Report of Systematic Zoology Lab Practicum, Volume 6: e18; August, 2015

16S rRNA partial sequence of the nudibranch Rostanga orientalis (Mollusca: Opisthobranchia) from Oshoro Bay, Hokkaido, Japan

Nagi Matsushita and Hiroko Adachi

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

Material and Methods
A dorid nudibranch was collected from under stone in the intertidal zone of Oshoro Bay, Hokkaido, Japan, about 43°12′N, 140°51′E, on 25 May 2015 by Nagi Matsushita, photographed by Shinri Tomioka, and fixed in 99% EtOH. The specimen was identified by Hiroshi Kajihara based on the photographs as Rostanga orientalis Rudman and Avern, 1989, according to Hamatani (1994). Total DNA was extracted from the middle part of the body using the silica method (Boom et al. 1990) with some modifications. The 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 ICHU2130889 (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 total of 440 bp of partial sequence of the mitochondrial 16S rRNA gene was determined from ICHU2130889, identified as Rostanga orientalis (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 AY345044 (E value = 0.0), a sequence from a dorid nudibranch identified as Rostanga pulchra MacFarland, 1905 collected in California, USA (Grande et al. 2004).


Taxonomy
Phylum Mollusca
Class Gastropoda
Subclass Heterobranchia
Infraclass Opisthobranchia
Order Nudibranchia
Infraorder Doridacea
Family Discodorididae
Genus Rostanga Bergh, 1879
Rostanba orientalis Rudman and Avern, 1989
[Japanese name: isoumiushi]
(Fig. 1)


Fig. 1. Rostanga orientalis Rudman and Avern, 1989 (ICHU2130889), dorsal view, taken in life.




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.