Contribution and pathways of diazotroph-derived nitrogen to zooplankton during the VAHINE mesocosm experiment in the oligotrophic New Caledonia lagoon

In oligotrophic tropical and subtropical oceans, where strong stratification can limit the replenishment of surface nitrate, dinitrogen (N-2) fixation by diazotrophs can represent a significant source of nitrogen (N) for primary production. The VAHINE (VAriability of vertical and tropHIc transfer of fixed N-2 in the south-wEst Pacific) experiment was designed to examine the fate of diazotroph-derived nitrogen (DDN) in such ecosystems. In austral summer 2013, three large ( similar to aEuro parts per thousand aEuro-50aEuro-m(3)) in situ mesocosms were deployed for 23 days in the New Caledonia lagoon, an ecosystem that typifies the low-nutrient, low-chlorophyll environment, to stimulate diazotroph production. The zooplankton component of the study aimed to measure the incorporation of DDN into zooplankton biomass, and assess the role of direct diazotroph grazing by zooplankton as a DDN uptake pathway. Inside the mesocosms, the diatom-diazotroph association (DDA) het-1 predominated during days 5-15 while the unicellular diazotrophic cyanobacteria UCYN-C predominated during days 15-23. A Trichodesmium bloom was observed in the lagoon (outside the mesocosms) towards the end of the experiment. The zooplankton community was dominated by copepods (63aEuro-% of total abundance) for the duration of the experiment. Using two-source N isotope mixing models we estimated a mean similar to aEuro parts per thousand aEuro-28aEuro-% contribution of DDN to zooplankton nitrogen biomass at the start of the experiment, indicating that the natural summer peak of N-2 fixation in the lagoon was already contributing significantly to the zooplankton. Stimulation of N-2 fixation in the mesocosms corresponded with a generally low-level enhancement of DDN contribution to zooplankton nitrogen biomass, but with a peak of similar to aEuro parts per thousand aEuro-73aEuro-% in mesocosm 1 following the UCYN-C bloom. qPCR analysis targeting four of the common diazotroph groups present in the mesocosms (Trichodesmium, het-1, het-2, UCYN-C) demonstrated that all four were ingested by copepod grazers, and that their abundance in copepod stomachs generally corresponded with their in situ abundance. N-15(2) labelled grazing experiments therefore provided evidence for direct ingestion and assimilation of UCYN-C-derived N by the zooplankton, but not for het-1 and Trichodesmium, supporting an important role of secondary pathways of DDN to the zooplankton for the latter groups, i.e. DDN contributions to the dissolved N pool and uptake by nondiazotrophs. This study appears to provide the first evidence of direct UCYN-C grazing by zooplankton, and indicates that UCYN-C-derived N contributes significantly to the zooplankton food web in the New Caledonia lagoon through a combination of direct grazing and secondary pathways