Report of Systematic Zoology Lab Practicum, August, 2012

28S rDNA partial sequence of Dynoides dentisinus (Crustacea: Isopoda: Sphaeromatidae)

Atsushi Saito

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

Material and Methods
A sphaeromatid isopod was obtained in subtidal area at Oshoro Bay, Hokkaido, Japan, about 43°12′N, 140°51′E, on 28 May 2012 by Atsushi Saito, photographed and identified by Hiroshi Kajihara as Dynoides dentisinus (Shen, 1929), and fixed in 99% EtOH. 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.Remaining morphological voucher specimen has been deposited at the Hokkaido University Museum under the catalogue number ICHU 22100175 (contact: Dr. Hiroshi Kajihara, kazi@mail.sci.hokudai.ac.jp).
      Amplification of mitochondrial cytochrome c oxidase subunit I gene (COI) using LCO1490 (5′-GGTCAACAAATCATAAAGATATTGG-3′) and HCO2198 (5′-TAAACTTCAGGGTGACCAAAAAATCA-3′) (Folmer et al. 1994) was unsuccessful.
      An about 1.2 K-bp fragment of mitochondrial 28S rRNA gene was amplified by polymerase chain reaction (PCR) using LSU5 (5′-ACCCGCTGAAYTTAAGCA-3′) and LSU3 (5′-TCCTGAGGGAAACTTCGG-3′) (Littlewood DT. 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 and an internal primer set D2F(5′-CTTGAAGAGAGAGTTC-3′)(Littlewood. 1994) and 28z(5′-CTTGGTCCGTGTTCAAGAC-3′)(Hillis and Cixon.1991) . Sequencing was performed with ABI Prism 3730 DNA Analyzer (Applied Biosystems). Chromatogram and sequence data were operated with MEGA v4 software (Tamura et al. 2011).

Results
A total of 1778 bp of 28S rDNA sequence was determined from Dynoides dentisinus (see Appendix).


Taxonomy
Order Isopoda
Family Shaeromatidae Latreille. 1825
Genus DynoidesBarnard. 1914
Dynoides dentisinus Shen, 1929
(Fig. 1)



Fig. 1. Dynoides dentisinus, ICHU22100175, dorsal 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 Microbiology28: 495–503.