Unraveling the link between copepods and diazotrophs in the South China Sea

Abstract

Biological N₂ fixation is a crucial process in the marine nitrogen (N) cycle, driving primary productivity and biogeochemical cycling. As the dominant marine zooplankton, copepods play a critical role in biogeochemical fluxes and may potentially influence N₂ fixation. However, the potential interactions between copepods and diazotrophs remain poorly understood. In this study, we combined natural isotope abundance (δ¹⁵N) analysis, ¹⁵N tracer assays, and nitrogenase gene (nifH) sequencing to examine the potential link between N₂ fixation and copepods in the South China Sea during summer 2020. Notably, while high-throughput sequencing detected diverse nifH gene sequences (primarily Trichodesmium and proteobacteria) in copepod samples, no detectable copepod-associated N₂ fixation activity was observed using the ¹⁵N₂ tracer method. This decoupling between gene presence and detectable activity suggests that the copepod-associated nifH signals more likely reflected ingestion or transient retention rather than active N₂ fixation within or on copepods. Molecular evidence, especially the high similarity between copepod-associated and seawater nifH sequences, is consistent with a potential trophic linkage between copepods and diazotrophs. This interpretation is also supported by isotopic patterns: copepod δ¹⁵N values were positively correlated with suspended particulate δ¹⁵N, and both pools showed clear spatial gradients decreasing from nearshore to offshore regions. These results suggest that copepods may interact with diazotrophs and diazotroph-associated particulate matter through grazing pathways. Our findings suggest that copepods may provide a potential pathway for diazotroph-derived nitrogen to enter the food web, thereby influencing the fate of newly fixed nitrogen in oligotrophic waters.