATCAATATATACTTTTAGCGTAGCAGCTCGACGCTTTTCCGAAGAAAACGGCAGTCGGCTTTTACGATGGGATCGGTCCAACAAGGTTTTATCAACCCATGAATCGAAACGAGTTTCGATAATTACATTACTTTCGTTGCGCCATCTGAGGTTTAGTGAACCCTTATGACTCGCGCACTTGCTAGACTCCTTGGTCCGTGTTTCAAGACGGGACGAATGGACGTACGCCGTATCTATGCGCCGCGCACGGGTCGGTAGCGTTTTACTCGAGGTTTAGGGGAAGTACGTAAAGCACTACACACCCGCACACTCCGCTTTCGTGAACCGACCCGTCTGCTGTCAGTAACGTCGCAGGCGTCCGTCGTATTCCCAATGGCGTCCCCGACTCGTACTCGCGGACCACCGCACCCGTG