Report of Systematic Zoology Lab Practicum, August, 2011


28S rDNA partial sequence of the crab Gaetice depressus (De Haan, 1833) (Crustacea: Decapoda: Bracyura: Varunidae)


Yasuyuki NIKAMI

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



Material and Methods
A crab specimen was obtained among laminarian holdfast at Oshoro Bay, Hokkaido, Japan, about 43°12’N, 140°51’E, on 7 May 2011 by Yasuyuki Nikami, photographed and identified by Hiroshi Kajihara as Gaetice depressus (De Haan, 1833) based on Sakai (1976). The anterior half of the specimen was fixed in 10% formalin seawater, while the posterior half was in 99% EtOH. DNA was extracted from the EtOH-fixed tissue, 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 ?E0°C.. Remaining morphological voucher specimen has been deposited at the Hokkaido University Museum under the catalogue number ICHU22090037 (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 1.2K-bp fragment of 28S rRNA gene was amplified by polymerase chain reaction (PCR) using LSU5 (5′-ACCCGCTGAAYTTAAGCA-3′) and LSU3 (5′-TCCTGAGGGAAACTTCGG-3′) (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 5 software (Tamura et al. 2011). Results A total of 346 bp of 28S rDNA sequence was determined from ICHU220900037, identified as Gaetice depressus (De Haan, 1883) (see Appendix).

Taxonomy
Order Decapoda
Family Varunidae H. Milne Edwards, 1853
Genus Gaetice Gistel, 1848
Gaetice depressus (De Haan, 1833)




Fig. 1. ICHU22090037, identified as Gaetice depressus (De Haan, 1833).






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?E03.

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?E99.

Sakai, T. 1976. Crabs of Japan and the Adjacent Seas. Kodansha, Tokyo. xxix+773 pp [English]; 461 pp [Japanese]; 251 pls.

Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. 2011. MEGA5: Molecular Evolutionary Genetics Analysis Using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods. Molecular Phylogenetics and Evolution 24: 1596–1599.




Appendix
Fragment of 28S rDNA (346 bp) from ICHU22090037, identidied as Gaetice depressus (De Haan, 1883).

GGTAAACTCCATCTAAGGCTAAATATGACCACGAGACCGATAGCGAACAAGTACCGTGAGGGAAAGTTGAAAAGAACTTTGAAGAGAGAGTTCAAGAGTACGTGAAACCGTTAAGAGCCAAACGGGTGGGACCTCGAAGGTCGAACCGAGGGGATTCAGCTCGTCGGCCAAAGCTGGCTGGGCGTCGGGATTCGCTTATGCGACCCAGGAACCCTTGGGCGTGCCCGCGTCCAAGCCGGCGCCGGCGGGCGTATTTCCCCTCGTGATGTAGGTCGCCGCGACCCGTTGCTGGGGACGTCGAAGGCCCAGGTGGATTGGTACCCCGACGTCTGCGTTTACGCGGTCG