ORIGINAL RESEARCH published: 14 August 2019 doi: 10.3389/fmicb.2019.01860 Divergent Capacity of Scleractinian and Soft Corals to Assimilate and Transfer Diazotrophically Derived Nitrogen to the Reef Environment Chloé A. Pupier1,2* †, Vanessa N. Bednarz1†, Renaud Grover1, Maoz Fine 3,4, Jean-François Maguer 5 and Christine Ferrier-Pagès1 1Marine Department, Centre Scientifique de Monaco, Monaco, Monaco, 2Collège Doctoral, Sorbonne Université, Paris, France, 3The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan, Israel, 4The Interuniversity Institute for Marine Science in Eilat, Eilat, Israel, 5Laboratoire de l’Environnement Marin (LEMAR), UMR 6539, UBO/CNRS/ IRD/IFREMER, Institut Universitaire Européen de la Mer, Plouzané, France Corals are associated with dinitrogen (N2)-fixing bacteria that potentially represent an additional nitrogen (N) source for the coral holobiont in oligotrophic reef environments. Edited by: Zhiyong Li, Nevertheless, the few studies investigating the assimilation of diazotrophically derived Shanghai Jiao Tong University, China nitrogen (DDN) by tropical corals are limited to a single scleractinian species (i.e., Stylophora Reviewed by: pistillata). The present study quantified DDN assimilation rates in four scleractinian and Christina A. Kellogg, three soft coral species from the shallow waters of the oligotrophic Northern Red Sea United States Geological 15 Survey (USGS), United States using the N2 tracer technique. All scleractinian species significantly stimulated N2 fixation Tuo Shi, in the coral-surrounding seawater (and mucus) and assimilated DDN into their tissue. Xiamen University, China Interestingly, N2 fixation was not detected in the tissue and surrounding seawater of soft *Correspondence: Chloé A. Pupier corals, despite the fact that soft corals were able to take up DDN from a culture of free- [email protected] living diazotrophs. Soft coral mucus likely represents an unfavorable habitat for the †These authors have contributed colonization and activity of diazotrophs as it contains a low amount of particulate organic equally to this work matter, with a relatively high N content, compared to the mucus of scleractinian corals. Specialty section: In addition, it is known to present antimicrobial properties. Overall, this study suggests This article was submitted to that DDN assimilation into coral tissues depends on the presence of active diazotrophs Microbial Symbioses, in the coral’s mucus layer and/or surrounding seawater. Since N is often a limiting nutrient a section of the journal Frontiers in Microbiology for primary productivity in oligotrophic reef waters, the divergent capacity of scleractinian Received: 05 June 2019 and soft corals to promote N2 fixation may have implications for N availability and reef Accepted: 29 July 2019 biogeochemistry in scleractinian versus soft coral-dominated reefs. Published: 14 August 2019 Citation: Keywords: dinitrogen fixation, diazotrophs, scleractinian corals, soft corals, Red Sea Pupier CA, Bednarz VN, Grover R, Fine M, Maguer J-F and Ferrier-Pagès C (2019) Divergent INTRODUCTION Capacity of Scleractinian and Soft Corals to Assimilate and Transfer Diazotrophically Derived Nitrogen Coral reefs are highly productive ecosystems despite thriving in oligotrophic waters, which contain to the Reef Environment. low levels of essential dissolved inorganic nutrients such as nitrogen (N) (de Goeij et al., 2013). Front. Microbiol. 10:1860. Corals, which are the main reef builders, can achieve high productivity thanks to their association doi: 10.3389/fmicb.2019.01860 with endosymbiotic dinoflagellates, which recycle the animal waste products and are efficient in Frontiers in Microbiology | www.frontiersin.org 1 August 2019 | Volume 10 | Article 1860 Pupier et al. Dinitrogen Fixation in Corals scavenging inorganic N dissolved in seawater (Wang and Douglas, and the soft coral Sarcophyton sp., which have been maintained 1999; Grover et al., 2003). Corals also profit from high rates in aquaria for years, and are not associated with active diazotrophs of N recycling in the water column and sediments via microbial (Bednarz, unpublished data; Kooperman et al., 2007). All processes (Carpenter and Capone, 2008), and from biological colonies were maintained under the same light (200 μmoles −2 −1 dinitrogen (N2) fixation of diazotrophic symbionts (Bednarz photons m s ) and temperature (25°C) conditions to ensure et al., 2017), which are heterotrophic bacteria and cyanobacteria comparability between the species during the following that convert atmospheric N2 into bioavailable N. In addition to experiment. For this purpose, a culture of diazotrophs being associated with benthic organisms (reviewed in Benavides (Crocosphaera watsonii) was prepared and diluted to obtain et al., 2017), diazotrophs can live pelagic, and colonize reef two cell concentrations of 100 and 1,000 cells ml−1, respectively, substrates (e.g., sediments, coral rubble). Many studies using matching concentrations of 3- to 20-μm size class phycoerythrin- the acetylene reduction assay (ARA) method have recorded high containing unicellular cyanobacteria under non-bloom and rates of N2 fixation either in reef waters or in the presence of bloom conditions (Campbell et al., 1997). C. watsonii corals (i.e., Bednarz et al., 2015; Rädecker et al., 2015; Cardini concentrations were determined using a hemocytometer et al., 2016). The ARA technique quantifies gross N2 fixation (Z1 particles counter, Beckman Coulter, USA). Four nubbins without providing insights into DDN (diazotrophically derived of each coral species per C. watsonii concentration were then nitrogen) assimilation by the coral-dinoflagellate symbiosis. Instead, individually placed in 320-ml gas-tight bottles completely filled 15 the use of labeled N2 gas measures DDN assimilation (net N2 with 280 ml of 0.22 μm-filtered seawater amended with the 15 fixation) and distinguishes between the different compartments adequate concentration of diazotrophs and 40 ml of N2 (i.e., seawater particles, tissue, dinoflagellate symbionts), but it enriched seawater. In addition, three nubbins of each species is still poorly known to what extent and under which conditions were incubated under the same above conditions but without corals profit from DDN. C. watsonii cells. At the end of the 24 h incubation, corals 15 Most of the research using the N2 method to assess DDN were removed and stored frozen until subsequent analysis. They assimilation in adult tropical corals has been directed toward were then processed as described below. only one species, Stylophora pistillata, belonging to the Pocilloporidae family. These studies showed that S. pistillata Field Experiment: Diazotrophically Derived can acquire DDN via grazing on a culture of diazotrophic Nitrogen/Diazotrophic Cells Assimilation cells (Benavides et al., 2016). However, under natural conditions, and Transfer to Seawater this coral species has variable capacities to stimulate diazotrophic Biological Material activity in seawater as well as variable DDN assimilation rates into the coral holobiont (Grover et al., 2014; Bednarz et al., The study was conducted during November 2017 at the 2017; Lesser et al., 2018). As DDN can play a major role in Interuniversity Institute (IUI) of Marine Sciences, Eilat, Red supplying N to corals (Cardini et al., 2016; Bednarz et al., Sea. Coral fragments were collected by SCUBA diving from 2017), the estimation of the capacity of different coral species different colonies of the reef adjacent to the IUI and were to take advantage of this nutrient source can help in brought back to the Red Sea Simulator facility (see Bellworthy understanding how they cope with nutrient limitation. In and Fine, 2018, for more details regarding the system) to addition, no study so far investigated DDN assimilation by recover for one day prior to starting the incubations. Scleractinian soft corals, although they represent together with scleractinian corals (Acropora eurystoma, n = 6; Pocillopora damicornis, corals the two most dominant benthic groups on many reefs n = 3; Goniastrea sp., n = 3; and Cynarina sp., n = 2) were worldwide. Soft corals can quickly colonize open space due sampled in shallow waters (8–10 m depth) and soft corals to their fast growth rates, high fecundity, and asexual reproduction (n = 5 for all species; Dendronephthya sp., Rhytisma fulvum and these opportunistic life history features may be facilitated fulvum and Litophyton sp.) in shallow waters and at upper by diazotrophs as additional N source (i.e., DDN). The aim mesophotic depths (40–45 m). Biological replicates for each of this study was to perform a multi-species comparison and species were derived from individual colonies and the different to investigate the capacity of different scleractinian and soft species were chosen in order to cover a broad range of families and morphological traits (Table 1). Three additional nubbins coral species to either assimilate DDN/diazotrophic cells and/or 15 transfer it to the surrounding seawater. per species per depth were collected to measure natural N abundance of the corals. At the collection time, the site was characterized by stable temperature (24–25°C) and nutrient MATERIALS AND METHODS levels (< 0.5 μM dissolved N and < 0.2 μM dissolved phosphorus) along the depth gradient (data from the Israel National Laboratory Experiment: Diazotrophically Monitoring program of the Gulf of Eilat, http://www.iui-eilat. Derived Nitrogen Assimilation