A TaqMan real-time PCR based assay targeting plaice (Pleuronectes platessa L.) DNA to detect predation by the brown shrimp (Crangon crangon L.) and the shore crab (Carcinus maenas L.)-Assay development and validation

摘要

We describe a protocol for the preservation, extraction, and detection of plaice (Pleuronectes platessa) DNA from the stomach contents of the brown shrimp, Crangon crangon and the shore crab, Carcinus maenas. These two predatory species are thought to be important sources of mortality of small juvenile plaice on inshore nursery grounds. Previous studies of predation on juvenile plaice have used visual examination of stomach contents but this is time consuming and may under-estimate true predation levels as remains may become unidentifiable due to maceration and digestion. Molecular based tools for detecting the presence of prey tissue in predator stomachs and scat are becoming increasingly used in marine ecology and provide an alternative or complementary approach to visual identification. We sequenced a part of the cytochrome-b region of plaice mitochondrial DNA and designed a species-specific, TaqMan real-time Polymerase Chain Reaction (PCR) based assay which was successfully tested for intra- and inter-species-specificity. For application to predator stomach contents, two tissue preservation and two DNA extraction methods were tested followed by a set of aquarium experiments to determine the effect of digestion time on detectability. The quality of the extracted DNA was comparable for the two preservation and two extraction methods tested and the detectability remained similar for all of them. However, levels of PCR inhibition were significant for samples from both predators but could be overcome using serial dilution and 1.25 μg/μl Bovine Serum Albumin to reduce the incidence of false negatives. Successful amplification and detection of plaice DNA from stomach contents was possible up to 24 h after ingestion for both predator species. For extracts of C. crangon stomachs the half-life detection rate (T50) was ～10 h at water temperatures of 14-16 ℃. The effects of a wider range of temperatures were tested for stomach contents of C. maenas where the T50s were ～7 h at 6-10 ℃ and ~6 h at 14-16 ℃ but only 2 h at 19-20 ℃. Our results indicate that the TaqMan method is applicable in field studies providing species-specific T50s, PCR inhibition and water temperatures are taken into account.