Report of Systematic Zoology Lab Practicum, Volume 6: e03; August, 2015


Observation of an unidentified holothurian (Echinodermata: Holothuroidea) in Oshoro Bay, Hokkaido, Japan


Yuri Sakai and Daisuke Aida

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



Material and Methods
A sea cucumber was collected intertidally in Oshoro Bay, Hokkaido, Japan, about 43°12′N, 140°51′E on 11 May 2015 by A. Eguchi and M. Nakatani, photographed by S. Tomioka, and fixed in 99% EtOH. Total DNA was extracted from a piece of body wall. 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, 16S ar-L (5′-CGCCTGTTTATCAAAAACAT-3′) and br-H (5′-CCGGTCTGAACTCAGATCACGT-3′) (Palumbi et al. 1991) for the mitochondrial 16S rRNA gene, H3aF (5′-ATGGCTCGTACCAAGCAGAC-3′) and H3aR (5′-ATATCCTTRGGCATRATRGTGAC-3′) (Colgan et al. 1998) for the nuclear histone H3 gene, and LSU5 (5′-ACCCGCTGAAYTTAAGCA-3′) and LSU3 (5′-TCCTGAGGGAAACTTCGG-3′) (Littlewood 1994) for the nuclear 28S rRNA gene. PCR products were visualized by electrophoresis in 1% agarose gel, but none of the four gene markers attempted were confirmed to be successfully amplified, probably due to a failure in DNA extraction.


Taxonomy
phylum Echinodermata
Holothuroidea fam., gen. et sp. indet.
(Fig. 1)

Material examined: ICHU2130031 (non-extant, completely destroyed for DNA extraction).




Fig. 1. Holothuroidea sp. (ICHU2130031), general shape.






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.

Littlewood, D. T. J. 1994. Molecular phylogenetics of cupped oysters based on partial 28S rRNA gene sequences. Molecular Phylogenetics and Evolution. 3: 221–229.

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.