GRAZING BY ZOOPLANKTON ON DIAZOTROPHS IN THE AMAZON RIVER PLUME AND WESTERN TROPICAL NORTH ATLANTIC

Organisms capable of fixing di-nitrogen (N₂), known as diazotrophs, are important primary producers and a potentially significant source for new nitrogen entering the planktonic food web. However, limited evidence exists for zooplankton grazing on diazotrophs compared to other primary producers. In the western tropical North Atlantic Ocean (WTNA), the Amazon River plume creates a niche for symbiotic diatom-diazotroph associations (DDAs) which can form large blooms. In adjacent non-plume-influenced waters, the colonial cyanobacterium Trichodesmium is abundant. In order to reveal zooplankton-diazotroph grazing interactions and determine the fate of newly fixed nitrogen, gut contents of zooplankton captured in these two regions were compared based on quantitative PCR (qPCR) assay of nitrogenase genes (nifH), and their microbiomes compared using next generation sequencing (NGS) analysis of 16S rRNA genes. We sampled individual copepods from discrete depth intervals (0-25m and 25-50m) and in two size classes (0.5-1mm and 1-2mm) for analysis. A modified DNA extraction protocol was developed and 54 extracts were used as templates in nifH qPCR assays for the larger size fraction diazotrophs (>10µm): Trichodesmium, and Hemiaulus or Rhizosolenia (diatoms)-Richelia (diazotroph) associations. Copepod gut content nifH copies ranged from 1.6 to 13.6 copies individual^-1 for the assay targeting the Hemiaulus-Richelia DDA and from 1.1 to 3.0 copies individual^-1 for Trichodesmium. 16S NGS conducted on 35 extracts with an Ion Torrent PGM and mothur revealed that cyanobacteria sequences accounted for up to 20% of sequences per extract. Our results show that both DDAs and Trichodesmium are prey for zooplankton, and that new nitrogen moves through the food web via these grazing interactions. These interactions should be considered in future explorations of the global ocean nitrogen cycle.