Mesopelagic fishes are a crucial component of the world’s oceans in terms of their abundance, biomass, and ecosystem function. These fishes are important contributors to the biological carbon pump via their feeding and behaviors, whereby they facilitate the transfer of carbon from shallow waters to the deep sea. Several species undertake diel vertical migration, feeding in shallower waters at night and moving to deeper waters during the day. This process actively expedites the downward flux of carbon. However, carbon budgets and climate models require accurate information regarding the depth distributions and migration patterns of these fishes, and environmental DNA (eDNA) analyses can provide this information. Here, we utilize eDNA approaches, generating taxonomically-informative COI and 12S reference barcodes for 80 species of mesopelagic fishes, which can be used to for species-level identification of eDNA sequences. Using these, along with a publicly available barcode database, we compare results from eDNA analysis with traditional net sampling and explore the ability of eDNA techniques to detect diel vertical migration in fishes from samples collected in Northwest Atlantic Slope Water. We found that eDNA and net samples often resulted in different species identifications, demonstrating that eDNA can detect species that would otherwise be missed with traditional methods. In our eDNA samples, we also detected more species (12) in our shallowest depth category (0–100 m) from nighttime samples than from daytime samples (3). This is consistent with increased diversity in shallow waters at night due to diel vertical migration. Based on the variability observed in sample duplicates, we suggest that future mesopelagic eDNA studies incorporate larger sample volumes and scaled-up sampling efforts. We also note the potential for eDNA analysis to address ecological questions such as predator-prey relationships and identification of foraging hotspots, yielding insights into carbon flow through the ocean’s midwaters.

The data are photographs of mesopelagic fish specimens that were used to obtain COI and 12S DNA barcodes. The barcodes have been deposited in GenBank and the GenBank accession numbers are provided in a supplementary metadata file.

The file name includes the fish species and the source identification numbers, which are also provided in the associated manuscript supplementary data, as well as in the GenBank records.

The images are .jpg files that can be opened with any standard image software.