Amino acid nitrogen isotopic compositions show seep copepods gain nutrition from host animals

Deep-sea chemosynthetic ecosystems harbour high biomass centred around animals with chemoautotrophic symbionts. Despite being intensively studied over the last 4 decades, microscopic animals associated with and/or parasitic on dominating holobionts remain understudied. Here, we combine bulk tissue isotope analysis for carbon and nitrogen and compound-specific isotope analysis of amino acids (CSIA-AA) for nitrogen to elucidate the relationship between 2 copepod-host pairs from the Off Hatsushima hydrothermal seep in Sagami Bay, Japan: the vesicomyid clam Phreagena okutanii and Hyphalion sagamiense living on its mantle, and the tubeworm Lamellibrachia columna and Dirivultus kaiko found on its plume. Bulk tissue isotope analyses found overall large variations in delta 13C and delta 15N values across both associations, and did not allow conclusions on the trophic relationship between each pair. In contrast, CSIA-AA for Phreagena-Hyphalion clearly showed trophic positions (TPGlu/Phe) increasing from gill (symbiont) to adductor muscle (host tissue) to H. sagamiense. In the case of Lamellibrachia-Dirivultus, a similar increase in TPGlu/Phe was found from the plume to D. kaiko. These results show that both H. sagamiense and D. kaiko are nutritionally dependent on their respective hosts and therefore should be considered parasites despite being from copepod families that are typically not recognised as parasitic. Our CSIA-AA results represent the first use of this technique to document host-parasite relationships in chemosynthetic ecosystems. Understanding the role of parasites is of great importance in reconstructing energy flow in ecosystems, and our results underscore the promising nature of CSIA-AA in revealing their otherwise hidden relationships.