Report of Systematic Zoology Lab Practicum, August, 2011


Observation of Phascolosoma sp. (Sipuncula)


Yukiko Mizuo

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



Material and Methods
A sipunculan was obtained subtidally at Oshoro Bay, Hokkaido, Japan, about 43°12′N, 140°51′E, on 23 May 2011 by Yukiko Mizuo, then photographed and identified by Hiroshi Kajihara as a member of the genus Phascolosoma; the body was fixed in 99% EtOH. DNA was extracted from the anterior part 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.
      An about 600-bp fragment of mitochondrial cytochrome c oxidase subunit I gene (COI) was amplified by polymerase chain reaction (PCR) using LCO1490 (5′-GGTCAACAAATCATAAAGATATTGG-3′) and HCO2198 (5′-TAAACTTCAGGGTGACCAAAAAATCA-3′) (Folmer et al. 1994). A hot start PCR was performed by a thermal cycler, iCycler (Bio-Rad), 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.
     

Results
The COI region was not amplified. It is probably due to the erroneous procedure in which DNA extraction was carried out with formalin-fixed specimen, instead of that fixed in EtOH.

Taxonomy
Order Phascolosomatida
Family Phascolosomatidae Stephen and Edmonds, 1972
Genus Phascolosoma Leuckart, 1828
Phascolosoma sp.
(Figs 1–3)



Fig. 1. Phascolosoma sp. (ICHU22090047), entire view.


Fig. 2. Phascolosoma sp. (ICHU22090047), entire view.


Fig. 3. Phascolosoma sp. (ICHU22090047), partially protruded tentacles.



References

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

Folmer, O., Black, M., Hoeh, W., Lutz, R. and Vrijenhoek, R. 1994. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Molecular Marine Biology and Biotechnology 3: 294–299.