Integrating multiple chemical tracers to elucidate the diet and habitat of Cookiecutter Sharks.

The Cookiecutter shark (Isistius brasiliensis) is an ectoparasitic, mesopelagic shark that is known for removing plugs of tissue from larger prey, including teleosts, chondrichthyans, cephalopods, and marine mammals. Although this species is widely distributed throughout the world's tropical and subtropical oceanic waters, like many deep-water species, it remains very poorly understood due to its mesopelagic distribution. We used a suite of biochemical tracers, including stable isotope analysis (SIA), fatty acid analysis (FAA), and environmental DNA (eDNA), to investigate the trophic ecology of this species in the Central Pacific around Hawaii. We found that large epipelagic prey constituted a relatively minor part of the overall diet. Surprisingly, small micronektonic and forage species (meso- and epipelagic) are the most important prey group for Cookiecutter sharks across the studied size range (17-43 cm total length), with larger mesopelagic species or species that exhibit diel vertical migration also being important prey. These results were consistent across all the tracer techniques employed. Our results indicate that Cookiecutter sharks play a unique role in pelagic food webs, feeding on prey ranging from the largest apex predators to small, low trophic level species, in particular those that overlap with the depth distribution of the sharks throughout the diel cycle. We also found evidence of a potential shift in diet and/or habitat with size and season. Environmental DNA metabarcoding revealed new prey items for Cookiecutter sharks while also demonstrating that eDNA can be used to identify recent prey in stomachs frozen for extended periods. Integrating across chemical tracers is a powerful tool for investigating the ecology of elusive and difficult to study species, such as meso- and bathypelagic chondrichthyans, and can increase the amount of information gained from small sample sizes. Better resolving the foraging ecology of these mesopelagic predators is critical for effective conservation and management of these taxa and ecosystems, which are intrinsically vulnerable to overfishing and exploitation.