Investigation on Natural Diets of Larval Marine Animals Using Peptide Nucleic Acid-Directed Polymerase Chain Reaction Clamping

Seinen Chow, Sayaka Suzuki, Tadashi Matsunaga, Shane Lavery, Andrew Jeffs, Haruko Takeyama

Research output: Contribution to journalArticle

39 Citations (Scopus)

Abstract

The stomach contents of the larvae of marine animals are usually very small in quantity and amorphous, especially in invertebrates, making morphological methods of identification very difficult. Nucleotide sequence analysis using polymerase chain reaction (PCR) is a likely approach, but the large quantity of larval (host) DNA present may mask subtle signals from the prey genome. We have adopted peptide nucleic acid (PNA)-directed PCR clamping to selectively inhibit amplification of host DNA for this purpose. The Japanese spiny lobster (Panulirus japonicus) and eel (Anguilla japonica) were used as model host and prey organisms, respectively. A lobster-specific PNA oligomer (20 bases) was designed to anneal to the sequence at the junction of the 18 S rDNA gene and the internal transcribed spacer 1 (ITS1) of the lobster. PCR using eukaryote universal primers for amplifying the ITS1 region used in conjunction with the lobster-specific PNA on a mixed DNA template of lobster and eel demonstrated successful inhibition of lobster ITS1 amplification while allowing efficient amplification of eel ITS1. This method was then applied to wild-caught lobster larvae of P. japonicus and P. longipes bispinosus collected around Ryukyu Archipelago, Japan. ITS1 sequences of a wide variety of animals (Ctenophora, Cnidaria, Crustacea, Teleostei, Mollusca, and Chaetognatha) were detected.

Original languageEnglish
Pages (from-to)305-313
Number of pages9
JournalMarine Biotechnology
Volume13
Issue number2
DOIs
Publication statusPublished - 2011 Apr 1

Keywords

  • Gut content analysis
  • Larval diet
  • PCR-clamping
  • PNA
  • Spiny lobster

ASJC Scopus subject areas

  • Biotechnology
  • Aquatic Science

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