해산어류의 위 내용물 분석을 위한 차세대 염기서열(Next Generation Sequencing) 분석법의 적용

Abstract

Diet study provides the basic information of feeding relationship among the different tropic levels in marine ecosystem. Traditional diet studies for the marine fish species have been conducted mostly by the direct observation of prey remains in stomach, which relies on microscopic morphological identification. Although it has been a useful strategy for its relatively low cost for the analysis and logistical ease, traditional method may involve the several potential problems including difficulty in identifying the partially digested prey items, different digestion time, and a considerable times and efforts for the analysis. DNA-based diet analysis can be a useful alternative strategy for the prey identification, which may eventually replace traditional microscopic observation. Compared with morphological analysis, molecular genetic identification shows several advantages including the faster analysis times, accurate species identification, and small amount of DNA samples for the analysis. Recent advance in the DNA sequencing, so called next generation sequencing (NGS), technology and the large amount of DNA sequences deposited in the database such as GenBank (http://www.ncbi.nlm.nih.gov/genbank) or the Barcode of Life Database (BOLD; http://www.boldsystems.org) enabled researchers to assess the biodiversity by massive sequencing of DNA barcodes that are amplified from environmental samples, a process known as “metabarcoding”. In present study, we established a system for the species identification using MiSeq platform, which is decided to be the most suitable NGS system for the stomach content analysis. As the first step, the universal COI primer set were designed targeting metazoan mitochondrial COI region, which were optimized for the MiSeq platform in the stomach contents analysis of marine fish. Reliability of the universal COI primer set was confirmed by the NGS result of the zooplankton samples collected from East Sea. Newly designed universal COI primer set generated 537 OTUs, which included 77 species, 62 genera, 24 orders, and 14 classes covering most of metazoan taxa found in ocean. In order to apply the system for the diet study of marine fish species, we chose three different species including conger pike, Muraenesox cinereus, common flying squid, Todarodes pacificus, and marbled flounder, Pseudopleuronectes yokohamae. According to the NGS data generated from the stomach of both conger pike and common flying squid, we were able to obtain the reliable information about their prey items. Relative amount of obtained contig numbers represented the relative amount of prey items, which suggests that NGS-based stomach content analysis also may provide quantitative data in diet study. Additional information could be obtained by the NGS analysis such as population or species identification of examined fish collected from difference locations. High OTU numbers belonging to Anisakis species showed that many fish species are now infected by various parasitic nematodes. Different from those in two pelagic fish species, high amount of ‘unidentified prey items’ were obtained in the stomach of the marbled flounder. It is postulated that P. yokohamae mainly preys on the benthic organisms such as polychaeta, nematodes, or sponges whose DNA sequence data are extremely low compared to those of pelagic animals. In order to establish better stomach content analysis system, more COI data of benthos should be supplemented. In conclusion, NGS technology can be applied for the stomach contents analysis of marine fish species and this techniques can replace the traditional morphological analysis with high accuracy and low cost and labors as long as high amount of DNA database are supplemented.