Journal of the Marine Biological Association of the UK

Research Article

Evaluation of PCR methods for fixed bivalve larvae

Hideki Sawadaa1, Hajime Saitoa2, Masatomi Hosoia1a3 and Haruhiko Toyoharaa1 c1

a1 Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Oiwake-cho, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan

a2 National Research Institute of Fisheries Engineering, Fisheries Research Agency, Hasaki, Kamisu, Ibaraki 314-0408, Japan

a3 Present address: School of Environmental Science, University of Shiga Prefecture, 2500 Hassaka-cho, Hikone-City, Shiga 522-8533, Japan

Abstract

Investigating the spatio-temporal patterns of planktonic larvae is fundamental to studies regarding stock assessment and larval dispersal of commercial and non-commercial, i.e. invasive or rare marine invertebrates. Because of the difficulty involved in morphological identification of marine invertebrate larvae, various molecular methods based on PCR have been used to enhance taxonomic resolution. In previous studies, different methods for the preservation or pretreatment of larvae were applied in each case. However, no comparative studies have been conducted to determine the optimal method for PCR testing for bivalve larvae, and no information is available regarding the selection of an appropriate method.

This study compared the PCR success rate of 6 pretreatment methods for larvae of the Mediterranean blue mussel, which was preserved using different fixatives (70% ethanol, 100% ethanol, 70% acetone and 10% formalin). The results revealed that the success rate of PCR was different for each pretreatment; moreover, the use of ammonium sulphate and Tween 20 buffer with proteinase K digestion was found to be the most effective method. Some pretreatments showed lower success rates for long-fixed larvae than for short-fixed larvae for formalin-fixed larvae; however, the success rate of PCR amplification for ethanol-fixed larvae pretreated by this method did not decrease through 1-year fixation. In addition, this pretreatment showed a high success rate for different fixation periods. These findings suggest that the selection of the pretreatment method is critically important for successfully amplifying larval DNA and that the pretreatment involving the use of ammonium sulphate prior to PCR amplification enables the use of fixatives for preserving bivalve larvae. This method will be utilized in various field studies and molecular genetic studies.

(Received January 22 2008)

(Accepted April 03 2008)

(Online publication July 08 2008)

Correspondence:

c1 Correspondence should be addressed to: Haruhiko Toyohara, Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Oiwake-cho, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan email: toyohara@kais.kyoto-u.ac.jp