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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. 

Fish diversity and ecology in the ocean’s mesopelagic zone are understudied compared to other marine regions despite growing interest in harvesting these potential resources. Otoliths can provide a wealth of taxonomic and life history information about fish, which can help fill these knowledge gaps; however, there has been relatively little research to date on the otoliths of mesopelagic species. Here, a species-specific image library was assembled of sagittal otoliths from 70 mesopelagic fishes belonging to 29 families collected in the western North Atlantic Ocean. Images of adult sagittal otoliths from 12 species were documented and photographed for the first time. The fish were identified to species with a combination of morphological characters and DNA barcoding. Regressions between otolith size and fish length are presented for the six species with the largest sample sizes in this study. This otolith image library, coupled with otolith-length and width to fish-length relationships, can be used for prey identification and back-calculation of fish size, making it a valuable tool for studies relating to food webs in the important yet poorly understood mesopelagic zone. In addition, the 44 fish barcodes generated in this study highlight the benefit of using an integrative taxonomic approach to studies of this nature, as well as add to existing public databases that enable cryptic species and metabarcoding analyses of mesopelagic species.