Mar Benavides

Chloé Martias

Hila Elifantz

Ilana Berman-Frank

Cécile Dupouy

Sophie Bonnet

Dissolved Organic Matter Influences N 2 Fixation in the New Caledonian Lagoon (Western Tropical South Pacific)

Keywords: non-cyanobacterial diazotrophs, nifH, DCMU, DOM, CDOM, FDOM

Specialized prokaryotes performing biological dinitrogen (N 2 ) fixation ("diazotrophs") provide an important source of fixed nitrogen in oligotrophic marine ecosystems such as tropical and subtropical oceans. In these waters, cyanobacterial photosynthetic diazotrophs are well known to be abundant and active, yet the role and contribution of non-cyanobacterial diazotrophs are currently unclear. The latter are not photosynthetic (here called "heterotrophic") and hence require external sources of organic matter to sustain N 2 fixation. Here we added the photosynthesis inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) to estimate the N 2 fixation potential of heterotrophic diazotrophs as compared to autotrophic ones. Additionally, we explored the influence of dissolved organic matter (DOM) on these diazotrophs along a coast to open ocean gradient in the surface waters of a subtropical coral lagoon (New Caledonia). Total N 2 fixation (samples not amended with DCMU) ranged from 0.66 to 1.32 nmol N L -1 d -1 . The addition of DCMU reduced N 2 fixation by >90%, suggesting that the contribution of heterotrophic diazotrophs to overall N 2 fixation activity was minor in this environment. Higher contribution of heterotrophic diazotrophs occurred in stations closer to the shore and coincided with the decreasing lability of DOM, as shown by various colored DOM and fluorescent DOM (CDOM and FDOM) indices. We tested the response of diazotrophs (in terms of nifH gene expression and bulk N 2 fixation rates) upon the addition of a mix of carbohydrates ("DOC" treatment), amino acids ("DON" treatment), and phosphonates and phosphomonesters ("DOP" treatment). While nifH expression increased significantly in Trichodesmium exposed to the DOC treatment, bulk N 2 fixation rates increased significantly only in the DOP treatment. The lack of nifH expression by gammaproteobacteria, in any of the DOM addition treatments applied, questions the contribution of non-cyanobacterial diazotrophs to fixed nitrogen inputs in the New Caledonian lagoon. While the metabolism and ecology of heterotrophic diazotrophs is currently elusive, a deeper understanding of their ecology and relationship with DOM is needed in the light of increased DOM inputs in coastal zones due to anthropogenic pressure.

INTRODUCTION

Biological dinitrogen (N 2 ) fixation provides an important source of fixed nitrogen to fuel primary production in the oceans [START_REF] Karl | Dinitrogen fixation in the world's oceans[END_REF], being especially critical in areas devoid of other significant fixed nitrogen sources such as the oligotrophic subtropical gyres. N 2 fixation is performed by specialized prokaryotic microbes called "diazotrophs." These include cyanobacterial and non-cyanobacterial (bacteria and archaea) groups, that are commonly detected and quantified via the presence of the nifH gene, which encodes for a subunit of the nitrogenase enzyme system [START_REF] Zehr | Nitrogen fixation by marine cyanobacteria[END_REF]. Traditionally, marine diazotrophs were hypothesized to be constrained by warm, low inorganic nitrogen waters [START_REF] Sohm | Emerging patterns of marine nitrogen fixation[END_REF]). Yet, several filamentous cyanobacteria are able to obtain energy from dissolved organic matter-DOM- [START_REF] Rippka | Generic assignments, strain histories and properties of pure cultures of cyanobacteria[END_REF], and several reports have shown that the most abundant photoautotrophs in the world's oceans (Prochlorococcus and Synechococcus) harbor the genes necessary to use amino acids, peptides and sugars [START_REF] Yelton | Global genetic capacity for mixotrophy in marine picocyanobacteria[END_REF]. As evidence accumulates, it seems that the ability of using DOM as an alternative source of nutrients under limiting situations is common in various planktonic groups [START_REF] Stoecker | Mixotrophy in the marine plankton[END_REF]. Among marine diazotrophs, unicellular cyanobacteria such as Cyanothece have been reported to grow on glycerol [START_REF] Feng | Mixotrophic and photoheterotrophic metabolism in Cyanothece sp. ATCC 51142 under continuous light[END_REF], and filamentous diazotrophs such as Trichodesmium may obtain as much carbon from amino acids assimilation as from CO 2 fixation in the field [START_REF] Benavides | Dissolved organic matter uptake by Trichodesmium in the Southwest Pacific[END_REF]. Non-cyanobacterial and nonphotosynthetic diazotrophs require external sources of DOM as energy sources [START_REF] Riemann | Nitrogenase genes in non-cyanobacterial plankton: prevalence, diversity and regulation in marine waters[END_REF]. The globally widespread non-cyanobacterial diazotroph Gamma A [START_REF] Langlois | Widespread distribution and expression of Gamma A (UMB), an uncultured, diazotrophic, γ-proteobacterial nifH phylotype[END_REF] upregulates nifH gene expression upon glucose + mannitol additions [START_REF] Moisander | Analogous nutrient limitations in unicellular diazotrophs and Prochlorococcus in the South Pacific Ocean[END_REF]. Previous work has also shown that N 2 fixation activity is stimulated by DOM additions [START_REF] Bonnet | Aphotic N 2 fixation in the eastern tropical South Pacific Ocean[END_REF][START_REF] Rahav | Dinitrogen fixation in aphotic oxygenated marine environments[END_REF][START_REF] Rahav | Heterotrophic and autotrophic contribution to dinitrogen fixation in the Gulf of Aqaba[END_REF], and correlates with labile DOM compounds in environments where non-cyanobacterial (non-photosynthetic) diazotrophs predominate, such as the aphotic mesopelagic layer (Benavides et al., 2015), or the surface ultraoligotrophic eastern Mediterranean Sea [START_REF] Rahav | Contribution of mono and polysaccharides to heterotrophic N 2 fixation at the eastern Mediterranean coastline[END_REF].

N 2 fixation and diazotroph community studies have mostly focused on populations in the North Atlantic [START_REF] Benavides | Five decades of N 2 fixation research in the North Atlantic Ocean[END_REF] and North Pacific Oceans, while other areas such as the Indian and the South Atlantic and Pacific Oceans have been chronically under-sampled [START_REF] Luo | Database of diazotrophs in global ocean: abundance, biomass and nitrogen fixation rates[END_REF]. The western tropical Southwest Pacific (WTSP) Ocean comprises warm, oligotrophic, trace metal rich waters that harbor among the high N 2 fixation rates measured at sea (>500 µmol N m -2 d -1; [START_REF] Bonnet | Hot spot of N 2 fixation in the western tropical South Pacific pleads for a spatial decoupling between N 2 fixation and denitrification[END_REF]. The activity and distribution of diazotrophs in the WTSP has been related to temperature, iron and inorganic nutrient availability thresholds (e.g., [START_REF] Bonnet | Nitrogen fixation in the western equatorial Pacific: rates, diazotrophic cyanobacterial size class distribution, and biogeochemical significance[END_REF][START_REF] Moisander | Unicellular cyanobacterial distributions broaden the oceanic N 2 fixation domain[END_REF]. In austral summer conditions, the relatively warmer and productive waters like the Arafura, Timor and Solomon Seas (∼26.5-30 • C) are dominated by Trichodesmium and UCYN-B [START_REF] Messer | High levels of heterogeneity in diazotroph diversity and activity within a putative hotspot for marine nitrogen fixation[END_REF], while UCYN-A remains confined to the colder and more oligotrophic Coral Sea (∼22-26.5 • C; [START_REF] Moisander | Unicellular cyanobacterial distributions broaden the oceanic N 2 fixation domain[END_REF][START_REF] Bonnet | Contrasted geographical distribution of N 2 fixation rates and nifH phylotypes in the Coral and Solomon Seas (southwestern Pacific) during austral winter conditions[END_REF][START_REF] Messer | High levels of heterogeneity in diazotroph diversity and activity within a putative hotspot for marine nitrogen fixation[END_REF]. Together with the high abundances of diazotroph phylotypes [START_REF] Moisander | Unicellular cyanobacterial distributions broaden the oceanic N 2 fixation domain[END_REF][START_REF] Bonnet | Contrasted geographical distribution of N 2 fixation rates and nifH phylotypes in the Coral and Solomon Seas (southwestern Pacific) during austral winter conditions[END_REF], the WTSP has been recently acknowledged as a hotspot of global importance for N 2 fixation activity [START_REF] Bonnet | Hot spot of N 2 fixation in the western tropical South Pacific pleads for a spatial decoupling between N 2 fixation and denitrification[END_REF]. Thus, understanding how environmental factors (besides temperature and inorganic nutrients) shape the distribution and activity of diazotrophs in the WTSP is of utmost importance to assess their potential to fuel primary production in these oligotrophic waters and their contribution to the oceanic nitrogen budget.

New Caledonia, situated at the eastern edge of the WTSP, is surrounded by a reef enclosing one of the largest coral lagoons in the world. Here, primary production is nitrogenlimited throughout the year [START_REF] Torréton | Variability of primary and bacterial production in a coral reef lagoon (New Caledonia)[END_REF], prompting diazotrophic activity [START_REF] Garcia | Seasonal nitrogen fixation and primary production in the Southwest Pacific: nanoplankton diazotrophy and transfer of nitrogen to picoplankton organisms[END_REF][START_REF] Biegala | High abundance of diazotrophic picocyanobacteria (<3 µm) in a Southwest Pacific coral lagoon[END_REF] and the presence of various cyanobacterial diazotroph phylotypes including Trichodesmium (often occurring as large blooms; [START_REF] Rodier | Population dynamics and environmental conditions affecting Trichodesmium spp. (filamentous cyanobacteria) blooms in the south-west lagoon of New Caledonia[END_REF], diatom-diazotroph symbioses (DDAs) as well the unicellular cyanobacteria UCYN-A1, UCYN-A2, UCYN-B, and UCYN-C [START_REF] Turk-Kubo | Diazotroph community succession during the VAHINE mesocosm experiment (New Caledonia lagoon)[END_REF]. However, non-cyanobacterial diazotrophs have been far less studied, and despite representatives such as the putative gammaproteobacterium γ-24774A11 that has been detected at abundances of 10 2 -10 3 nifH copies L -1 within the lagoon [START_REF] Turk-Kubo | Diazotroph community succession during the VAHINE mesocosm experiment (New Caledonia lagoon)[END_REF], their contribution to N 2 fixation in this environment remains unquantified. Moreover, the importance of DOM as a source of energy and nutrition for both cyanobacterial and non-cyanobacterial diazotrophs has never been explored in this environment. In this study we assess if the inhibition of photosynthesis triggers DOM nutrition in pelagic diazotrophs, and whether the N 2 fixation activity and/or nifH expression of these pelagic diazotrophs is enhanced by different model DOM molecules.

MATERIALS AND METHODS

Hydrography, Oxygen, Inorganic Nutrients, Chl α and DOC Measurements Sampling took place on 13 January 2016 approximately from 8:00 a.m. to noon at four stations along an anthropogenic pressure gradient in the New Caledonian lagoon onboard the R/V Archamia. Station 1 was close to the rim of the coral reef barrier and Station 4 near the harbor of the city of Nouméa (Figure 1, Table 1). This transect connects the passage of Dumbéa (the main canal for the entrance of ships into the lagoon) with the harbor. Sampling started at Station 1 at high tide and proceeded toward the coast as the tide ebbed.

At each station, temperature and salinity were measured by casting a SeaBird SB19 plus CTD probe from the surface to the bottom. Water samples for the measurement of oxygen, nutrients, chlorophyll a (Chl a) and DOC concentrations, and colored DOM (CDOM) and fluorescent DOM (FDOM) parameters (see below) were collected from the subsurface (∼3 m), using a 5 L Niskin bottle and transported to the lab for analysis in coolers equipped with icepacks within 2 h. Samples for the measurement of dissolved oxygen were taken in triplicate and analyzed ashore using an automated titration system with a photometric endpoint [START_REF] Langdon | Determination of dissolved oxygen in seawater by Winkler titration using the amperometric technique[END_REF]. Samples for the determination of nitrate plus nitrite (NO x ) and phosphate (PO 3- 4 ) concentrations were taken on acid-washed 20 mL polyethylene tubes, poisoned with 1% HgCl 2 , and stored at 4 • C until analysis. The samples were analyzed on a AA3 Bran+Luebbe autoanalyzer following JGOFS recommendations [START_REF] Jgofs | Protocols For The Joint Global Ocean Flux Study (JGOFS) core measurements[END_REF]. The detection limits were 50 and 10 nM for NO x and PO 3- 4 , respectively. Chl a concentrations were estimated from seawater samples (500 mL) filtered through 25 mm GF/F filters (Whatman) and stored at -80 • C until analysis, extracted in methanol on a AU Turner Designs bench fluorometer, previously calibrated with pure Chl a [START_REF] Herbland | Size structure of phytoplankton biomass in the equatorial Atlantic Ocean[END_REF]. Samples for DOC were collected on triplicate precombusted (450 • C, 6 h) glass ampoules and acidified to pH 2 with 50 µL of 50% phosphoric acid. The samples were analyzed by high temperature catalytic oxidation on a TOC-V analyzer [START_REF] Sohrin | Seasonal variation in total organic carbon in the northeast Atlantic in 2000-2001[END_REF].

FDOM and CDOM Analyses

Samples for the analysis of FDOM and CDOM were filtered through 0.2 µm polycarbonate filters in a custom-made filtration system pre-cleaned with 1 M HCl and Milli-Q water. Samples for FDOM analyses were stored at 4 • C until analysis (within 1 month), while samples for CDOM absorption analyses were directly measured on a liquid waveguide capillary flow cell (WPI) according to the method of [START_REF] Kowalczuk | Composition of dissolved organic matter along an Atlantic meridional transect from fluorescence spectroscopy and parallel factor analysis[END_REF]. Excitationemission matrix (EEM) spectra for analysis of FDOM were determined using a Hitachi F-7000 spectrofluorometer [START_REF] Tedetti | Characterization of dissolved organic matter in a coral reef ecosystem subjected to anthropogenic pressures (La Réunion Island, Indian Ocean) using multidimensional fluorescence spectroscopy[END_REF]. FDOM components were identified by parallel factor analysis (PARAFAC), a statistical decomposition technique used to extract the most representative fluorescent components within EEMs dataset. To increase the validity of the PARAFAC analysis, data from the present experiment were pooled with 39 other samples obtained from the same waters, 3 months before and after the experiment. Indeed, to avoid systematic biases, remove signals unrelated to fluorescence and to validate the number of FDOM components, a large dataset was needed [START_REF] Stedmon | Tracing dissolved organic matter in aquatic environments using a new approach to fluorescence spectroscopy[END_REF]. For example, in the case where two of the components had very similar fluorescence characteristics and/or were highly correlated in their appearance among samples, the PARAFAC approach would have difficulties to discern between the two. We then determined several spectral indices: the absorption at 442 nm as a proxy of phytoplankton production and the absorption at 350 nm as an indicator of terrestrial inputs in the bay [START_REF] Passow | A dye-binding assay for the spectrophotometric measurement of transparent exopolymer particles (TEP) in the ocean[END_REF], a biological index (BIX) of recent autochthonous FDOM contribution [START_REF] Huguet | Properties of fluorescent dissolved organic matter in the Gironde estuary[END_REF], a specific ultraviolet absorbance at 254 nm of DOC (SUVA 254 ), which is an indicator of aromatic DOM [START_REF] Weishaar | Evaluation of specific ultraviolet absorbance as an indicator of the chemical composition and reactivity of dissolved organic carbon[END_REF], and the ratio of spectral slopes (S r ) at 275-295 nm (S 275-295 ) and 350-400 nm (S 350-400 ) ranges, related to the molecular weight shift of DOM [START_REF] Helms | Absorption spectral slopes and slope rations as indicators of molecular weight, source, and photoleaching of chromophoric dissolved organic matter[END_REF]; Table 2).

N 2 and Carbon Fixation Rates

Seawater for the quantification of N 2 and carbon fixation rates was sampled using a Teflon pump connected to polyethylene tubing. Bulk N 2 and carbon fixation rates were measured at each transect station on triplicate 2.3 L transparent polycarbonate bottles filled to overflow and closed air-free with septum screwcaps. Once in the lab (within ∼2 h) the bottles were amended with stable isotopes to assay N 2 and carbon fixation simultaneously. Each bottle was amended with NaH 13 CO 3 99 atom% 13 C to a final ∼10 atom% enrichment, and 2.5 mL 98.9 atom% 15 N 2 gas (both isotopes acquired from Cambridge Isotope Laboratories) injected through the septum using a gastight syringe [START_REF] Montoya | A simple, highprecision, high-sensitivity tracer assay for N 2 fixation[END_REF]. Commercial 15 N 2 gas stocks have been recently reported to be contaminated with varying amounts of other nitrogenated forms such as nitrous oxide and nitrite [START_REF] Dabundo | The contamination of commercial 15 N 2 gas stocks with 15 N-labeled nitrate and ammonium and consequences for nitrogen fixation measurements[END_REF]. The 15 N 2 gas batch used here was ) tested for contamination levels at J. Granger's lab (University of Connecticut, CT, USA), which were found to be negligible and hence did not affect our N 2 fixation rates (Benavides et al., 2015).

NOx (µM) PO 3- 4 (µM) Chl a (µg L -1 ) DOC (µM) a442 (m -1 ) a350 (m -1
After isotope additions, the incubation bottles were inverted 20 times, transferred to running seawater incubators covered with blue light screening reproducing the light intensity at the corresponding sampling depth, and incubated for 24 h. To test for non-cyanobacterial (non-photoautotrophic) N 2 fixation activity, a second set of incubation bottles was amended with DCMU to a final concentration of 50 µM [START_REF] Rahav | Heterotrophic and autotrophic contribution to dinitrogen fixation in the Gulf of Aqaba[END_REF]. These bottles were covered with black fabric bags to keep them in the dark at the same conditions as described above.

After incubation, the samples were filtered through precombusted GF/F filters (Whatman), and the filters were stored at -20 • C until analysis. Background (time zero δ 15 N and δ 13 C) samples were taken at every station to allow an accurate calculation of N 2 and carbon fixation rates. The samples were analyzed by continuous-flow isotope ratio mass spectrometry (IRMS) using an Integra2 Analyser (Integra CN), calibrated with International Atomic Energy Agency standards IAEA 310-A for nitrogen (47.2‰) and IAEA 303-B for carbon (466‰). The minimum quantifiable N 2 fixation rate was 0.0041 nmol N L -1 d -1 , as determined by error propagation analysis [START_REF] Gradoville | Diversity and activity of nitrogen-fixing communities across ocean basins[END_REF]. We note that the use of the traditional 15 N 2 "bubble method" [START_REF] Montoya | A simple, highprecision, high-sensitivity tracer assay for N 2 fixation[END_REF] instead of the "dissolved method" [START_REF] Mohr | Methodological underestimation of oceanic nitrogen fixation rates[END_REF] may have resulted in an underestimation of the actual rates [START_REF] Grokopf | Doubling of marine dinitrogen-fixation rates based on direct measurements[END_REF], although the long incubation time (24 h) likely reduces this possibility.

DOM Addition Experiments

To test for the enhancement of N 2 fixation rates and/or nifH gene expression as a response to DOM enrichments, we sampled seawater in 25 extra 2.3 L polycarbonate bottles at Station 2. Five bottles were amended with a mix of carbohydrates -"DOC treatment"-(sodium pyruvate, sodium acetate and glucose), another five with a mix of amino acids -"DON treatment"-(alanine, leucine, and glutamic acid), and another five were amended with a mix of phosphomonoesters and phosphonates -"DOP treatment"-(methylphosphonic acid, 2aminoethylphosphonic acid and fructose 1,6-biphosphate). All DOM mixtures were prepared to be equimolar in carbon (all contained 4 µM carbon; Benavides et al., 2015). Five bottles were used as a control (no DOM additions), and another five bottles were used as a "time zero" (i.e., sacrificed at the start of the experiment to know the baseline conditions). All bottles were spiked with 2.5 mL 15 N 2 and incubated for 24 h as described above. At the end of the incubation period, samples for NO x , PO 3- 4 and DOC analyses were taken from three bottles of each treatment (i.e., DOC, DON, DOP) as described above. The fourth and fifth bottles from each treatment were kept for RNA analyses, respectively (see below).

DNA and RNA Sampling, Extraction and qPCR Assays

Samples for DNA extractions were obtained at each transect station in 2.3 L polycarbonate bottles. Once ashore, the samples were filtered through 0.2 µm Supor filters (PALL) using a peristaltic pump. Upon filtration the filters were transferred to bead beating tubes including ∼50 µL of a mixture of 0.1 mm and 0.5 mm diameter glass beads (BioSpec Products), and stored at -80 • C until analysis. DNA was extracted via the phenolchloroform method [START_REF] Massana | Vertical distribution and phylogenetic characterization of marine planktonic Archaea in the Santa Barbara channel[END_REF]. RNA samples were taken in duplicate from each treatment (see "DOM addition experiments" above), filtered through 0.2 µm Supor filters and stored in sterile bead beating tubes containing 350 µL RLT buffer (Invitrogen) and 3.5 µL ß-mercaptoethanol, and finally stored at -80 • C until analysis. The RNA was extracted by TRI Reagent (Invitrogen). 1 mL of Tri reagent was added to each sample that was disrupt and homogenized by pipetting and vortexing in order to break the cells. The cell lysate was incubated at room temperature for 3 h with occasional vortexing. The samples were then centrifuge at 12,000 g for 20 min at 4 • C. The supernatant was transferred to a new tube and 10% of Phase Separation Reagent, BCP (Molecular Research Center, Inc., Cincinnati, OH, USA) was added. After additional incubation (30 min) at room temperature and centrifugation (12,000 g, 15 min, 4 • C) the supernatant was transferred to a new tube and a cleanup and DNase treatment was performed using the RNA Clean and Concentrator-5 kit (Zymo Research, Irvin, CA, USA). Complementary DNA (cDNA) was created using the qScript TM cDNA Synthesis Kit (Quantabio, Beverly, MA, USA) using the reverse primers of the nifH (nifH2 and nifH3; [START_REF] Zani | Expression of nifH genes in natural microbial assemblages in lake george, New York, detected by reverse transcriptase PCR[END_REF]. Quantitative PCR (qPCR) assays were performed on a CFX-96 Touch Real-Time PCR Detection System (Bio-Rad) using the TaqMan Fast Advanced Master Mix (Applied Biosystems) and primers for nifH of UCYN-A, UCYN-B, Trichodesmium and γ-24774A11 (here referred to as gammaproteobacteria) [START_REF] Turk | Nitrogen fixation and nitrogenase (nifH) expression in tropical waters of the eastern North Atlantic[END_REF].

RESULTS

Hydrography, Nutrients and in Situ DOM

The temperature of surface waters increased from 26.97 to 28.23 • C moving onshore from Station 1 toward Station 4, while salinity remained close to 35.6 at all stations (Table 1). The concentration of oxygen decreased onshore from 201 to 194 µmol kg -1 , while inorganic nutrient concentrations (NO x and PO 3- 4 ) did not vary significantly along the transect, and neither did Chl a concentrations, with the exception of Station 3 where Chl a was slightly lower (Table 1). DOC concentrations remained between 80 and 90 µM along the transect, showing a peak of ∼98 µM at Station 2 (Table 1).

CDOM absorption at 442 nm and 350 nm (a 442 and a3 50 ) presented lower values at station 2 and 3 as compared to the values recorded at Stations 1 and 4 (Table 1) which indicated a decrease in CDOM concentrations. Moreover, the spectral slope S 275-295 and the spectral ratio S r were high with values >1 and >0.0.3 nm -1 , respectively, which indicated the presence of photodegraded DOM with low molecular weight. In addition, the SUVA 254 indices were <4 (this reference value is indicative of DOM with low aromaticity) with an average value of 1.09 ± 0.44. Then CDOM at 442 nm and 350 nm is strongly photodegraded with a low aromaticity. Three fluorescent DOM compounds were identified by the PARAFAC model: terrestrial humic-like (compound 1), tyrosine-like (compound 2) and tryptophanlike (compound 3; Table 3). Humic-like compound signatures were higher closer to the coast (Station 4) and decreased to values around ∼5 QSU at the other stations. This pattern is opposite to that of tyrosine-like compounds signals, which increased toward the barrier reef. Tryptophane-like compound concentrations did not follow any coast-open ocean pattern but presented the highest concentrations at Station 2. The BIX indices were homogenous and >1 (this value is indicative of an enrichment in DOM from bacterial origin) along the transect (Table 1). Overall, the optical analyses of DOM suggest that while river-derived refractory DOM was more abundant close to the coast, biological-derived labile DOM compounds were present homogeneously at all stations along the transect.

N 2 and Carbon Fixation Activity and Diazotroph Abundance

No clear spatial trends were observed in either N 2 or carbon fixation rates along the transect. N 2 fixation rates in samples not amended with DCMU ranged from 0.66 to 1.32 nmol N L -1 d -1 , with the highest rates measured at Station 3 (Figure 2A). Carbon fixation rates in samples without DCMU ranged from 1.50 to 2.14 µg C L -1 d -1 (Figure 2B). Both N 2 and carbon fixation were effectively inhibited in DCMU-amended samples (>90%; Figures 2A,B).

Among the diazotroph phylotypes quantified by qPCR, Trichodesmium and gammaproteobacteria showed the highest abundance, ranging between 8.4 × 10 4 and 2 × 10 6 , and 7.5 × 10 5 and 1.8 × 10 6 nifH copies L -1 , respectively (Table 1). UCYN-A and UCYN-B phylotypes were one to two orders of magnitude lower, and undetectable at station 3 (Table 1).

N 2 Fixation, Diazotroph Abundance and Optical Response to Model DOM Compound Additions

At Station 2 we tested whether the addition of DOC, DON or DOP upregulated nifH expression and/or enhanced N 2 fixation rates. N 2 fixation rates were similar in the control, DOC and DON treatments (∼0.8 nmol N L -1 d -1 ), yet they doubled to ∼2 nmol N L -1 d -1 when DOP was added (Figure 3). However, due to the variability among replicates, the enhancement of either N 2 fixation or expression of nifH genes was not statistically significant in any treatment (Kruskal-Wallis, p > 0.05).

The DOC, DON, and DOP treatments yielded slightly higher abundances of UCYN-A nifH transcripts compared to transcripts measured in the controls, yet similar transcript numbers were with obtained in each of the three treatments (∼5-6 × 10 3 nifH transcripts L -1 ; Figure 3). UCYN-B did not show differential expression among treatments, while Trichodesmium increased by one order of magnitude when exposed to the DOC treatment (Figure 3). Despite being the most abundant ambient group as determined by DNA qPCR counts, gammaproteobacteria did not show detectable nifH gene expression in our DOM enrichment experiments (including control bottles). The prolonged incubation (24 h) in polycarbonate bottles could have affected nifH expression in this group [START_REF] Moisander | Gammaproteobacterial diazotrophs and nifH gene expression in surface waters of the South Pacific Ocean[END_REF].

Regarding optical parameters, the DOC treatment resulted in a peak of tyrosine-like compounds concentrations (+51%) whereas no changes were recorded in humic-like and tryptophan-like compounds concentrations (Table 4). The effect of DON and DOP treatments on concentrations of fluorescent compounds are not shown as FDOM results were considered outliers in the validation of PARAFAC model.

DISCUSSION

Spatial Variability of N 2 Fixation Rates and Diazotrophic Phylotypes

Gammaproteobacteria and Trichodesmium were the most abundant diazotrophs among the four phylotypes quantified (Table 1). The nifH copies of these two phylotypes were higher than in a previous study performed in the lagoon during the austral summer. The abundances, reported by Turk-Kubo et al. in nifH copies, ranged from 2.8 × 10 3 to 6.4 × 10 4 (UCYN-A), 3.5 × 10 2 to 2.7 × 10 3 (UCYN-B), 3.4 × 10 2 and 1.4 × 10 5 (Trichodesmium), and 4 × 10 2 to 1.9 × 10 3 (gammaproteobacteria) nifH copies L -1 . Typically, the nifH copy numbers reported here are similar to published numbers from [START_REF] Berthelot | High-nitrogen fixation rates in the particulate and dissolved pools in the Western Tropical Pacific (Solomon and Bismarck Seas)[END_REF] who reported abundances up to 1.3 × 10 3 (UCYN-A), 7.5 × 10 4 (UCYN-B), 1.6 × 10 5 (Trichodesmium), and 2.7 × 10 4 (gammaproteobacteria) nifH copies L -1 from the Solomon Sea.

DCMU blocks photosynthesis and thus the energy source required by autotrophic cyanobacterial diazotrophs to fix N 2 . Hence, measured N 2 fixation rates from DCMU-amended samples should be representative of non-cyanobacterial N 2 fixation. The lack of nifH expression in gammaproteobacteria in our DOM addition experiments (see below) questions whether this specific group was active. Nevertheless, other non-cyanobacterial diazotrophs such as Bradyrhizobium and Mesorhizobium, previously detected in the lagoon [START_REF] Pfreundt | Succession within the prokaryotic communities during the VAHINE mesocosms experiment in the New Caledonia lagoon[END_REF], could be responsible for the measured N 2 fixation rates in DCMU-amended bottles. N 2 fixation rates in DCMU-amended incubations represented 4-7% of N 2 fixation measured without amendments. The New Caledonian lagoon is dominated by autotrophic cyanobacterial diazotrophs [START_REF] Rodier | Population dynamics and environmental conditions affecting Trichodesmium spp. (filamentous cyanobacteria) blooms in the south-west lagoon of New Caledonia[END_REF][START_REF] Turk-Kubo | Diazotroph community succession during the VAHINE mesocosm experiment (New Caledonia lagoon)[END_REF], where noncyanobacterial phylotypes have been reported to represent <2% of the diazotrophic community [START_REF] Turk-Kubo | Diazotroph community succession during the VAHINE mesocosm experiment (New Caledonia lagoon)[END_REF][START_REF] Pfreundt | Succession within the prokaryotic communities during the VAHINE mesocosms experiment in the New Caledonia lagoon[END_REF]. Hence, such a low contribution to total N 2 fixation rates may not be surprising, but deserves further study in other parts of the lagoon and other times of the year. Still, N 2 fixation rates in DCMU-amended bottles ranged between 0.04 and 0.11 nmol N L -1 d -1 , which is in the range of N 2 fixation rates measured in environments where non-cyanobacterial diazotrophs prevail [START_REF] Moisander | Chasing after non-cyanobacterial nitrogen fixation in marine pelagic environments[END_REF].

It has to be noted that putative non-cyanobacterial N 2 fixation was higher at stations closer to the coast (Stations 3 and 4), which may be linked to the Dumbéa river inputs importing bioavailable CDOM at 350 nm and terrestrial humiclike DOM compounds into the bay. Spectral indices (Sr and S 275-295 ) revealed low molecular weight and photodegraded terrestrial CDOM/FDOM at Station 4 (the most impacted by the river), which has been seen to enhance its availability to heterotrophic bacteria in oligotrophic waters [START_REF] Su | Comparison of bacterial growth in response to photodegraded terrestrial chromophoric dissolved organic matter in two lakes[END_REF]. At Station 1, DOM sources come from autochthonous phytoplankton production), which is also available for bacterial growth (based on a 442 values >0.1 as compared to Stations 2 and 3; [START_REF] Steinberg | Production of chromophoric dissolved organic matter (CDOM) in the open ocean by zooplankton and the colonial cyanobacterium Trichodesmium spp[END_REF]. The observed relationship between DOM lability proxies and non-cyanobacterial N 2 fixation activity in the present study support the findings of previous studies in the WTSP and the Mediterranean Sea (Benavides et al., 2015[START_REF] Benavides | Basin-wide N 2 fixation in the deep waters of the Mediterranean Sea[END_REF][START_REF] Rahav | Contribution of mono and polysaccharides to heterotrophic N 2 fixation at the eastern Mediterranean coastline[END_REF]. Nevertheless, a mechanistic understanding of how non-cyanobacterial diazotrophs benefit from different DOM molecules (e.g., metabolic pathways, affinity for different substrates) is still lacking [START_REF] Bombar | Marine non-cyanobacterial diazotrophs: moving beyond molecular detection[END_REF].

Diazotrophic Responses to DOM Additions

The influence of the three different DOM treatments applied (DOC, DON, and DOP) differentially influenced nifH gene expression of different phylotypes detected, with transcript abundance increasing similarly in UCYN-A for all amendments as compared to the controls (from ∼3 to 5-6 × 10 3 nifH transcripts L -1 ), while no differences were observed between controls and amendments for UCYN-B transcripts (Figure 3). UCYN-B fixes carbon during the day and N 2 at night to avoid nitrogenase enzyme inhibition by photosynthetically derived oxygen. Thus, nifH expression in UCYN-B is typically higher at night [START_REF] Church | Temporal patterns of nitrogenase gene (nifH) expression in the oligotrophic North Pacific Ocean[END_REF][START_REF] Zehr | Experiments linking nitrogenase gene expression to nitrogen fixation in the North Pacific subtropical gyre[END_REF]. Hence, the absence of nifH expression in UCYN-B during the day (when our RNA samples were taken) is not surprising, even in the presence of external DOM sources. Similarly, [START_REF] Moisander | Analogous nutrient limitations in unicellular diazotrophs and Prochlorococcus in the South Pacific Ocean[END_REF] observed that nifH expression was not upregulated in UCYN-B when incubated with a mixture of glucose and mannitol. UCYN-A do not have a tricarboxylic acid pathway [START_REF] Tripp | Metabolic streamlining in an open-ocean nitrogen-fixing cyanobacterium[END_REF], and thus require reduced organic carbon sources provided by their hosts [START_REF] Thompson | Unicellular cyanobacterium symbiotic with a single-celled eukaryotic alga[END_REF]. It is therefore plausible that DOM additions in our experiments enhanced the diazotrophic activity of UCYN-A, similar to the experiments of [START_REF] Moisander | Analogous nutrient limitations in unicellular diazotrophs and Prochlorococcus in the South Pacific Ocean[END_REF], who demonstrated upregulated nifH gene expression in UCYN-A upon the addition of a glucose and mannitol mixture. In our experiments, however, a preference for DOC, DON or DOP was not observed and the nifH expression response was similar across treatments (Figure 3).

Active assimilation of both DOC and DON was measured in natural Trichodesmium colonies from the WTSP open ocean waters in similar experiments [START_REF] Benavides | Dissolved organic matter uptake by Trichodesmium in the Southwest Pacific[END_REF]. However, in that previous study only the addition of DON resulted in an increase of filament-specific N 2 fixation rates [START_REF] Benavides | Dissolved organic matter uptake by Trichodesmium in the Southwest Pacific[END_REF]. The bulk water N 2 fixation measurements presented here do not allow us to discern the contribution of each diazotroph phylotype. Nevertheless, while bulk N 2 fixation rates were highest in the DOP treatment, nifH gene expression by Trichodesmium was highest in the DOC treatment (Figure 3). The peak of tyrosine-like compounds coinciding with the highest nifH expression of Trichodesmium observed in the DOC treatment may be related to the production of CDOM by this cyanobacterium [START_REF] Steinberg | Production of chromophoric dissolved organic matter (CDOM) in the open ocean by zooplankton and the colonial cyanobacterium Trichodesmium spp[END_REF]. Surprisingly, DOM additions did not result in any nifH expression in the group of non-cyanobacterial diazotrophs tested in this study. In marine waters, maximum nifH expression of gammaproteobacteria typically reaches × 10 4 nifH transcripts L -1 [START_REF] Luo | Database of diazotrophs in global ocean: abundance, biomass and nitrogen fixation rates[END_REF][START_REF] Langlois | Widespread distribution and expression of Gamma A (UMB), an uncultured, diazotrophic, γ-proteobacterial nifH phylotype[END_REF]. For example, [START_REF] Church | Temporal patterns of nitrogenase gene (nifH) expression in the oligotrophic North Pacific Ocean[END_REF] reported an average of 4 × 10 2 nifH transcripts L -1 for gammaproteobacteria in a diel cycle study at station ALOHA in the North Pacific Ocean, which is in the same order of magnitude as in a study in the WTSP by [START_REF] Moisander | Gammaproteobacterial diazotrophs and nifH gene expression in surface waters of the South Pacific Ocean[END_REF].

Nevertheless, a study including samples from a wide range of oceanographic cruises across the North and South Atlantic and Pacific Oceans reported up to ×10 5 gammaproteobacterial nifH transcripts per liter in sunlit surface waters [START_REF] Langlois | Widespread distribution and expression of Gamma A (UMB), an uncultured, diazotrophic, γ-proteobacterial nifH phylotype[END_REF].

In offshore waters of the WTSP, the addition of glucose and mannitol upregulated nifH expression in gammaproteobacteria [START_REF] Moisander | Analogous nutrient limitations in unicellular diazotrophs and Prochlorococcus in the South Pacific Ocean[END_REF].

The lack of significant responses to DOM additions in this study could reflect sufficient DOM availability (based on Sr,S 275-295 and SUVA 254 indexes) potentially provided by corals in the lagoon which are known for producing tryptophane-like compounds (available DOM sources for bacteria; [START_REF] Weishaar | Evaluation of specific ultraviolet absorbance as an indicator of the chemical composition and reactivity of dissolved organic carbon[END_REF]. However, the absence of detected gammaproteobacterial nifH transcripts, also in the control bottles, suggests that bottle confinement effects may have inhibited this group [START_REF] Moisander | Gammaproteobacterial diazotrophs and nifH gene expression in surface waters of the South Pacific Ocean[END_REF]. In addition to the diel changes in nifH gene expression between phylotypes [START_REF] Church | Temporal patterns of nitrogenase gene (nifH) expression in the oligotrophic North Pacific Ocean[END_REF][START_REF] Turk | Nitrogen fixation and nitrogenase (nifH) expression in tropical waters of the eastern North Atlantic[END_REF], it is possible that the nifH expression measured in our samples does not reflect the immediate response of diazotrophs to DOM additions. It must be noted that while RNA samples represent a snapshot of nifH expression at the time of sampling, N 2 fixation rates integrate the nitrogenase activity that has taken place over a period of 24 h. Such differences probably also account for the uncoupling observed between N 2 fixation rates and nifH gene expression.

N 2 Fixation Rates Enhanced by DOP Additions

Diazotrophs differ in how they access DOP. For example, Crocosphaera watsonii (here represented by the UCYN-B phylotype) obtains inorganic phosphorus using high affinity phosphate binding systems, and organic phosphorus sources such as phosphomonoesters utilizing alkaline phosphatases (Dyhrman and Haley, 2006). In addition to these phosphorus sources, Trichodesmium is also able to grow on phosphonates and use phosphite (Dyhrman et al., 2006;[START_REF] Polyviou | Phosphite utilization by the globally important marine diazotroph Trichodesmium[END_REF]. It is intriguing why the nifH gene expression of Trichodesmiumor of any of the other diazotroph phylotypes tested-was not enhanced when DOP was enriched, and deserves further study. However, an enhancement of N 2 fixationactivity was observed in the DOP treatment (Figure 3), and could be due to inorganic phosphorus limitation alleviation. Unfortunately, we do not have alkaline phosphatase activity data to confirm whether the DOP added to our incubations was ultimately transformed to inorganic phosphorus compounds, but PO 3- 4 concentrations nearly doubled in DOP-amended samples with respect to the control (data not shown), suggesting that active alkaline phosphatases were functioning. The fact that N 2 fixation rates enhanced by DOP additions was not reflected in a significant enhancement of nifH expression of any of the diazotroph phylotypes tested suggests that the N 2 fixation activity measured may be provided by a diazotroph not accounted for in our nifH gene q-RT-PCR assays. Alternatively, transcription may be interrupted when the number of transcripts is enough to activate the nitrogenase protein, resulting in an uncoupling between expression and nitrogenase activity, as previously reported in Trichodesmium cultures [START_REF] Levitan | Regulation of nitrogen metabolism in the marine diazotroph Trichodesmium IMS101 under varying temperatures and atmospheric CO2concentrations[END_REF].

CONCLUSIONS

Although cyanobacterial diazotrophs predominate in the New Caledonian lagoon, here we demonstrate that non-cyanobacterial diazotrophs contribute ∼10% of total N 2 fixation rates. Comparable to previous findings (see [START_REF] Moisander | Chasing after non-cyanobacterial nitrogen fixation in marine pelagic environments[END_REF] gammaproteobacteria were the most abundant based on DNAderived qPCR nifH gene counts, yet we did not detect any nifH gene expression for this group throughout the experiments. This lack of detectable expression together with the low N 2 fixation rates measured in DCMU-treated samples questions the contribution of non-cyanobacterial diazotrophs to biologically fixed nitrogen inputs in the lagoon. Yet, the response of the cyanobacterial diazotroph phylotypes to the amended DOM treatments suggests that mixotrophic nutrition may be important in these organisms and should be investigated further.

FIGURE 1 |

 1 FIGURE 1 | Map of stations sampled in the New Caledonian lagoon. The yellow line represents the coral reef barrier.

  Standard deviation values ( ± SD) are provided where available.

FIGURE 2 |

 2 FIGURE 2 | (A) N 2 and (B) carbon fixation rates in DCMU and non-DCMU amended samples.

FIGURE 3 |

 3 FIGURE 3 | N 2 fixation rates (line) and nifH gene expression (nifH transcripts L -1 ; bars) in control, DOC, DON, and DOP treatments performed at Station 2. Error bars indicate the standard deviation of the mean.

TABLE 1 |

 1 Position, core parameters, CDOM and FDOM information obtained from each transect station.

	Salinity Oxygen	(µmol	kg -1
	Temperature	( • C)
	Station Bottom	depth	(m)

TABLE 2 |

 2 Spectral indices investigated in this study.

	Abbreviation Name	Formula
	HIX	Humification Index	Ex: 255 nm Em:	480-434 344-300
	BIX SUVA 254 Sr	Biological Index Specific UV Absorbance (254 nm) Spectral ratio	Ex: 255nm Em: 310-380 310-430 ABS (254) DOC S 275-295 S 350-400

TABLE 3 |

 3 Excitation and Emission maxima (Ex/Em) of three CDOM fluorescent components validated by PARAFAC model and identification by comparison with the literature data.

		This study			Previous study		FDOM sources
	Comp.	Ex(nm)	Em(nm)	Comp.	Ex(nm)	Em(nm)	Reference
	COMPOUND 1 Humic like	<220	454(322) Humic-like (A)	230-260 400-480 Coble, 1996	Allochtonous: terrestrial
								inputs from river
				Terrestrial humic-like	<260	454	Yamashita et al., 2010
				HumiC-like (A)	230	476	Tedetti et al., 2016
	COMPOUND 2 Tyrosine like	225(275)	304	C7	240(300)	338	Murphy et al., 2007	Autochtonous:
								heterotrophic bacterial
								degradation of DOM
				Component 8	275	304	Stedmon and Markager, 2005
				Tyrosine-like (B)	225(275)	304	Tedetti et al., 2016
	COMPOUND 3 Tryptophane like 270(335)	340	Tryptophane (T1)	220-235 334-360 Coble, 1996	Autochtonous: coral
								production,
								heterotrophic bacterial
								degradation of DOM
					230(300)	352	Martias et al., 2018

TABLE 4 |

 4 NO x , PO3- 4 , DOC concentrations and values of FDOM-related ratios for the different DOM additions (DOC, DON, and DOP treatments) performed at Station 2.

	Treatment	NO x (µM)	PO 3-4 (µM)	DOC (µM)	Humic-like (QSU)	Tyrosine-like (QSU)	Tryptophan-like (QSU)
	Control	0.1 ± 0.0	0.1 ± 0.0	90.6 ± 15.5	6.54	44.79	11.71
	DOC	0.2 ± 0.1	0.1 ± 0.0	98.6 ± 3.3	6.57	67.45	11.53
	DON	0.2 ± 0.0	0.1 ± 0.0	104.1 ± 30.6	n/d	n/d	n/d
	DOP	0.2 ± 0.0	0.3 ± 0.0	107.3 ± 7.5	n/d	n/d	n/d
	Standard deviation values ( ± SD) are provided where available.

Frontiers in Marine Science | www.frontiersin.org

March 2018 | Volume 5 | Article 89

ACKNOWLEDGMENTS

We thank the captain of R/V Archamia for his invaluable help at sea. We would like to thank O. Grosso for IRMS analyses. We are also grateful to P. Gérard and L. Jamet from the Laboratoire des Moyens Analytiques (LAMA) at IRD in Nouméa (New Caledonia) for oxygen, nutrient and DOC analyses.

FUNDING MB was funded by the People Programme (Marie Skłodowska-Curie Actions) of the European Union's Seventh Framework Programme (FP7/2007-2013) under REA grant agreement number 625185. Funding was obtained for IB-F through a collaborative grant from Ministry of Science and Technology (MOST) Israel and the High Council for Science and Technology (HCST)-France (2013). CM and SB were funded by the Institut de Recherche pour le Développement (IRD, France).

AUTHOR CONTRIBUTIONS

Conceived and designed the experiments: MB, CM, SB; Performed the experiments: MB, CM; Analyzed the data: MB, CM, HE; Contributed reagents, materials, analysis tools: CM, SB, HE, CD, IB-F; Wrote the paper: MB, CM, CD, HE, IB-F, SB.

Conflict of Interest Statement:

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The reviewer AF-C and handling Editor declared their shared affiliation.

Copyright © 2018 Benavides, Martias, Elifantz, Berman-Frank, Dupouy and Bonnet. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.