Report of Systematic Zoology Lab Practicum, Volume 6: e21; August, 2015


Partial sequence of the mitochondrial 16S rRNA gene from the hermit crab Pagurus nigrofascia (Crustacea: Anomura: Paguridae), collected in Oshoro Bay, Hokkaido, northern Japan


Yoji Kakuya and Yuto Nakagawa

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


Material and Methods
A hermit crab was collected in Oshoro Bay, Hokkaido, Japan, about 43°12′N, 140°51′E, on 1 June 2015 by Yoji Kakuya and Yuto Nakagawa, photographed and fixed in 99% EtOH by Shinri Tomioka; the specimen was identified by Hiroshi Kajihara as Pagurus nigrofasciata Komai, 1996, based on Komai (1996). Total DNA was extracted from one of the legs 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 ICHU2132067 (contact: Dr. Hiroshi Kajihara, kazi@mail.sci.hokudai.ac.jp).
      PCR amplification was attempted with the primer pairs LoboF1 (5′-KBTCHACAAAYCAYAARGAYATHGG-3′) and LoboR1 (5′-TGTTTYTTYGGWCAYCCWGARGTTTA-3′) (Lobo et al. 2013) for the mitochondrial cytochrome c oxidase subunit I gene (COI) and 16S ar-L (5′-CGCCTGTTTATCAAAAACAT-3′) and br-H (5′-CCGGTCTGAACTCAGATCACGT-3′) (Palumbi et al. 1991) for the 16S rRNA gene . PCR products were visualized by electrophoresis in 1% agarose gel. Of the two gene markers that were attempted, only 16S rRNA gene was confirmed to be successfully amplified. The 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°C and a final elongation at 72°C for 7 min.
      The PCR products of the 16S rRNA gene 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 v5.2 software (Tamura et al. 2011).


Results
A 528-bp partial sequence of the mitochondrial 16S rRNA gene was determined from ICHU213067, identified as Pagurus nigrofascia (see Appendix). A nucleotide BLAST search (Altschul et al. 1997) at the NCBI website (https://blast.ncbi.nlm.nih.gov/) resulted in that our sequence from Oshoro showed 95% similarity with AF425335 (E value = 0.0; 94% in query coverage), a sequence from a hermit crab identified as Pagurus bernhardus (Linnaeus, 1758) collected in Roscoff, France (Zaklan and Cunningham, unpublished).


Taxonomy
Phylum Arthropoda
Class Malacostraca
Order Decapoda
Family Paguridae Latreille, 1802
Genus Pagurus Fabricius, 1775
Pagurus nigrofascia Komai, 1996
[Japanese name: yomogi-hon-yadokari]
(Figs 1, 2)


Fig. 1. Pagurus nigrofascia Komai, 1996 (ICHU2132067), dorsal view, showing the white longitudinal stripe on the charapace, photographed by Shinri Tomioka.


Fig. 2. Pagurus nigrofascia Komai, 1996 (ICHU2132067), showing the characteristic black band at the proximal portion of the dactylus in each ambulatory pereopod, photographed by Shinri Tomioka.

References

Altschul, S. F., Madden, T. L., Schäffer, A. A., Zhang, J., Zhang, Z., Miller, W., and Lipman, D. J. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research 25: 3389–3402.

Boom, R., Sol, C. J., Salimans, M. M., Jansen, C. L., Wertheim-van Dillen, P. M., and Van der Noordaa, J. 1990. Rapid and simple method for purification of nucleic acids. Journal of Clinical Microbiology 28: 495–503.

Komai, T. 1996. Pagurus nigrofascia, a new species of hermit crab (Decapoda: Anomura: Paguridae) from Japan. Crustacean Research 25: 59–72.

Lobo, J., Costa, P. M., Teixeira, M. A. L., Ferreira, M. S. G., Costa, M. H, and Costa, F. O. 2013. Enhanced primers for amplification of DNA barcodes from a broad range of marine metazoans. BMC Ecology 13:34. DOI: 10.1186/1472-6785-13-34.

Palumbi, S., Martin, A., Romano, S., McMillan, W. O., Stice, L., Grabowski, G. 1991. The Simple Fools Guide to PCR, Ver. 2. Department of Zoology and Kewalo Marine Laboratory, University of Hawaii, Honolulu, 45 pp.

Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., and Kumar, S. 2011. MEGA5: molecullar evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution 28: 2731–2739.



Appendix
A 528-bp partial sequence of the mitochondrial 16S rRNA gene determined from ICHU2132067, identified as Pagurus nigrofascia Komai, 1996.

GTCTATATGAAGGTGTATATAGTTTAGCCTGCTCACTGATTATATTAAAGAGCCGCAGTATTCTGACTGTGCGAAGGTAGCATAATAAATTGTCTTTTAAATGAAGGCTTGAATGAAAGGTTGGACAAAGTGTCATCTGTTTCTTAAATATTTATTGAATTTGACTTTCAAGTGAAAAGGCTTGAATGAAACAAAAAGACGATAAGACCCTATAAATCTTTACAGTAAACATATTTTATATCTTAATTTATAAGTATATAAGATATTAAATATGTAATTGTTTCGCTGGGGCGGCGTAGATATATAAACAAACTGTCTACACATTAAATTCAATAATTATTGATTAATAAAATTGATCCTTAAATAGATTAAGAGATTAAGATACTTTAGGGATAACAGCGTTATTTTTTCTGAGAGTTCTTATCGAAGAAAAAGTTTGCGACCTCGATGTTGAATTAAAGTATCTTTATGGTGCAGCCGCTATAAGAGAGAGTCTGTTCGACTTTTAATACTTTACGTGATCTGAGT