Report of Systematic Zoology Lab Practicum, Volume 6: e16; August, 2015

Partial sequence of the nuclear histone H3 gene of the barnacle Semibalanus cariosus collected in Oshoro Bay, Hokkaido, Japan

Asami Takahashi

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

Material and Methods
A barnacle was collected on rocky shore in the intertidal zone of Oshoro Bay, Hokkaido, Japan, about 43°12′N, 140°51′E, on 25 May 2015 by Asami Takahashi, photographed and fixed in 99% EtOH by Shinri Tomioka; the specimen was identified by Hiroshi Kajihara as Semibalanus cariosus (Pallas, 1788) based on Takeda (1994). Total DNA was extracted from a piece 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 ICHU2131062 (contact: Dr. Hiroshi Kajihara, kazi@mail.sci.hokudai.ac.jp).
      PCR amplification was attempted for four gene markers, using 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), 16S ar-L (5′-CGCCTGTTTATCAAAAACAT-3′) and br-H (5′-CCGGTCTGAACTCAGATCACGT-3′) (Palumbi et al. 1991) for the 16S rRNA gene, H3 aF (5′-ATGGCTCGTACCAAGCAGAC-3′) and aR (5′-ATATCCTTRGGCATRATRGTGAC-3′) for the histone H3 (Colgan et al. 1998), and LSU5 (5′-ACCCGCTGAAYTTAAGCA-3′) and LSU3 (5′-TCCTGAGGGAAACTTCGG-3′) (Littlewood 1994) for the 28S rRNA gene. PCR products were visualized by electrophoresis in 1% agarose gel. Of the four gene markers attempted, only H3 was 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 histone H3 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 331-bp partial sequence of the nuclear histon H3 gene was determined from ICHU22100080, identified as Semibalanus cariosus (Pallas, 1788) (see Appendix). A nucleotide BLAST search (Altschul et al. 1997) at the NCBI website (https://blast.ncbi.nlm.nih.gov/) resulted in that my sequence from Oshoro completely matched with AY520695 (100% identity; E value = 7e–170; 99% in query coverage), which is a sequence of the same species from Monterey Bay, California, USA (Pérez-Losada et al. 2004).


Taxonomy
Phylum Arthropoda
Subphylum Crustacea
Superclass Multicrustacea
Infraclass Cirripedia
Family Archaeobalanidae
Genus Semibalanus (Pilsbry, 1916)
Semibalanus cariosus (Pallas, 1788)
[Japanese name: chishima-fujitsubo]
(Fig. 1)


Fig. 1. Semibalanus cariosus (Pallas, 1788), ICHU2131062, photographed by Shinri Tomioka.


References

Colgan, D. J., McLauchlan, A., Wilson, G. D. F., Livingston, S. P., Edgecombe, G. D., Macaranas, J., Cassis, G., and Gray, M. R. 1998. Histone H3 and U2 snRNA DNA sequences and arthropod molecular evolution. Austrarian Journal of Zoology. 46(5): 419–437.