Constant nitrogen limitation is the foundation of a stable coral-algal symbiosis. Diazotrophs, i.e., prokaryotes capable of fixing N₂ into ammonia, are key players in coral holobionts. Recent studies on reef-building corals have shown that labile dissolved organic carbon (DOC) enrichment or heat stress stimulated the growth and activity of diazotrophs, resulting in excess nitrogen availability. However, the (a)biotic drivers of diazotrophs in octocorals are still poorly understood. We investigated diazotroph abundance (assessed via relative quantification of nifH gene copy numbers) in two symbiotic octocorals, the more mixotrophic soft coral Xenia umbellata and the more autotrophic gorgonian Pinnigorgia flava, under a) labile DOC enrichment for 21 days, followed by b) combined labile DOC enrichment and heat stress for 24 days. In the absence of heat stress, relative diazotroph abundances in X. umbellata and P. flava were unaffected by DOC enrichment. During heat stress, DOC enrichment (higher than 20 mg glucose L_-1) increased the relative abundances of diazotrophs by 6-fold in X. umbellata and 4-fold in P. flava, compared to their counterparts without excess DOC. Our data suggest that labile DOC enrichment and concomitant heat stress could disrupt the nitrogen limitation in octocorals through stimulating diazotroph proliferations. Reducing labile DOC loading may effectively help octocorals cope with future ocean warming scenarios.

We investigated diazotroph abundance (assessed via relative quantification of nifH gene copy numbers) in two symbiotic octocorals, the more mixotrophic soft coral Xenia umbellata and the more autotrophic gorgonian Pinnigorgia flava, under a) labile DOC enrichment for 21 days, followed by b) combined labile DOC enrichment and heat stress for 24 days. Specifically, a fragment of the nifH gene that encodes for the iron protein subunit of nitrogenase was used as a marker to assess their-associated diazotroph communities. Eight different primer combinations were evaluated for their ability to amplify fragments of the nifH gene in the DNA extracted from octocoral holobionts. The best primer pair was subsequently used for the nifH gene qPCR to quantify diazotroph abundance in two octocorals.

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