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      DNA barcoding identifies a cosmopolitan diet in the ocean sunfish

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          Abstract

          The ocean sunfish ( Mola mola) is the world’s heaviest bony fish reaching a body mass of up to 2.3 tonnes. However, the prey M. mola consumes to fuel this prodigious growth remains poorly known. Sunfish were thought to be obligate gelatinous plankton feeders, but recent studies suggest a more generalist diet. In this study, through molecular barcoding and for the first time, the diet of sunfish in the north-east Atlantic Ocean was characterised. Overall, DNA from the diet content of 57 individuals was successfully amplified, identifying 41 different prey items. Sunfish fed mainly on crustaceans and teleosts, with cnidarians comprising only 16% of the consumed prey. Although no adult fishes were sampled, we found evidence for an ontogenetic shift in the diet, with smaller individuals feeding mainly on small crustaceans and teleost fish, whereas the diet of larger fish included more cnidarian species. Our results confirm that smaller sunfish feed predominantly on benthic and on coastal pelagic species, whereas larger fish depend on pelagic prey. Therefore, sunfish is a generalist predator with a greater diversity of links in coastal food webs than previously realised. Its removal as fisheries’ bycatch may have wider reaching ecological consequences, potentially disrupting coastal trophic interactions.

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          A new versatile primer set targeting a short fragment of the mitochondrial COI region for metabarcoding metazoan diversity: application for characterizing coral reef fish gut contents

          Introduction The PCR-based analysis of homologous genes has become one of the most powerful approaches for species detection and identification, particularly with the recent availability of Next Generation Sequencing platforms (NGS) making it possible to identify species composition from a broad range of environmental samples. Identifying species from these samples relies on the ability to match sequences with reference barcodes for taxonomic identification. Unfortunately, most studies of environmental samples have targeted ribosomal markers, despite the fact that the mitochondrial Cytochrome c Oxidase subunit I gene (COI) is by far the most widely available sequence region in public reference libraries. This is largely because the available versatile (“universal”) COI primers target the 658 barcoding region, whose size is considered too large for many NGS applications. Moreover, traditional barcoding primers are known to be poorly conserved across some taxonomic groups. Results We first design a new PCR primer within the highly variable mitochondrial COI region, the “mlCOIintF” primer. We then show that this newly designed forward primer combined with the “jgHCO2198” reverse primer to target a 313 bp fragment performs well across metazoan diversity, with higher success rates than versatile primer sets traditionally used for DNA barcoding (i.e. LCO1490/HCO2198). Finally, we demonstrate how the shorter COI fragment coupled with an efficient bioinformatics pipeline can be used to characterize species diversity from environmental samples by pyrosequencing. We examine the gut contents of three species of planktivorous and benthivorous coral reef fish (family: Apogonidae and Holocentridae). After the removal of dubious COI sequences, we obtained a total of 334 prey Operational Taxonomic Units (OTUs) belonging to 14 phyla from 16 fish guts. Of these, 52.5% matched a reference barcode (>98% sequence similarity) and an additional 32% could be assigned to a higher taxonomic level using Bayesian assignment. Conclusions The molecular analysis of gut contents targeting the 313 COI fragment using the newly designed mlCOIintF primer in combination with the jgHCO2198 primer offers enormous promise for metazoan metabarcoding studies. We believe that this primer set will be a valuable asset for a range of applications from large-scale biodiversity assessments to food web studies.
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            The Effects of Fishing on Marine Ecosystems

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              DNA metabarcoding multiplexing and validation of data accuracy for diet assessment: application to omnivorous diet.

              Ecological understanding of the role of consumer-resource interactions in natural food webs is limited by the difficulty of accurately and efficiently determining the complex variety of food types animals have eaten in the field. We developed a method based on DNA metabarcoding multiplexing and next-generation sequencing to uncover different taxonomic groups of organisms from complex diet samples. We validated this approach on 91 faeces of a large omnivorous mammal, the brown bear, using DNA metabarcoding markers targeting the plant, vertebrate and invertebrate components of the diet. We included internal controls in the experiments and performed PCR replication for accuracy validation in postsequencing data analysis. Using our multiplexing strategy, we significantly simplified the experimental procedure and accurately and concurrently identified different prey DNA corresponding to the targeted taxonomic groups, with ≥ 60% of taxa of all diet components identified to genus/species level. The systematic application of internal controls and replication was a useful and simple way to evaluate the performance of our experimental procedure, standardize the selection of sequence filtering parameters for each marker data and validate the accuracy of the results. Our general approach can be adapted to the analysis of dietary samples of various predator species in different ecosystems, for a number of conservation and ecological applications entailing large-scale population level diet assessment through cost-effective screening of multiple DNA metabarcodes, and the detection of fine dietary variation among samples or individuals and of rare food items.
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                Author and article information

                Journal
                Sci Rep
                Sci Rep
                Scientific Reports
                Nature Publishing Group
                2045-2322
                04 July 2016
                2016
                : 6
                : 28762
                Affiliations
                [1 ]CIBIO – Universidade do Porto, Centro de Investigação em Biodiversidade e Recursos Genéticos, Campus Agrário de Vairão, Rua Padre Armando Quintas , 4485-668 Vairão, Portugal
                [2 ]Marine Biological Association of the United Kingdom, The Laboratory , Citadel Hill, Plymouth PL1 2PB, UK
                [3 ]Ocean and Earth Science, University of Southampton, Waterfront Campus, European Way , Southampton SO14 3ZH, UK
                [4 ]School of Biological Sciences, Cardiff University , Cardiff, CF10 3AX, UK
                [5 ]Faculty of Sciences, University of Porto , Rua do Campo Alegre s/n, 4169-007 Porto, Portugal
                [6 ]MARE – Marine and Environmental Sciences Centre, Laboratório Marítimo da Guia, Faculdade de Ciências da Universidade de Lisboa , Av. Nossa Senhora do Cabo, 939, 2750-374 Cascais, Portugal
                [7 ]Centre for Biological Sciences, Building 85, University of Southampton, Highfield Campus , Southampton SO17 1BJ, UK
                Author notes
                Article
                srep28762
                10.1038/srep28762
                4931451
                27373803
                1b1dd447-cd20-455b-ae62-df7f9377972c
                Copyright © 2016, Macmillan Publishers Limited

                This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

                History
                : 15 March 2016
                : 07 June 2016
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