Report of Systematic Zoology Lab Practicum, August, 2010

Cytochrome c oxidase subunit I partial sequence from an egg mass of an unidentified gastropod (Mollusca: Gastropoda)

Midori Iwama

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

Material and Methods
A flattended, yellow molluskan egg mass contained in transparent jelly, about 3 mm in width, laid on a leaf of the seagrass Phyllospadix iwatensis Makino, 1931, was obtained at Oshoro Bay, Hokkaido, Japan, about 43°12′N, 140°51′E, on 10 May 2010 by Midori Iwama, photographed by Hiroshi Kajihara, and fixed in 99% EtOH. DNA was extracted from the eggs 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.
      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 and Discussion
A total of 625 bp of COI sequence was determined from the egg-mass (see Appendix). A BLAST search at the National Center for Biotechnology Information at the date of 16 August 2010 resulted no candidate species with the maximum percentage identity value more than 90%. The sequence in GenBank that showed the highetst homology between the present sequence was AJ488604.1, a sequence from a gastropod identified as Lacuna pallidula (Littorinoidea: Lacunidae), with the maximum percentage identity value being 87%; the next highest was the following three sequences with the maximum percentage identity value being 85%: AY2968335.1, identified as Ataxocerithium sp. (Triphoroidea: Certhiopsidae); GU575391.1, identified as Vitularia milaris (Muricoidea: MuricidaeI; and AY161634.1, as Simnia aequalis (Cypraeoidea: Ovulidae). A more intensive COI-sequence depositories of gastropods from Oshoro Bay in the future, or a direct observation of oviposition in situ, will reveal the adult species of this egg-mass.

Taxonomy
Phylum Mollusca
Class Gastropoda
(Fig. 1)



Fig. 1. An egg mass (ICHU22080008) laid on the seagrass Phyllospadix iwatensis by an unidentified species of gastropod.



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.