Report of Systematic Zoology Lab Practicum, Volume 4: e19; August, 2013

An attemt to PCR-amplify the 28S large subunit ribosomal RNA gene and the cytochromec oxidase subunit I in the hermit crab Paguristes ortmanni (Crustacea: Anomura: Diogenidae) collected in Oshoro Bay, Hokkaido, Japan

Shuhei Kawabata and Kazuya Kito

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

Material and Methods
A hermit crab was obtained in Oshoro Bay, Hokkaido, Japan on 12 June 2013 by Shuhei Kawabata and Kazuya Kito, photographed and identified by Hiroshi Kajihara as Paguristes ortmanni Miyake, 1978, 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 ICHU22100168 (contact: Dr. Hiroshi Kajihara, kazi@mail.sci.hokudai.ac.jp).
      We attempted to amplify the mitochondrial cytochrome c oxidase subunit I gene (COI) was amplified by polymerase chain reaction (PCR) using LCO1490 (5′-GGTCAACAAATCATAAAGATATTGG-3′) and HCO2198 (5′-TAAACTTCAGGGTGACCAAAAAATCA-3′) (Folmer et al. 1994) and the nuclear 28S rRNA gene using LSU5 (5′-ACCCGCTGAAYTTAAGCA-3′) and LSU3 (5′-TCCTGAGGGAAACTTCGG-3′) (Littlewood et al. 1994). A hot start PCR was performed by a thermal cycler, 2720 Thermal Cycler(Applied Biosystems), 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 (COI) and 90 sec (28S); and a final elongation at 72°C for 7 min.


Results
Phylum Arthropoda
SubphylumCrustacea
Class Malacostraca
Order Decapoda
SuborderPleocyemata
InfraoderAnomura
SuperfamilyPaguroidea
Family Diogenidae
Genus Paguristes Dana, 1851
Paguristes ortmanni Miyake, 1978
[Japanese name: kebuka-hime-yoko-basami]
(Figs 1, 2)

Due to the poor chromatogram data, no reliable sequence was obtained for both COI and 28S.



Fig. 1. Paguristes ortmanni Miyake, 1978 (ICHU22100168), from Oshoro Bay, photograph taken in life.


Fig. 2. Paguristes ortmanni Miyake, 1978 (ICHU22100168), magnification of the head, 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 Microbiology 28: 495–503.