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DNA metabarcoding and morphological taxonomic (microscopic) analysis of the gut contents was used to examine diet diversity of seven species of fishes collected from mesopelagic depths (200-1000 m) in the NW Atlantic Ocean Slope Water during Summer 2018 and 2019. Metabarcoding used two gene regions: V9 hypervariable region of nuclear 18S rRNA and mitochondrial cytochrome oxidase I (COI). V9 sequences were classified into 14 invertebrate prey groups, excluding fish due to predator swamping. Ecological network analysis was used to evaluate relative strengths of predator-prey linkages. Multivariate statistical analysis revealed consistently distinct diets of four fish species in 2018 and/or 2019:Argyropelecus aculeatus, Chauliodus sloani, Hygophum hygomii, andSigmops elongatus. Three other species analyzed (Malacosteus niger, Nemichthys scolopaceus, andScopelogadus beanii) showed more variability between sampling years. COI sequences were classified into eight invertebrate prey groups, within which prey species were detected and identified. Considering all predator species together, a total of 77 prey species were detected with a minimum of 1,000 COI sequences, including 22 copepods, 18 euphausiids, and 7 amphipods. Morphological prey counts were classified into seven taxonomic groups, including a gelatinous group comprised of soft-bodied organisms. The ocean twilight zone or is home to exceptional diversity and biomass of marine fish, which are key players in deep sea food webs. This study used integrative morphological-molecular analysis to provide new insights into trophic relationships and sources of productivity for mesopelagic fishes, including identification of key prey species, recognition of the importance of gelatinous prey, and characterization of differences in diet among fish predators in the NW Atlantic Slope Water. 

Marine zooplankton are rapid-responders and useful indicators of environmental variability and climate change impacts on pelagic ecosystems on time scales ranging from seasons to years to decades. The systematic complexity and taxonomic diversity of the zooplankton assemblage has presented significant challenges for routine morphological (microscopic) identification of species in samples collected during ecosystem monitoring and fisheries management surveys. Metabarcoding using the mitochondrial Cytochrome Oxidase I (COI) gene region has shown promise for detecting and identifying species of some – but not all – taxonomic groups in samples of marine zooplankton. This study examined species diversity of zooplankton on the Northwest Atlantic Continental Shelf using 27 samples collected in 2002-2012 from the Gulf of Maine, Georges Bank, and Mid-Atlantic Bight during Ecosystem Monitoring (EcoMon) Surveys by the NOAA NMFS Northeast Fisheries Science Center. COI metabarcodes were identified using the MetaZooGene Barcode Atlas and Database ( https://metazoogene.org/MZGdb ) specific to the North Atlantic Ocean. A total of 181 species across 23 taxonomic groups were detected, including a number of sibling and cryptic species that were not discriminated by morphological taxonomic analysis of EcoMon samples. In all, 67 species of 15 taxonomic groups had ≥ 50 COI sequences; 23 species had >1,000 COI sequences. Comparative analysis of molecular and morphological data showed significant correlations between COI sequence numbers and microscopic counts for 5 of 6 taxonomic groups and for 5 of 7 species with >1,000 COI sequences for which both types of data were available. Multivariate statistical analysis showed clustering of samples within each region based on both COI sequence numbers and EcoMon counts, although differences among the three regions were not statistically significant. The results demonstrate the power and potential of COI metabarcoding for identification of species of metazoan zooplankton in the context of ecosystem monitoring. 

ABSTRACT DNA metabarcoding of zooplankton biodiversity is used increasingly for monitoring global ocean ecosystems, requiring comparable data from different research laboratories and ocean regions. The MetaZooGene Intercalibration Experiment (MZG‐ICE) was designed to examine1 and analyse patterns of variation of DNA sequence data resulting from multi‐gene metabarcoding of 10 zooplankton samples carried out by 10 research groups affiliated with the Scientific Committee for Ocean Research (SCOR). Aliquots of DNA extracted from the 10 zooplankton samples were distributed to MZG‐ICE groups for metabarcoding of four gene regions: V1‐V2, V4 and V9 of nuclear 18S rRNA and mitochondrial COI. Molecular protocols and procedures were recommended; substitutions were allowed as necessary. Resulting data were uploaded to a common repository for centralised statistics and bioinformatics. Based on proportional sequence numbers for abundant phyla, overall patterns of variation were consistent across many—but not all—MZG‐ICE groups. V9 showed highest similarity, followed (in order) by V4, V1‐V2, and COI. Outlier data were hypothesised to result from the use of different PCR protocols and sequencing platforms, and possible contamination. MZG‐ICE results indicated that DNA metabarcoding data from different laboratories and research groups can provide reliable, accurate and valid descriptions of biodiversity of zooplankton throughout the ocean. Recommendations included: pre‐screening QA/QC of raw data, detailed records for laboratory protocols, reagents, and instrumentation, and centralised bioinformatics and multivariate statistics. In the absence of universal agreement on standardised protocols or best practices, intercalibration is the best way forward toward validation of DNA metabarcoding of zooplankton diversity for global ocean monitoring.