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

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.

DNA identification of the phyllosoma diet ofJasus edwardsiiandScyllarussp. Z

“…Cnidaria are gelatinous zooplankton and fit the criteria for what earlier studies suggest are likely prey for spiny lobster phyllosomas (Cox & Bruce 2003;Cox & Johnston 2003a, b;Jeffs 2007). Cnidaria were also identified in the earlier molecular diet studies from scyllarid phyllosomas from Japanese waters (Suzuki et al 2007(Suzuki et al , 2008 and from palinurid phyllosomas (Suzuki et al 2006(Suzuki et al , 2007(Suzuki et al , 2008Chow et al 2010;O'Rorke et al 2014). Gadiforme DNA was identified from two of the phyllosomas in this present study, J. edwardsii stage X and Scyllarus sp.…”

“…Chimeras may consist of a combination of short DNA sequences from various samples, including primers (Judo et al 1998;Qiu et al 2001;Wang & Wang 1996, 1997. Previous studies attempting to identify the prey of phyllosoma have also had chimeras/unidentified sequences present in their results (Suzuki et al 2006(Suzuki et al , 2007(Suzuki et al , 2008Chow et al 2010). Sequences of both COI and 16S from the zooplankton sample that was morphologically described as a salp did not match any current GenBank or Barcode of Life sequences.…”

“…The DNA sequence was different to that of the host slipper lobster, but as it did not exactly match the reference sequence, it cannot be confirmed to be brachyuran in origin, so it was classed (conservatively) as Decapoda. Chow et al (2010) also identified crustacean DNA among the prey items found in the gut of a palinurid phyllosoma, but identified these as being from slipper lobster (scyllarid). In comparison to the previous prey studies, this present study achieved a greater level of taxonomic identification than studies using highthroughput amplicon sequencing of 18S rDNA (O'Rorke et al 2012a, a similar level to studies using universal primers on 18S rDNA (Suzuki et al 2006(Suzuki et al , 2007(Suzuki et al , 2008, but was not as specific as that achieved in Chow et al (2010) using the ultra-variable ITS1 region in combination with peptide nucleic acid-directed PCR clamping.…”

“…The majority of the earlier molecular genetic diet studies on spiny lobster larvae have used universal primers (Suzuki et al 2006(Suzuki et al , 2007(Suzuki et al , 2008Chow et al 2010;O'Rorke et al 2012a). The rationale for their use is their ability to amplify a larger variety of prey items, although at the cost of also amplifying the host lobster (Suzuki et al 2006(Suzuki et al , 2007(Suzuki et al , 2008.…”

Dietary analysis on the shallow-water hydrothermal vent crab Xenograpsus testudinatus using Illumina sequencing

“…This method is sensitive and unaffected by the size and digestibility of the prey. While PCR using universal eukaryotic primers may amplify gut content DNA as well as host DNA, the problem can be partially solved by the addition of predator-specific blocking probes into PCR (Terahara et al 2011;Chow et al 2011).…”