Report of Systematic Zoology Lab Practicum, July, 2012


28S large subunit ribosomal RNA gene partial sequence of Notocomplana humilis (Platyhelminthes: Rhabditophora: Polycladida: Leptoplanoidea: Notoplanidae)


Hiroki Kawamoto and Ippei Kojima

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


Material and Methods
A flatworm was obtained from a tide pool in Oshoro Bay, Hokkaido, Japan, about 43°22′N, 140°85′E, on 21 May 2012 by Hiroki Kawamoto and Ippei Kojima, photographed and identified by Hiroshi Kajihara as Notocomplana humilis (Stimpson, 1857), and fixed in 99% EtOH. DNA was extracted from the posterior half 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. Remaining morphological voucher specimen has been deposited at the Hokkaido University Museum under the catalogue number ICHU2210066 (contact: Dr. Hiroshi Kajihara, kazi@mail.sci.hokudai.ac.jp).
      An about 1000-bp fragment of 28S rDNA fragment 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, DNA Engine, 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 and new primer set, D2F (5′-CTTTGAAGAGAGAGTTC-3′) (Littlewood et al. 1994) and 28z (5′-CTTGGTCCGTGTTTCAAGAC-3′) (Hillis and Dixon et al. 1991) as the initial PCR amplification. Sequencing was performed with ABI Prism 3730 DNA Analyzer (Applied Biosystems). Chromatogram and sequence data were operated with MEGA v.5 software (Tamura et al. 2011).


Results
A total of 900 bases of 28S rDNA partial sequence was determined from ICHU2210066, identified as Notocomplana humilis (Stimpson, 1857) (see Appendix).


Taxonomy
Phylum Platyhelminthes
Class Rhabditophora
Order Polycladida
Family Notoplanidae Marcus & Marcus, 1966
Genus Notocomplana Pucelis Marcus, 1947
Notocomplana humilis (Stimpson, 1857)
(Fig. 1)


Fig. 1. Notocomplana humilis (Stimpson, 1857), ICHU22100066, photograph taken in life.




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.

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

Hills, D. M. and Dixon, M.T. 1991. Ribosomal DNA: molecular evolution and phylogenetic inference. Quarterly Review of Biology 66 441-453

Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., and Kumar, S. 2011. MEGA5: Molecullar Evolutionary Genetics Analysis (MEGA) software version 5.0. Molecular Phylogenetics and Evolution 28: 2731-2739.



Appendix
A 900-base partial sequence of 28S rDNA sequence from ICHU22100066, identidied as Notocomplana humilis (Stimpson, 1857).

AGAGAGGGTGTAAGGCCCGTGGAGGCGAGTCGTCCGGTGCCCGGCACGTCCTTAGAGTCGGGTTGTTTGGGAATGCAGCCCAAAGCGGGTGGTAAACTCCATCCAAGGCTAAATACTAGCACGAGTCCGATAGCGAAGAAGTACCGCGAGGGAAAGTTGAAAAGAACTTTGAAGAGAGAGTTCAAGAGTACGTGAAACCGCTGAGAGGCAAACTGGTGGAGCTGAAATGGTCTTGAGGAATTCAGCCCTTCCTGCTAGAGTTGACGTCGGTTGTGAGATCTCACGACTCCGCTGGCGGCATCTTCGTGAAGGATAGGTGCACTTTCTTCTTGATCGGACCACGACCAGCGACTTGCTTGCCTGACATGGGTCGGAAGGTAGCGTTGGCTTCGGCGAACGTGTTATAGCCGCCTATTCGGTAGTTGAGCGAGGCGTTGGATGCGATGAGTTGGCGCTTGCCCTCTGGGCTCGTACGTTGCCTCTTTCAGGCGTGTCGCCGATTTCACTGCCTTTGGCAGGGGTGTCGAAGATGTGTCGGTTAGAATGGTGGCCTCTGCGATTGGCGTCGAAAGTCTGTGGTTGTGAAGTAGGCAGTCCACCTGACCCGTCTTGAAACACGGACCAAGGAGTCTAACATGTGCGCGAGTCATTGGGCTCTACGAAACCTAAAGGCGAAGTGAAAGTAAAGATCCATTTCCATGGATTGAGGTGGGATCCTGAGTCACGTGCTCGGGCGCACTACCGGCCCGTTCGACATGTCTTCATGCGGGCGGAGCAAGAGCGTACACGTTGGGACCCGAAAGATGGTGAACTATGCTTGCGCAGGTCGAAGCCAGAGGAAACTCTGGTGGAGGACCGTAGCGATTCTGACGTGCAAATCGATCGTCAAACGTGAGTATA