Report of Systematic Zoology Lab Practicum, August, 2011

28S ribosomal RNA gene partial sequences of Ammothea hilgendorfi (Pycnogonida: Pantopoda: Ammotheidae)

Naomi Takahashi

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

Material and Methods
A sea spider specimen was obtained among Mytilus bed at Oshoro Bay, Hokkaido, Japan, about 43°12′N, 140°51′E, on 6 June 2011 by Naomi Takahashi, photographed and identified by Hiroshi Kajihara, and fixed in 99% EtOH. DNA was extracted from the four legs, 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 ICHU22090237 (contact: Dr. Hiroshi Kajihara, kazi@mail.sci.hokudai.ac.jp).
      Amplification of mitochondrial cytochrome c oxidase subunit I gene (COI) using LCO1490 (5′-GGTCAACAAATCATAAAGATATTGG-3′) and HCO2198 (5′-TAAACTTCAGGGTGACCAAAAAATCA-3′) (Folmer et al. 1994) was unsuccessful. About 1.2 Kbp fragment of 28S rRNA gene was amplified by polymerase chain reaction (PCR) using LSU5 (5′-ACCCGCTGAAYTTAAGCA-3′) and LSU3 (5′-TCCTGAGGGAAACTTCGG-3′) (Littlewood 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.
      The PCR product 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 v4 software (Tamura et al. 2007).

Results
Two regions of 28S rDNA, 506 bp and 526 bp, respectively, were determined from the specimen identified as Ammothea hilgendrfi (Böhm, 1879) (see Appendix).

Taxonomy
Phylum Arthropoda
Class Pycnogonida
Order Pantopoda
Family Ammotheidae Dohrn, 1881
Genus Ammothea Leach, 1814
Ammothea hilgendorfi (Böhm, 1879)
(Figs 1,2)



Fig. 1. Ammothea hilgendorfi (Böhm, 1879) (ICHU22090237), dorsal view.



Fig. 2. Ammothea hilgendorfi (Böhm, 1879) (ICHU22090237), magnification of back.


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.