Report of Systematic Zoology Lab Practicum, Volume 5: e14; August, 2014


28S rDNA partial sequence of Pseudopotamilla occelata (Annelida: Sabellidae) from Oshoro Bay, Hokkaido, northern Japan


Akira Matuura and Haruka Yamaguchi

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


Material and Methods
A feather duster worm was obtained among laminarian holdfast in Oshoro Bay, Hokkaido, Japan, about 43°21'N, 140°85'E, on 9 June 2014 by Akira Matuura and Haruka Yamaguchi. It was identified as Pseudopotamilla occelata Moore, 1905, then photographed and fixid in 99% EtOH by Takumi Onishi. DNA was extracted from the posterior quarter 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 ICHU2121050 (contact: Dr. Hiroshi Kajihara, kazi@mail.sci.hokudai.ac.jp).
      PCR amplification was attempted using the primer pair LCO1490 (5′-GGTCAACAAATCATAAAGATATTGG-3′) and HCO2198 (5′-TAAACTTCAGGGTGACCAAAAAATCA-3′) (Folmer et al. 1994) for the mitochondrial cytochrome c oxidase subunit I (COI) gene, which, however, was unsuccessful. We obtained an about 1000-bp fragment of the nuclear 28S rRNA gene that was amplified by using LSU5 (5′-ACCCGCTGAAYTTAAGCA-3′) and LSU3 (5′-TCCTGAGGGAAACTTCGG-3′) (Littlewood 1994). A hot start PCR was performed by a thermal cycler, 2720 Thermal Cycler (Applied Biosystem), 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 1 min (for COI) or 3 min (for 28S), 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 as the initial PCR amplification, as well as D2F (5′-CTTTGAAGAGAGAGTTC-3′) (Littlewood 1994) and 28z (5′-CTTGGTCCGTGTTTCAAGAC-3′) (Hillis and Dixon 1991) as internal primers. 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
Phylum Annelida
Family Sabellidae Latreille, 1825
Genus Psuedopotamilla Bush, 1905
Pseudopotamilla occelata Moore, 1905
[Japanese name: erako]
(Figs 1, 2)

The chromatogram data generated by the sequencer were mostly dirty. Due to the low quality, we were able to reliably determine only 225 bases near the 5′ end (see Appendix). A nucleotide BLAST search (Altschul et al. 1997) at the NCBI website (https://blast.ncbi.nlm.nih.gov/) showed that our sequence from Oshoro was 95% identical with HM801003 (91% in query coverage; E value = 5e–85), a sequence from unidentified specimen in the genus Pseudopotamilla by Capa et al. (2011), whereas it was most similar to that of the confamilial, but in a different genus, Schizobranchia insignis (100% query coverage; E value = 7e–104; 98% identity) by Struck et al. (2005). There was no GenBank entry of the 28S rRNA gene sequence from P. occelata as of writing.


Fig. 1. Pseudopotamilla occelata Moore, 1905 (ICHU2121050) from Oshoro, Hokkaido, Japan, magnification of the radioles that have black ocelli.


Fig. 2. Pseudopotamilla occelata Moore, 1905 (ICHU2121050) from Oshoro, Hokkaido, Japan, showing overall shape.



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.

Capa, M., Hutchings, P., Teresa Aguado, M., and Bott, N. J. 2011. Phylogeny of Sabellidae (Annelida) and relationships with other taxa inferred from morphology and multiple genes. Cladistics 27: 449–469.

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.

Hillis, D. M. and Dixon, M. T. 1991. Ribosomal DNA: molecular evolution and phylogenetic inference. Quarterly Riview of Biology 66: 411–453.

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

Struck, T. H., Halanych, K. M., and Purschke, G. 2005. Dinophilidae (Annelida) is most likely not a progenetic Eunicida: evidence from 18S and 28S rDNA. Molecular Phylogenetics and Evolution 37: 619–623.

Tamura, K.,Peterson, D., Peterson, N., Stecher, G., Nei, M., and Kumar, S. 2011. MEGA5: molecullar evolutionary genetics analysis using maximum likelihood, evolutionary distance, and mamaximum parsimony methods. Molecular Biology and Evolution 28: 2731–2739.


Appendix
28S rDNA sequence from ICHU2121050 identidied as Pseudopotamilla occelata Moore, 1905

GTCTTTCGCCCCTATACCCAAGTTTGACGATCGATTTGCACGTCAGAATCGCTACGGTCCTCCATCAGAGTTTCCTCTGACTTTAACCTACTCAGGTATAGTTCACCATCTTTCGGGTCCCAACGTGTACGCTCTTGCTCCGCCTCATCAGTTACGATAGAGACAGGCCAGTGGTGCGCCCGCCCCGAAGGACAGGATCCCACCTAAGCCCACCTAGAGGAAGGC