Report of Systematic Zoology Lab Practicum 8: e9 (August 2017)

Partial sequence of the mitochondrial 16S rRNA gene of the polychaete Acrocirrus validus (Annelida: Acrocirridae) from Oshoro, Hokkaido, Japan

Hayato Kojima and Airi Yoshikawa

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

Material and Methods
A kumanoashituki specimen was collected intertidally under stone in Oshoro Bay, Hokkaido, Japan, about 43°12′N, 140°51′E, on 22 May 2017 by Hayato Kojima and Airi Yoshikawa, photographed, fixed in 99% EtOH by Naoto Jimi; the specimen was identified by Hiroshi Kajihara as Acrocirrus validus Moore, 1905. DNA was extracted from palp assembly 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 ICHU2150616 (contact: Dr. Hiroshi Kajihara, kazi@mail.sci.hokudai.ac.jp).
      PCR amplification was attempted with the primer pair 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; 35 cycles of denaturation at 95°C for 30 sec, annealing at 47°C for 45 sec, and elongation at 72°C for 45 sec and a final elongation at 72°C for 7 min.
      The PCR products of the 16S rRNA gene were 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 and Taxonomy
Phylum Annelida
Class Polychaeta
Order Terebellida
Family Acrocirridae Banse, 1969
Genus AcrocirrusGrube, 1873
Acrocirrus validus Marenzeller, 1879
[Japanese name: kumanoashituki]

(Fig. 1)

A total of 404 bp of the mitochondrial 16S rRNA gene sequence was determined from ICHU2150616 (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 showed 79% similarity with HQ326957 (E value = 4e–60; 91% in query coverage).



Fig. 1. Acrocirrus validus Marenzeller, 1879 (ICHU2150616) from Oshoro, Hokkaido, Japan, photographed by Naoto Jimi.




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.