Report of Systematic Zoology Practicum, August, 2010

28S rRNA partial sequence of an undetermined gammarid of the family Pleustidae (Crustacea: Amphipoda: Gammaridea)

Koichi Tone

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

Material and Methods
      A gammarid specimen was obtained intertidally at Oshoro Bay, Hokkaido, Japan, about 43°12′N, 140°51′E, on 10 May 2010 by Koichi Tone, photographed by Hiroshi Kajihara, before fixed in 99% EtOH. The specimen was later identified by Ko Tomikawa (Hiroshima University) based on the photographs as Pleustidae gen. et sp. DNA was extracted from the whole 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.
      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 1.5K-bp fragment of 28S rRNA gene was amplified by polymerase chain reaction (PCR) using LSU5 (ACCCGCTGAAYTTAAGCA) and LSU3 (TCCTGAGGGAAACTTCGG) (Littlewood 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). 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 v4 software (Tamura et al. 2007).

Results
A total of 922 bp of 28S rRNA sequence was determined from Pleustidae gen. et sp. (see Appendix).

Taxonomy
Class Crustacea
Order Amphipoda
Suborder Gammaridea
Family Pleustidae
Pleustidae sp. (Figs 1–2)



Fig. 1. Pleustidae gen. et sp. (ICHU22070191), lateral view.



Fig. 2. Pleustidae gen. et sp. (ICHU22070191), ventro-lateral 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.