Report of Systematic Zoology Lab Practicum, August, 2011

Observation of an unidentified species of Caprellidea (Crustacea: Amphipoda)

Makoto Someya

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

Material and Methods
A skeleton shrimp was obtained subtidally at Oshoro Bay, Hokkaido, Japan, about 43°12′N, 140°51′E, on 23 May 2011 by Makoto Someya, photographed and identified by Hiroshi Kajihara as Caprellida sp. based on Uchida (1992: 334, pl. 66, fig. 3), and fixed in 99% EtOH. DNA was extracted from the part of a leg 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 ICHU22090030 (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 5 software (Tamura et al. 2011).

Results
      Due to the poor chromatogram data, only a fragment of 379 bp was reliably sequenced (see Appendix). However, a blast search at the National Center for Biotechnology Information (http://blast.ncbi.nlm.nih.gov/Blast.cgi) indicated that the obtained sequence likely represents Pauliella, a genus of photosyntheitc heterokonts in the phylum Ochrophyta.

Taxonomy
Phylum Arthropoda Siebold & Stannius, 1845
Class Crustacea Pennant, 1777
Order Amphipoda Latreille, 1814
Infraorder Caprellida (Figs 1, 2)



Fig. 1. Caprellida sp. (ICHU22090030).


Fig. 2. Caprellida sp. (ICHU22090030).



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.