Isabelle C Biegala
email: biegala@univmed.fr

Patrick Raimbault

High abundance of diazotrophic picocyanobacteria (<3 µm) in a Southwest Pacific coral lagoon

Keywords: Diazotrophs, Cyanobacteria, Picoplankton, FISH, Pacific, Coral lagoon, Symbiosis

In the present study we detected unicellular diazotrophic cyanobacteria along a nutrient gradient in New Caledonia's coral lagoon (24 000 km 2 ) using whole-cell hybridization of specific Nitro821 probes (TSA-FISH, Tyramide Signal Amplification of Fluorescent In Situ Hybridization). The specificity of this probe was confirmed on cultures and in the natural environment. Surprisingly, the community of Nitro821-targeted cells was numerically dominated by picocyanobacteria (97%, 1 to 1.5 µm). These organisms were either free living (63%), recovered with the 0.2 to 3 µm size fraction or associated (37%) to particles or planktonic dinoflagellates from larger size fractions (3 to 10 µm and >10 µm). Diazotrophic picocyanobacterial abundance ranged from 3 to 140 cells ml -1 along the nutrient gradient and was highest at the oligotrophic lagoonal station. These cells may contribute to N 2 fixation from the <10 µm size fraction (4.4 to 8 nmol N -1 24 h -1 ), which was 4 times higher than the >10 µm size fraction (0 to 1.9 nmol N -1 24 h -1 ). In addition, in the middle of the lagoon daylight 15 N 2 fixation could explain 78% of 15 N 2 fixation over 24 h. The results presented in the present study reveal for the first time a significant concentration of photosynthetic diazotrophs within the marine picoplankton community.

INTRODUCTION

Marine diazotrophs are a functional group of heterotrophic prokaryotes and cyanobacteria, which are able to fix dissolved dinitrogen (N 2 ) from the atmosphere. This function offers them an important ecological advantage compared to other microorganisms living in nitrogen-depleted waters. In oceanic and coastal oligotrophic waters, up to 50% of new primary production can be explained by diazotrophic cyanobacteria [START_REF] Karl | The role of nitrogen fixation in biogeochemical cycling in the subtropical North Pacific Ocean[END_REF]. Highest rates of N 2 fixation were detected on tropical coastal areas from cyanobacterial mats, but their restricted colonized areas do not make them significant contributors to total N 2 fixation [START_REF] Herbert | Nitrogen cycling in coastal marine ecosystems[END_REF]. Since the discovery of diazotrophy in the ubiquitous filamentous cyanobacteria Trichodesmium sp., it was believed that this genus was responsible for the major-ity of N 2 fixation in tropical and subtropical oceanic waters as it can form massive blooms [START_REF] Capone | Trichodesmium, a globally significant marine cyanobacterium[END_REF]. Other oceanic cyanobacteria that are known to significantly contribute to N 2 fixation are intracellular symbionts of diatoms (Richelia sp.) [START_REF] Carpenter | Extensive bloom of a N 2fixing diatom/cyanobacterial association in the tropical Atlantic Ocean[END_REF].

Many recent biogeochemical studies have concluded that previous rates of global ocean N 2 fixation have been significantly underestimated and cannot be reconciled with observed Trichodesmium sp. and Richelia sp. abundance [START_REF] Gruber | Global patterns of marine nitrogen fixation and denitrification[END_REF], Capone & Carpenter 1999). It has been recently recognized that unicellular pico-and nanoplanktonic organisms (<10 µm) can support a significant amount of N 2 fixation [START_REF] Montoya | High rates of N 2 fixation by unicellular diazotrophs in the oligotrophic Pacific Ocean[END_REF][START_REF] Garcia | Nanoplanktonic diazotrophy and trophic transfer of newly fixed nitrogen in oceanic and lagoonal waters off Southwest Pacific (New Caledonia)[END_REF]). These organisms have been identified as unicellular nanoplanktonic cyanobacteria and heterotrophic Bacteria and Archaea, which are either free living or associated with other organisms [START_REF] Zehr | Unicellular cyanobacteria fix N 2 in the subtropical North Pacific Ocean[END_REF], Falcón et al. 2004b, Foster et al. 2006a). Some of these nanoplanktonic cyanobacteria are maintained in culture collections (e.g. Cyanothece sp. ATCC51142 and Crocosphaera watsonii WH8501), while others have not been observed or cultivated so far, such as Group A. Groups A, B and C represent clusters of mainly unidentified nifH sequences, which are closely connected to Crocosphaera sp. and Cyanothece sp. for Groups B and C, respectively, when group A is weakly affiliated to Group C [START_REF] Zehr | Unicellular cyanobacteria fix N 2 in the subtropical North Pacific Ocean[END_REF]. Although these cells seem to have a significant effect on nitrogen fluxes, they are 2 or 3 orders of magnitude less abundant than nondiazotrophic marine Synechococcus sp. and Prochlorococcus sp. [START_REF] Campbell | Picoplankton community structure within and outside a Trichodesmium bloom in the southwerstern Pacific Ocean[END_REF], Zehr et al. 2007). Another peculiar characteristic is that Group A has been recently suspected to fix N 2 during the day, while the other unicellular cyanobacteria fix N 2 during the night [START_REF] Church | Temporal patterns of nitrogenase gene (nif H) expression in the oligotrophic North Pacific Ocean[END_REF], Zehr et al. 2007). This feature is very surprising within unicellular diazotrophic cyanobacteria, as it is known that nitrogenase, which catalyses N 2 fixation, is irreversibly inhibited by O 2 [START_REF] Burgess | Mechanism of molybdenum nitrogenase[END_REF]. As such, diazotrophic cyanobacteria have segregated in time and/or space the O 2 -generating photosynthesis from nitrogen fixation. While most filamentous cyanobacteria have differentiated cells in heterocyst to fix N 2 , unicellular cyanobacteria show photosynthetic activity during the day and diazotrophic activity at night [START_REF] Zehr | Experiments linking nitrogenase gene expression to nitrogenase fixation in the North Pacific subtropical gyre[END_REF].

The Southwest Pacific harbors regular massive blooms of Trichodesmium sp., which share or alternate in space with Crocosphaera-like cells [START_REF] Campbell | Picoplankton community structure within and outside a Trichodesmium bloom in the southwerstern Pacific Ocean[END_REF]. Dominant southeast trade winds drive these oceanic waters into Noumea's coral lagoon (New Caledonia), and after 3 to 11 d of residential time they are washed through the coral reef passes [START_REF] Jouon | Calculations of hydrodynamic time parameters in a semi-open coastal zone using a 3D hydrodynamic model[END_REF]. New Caledonia's coral lagoon is one of the largest in the world (24 000 km 2 and 25 m deep on average). It is mainly oligotrophic (0.3 µg chl a l -1 ), but an important concentration of dissolved nitrogen is regularly discharged from Noumea city (132 000 habitants), which increases its chl a concentration 10-fold (3 µg chl a l -1 ) [START_REF] Jacquet | Response of phytoplankton communities to increased anthropogenic influences (south-western lagoon, New Caledonia)[END_REF]) and maintains detectable nutrient concentrations (0.9 µg DIN l -1 , 0.15 µg DIP l -1 , [START_REF] Jacquet | Response of phytoplankton communities to increased anthropogenic influences (south-western lagoon, New Caledonia)[END_REF]. Filamentous cyanobacteria (Trichodesmium sp. and Richelia sp), as well as planktonic N 2 -fixation activity have been regularly detected inside and outside the lagoon both from >10 µm and <10 µm fractions [START_REF] Garcia | Nanoplanktonic diazotrophy and trophic transfer of newly fixed nitrogen in oceanic and lagoonal waters off Southwest Pacific (New Caledonia)[END_REF]). However, unicellular planktonic diazotrophs have never been observed.

To determine the relative contribution of these various groups of unicellular diazotrophs to global oceanic N 2 fixation, it is important to estimate their abundance while simultaneously expressing their specific diazotrophic activity. So far 2 groups of techniques have been used on marine diazotrophs to identify and quantify cells while expressing their N 2 -fixing activities. The first one uses quantitative PCR (qPCR) on nifH genes and transcripts, which code for nitrogenase [START_REF] Zehr | Unicellular cyanobacteria fix N 2 in the subtropical North Pacific Ocean[END_REF], Falcón et al. 2004a), and the second one uses transmission electronic microscopy (TEM) associated with nitrogenase immunolocalization (Falcón et al. 2004b, Foster et al. 2006a). Recently [START_REF] Mazard | PCR analysis of the distribution of unicellular cyanobacterial diazotrophs in the Arabian sea[END_REF] designed a PCR 16S rDNA primer (Nitro821R coupled with a general cyanobacteria primer Cya359F) to specifically target a group of unicellular diazotrophic cyanobacteria (i.e. UCN 2 -Fix lineage). This was possible as nifH is one of the oldest functional genes and has coevolved with 16S rDNA [START_REF] Hennecke | Concurrent evolution of nitrogenase genes and 16S ribosomal-RNA in Rhizobium species and other nitrogen fixing bacteria[END_REF]. Therefore phylogenetic trees based on 16S and nifH genes have strong similarity [START_REF] Zehr | Nitrogenase gene diversity and microbial community structure: a cross-system comparison[END_REF]. With this novel approach in the field of unicellular diazotrophs, [START_REF] Mazard | PCR analysis of the distribution of unicellular cyanobacterial diazotrophs in the Arabian sea[END_REF] successfully detected and quantified the targeted cells along an oceanic transect in the Arabian Sea.

In the present study the use of Nitro821R primer was first tested as a fluorescent probe on unicellular cyanobacterial cultures and environmental samples using the TSA-FISH (Tyramide Signal Amplification of Fluorescent In Situ Hybridization) technique. In order to find out if the Nitro821-targeted cells could be active diazotrophs, cell abundance was compared with 15 N 2fixing activity from different size fractions. A second aspect of our work was to investigate whether the nutrient gradient from Noumea's lagoon influences the abundance and distribution of unicellular diazotrophic cyanobacteria. Finally, by processing different size fractions, we described whether the cells were free living or associated with particles or organisms. Two size fractions were collected, >10 µm and 3 to 10 µm, for filamentous and nanoplanktonic cells, respectively. The filtrates from the 3 µm filter were also collected on a 0.2 µm pore size filter as a negative control for the Nitro821 probe, as this probe was first suspected to target only nanoplanktonic unicellular cyanobacteria. + l -1 and 3 to 0.3 µg chl a l -1 [START_REF] Jacquet | Response of phytoplankton communities to increased anthropogenic influences (south-western lagoon, New Caledonia)[END_REF]). The most eutrophic Stns A and B are located in the middle of Sainte Marie's bay, 1.5 km away from land, and just outside the bay, respectively. Sainte Marie's bay is not influenced by any direct release of sewage, but receives small polluted rivers and city runoff waters. Temperature and salinity were measured with a profiler (CTD SBE 19, Seabird) and were ranged along the nutrient gradient from 23 to 25 and 35.6 to 35.9°C, respectively. For Stns D and E, CTD profiles were homogeneous throughout the water column, when weak stratifications were observed at Stns A, B and C.

MATERIALS AND METHODS

Strains

For 3 & 4), water samples were collected at 3 different depths when possible (0.5, 10 or 20 m, Fig. 4). For N 2 -fixation experiments over 24 h (Fig. 5), samples were taken at Stns B, C, D and E at 3 different depths (0.5, 10 and 20 m), apart from Stn E where only subsurface water was sampled (0.5 m). For additional N 2 -fixation experiments over the daylight period (12 h), samples were taken only at Stns C and D at 3 depths (Fig. 5). Apart from the 12 h incubation experiment on Stn D all other samples for both TSA-FISH and 15 N 2 experiments were collected simultaneously.

TSA-FISH. Before hybridization, cyanobacterial cultures were collected on 0.2 µm pore-size ISO-PORE TM 47 mm diameter membrane filters (Millipore), and water samples were size fractionated. According to oligotrophic status, filters were more or less quickly saturated and different volumes of waters were filtered through the 10 or 3 µm membranes by gravity. The amount of seawater to be filtered was defined by changes of flow through membranes. Between 400 ml and 4 l were filtered by gravity through 10 µm ISO-PORE TM 47 mm filters. Between 200 ml and 1 l of filtrate were then collected by gravity on 3 µm ISO-PORE TM 47 mm filters. Finally, 200 ml of filtrate were collected under 200 mm Hg vacuum on 0.2 µm ISO-PORE™ 47 mm filters. Once collected, cells were fixed with 1% paraformaldehyde (pH 7.2, buffered with PBS and clarified by filtration, Sigma-Aldrich) at room temperature for 15 min. Cells were subsequently dehydrated with 100% EtOH molecular grade at room temperature for 10 min and stored at -80°C or in liquid nitrogen until further analysis.

Hybridizations were done according to [START_REF] Biegala | Identification of bacteria associated with dinoflagellates (Dinophyceae) Alexandrium spp. using tyramide signal amplification-fluorescent in situ hybridization and confocal microscopy[END_REF] with slight modifications. Probes (Table 1) were purchased HRP (Horse Radish Peroxidase) labeled (Thermo Electron Corporation). Before hybridization, filters were thawed and quickly dipped in warm 0.07% agarose (1500 g cm -2 , Eurogentec) and dried at 37°C for 10 min to avoid cell loss. Cells on filters were either stored back at -80°C or further processed. To allow 16S rRNA in most bacteria GCT GCC TCC CGT AGG AGT Amann et al. (1990) Table 1. Oligonucleotide probes used in the present study HRP-labeled probes to reach their 16S rRNA target, cells were perforated by successive enzymatic incubations: in 10 mg ml -1 lysozyme (47 000 U mg -1 , Sigma-Aldrich) at 37°C for 1 h and in 60 U ml -1 acromopeptidase (3200 U mg -1 , Sigma-Aldrich) at 37°C for 30 min. Each enzyme treatment was followed by 3 rinses in Milli-Q sterile water, 10 min in 100% EtOH and drying at 37°C. At the latter step, cells could be either stored back at -80°C or processed for hybridization steps and TSA reactions. To check empiric Nitro821 probe specificity, a range of stringent hybridization conditions was tested on cyanobacteria cultures (hybridization temperature and salt concentrations were kept constant, when formamide concentration varied from 20 to 65%, data not shown). The best compromise between fluorescence intensity from positive hybridization and probe specificity was achieved at 50% of formamide concentration in the hybridization buffer. This stringent condition was then used for hybridizations on cultures and natural samples (Figs. &2). Differences between FITC-labeled cells and remaining autofluorescence of phytoplanktonic cells were checked under a different dichroïc filter (480 ± 20 ex., 535 ± 40 short pass em.), which removed the entire orange-or red-emitted wavelength and allowed only the green stain to be seen.

The entire surface of each filter portion was counted, corresponding to 20-25 microscopic fields. According to oligotrophic conditions, filter surfaces corresponded to 7-12 ml of water for the 0.2 to 3 µm size fraction, 9-56 ml for 3 to 10 µm and 27-250 ml for >10 µm. For all 3 size fractions, diazotroph counts were acquired with the 40× objective. A second count was done with the 10× objective on the 3 to 10 µm and >10 µm size fractions to count only nanoplanktonic unicellular diazotrophs. At that magnification picoplanktonic cells were not seen. Picoplanktonic cell counts were obtained by subtracting counts (Fig. 4).

Nitrogen fixation. Nitrogen-fixation rates were determined on prefiltered (<10 µm or >10 µm) seawater using the high-sensitive 15 N 2 tracer method (Fig. 5) as described by [START_REF] Garcia | Nanoplanktonic diazotrophy and trophic transfer of newly fixed nitrogen in oceanic and lagoonal waters off Southwest Pacific (New Caledonia)[END_REF]. The detection limit of the 15 N 2 tracer method was estimated at 0.3 nmol l -1 24 h -1 . The day and night incubations were 1). Hybridized cells were stained with FITC (green fluorescence) using the TSA-FISH technique.

Arrows point to hybridized heterotrophic bacteria from non axenic cultures. Scale bars = 10 µm Fig. 3. Different size-fractionated plankton hybridized with the Nitro821 probe, specific for unicellular cyanobacterial diazotroph clusters (A 1 , B 1 to C 4 ) (Table 1), or with the general cyanobacterial probe (A 2 : Cya664) (Table 1). ( C done over 24 h, when the daylight experiment was run for 12 h from sunrise to sunset. Incubations were performed in simulated environmental conditions in an opaque incubator equipped with a light screen which allowed 50% light penetration.

RESULTS

Probe specificity

To test the specificity of the Nitro821 probe (Table 1), which targets unicellular cyanobacterial diazotrophs, 3 non-axenic unicellular cyanobacteria cultures (Cyanothece sp., Gleocapsa sp. and Synechocystis sp.) were used. Among the 3 strains, only the nif H containing Cyanothece sp. was successfully hybridized by the Nitro821 probe (Fig. 2). A positive control for hybridization was done using the general Bacteria probe Eub338 (Table 1). All 3 cyanobacterial strains, as well as heterotrophic bacteria, showed a positive signal with the Eub338 probe (Fig. 2).

In the natural environment the Nitro821 probe hybridized to unicellular cyanobacteria from an unex-pectedly wide range of cell sizes (1 to 1.5 µm and up to 13 µm, Fig. 3A 1 -C 4 ), while the filamentous cyanobacteria Richelia sp. and Trichodesmium sp. showed only remnants of natural orange autofluorescence as they were not hybridized by the Nitro821 probe (Fig. 3C 5 ,C 6 ). To confirm that the Nitro821 probe targets were cyanobacteria, a double hybridization experiment was done on 3 size fractions, with the general Cya664 probe, specific for cyanobacteria (Table 1). Double hybridization was positive for every Nitro821 probe target (data shown only for the picoplankton size fraction 0.2 to 3 µm, Fig. 3A 1 ,A 2 ).

Cell abundance

In Noumea's coral lagoon, the abundance of Nitro821 probe targets ranged from 3 to 140 cells ml -1 along the nutrient gradient (Fig. 4), while hardly any larger filamentous diazotrophs (Trichodesmium sp. and Richelia sp.) were observed (Fig. 3C 5 ,C 6 ). Picoplanktonic (< 3 µm) cyanobacteria largely dominated the targeted community, representing 97% of the total abundance. These small cells were recovered in all size fractions investigated. The remaining 3% were nanoplanktonic (> 3 µm), two-thirds of them were in the 3 to 10 µm fraction and one-third in the >10 µm fraction (Fig. 4). Among the picoplankton cyanobacterial diazotrophs, 63% were free living and 37% were associated to inert particles (Fig. 3B 1 ,C 1 ) or intracellular of eukaryotes (Fig. 3B 2 ,B 4 ,C 3 ). These organisms were mainly dinoflagellates, which were easily recognized by their high DNA content (DAPI micrographs in Fig. 3B 2 ,B 4 ,C 3 ) and condensed chromosomes (data not shown). Within dinoflagellates, targeted cells had different shapes and sizes, either grouped in clusters around the nucleus (Fig. 3B 4 ) or spread all over the eukaryote's cytoplasm (Fig. 3C 3 ). Up to 60 cells were detected within a dinoflagellate. The distribution of picoplanktonic cyanobacterial diazotrophs was heterogeneous in the water column and along the nutrient gradient, reaching a maximum and a minimum in the middle and outside Noumea's lagoon, respectively (Stns D and E,Figs. 1 & 4).

Diazotrophic activity

Results from the 15 N-tracer experiments showed significant nitrogen fixation in the <10 µm fraction, which was 4 times higher than the >10 µm fraction (Fig. 5). This was true whether the rates were measured over 24 h incubation or over 12 h, from sunrise to sunset (Fig. 5). Although the 12 and 24 h incubations were done simultaneously only for Stn C and with 1 d delay for Stn D, N 2 fixation by day was high (average 5.3 nmol N l -1 12 h -1 , n = 12) and could explain 78% of the average activity measured over 24 h (6.8 nmol N l -1 24 h -1 , n = 12). Along the nutrient gradient, slightly higher rates were measured over 24 h at nutrient-rich Stns B and C (4.5 to 8 nmol N l -1 24 h -1 ) compared to oligotrophic Stns D and E (4.4 to 6 nmol N l -1 24 h -1 ). Diazotrophic activity did not correlate with the Nitro821-targeted cell number from both the <10 µm and >10 µm size fractions.

DISCUSSION

In the present study we confirmed that the Nitro821 probe was specific to aerobic unicellular diazotrophic cyanobacteria, such as Cyanothece sp. PCC 8801 (also called Synechococcus sp. RF-1), and to other Crocosphaera-like cells recovered from the natural environment (Zehr et al. 2001, Fig. 3B 3 ,C 2 ). As predicted, the Nitro821 probe did not label cultures of pico-and nanoplanktonic non-diazotrophic cyanobacteria (Gleocapsa sp. and Synechocystis sp., Fig. 2), nor filamentous diazotrophic cyanobacteria from Noumea's la-goon (Trichodesmium sp. and Richelia sp., Fig. 3C 5 ,C 6 ). Furthermore, the general cyanobacterial probe Cya664 double labeled the Nitro821-targeted cells (Fig. 3A 1 ,A 2 ). Since [START_REF] Mazard | PCR analysis of the distribution of unicellular cyanobacterial diazotrophs in the Arabian sea[END_REF]'s first description, the specificity of the Nitro821 probe was checked over the entire 16S and 18S rDNA GenBank database and remained stable. The probe showed several mismatches with heterotrophic Bacteria or Archaea and marine filamentous cyanobacteria. As such, it is unlikely that Nitro821-labeled picocyanobacteria would be spores or baeocytes from benthic filamentous cyanobacteria or heterotrophic picodiazotrophs.

The population of cells targeted by the Nitro821 probe, specific for the unicellular diazotrophic cyanobacteria, belonged essentially to the picoplanktonic size fraction. This result was surprising as the nif H gene has never been recovered from the marine picocyanobacteria Prochlorococcus sp. or Synechococcus sp. [START_REF] Palenik | The genome of a motile marine Synechococcus[END_REF][START_REF] Dufresne | Accelerated evolution associated with genome reduction in a freeliving prokaryote[END_REF]. Both genera are known to be abundant in the Southwest Pacific oligotrophic waters [START_REF] Campbell | Picoplankton community structure within and outside a Trichodesmium bloom in the southwerstern Pacific Ocean[END_REF][START_REF] Jacquet | Response of phytoplankton communities to increased anthropogenic influences (south-western lagoon, New Caledonia)[END_REF]). To support the presence of pico-and nanoplanktonic unicellular diazotrophs in Noumea's coral lagoon, a significant N 2 fixation was measured in the <10 µm fraction (Fig. 5). Similar rates of N 2 fixation from the <10 µm fraction have previously been measured in adjacent oceanic waters at similar times of the year [START_REF] Garcia | Nanoplanktonic diazotrophy and trophic transfer of newly fixed nitrogen in oceanic and lagoonal waters off Southwest Pacific (New Caledonia)[END_REF], from the Northern Pacific, Arafura Sea and Northern Atlantic (Falcón et al. 2004a[START_REF] Montoya | High rates of N 2 fixation by unicellular diazotrophs in the oligotrophic Pacific Ocean[END_REF], Zehr et al. 2007). These rates were assigned to diazotrophic unicellular cyanobacteria from Groups A, B or C and to a lesser extent heterotrophic Proteobacteria. In addition, it is interesting to stress that 78% of the 15 N 2 fixation was done during the daylight period, at least for Stns C and D (Fig. 5). However, all known nanoplanktonic diazotrophic cyanobacteria affiliated to Groups B or C are diazotrophically active at night in the natural environment, when Group A and heterotrophic bacteria have been shown to express their nif H at day [START_REF] Church | Temporal patterns of nitrogenase gene (nif H) expression in the oligotrophic North Pacific Ocean[END_REF], Foster et al. 2006b). As the newly discovered Nitro821-labeled picocyanobacteria numerically dominated the <10 µm fraction (Fig. 4), it is probable that they contributed to the daylight N 2 fixation from this small fraction. However, changes in N 2 fixation and Nitro821-targeted cell abundance were not correlated. This may reveal either the presence of organisms not targeted by the Nitro821 probe, such as Proteobacteria, and/or important single-cell variability in N 2 -fixing activity. This latter hypothesis is probable as a lack of correlation between nif H copy and transcript numbers have been shown previously within cyanobacterial Groups A and B [START_REF] Zehr | Experiments linking nitrogenase gene expression to nitrogenase fixation in the North Pacific subtropical gyre[END_REF]). The high numbers of Nitro821-labeled picocyanobacteria in all size fractions and the significant daylight 15 N 2 fixation allow us to hypothesize that the newly discovered picocyanobacteria from the present study could be affiliated to Group A. Group A diazotrophic cyanobacteria were recently suspected to belong to the picoplanktonic size fraction and to have their 16S rRNA amplified by Nitro821 and Cya359 primers [START_REF] Mazard | PCR analysis of the distribution of unicellular cyanobacterial diazotrophs in the Arabian sea[END_REF], Zehr et al. 2007).

The picoplanktonic Nitro821-targeted cells reached a maximum of 10 2 cells ml -1 , which makes them difficult to detect among the non-diazotrophic picocyanobacteria which are major contributors to the lagoon chl a (10 4 to 10 5 cells ml -1 , Figs. 3A 2 & 4) [START_REF] Jacquet | Response of phytoplankton communities to increased anthropogenic influences (south-western lagoon, New Caledonia)[END_REF]. The concentration of fluorescently labeled picocyanobacteria agrees with the concentration of nif H copy numbers of unicellular cyanobacteria from various oceanic and coastal waters, including Australian coral lagoon [START_REF] Mazard | PCR analysis of the distribution of unicellular cyanobacterial diazotrophs in the Arabian sea[END_REF], Zehr et al. 2007, Hewson et al. 2007). Although picocyanobacteria were numerically dominating the diazotrophic cyanobacterial community at every station, their highest concentration was detected at the most oligotrophic station from the lagoon (Stn D,Figs. 1 & 4), where DIN and chl a concentrations were typically 0.2 µmol l -1 and 0.25 µg l -1 , respectively [START_REF] Jacquet | Response of phytoplankton communities to increased anthropogenic influences (south-western lagoon, New Caledonia)[END_REF]. This high concentration was expected as diazotrophs can sustain their nitrogen needs by fixing dissolved N 2 from the atmosphere, when other plankters will be limited by these oligotrophic conditions. However, similar oligotrophic conditions are present outside the reef barrier (Stn E, 0.2 µmol DIN l -1 and 0.2 µg chl a l -1 ) [START_REF] Jacquet | Response of phytoplankton communities to increased anthropogenic influences (south-western lagoon, New Caledonia)[END_REF], but low concentrations of diazotrophic picocyanobacteria were detected. Both stations can receive sporadic nutrient inputs either from Noumea city and nearby river discharge (Stn D) or from local barrier reef upwelling (Stn E). These sources of enrichment are mainly driven by wind force and direction and do not necessarily occur simultaneously [START_REF] Jouon | Calculations of hydrodynamic time parameters in a semi-open coastal zone using a 3D hydrodynamic model[END_REF]). These inputs might carry phosphorus, which is important for diazotrophic cyanobacterial growth [START_REF] Mills | Iron and phosphorus co-limit nitrogen fixation in the eastern tropical North Atlantic[END_REF]). Beside a bottom-up control of the unicellular cyanobacterial diazotrophs, a top-down control of this community by predation activity may contribute to local changes in Nitro821-targeted cell concentration.

Among the diazotrophic picocyanobacteria we show that one-third of their concentration was associated with inert particles or intracellular of dinoflagellates within the 3 to 10 µm and >10 µm size fractions. Most of these associations were recovered in the nutrientrich Stns A, B and C (Fig. 4). This is probably because these stations have a higher concentration of particulate organic matter than the most oligotrophic ones [START_REF] Jacquet | Response of phytoplankton communities to increased anthropogenic influences (south-western lagoon, New Caledonia)[END_REF]). The cells are either passively trapped on inert particles (Fig. 3B 1 ,C 1 ) or may be chemically attracted towards these microenvironments as they may have found essential nutrients for growth, such as phosphorus and biologically available iron. Both elements have been shown to control diazotrophic activity [START_REF] Mills | Iron and phosphorus co-limit nitrogen fixation in the eastern tropical North Atlantic[END_REF]). Nitro821-labeled cells have also frequently been observed intracellular of dinoflagellates, with up to 60 targeted cells spread over a eukaryote cytoplasm (Fig. 3C 3 ). Based on the fact that a high fluorescent signal was recovered with the rRNA-targeting Nitro821 probe, it is argued that cells were in a good physiological state at the time of sampling and were probably cyanobionts. Similar signal intensity has been previously detected in eukaryotes harboring intra-cytoplasmic or intra-nuclear prokaryote symbionts (Alverca et al. 2002[START_REF] Biegala | Hybridization of picoeukaryotes by eubacterial probes is widespread and may introduce bias in biodiversity researches or reveal intimate relationship between both types of organisms[END_REF]. Non-thecate dinoflagellates are abundant in Noumea's lagoon [START_REF] Jacquet | Response of phytoplankton communities to increased anthropogenic influences (south-western lagoon, New Caledonia)[END_REF]) and cyanobionts from pico-and nanoplanktonic sizes were recently revealed in Histoneis sp., a tropical non-thecate dinoflagellate (Foster et al. 2006a). Foster et al. (2006a,b) demonstrated that nitrogenase was expressed at night at least in one nanoplanktonic cell, which looked like Cyanothece sp. ATCC51142. The present study provides the first evidence of potential N 2 -fixing picoplanktonic cyanobionts (Fig. 3C 3 ).

In conclusion, the use of whole-cell hybridization of specific Nitro821 fluorescent probes proved to be a useful technique in the field of marine nitrogen fixation by revealing for the first time the presence of diazotrophic picocyanobacteria. In Noumea's lagoon these picocyanobacteria were numerically dominating the community of cyanobacterial diazotrophs. They were mainly free-living organisms but also associated to particles and probably symbionts of non-thecate dinoflagellates. In addition, they were present at high concentrations over a large range of nutrient levels during austral spring 2005 when other planktonic diazotrophs were not abundant. Further studies will be necessary to prove the identity of these Nitro821targeted picocyanobacteria and quantify their specific contribution to global N 2 fixation.

  , cultivation, study site and sampling. To test probe and primer specificity, 3 non-axenic cyanobacterial strains -Cyanothece sp. (PCC 8801), Gleocapsa sp. (PCC 73106) and Synechocystis sp. (PCC 6803)were grown in culture media BG11, at 28°C, under a light:dark cycle of 12:12 h. Natural samples were collected with 5 l Niskin bottles within 1 wk (from 21 to 27 October 2005) inside and outside Noumea's coral lagoon (New Caledonia, France, Southwest Pacific) at 5 different stations (Fig. 1): A (22°17' S, 166°28' E), B (22°19' S, 166°28' E), C (22°20' S, 166°28' E), D (22°21' S, 166°23' E) and E (22°22' S, 166°13' E), following a nutrient and chl a gradient ranging from 0.3 to 0.03 µmol NH 4

Fig. 1 .

 1 Fig. 1. Sampling stations (A to E) in Noumea's southwest coral lagoon (New Caledonia, N.C., France, Southwest Pacific; N.Z.: New Zealand). Dark grey means permanently emerged coral reef or land and light grey shows the presence of totally or temporarily immersed coral reef

  2 & 3). Microscopy and cell counts. Images were acquired with an epifluorescence BX61 microscope (Olympus Optical) equipped with a mercury lamp (HBO 100W/2, Osram), 2 objectives -10× (NA 0.3 N Plan Fluor DT: 10 mm, Olympus) and 40× (NA 0.75 N Plan Fluor PH2 DT: 0.51 mm, Olympus) -excitation (ex.) and emission (em.) dichroïc filters (360 ± 20 ex., 410 ± 5 em. for the DAPI [blue fluorescence], 480 ± 20 ex., 510 long pass em. for the FITC associated with Nitro821 probe [green fluorescence] and for the remaining chlorophyll fluorescence [orange fluorescence], 540 ± 15 ex., 605 ± 25 short pass em. for the CY3 associated with Cya664 probe [red fluorescence]) and a digital camera (Coolpix 5400, Nikon, Figs. 1

48Fig. 2 .

 2 Fig. 2. Cyanobacterial cultures Cyanothece sp. (PCC 8801), Gleocapsa sp. (PCC 73106) and Synechocystis sp. (PCC 6803) hybridized with either the Nitro821 probe, specific for unicellular diazotroph cyanobacterial cluster, or with the general bacterial probe Eub338 (Table1). Hybridized cells were stained with FITC (green fluorescence) using the TSA-FISH technique.Arrows point to hybridized heterotrophic bacteria from non axenic cultures. Scale bars = 10 µm

  Fig.3. Different size-fractionated plankton hybridized with the Nitro821 probe, specific for unicellular cyanobacterial diazotroph clusters (A 1 , B 1 to C 4 ) (Table1), or with the general cyanobacterial probe (A 2 : Cya664) (Table1). (C5 , C 6 ) Orange autofluorescence from filamentous cyanobacteria Richelia sp. (C 5 ) and Trichodesmium sp. (C 6 ), not hybridized by Nitro821. Hybridized cells were labeled with FITC (green fluorescence) or CY3 (red fluorescence) using the TSA-FISH technique. Corresponding micrographs in blue fluorescence showing cells DNA-stained with DAPI. Unicellular diazotrophs were either free living (A 1 , B 2 , B 3 & C 4 ), associated with inert particles (B 1 , C 1 , C 2 ) or intracellular of dinoflagellates (B 2 cell on the right; B 4 , C 3 ). Arrows point to picoplankton cyanobacteria diazotrophs either free living (A 1 , A 2 ) or attached to inert particles (C 1 ). Scale bars = 10 µm

Fig. 4 .Fig. 5 .

 45 Fig. 4. Cell concentration of Nitro821-targeted unicellular cyanobacterial diazotrophs from pico-or nanoplanktonic sizes. Counts were recovered from 3 size fractions (0.2 to 3 µm, 3 to 10 µm and >10 µm) and 3 depths (0.5, 10 and 20 m), when possible, along a nutrient gradient from Noumea's coral lagoon (see Fig.1)

Acknowledgements.Editorial

  This work was financed by the French national program PNEC on coastal oceanography and IRD (French Institute for Research and Development). The crews of the IRD RVs 'Coris' and 'Aldrich' are thanked for their help collecting samples. Dr. D. Vaulot is also thanked for providing comments on this work. LITERATURE CITED Alverca E, Biegala IC, Kennaway GM, Lewis J, Franca S (2002) In situ identification and localization of bacteria associated with Gyrodinium instriatum (Gymnodiniales, Dinophyceae) by electron and confocal microscopy. Eur J Phycol 37:523-530


Debany Virginie Riou

Fonseca Batista

Arnout Roukaerts

Isabelle C Biegala

Ram Shree

Clara Magalhães Prakya

Mariana Loureiro

Angel E Santos

M Muniz-Piniella

Mara Schmiing

Marc Elskens

Debany Fonseca Batista

Shree Ram Prakya

Clara Magalhães Loureiro

Mariana Santos

Angel E M Muniz-Piniella

Natacha Brion

M Ana Martins

Frank Dehairs

Supporting Online Material (SOM) for: Impact of contrasting conditions at the North-Western Azores Current on carbon-and N 2fixation rates and the abundance of diazotrophic unicellular cyanobacteria

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Materials and Methods:

During the DIAPICNA cruise (25 July-3 August 2011) aboard R.V. "NRP Dom Carlos I", five stations were sampled from 31.5°N-33.0°W to 36.2°N-33.9°W (Station A: 31°29'53''N, 33°00'01''W, 4360 m bottom depth (b.d.); Station B: 32°11'58''N, 33°00'01''W, 3740 m b.d.; Station C: 33°42'01''N, 32°59'57''W, 3324 m b.d.; Station D: 35°00'00''N, 33°00'02''W, 3091 m b.d.; Station E: 36°13'58''N, 33°54'02 ''W, 2334 m b.d.). At each station, the water column physical properties were recorded down to 500-2000m depth using in situ sensors. The upper 500m of the water column was sampled at 12 discrete depths for the measurement of nutrient concentrations. Particulate nitrogen (PN) and organic carbon (POC) concentrations, natural NO 3 -isotopic signatures, carbon-and N 2 fixation rates and the abundance of Unicellular CYanobacterial N 2 -fixers (UCYN) were assessed on water samples sequentially collected during four additional casts at four chosen depths within the euphotic zone. All longitudinal cross-sections were obtained using the Ocean Data View software (Schlitzer, 2008).

N 2 -and C-fixation incubations: N 2 fixation rates were measured using the dissolved 15 N 2 tracer method (Mohr et al., 2010;Wilson et al., 2012). Spiked solutions of 15 N 2 were prepared prior to the cruise, using degassed (by vacuum pumping under magnetic stirring) 0.2 µm-filtered low nutrient seawater (Osil, UK), injected with 2.5 mL of 15 N 2 -gas (98+ Atom% 15 N, Eurisotop, France) and shaken for 24 h. These spiked solutions were conditioned in 285 mL borosicilate vials crimp sealed with Teflon lined silicone septa (Wheaton, US).

Prior to the cruise, tests were conducted in the lab using five replicate spiked solutions in separate 285 mL borosilicate vials sampled respectively after 0, 2, 4, 7 and 9 days incubation at room temperature. They showed that after an initial decrease from 70% to 50% 15 N atom% between days 0 and 4, the 50% enrichment remained stable for up to 9 days. Final enrichment in incubation bottles were also conducted in triplicates: three acid-washed 4.5-L Nalgene polycarbonate bottles were filled with 4.3L of non-degassed 0.2 µm-filtered low nutrient seawater (OSIL incubation seawater) to which 285 mL 15 N 2 spiked solutions (previously homogenised for 24h) were added. Nalgene bottles were filled to the very rim with the OSIL incubation seawater, capped with septum equipped screw caps (PTFE; Nalgene) and incubated for 24 h at room temperature (23°C) in the lab, before they were sampled for dissolved N 2 15 N atom%. Triplicate 12 mL aliquots were transferred from each incubation bottle through the PTFE septa, applying a slight He overpressure and using 2-way needles, into helium pre-flushed exetainers (Labco, UK) poisoned with 100 µL saturated HgCl 2 solution. Dissolved N 2 isotopic composition was measured from equilibrated headspaces prepared by the removal of 3 mL seawater from the 12 mL exetainer vials, using He pressure. Analyses were performed using a Flash EA 1112 Elemental Analyzer coupled to a DELTA V Isotope Ratio Mass Spectrometer via a Conflo III interface (EA-IRMS, Thermo Instruments) equipped with a custom made manual gas injection port. The injections of 300 µL headspace were done upstream of the EA reduction column, under helium flow. These analyses revealed final dissolved N 2 15 N atom% of 5.4 ± 0.4 % in the incubated seawater.

During the cruise, seawater samples were taken at each station at the surface (11-16m), above the Deep Chlorophyll Maximum (DCM; 45-48m), at the DCM (86-112m) and in the upper mesopelagic zone (200-217m), from which 4.5 L were immediately filtered to determine natural particulate organic Carbon and Nitrogen (POC, PN) concentrations and their 13 C and 15 N isotopic compositions. Further duplicate samples from each depth (approx. 4.3 L) were transferred to acid-washed 4.5-L Nalgene polycarbonate bottles to which 285 mL 15 N 2 spike solution were added, together with 0.73 mmol of dissolved NaH 13 CO 3 each (99 Atom% 13 C, Eurisotop, France), to reach theoretical enrichments of 5 Atom% 15 N and 10 Atom% 13 C. Nalgene bottles were then filled to the very rim with ambient seawater and capped with a screw cap fitted with a septum (PTFE; Nalgene). These spiked samples were incubated for 24 h in on-deck incubators wrapped in blue filters (Rosco, UK) and bathed in continuously renewed surface seawater to ascertain a constant temperature (around 24°C). The light filters were selected to mimic in situ daylight attenuation in Jerlov type OIA waters (Piazena et al., 2002): no filter for surface samples (100% surface light), "No color Blue" filter for above the DCM samples (#144 ≈30% light transmission), "Dark Blue" filter for DCM samples (around 100 m, #119≈3% light transmission) and an overlap of "Alice Blue" and "Tokyo Blue" filters for upper mesopelagic samples (#197+#071≈0.1% light transmission). At the end of the incubation period, six replicate 12 mL aliquots were transferred through the PTFE septa, applying a slight He overpressure and using 2-way needles, into helium pre-flushed exetainers (Labco, UK) poisoned with 100 µL saturated HgCl 2 solution for the assessment of the dissolved N 2 15 N atom% and Dissolved Inorganic Carbon (DIC) 13 C atom%. Triplicate dissolved N 2 and DIC isotopic compositions were measured from equilibrated headspaces prepared by the removal of 3 mL seawater from the 12 mL exetainer vials, using He pressure. For DIC we subsequently added 200µl of 99% phosphoric acid. Analyses were performed using a Flash EA 1112 Elemental Analyzer coupled to a DELTA V Isotope Ratio Mass Spectrometer via a Conflo III interface (EA-IRMS, Thermo Instruments) equipped with a custom made manual gas injection port. The injections of 300 µL headspace were done upstream of the EA reduction column, under helium flow. Natural and enriched particles were size-fractionated by serial filtration onto 25 mm diameter membranes of 3.0 µm and 0.3 µm porosities, made of silver (Sterlitech) and pre-combusted glass fiber (GF75, Advantec MFS Inc.), respectively. The filters were dried overnight at 60°C and stored over desiccant until analysis. After carbonate acidification overnight under saturated HCl vapours and drying at 60°C, POC and PN concentrations and  13 C and  15 N signatures were measured using the EA-IRMS. Standards and blanks were run under the same analytical conditions as the samples. POC and PN areas were corrected for blank areas and converted to moles using acetanilide standard calibration (Merck, 71.09%C, 10.36%N). IRMS  13 C and  15 N values were corrected for the influence of blanks and for the offset of the IAEA standards from their certified values (IAEA-CH6,  13 C=+10.449‰ vs. Vienna Pee Dee Belemnite; IAEA-N1,  15 N=+0.43‰ vs. atmospheric N 2 ). The uncertainties associated with the calibrations are reported in Tables S1, S2 and S3 under "t=0 Natural PN,  15 N, E", "t=24h Natural PN,  15 N, E" and "t=24h Natural PX, Concentration, E". δ H X values (i.e. δ 15 N or δ 13 C) were converted to H X atom% using the following formulas: R = ( H X/ L X) sample = [(δ H X/1000)+1] x ( H X/ L X) reference and H X atom% = R/[1+R] Where H is the heavy and L is the light isotope and references are atmospheric N 2 and Vienna Pee Dee Belemnite for N and C, respectively.

N 2 -and C-fixation rates and error calculations:

The final A substrate term for DIC was corrected by the equation of Miyajima et al. (1995), and that for dissolved 15 N 2 was calculated using the combination of IRMS masses M 28 ( 14 N 2 ), M 29 ( 14 N 15 N) and M 30 ( 15 N 2 ) areas (after correction of the machine bias for M 30 ): final A N2 = [M 30 + (M 29 /2)]/[M 28 + M 29 + M 30 ] N 2 and C-uptake rates were not considered significant (grey police in Tables S1, S2 andS3) when: (i) particle enrichment ("PX Enrichment") was lower than three times the standard deviation of the natural PX isotopic signature calculated for each depth ("t=0 Natural PX Depth 3xSD"), and/or the particle enrichment after incubation in atom% was lower than the error calculated on the enrichment ("PX enrichment E"); (ii) Substrate enrichment in atom% ("Substrate enrichment") was lower than the highest error on final A substrate triplicate measurements (i.e., 15 N 2 <0.0908); (iii) The error on the uptake rate calculation ("Uptake Rate E"), calculated from the propagation of the errors or uncertainties associated with each term used for the calculation, was higher than the uptake rate value itself.

Unicellular cyanobacterial diazotroph (UCYN) cellular abundance:

Cells were collected on the 0.2, 3.0 and 10 µm porosity PCTE membranes, applying light vacuum as for the POC/PN filters with a water jet pump (Medline, Model VE-11) reaching an average filtration speed of 100 mL min -1 . Collected cells were then fixed with 10 mL of 1% paraformaldehyde (pH 7.2 buffered with PBS and clarified by filtration, Sigma-Aldrich, France) for 15 min at room temperature (RT), desiccated in 100% ethanol molecular grade (Sigma-Aldrich, France) for 10 min at RT, and immediately preserved in liquid N 2 until the end of the cruise. During transfer and at the lab, filters were stored between -20°C and -80°C until analysis.

Nitrate dual isotopic composition (δ 15 N and δ 18 O) measurements:

Isotope signatures for nitrate were measured using the denitrifier method (Sigman et al., 2001;Casciotti et al., 2002). Nitrate was converted to N 2 O by adding sample to a solution of denitrifying bacteria (Pseudomonas aureofaciens) that lack the active N 2 O reductase. The produced N 2 O was then cryogenically isolated and purified from the sample headspace for subsequent isotopic analysis for δ 15 N and δ 18 O using IRMS (DELTA, Thermo Instruments). Nitrate concentrations down to 1 µmol L -1 could be analysed.

Results: C-fixation integration from <3µm + >3µm size fractions

StA: day 2.42+3.16=5.58 mmolC m -² d -1 =66.96 mgC m -² d -1 ; 43% <3µm night 2.67+3.78=6.45 mmolC m -² d -1 =77.4 mgC m -² d -1 ; 41% <3µm StB: day 1.86+3.20=5.06 mmolC m -² d -1 =60.72 mgC m -² d -1 ; 37% <3µm StC: day 1.97+3.01=4.98 mmolC m -² d -1 =59.76 mgC m -² d -1 ; 40% <3µm StD: day 3.76+6.47=10.23 mmolC m -² d -1 =122.16 mgC m -² d -1 ; 37% <3µm StE: day 3.98+4.84=8.82 mmolC m -² d -1 =105.84 mgC m -² d -1 ; 45% <3µm N 2 -

fixation measurements and corrections

The decrease in 15 N 2 final incubation enrichment level from 5.0 15 N Atom% for the test incubations prior to the cruise, down to 0.4-1.0 15 N Atom% for the samples incubated during the cruise (S5 & S7 Tables) indicates that the Teflon-lined silicon septa used to seal the serum bottles with 15 N 2 spike did not remain gas tight over prolonged periods. This caused loss of 15 N 2 during transportation and storage before the cruise. This problem was solved a posteriori, using bromobuthyl septa that avoided the loss of 15 N 2 enrichment for up to 2 months at 5-30°C. We did not take into consideration the incubations with substrate enrichments lower than 0.0908% above t=0 A particle , which was the largest measurement error on replicates for dissolved N 2 15 N Atom%. Some samples were 'corrected' for insufficient particle enrichment (i.e. lower than 3 times the natural δ 15 N PN standard deviation) using the value of half the standard deviation of natural isotopic signatures from all stations at the same depth (Tables S1 andS3). Since resulting corrected N 2 fixation rates presenting uncertainties (E) inferior to the value itself could reach values of 0.5±0.4 µmol N m -3 d -1 , rates below this value should be considered with care.

References:

Casciotti, K.L., Sigman, D.M., Hastings, M.G., Böhlke, J.K., Hilkert, A., 2002. Measurement of the oxygen isotopic composition of nitrate in seawater and freshwater using the denitrifier method. Anal. Chem. 74(19), 4905-4912. Krupke, A., Lavik, G., Halm, H., Fuchs, B.M., Amann, R.I., Kuypers, M.M.M., 2014. Distribution of a consortium between unicellular algae and the N 2 fixing cyanobacterium UCYN-A in the North Atlantic Ocean. Environ. Microbiol. 16(10), 3153-3167. doi:10.1111/1462-2920.12431 Luo, Y.W., Doney, S.C., Anderson, L.A., Benavides, M., Bode, A., Bonnet, S., et al., 2012. Database of diazotrophs in global ocean: abundances, biomass and nitrogen fixation rates. Earth Syst. Sci. Data Discuss. 5, 47-106. Pangaea database Miyajima, T., Yamada, Y., Hanba, Y., Yoshii, K., Koitabashi, T., Wada, E., 1995. Determining the stableisotope ratio of total dissolved inorganic carbon in lake water by GC/C/IRMS. Limnol. Oceanogr. 40(5), 994-1000. Mohr, W., Grosskopf, T., Wallace, D.W.R., LaRoche, J., 2010. Methodological underestimation of oceanic nitrogen fixation rates. PLoS ONE 5, e12583. Moore, C.M., Mills, M.M., Achterberg, E.P., Geider, R.J., LaRoche, J., Lucas, M.I.

  , et al., 2009. Largescale distribution of Atlantic nitrogen fixation controlled by iron availability. Nature Geosci. 2, 867-871. Piazena, H., Perez-Rodrigues, E., Häder, D.P., Lopez-Figueroa F., 2002. Penetration of solar radiation into the water column of the central subtropical Atlantic Ocean-optical properties and possible biological consequences. Deep-Sea Res. II 49, 3513-3528. Rijkenberg, M.J.A., Langlois, R.J., Mills, M.M., Patey, M.D., Hill, P.G., Nielsdóttir, M.C., et al., 2011. Environmental Forcing of Nitrogen Fixation in the Eastern Tropical and Sub-Tropical North Atlantic Ocean. PLoS ONE 6(12), e28989. doi:10.1371/journal.pone.0028989 Schlitzer (2004) Ocean Data View, http://odv.awi-bremerhaven.de Sigman, D.M., Casciotti, K.L., Andreani, M., Barford, C., Galanter, M., Böhlke, J.K., 2001. A bacterial method for the nitrogen isotopic analysis of nitrate in seawater and freshwater. Anal.Chem. 73(17),[4145][4146][4147][4148][4149][4150][4151][4152][4153] doi:10.1021/ac010088e. Wilson, S.T., Böttjer, D., Church, M.J., Karl, D.M., 2012. Comparative assessment of nitrogen fixation methodologies conducted in the oligotrophic North Pacific Ocean.Appl. Environ. Microbiol. 78, 6516


Nathalie Simon
email: simon@sb-roscoff.fr

Isabelle C Biegala

Elizabeth A Smith

Daniel Vaulot

Kinetics of attachment of potentially toxic bacteria to Alexandrium tamarense

INTRODUCTION

The relationships between phytoplankton and bacteria in the aquatic environment play a crucial role in important processes such as carbon fluxes and nutrient regeneration [START_REF] Cole | Bacterial production in fresh and saltwater ecosystems: a cross-system overview[END_REF], Azam 1998). Major fluxes of organic matter produced by phytoplankton are transferred via dissolved organic matter (through exudates and leakage from broken cells) to bacteria and the microbial loop (Azam 1998), while remineralisation by bacteria supplys nutrients to phytoplankton. Bacteria and phytoplankton cells can also compete in conditions where nutrients are limiting for both organisms [START_REF] Cole | Interactions between bacteria and algae in aquatic ecosystems[END_REF][START_REF] Gurung | Regulation of the relationship between phytoplankton Scenedesmus acutus and heterotrophic bacteria by the balance of light and nutrients[END_REF]). In addition, phytoplankton cells and bacteria can release antagonistic or stimulating substances such as antibiotics or vitamins [START_REF] Cole | Interactions between bacteria and algae in aquatic ecosystems[END_REF][START_REF] Manage | Algicidal effect of the bacterium Alcaligenes denitrificans on Microcystis spp[END_REF]. Interactions such as these are of prime importance in the regulation of algal populations.

In the context of toxic events and blooms, bacterialalgal interactions may also be involved in the induction and control of toxin production. [START_REF] Osada | Gluconic acid/gluconolactone: physiological influrences on domoic acid production by bacteria associated with Pseudo-nitzschia multiseries[END_REF] suggested the indirect involvement of non-toxic bacteria in the enhancement of the production of the toxin domoic acid by the diatom Pseudo-nitzschia multiseries. In this case, bacteria, through the production of gluconic acid that reportedly shows powerful sequestering properties for nutrients, are thought to induce the algae to produce domoic acid, which is argued to be a counter chelating agent. The role of bacteria in the production of paralytic shellfish toxins (PST) by Alexandrium spp. has been highly debated. The demonstration of PST production after inoculation of a bacterial strain into non-toxic dinoflagellate cultures [START_REF] Silva | Experimental work on the dinoflagellate toxin production[END_REF][START_REF] Silva | Intracellular bacteria: the origin of dinoflagellate toxicity[END_REF]), the modification of the toxin profile after a bactericidal treatment of a toxic dinoflagellate culture [START_REF] Hold | Comparison of paralytic shellfish toxin (PST) production by the dinoflagellates Alexandrium lusitanicum NEPCC 253 and Alexandrium tamarense NEPCC 407 in the presence and absence of bacteria[END_REF]) and the ABSTRACT: Interactions between bacteria and harmful algae are potentially important regulating factors for population dynamics of both organisms and for toxin production. These interactions are still poorly understood. To monitor the physical associations between potentially toxic bacteria and dinoflagellates in controlled conditions, we inoculated an axenic non-toxic strain of Alexandrium tamarense (Dinophyceae) together with reputed paralytic shellfish toxin (PST)-producing bacteria belonging to the genus Alteromonas (γ subdivision of the division Proteobacteria) and to the clade Roseobacter (α subdivision of the division Proteobacteria). The attachment behavior of both bacterial strains was monitored using TSA-FISH (tyramide signal amplification and fluorescent in situ hybridization) and confocal microscopy. Our results suggest that ageing dinoflagellate cultures stimulate both free bacterial growth and attachment. However, toxin production by originally non-toxic dinoflagellate cells was not induced by the physical interaction of either of the bacterial strains with the dinoflagellate cells. This does not support the hypothesis that toxic bacteria could simply control toxin production by attachment to particle surfaces such as eukaryotic organisms. autonomous bacterial production of phycotoxins [START_REF] Gallacher | Evidence for production of paralytic shellfish toxins by bacteria associated with Alexandrium spp. (Dinophyta) in culture[END_REF], Doucette et al. 1998) has, in some cases, cast doubt that these toxins are indeed produced by the algae alone (see reviews by Rausch de [START_REF] Rausch De Traubenberg | Dinoflagellate toxicity: are marine bacteria involved? Evidence from the literature[END_REF], Doucette et al. 1998). Nevertheless, several authors have reported on the production of PST by 'axenic' dinoflagellate cultures with levels of toxicity similar to those observed for non-axenic strains [START_REF] Dantzer | Bacterial influence on the production of paralytic shellfish toxins by dinoflagellate algae[END_REF][START_REF] John | Amino acid uptake by the toxic dinoflagellate Alexandrium fundyense[END_REF] but the absence of bacteria (free, attached or endocellular) was not clearly demonstrated. [START_REF] Tosteson | Associated bacterial flora, growth, and toxicity of cultured benthic dinoflagellates Ostreopsis lenticularis and Gamberdiscus toxicus[END_REF] and Doucette et al. (1998) suggested that bacteria could modify algal toxicity via attachment. However, mechanisms by which physical interactions can influence toxin production levels for the partners of the bacteria-dinoflagellate association remain unknown. Indeed, techniques have been lacking to identify specifically the bacteria associated to Alexandrium spp. and to study their spatial relationships.

Bell & Mitchell (1972) created the term 'phycosphere' to qualify a zone surrounding algal cells in which bacteria are influenced by algae and conversely algae by bacteria. Bacteria can be free in the phycosphere [START_REF] Blackburn | Microscale nutrients patches in planktonic habitats shown by chemotactic bacteria[END_REF], attached to the surface of algal cells [START_REF] Kogure | Bacterial attachment to phytoplankton in sea water[END_REF][START_REF] Vaqué | Influence of algal population dynamics on phytoplankton colonization by bacteria: evidence from two diatom species[END_REF][START_REF] Worm | Dynamics of heterotrophic bacteria attached to Microcystis spp. (Cyanobacteria)[END_REF] or intracellular [START_REF] Silva | The association dinoflagellatebacteria: their ultrastructural relationship in two species of dinoflagellates[END_REF][START_REF] Lewis | Bacterial dinoflagellate interactions: investigative microscopy of Alexandrium spp. (Gonyaulacales, Dinophyceae)[END_REF]. Few investigators have examined the process of attachment to algal cells [START_REF] Kogure | Bacterial attachment to phytoplankton in sea water[END_REF][START_REF] Vaqué | Influence of algal population dynamics on phytoplankton colonization by bacteria: evidence from two diatom species[END_REF][START_REF] Worm | Dynamics of heterotrophic bacteria attached to Microcystis spp. (Cyanobacteria)[END_REF]. Methods used by these authors have included phase contrast and epifluorescence microscopy to directly detect and enumerate attached bacteria on algal cell surfaces, with a preliminary filtration or centrifugation step sometimes used to exclude free bacteria. Indirect methods have also been proposed, with the number of attached bacteria estimated by subtracting the total number of bacteria collected before and after chemical treatment and sonication of cultures (Albright et al. 1986). These treatments dispersed attached bacteria in the medium. Biegala et al. (2002) recently proposed a method involving fluorescent in situ hybridization with group-specific oligonucleotide probes and tyramide signal amplification (TSA-FISH), used in conjunction with confocal laser scanning microscopy (CLSM) to monitor bacterial attachment to phytoplankton cells. This method presents several advantages. First, individual bacterial strains can be distinguished in a mixture of taxa and their specific behavior can be monitored. Second, with CLSM, attached bacteria surrounding the entire cell (including in the sulcus and cingulum) can be enumerated (while only the top half of the cell can be examined using epifluorescence microscopy) and intracellular bacteria can be detected.

To study toxic bacteria-dinoflagellate interactions in controlled conditions, we incubated an axenic nontoxic strain of Alexandrium tamarense with bacterial isolates belonging or closely related to the genera Alteromonas and Roseobacter (respectively γ and α subdivisions of the Proteobacteria). Previous mouse neuroblastoma (MNB) assays suggested that these bacteria, isolated from toxic Alexandrium strains, were capable of synthesizing sodium channel blocking (SCB) toxins (Gallacher et al. 1997, Gallacher & Smith unpubl. data). The chemical nature of the toxins was determined in the Alteromonas isolate as PST by high performance liquid chromatography (HPLC) and capillary electrophoresis-mass spectrometry (CE-MS) [START_REF] Gallacher | Evidence for production of paralytic shellfish toxins by bacteria associated with Alexandrium spp. (Dinophyta) in culture[END_REF]. Both the genera Alteromonas and Roseobacter are common in marine environments and are frequently found in association with algae [START_REF] Lafay | Roseobacter algicola sp. nov., a new marine bacterium isolated from the phycosphere of the toxin-producing dinoflagellate Prorocentrum lima[END_REF][START_REF] Holmström | Marine Pseudoalteromonas species are associated with higher organisms and produce biologically active extracellular agents[END_REF]. The questions we addressed were: (1) Do potentially toxic bacterial isolates attach to dinoflagellates? (2) What is the specific attachment behavior of those bacterial strains?

(3) Does attachment of potentially toxic bacteria to cells of A. tamarense induce the algal cells to produce PSTs? Using fluorescent whole-cell hybridization with group-specific oligonucleotide probes and confocal microscopy, we were able to monitor the physical association of each potentially toxic bacterial strain to A. tamarense cells throughout the dinoflagellates' growth. The ability of these bacteria to induce detectable toxin production by the algae, especially through attachment to the cells, was also tested.

MATERIALS AND METHODS

Strains and cultivation.

An axenic non-toxic culture of Alexandrium tamarense (strain CCMP 1771, Provasoli-Guillard Culture Center for Marine Phytoplankton, Boothbay Harbor, ME, USA), and 2 different bacterial strains (253-11 and 407-2 provided by the Fisheries Research Services, Marine Laboratory, Aberdeen, UK) were investigated in this study. The bacterial strain 253-11 belongs to the Roseobacter clade (α-Proteobacteria) and was isolated from toxic Alexandrium lusitanicum (NEPCC 253, North East Pacific Culture Collection, Vancouver, BC, Canada) whereas 407-2 belongs to the genus Alteromonas (γ-Proteobacteria) and was isolated from toxic A. tamarense (NEPCC 407;[START_REF] Gallacher | Evidence for production of paralytic shellfish toxins by bacteria associated with Alexandrium spp. (Dinophyta) in culture[END_REF]. Both 407-2 and 253-11 isolates had been previously shown to produce SCB toxins (Gallacher et al. 1997, S. Gallacher pers. comm.). Inocula of dinoflagellate and bacterial strains were prepared respectively in Guillard's f/2 medium (Sigma-Aldrich) and marine broth (DIFCO). Autoclaved and sterile filtered natural seawater collected off Roscoff was used to prepare the f/2 medium. Bacteria were cultured in the dark at 15°C in polycarbonate 10 ml tubes (Nalgene) whereas dinoflagellates were grown in 1 l polycarbonate bottles (Nalgene) at 15°C, under a light:dark cycle of 14:10 h, with an intensity of 54 µmol photons m -2 s -1 .

Experimental conditions and sampling. Prior to inoculation in f/2 medium, bacteria (strains 253-11 and 407-2 in late exponential growth phase) were pelleted (600 × g, 3 min) and washed in f/2 medium. Three different combinations of cell association were tested: Alexandrium tamarense and 253-11, A. tamarense and 407-2, A. tamarense with both 253-11 and 407-2. In addition, 4 control cultures were prepared: A. tamarense alone, 253-11 alone, 407-2 alone or 253-11 together with 407-2. All cultures (1 l each) were monitored for 34 d. Sampling (1 ml) for cell enumeration by flow cytometry was performed every other day for the first 2 wk and then every week. Samples were fixed with 0.25% glutaraldehyde, frozen in liquid nitrogen and stored at -20°C until further analysis. For FISH (fluorescent in situ hybridization), sampling (10 to 20 ml) was achieved every 4 d for the first 2 wk and then every week. Samples were fixed with 1% paraformaldehyde at 4°C for 24 h, immobilized onto inorganic filter membranes (Anodisc, Whatman), dehydrated in an ethanol series (50, 80, 100%, 3 min each; Amann 1995), dried and kept at room temperature in the dark until FISH was performed.

Flow cytometry. Dinoflagellates and total free-living bacteria were enumerated using a FACSort flow cytometer (Becton Dickinson). Dinoflagellates were identified according to their red autofluorescence and their scattering properties, whereas bacteria were stained with SYBR Green I (Molecular Probes) prior to analysis [START_REF] Marie | Enumeration and cell cycle analysis of natural populations of marine picoplankton by flow cytometry using the nucleic acid stain SYBR Green I[END_REF].

Probes. Oligonucleotide probes (Table 1) were purchased with a 5'-aminolink (C6) from Interactiva and labeled with the enzyme horseradish peroxidase (HRP, Boehringer Mannheim) according to [START_REF] Urdea | A comparison of non-radioisotopic hybridization assay methods using fluorescent, chemiluminescent, and enzyme labeled oligodeoxyribonucleotide probes[END_REF] and Amann et al. (1992). ROSEOC536R was used as a competitor [START_REF] Manz | Phylogenetic oligodeoxynucleotide probes for the major subclasses of proteobacteria: problems and solutions[END_REF]) and was not labeled with HRP.

Fluorescent in situ hybridization. In situ hybridization with TSA amplification was performed according to the protocol described by Biegala et al. (2002) modified from [START_REF] Schönhuber | In situ identification of cyanobacteria with horseradish peroxidase-labeled, rRNA-targeted oligonucleotide probes[END_REF]. Briefly, the bacterial cell wall is permeabilized with lysozyme prior to hybridization with HRP-labeled probes. Probes in excess are removed by several washing steps and cells are further incubated in a buffer containing fluorophore-labeled tyramides (TSA™-Direct kit, NEN™ Life Science Product). HRP catalyzes the rapid deposition of fluorophore-tyramides in the close environment of the enzyme. To identify bacterial types in mixed cultures, a double hybridization was performed (Biegala et al. 2002). The HRP-labeled probes AMAC137R and EUB338R were used successively in combination with tyramide-fluorescein and tyramide-coumarin as substrates for HRP. To monitor bacterial attachment onto dinoflagellate theca, a single hybridization was performed either with AMAC137R or ROSEO536R together with the competitor ROSEOC536R. For single hybridizations, tyramide-fluorescein was used as the substrate for HRP and cells were then further stained with the DNA-specific dye DAPI (4', 6-diamidino-2phenylindole, Sigma-Aldrich) and the cellulose specific dye calcofluor (Sigma-Aldrich) as described in Biegala et al. (2002). Calcofluor stained the dinoflagellate theca and helped to visualize precisely bacterial attachment, whereas DAPI served to visualize the nucleus. Both dyes helped distinguish morphologically intact cells from empty theca and broken cells. DAPI staining and in situ hybridization with TSA amplification and the general eubacterial probe also allowed us to confirm the axenic status of the dinoflagellate strain chosen in this study; no free, attached or intracellular bacteria were observed in the control culture.

Microscopy. A BH-2 epifluorescence microscope (Olympus Optical) was used to enumerate the 2 freeliving bacterial types in the cultures where both 253-11 and 407-2 were inoculated. Excitation and emission filters used were, respectively, a 405 nm band pass and a 455 nm long pass. After double hybridization, bacteria labeled by AMAC137R (407-2) and stained with fluorescein appeared green and bacteria labeled with EUB 338R (both 253-11 and 407-2) and stained with coumarin appeared blue (Fig. 1). A total of 50 to 500 cells were enumerated per sample. Percentages of 407-2 cells relative to total bacteria (407-2 and 253-11 bacteria) were calculated and specific cell densities were estimated from flow cytometry counts.

To enumerate bacteria attached to dinoflagellates, optical sections of hybridized cells (either with AMAC-137R or ROSEO536R) were obtained using confocal microscopy: a minimum of 5 dinoflagellates were examined per sampling time. When the density of cells was less than 5 filter -1 (Days 0 and 4), all morphologically intact dinoflagellate cells were analyzed. An Olympus Fluoview confocal microscope equipped with 2 lasers was used to study bacterial attachment: an argon-krypton laser (634R-OLYM-A03 Omnichrome, Melles Griot) set at 488 nm for FITC excitation and a pulsed laser (Coherent mira 900) set at 360 nm for DAPI and calcofluor excitation. Counts of attached bacteria were obtained by visualizing every optical slice acquired (Biegala et al. 2002). The percentages of attached bacteria cells relative to total bacterial cells was calculated as B a × D/[B f + B a ] × 100, where B a = mean number of attached bacteria per dinoflagellate cell, D = number of dinoflagellate cells m -1 and B f = number of free bacteria m -1 .

Toxicity. The toxicity of the Alteromonas and Roseobacter strains 407-2 and 253-11 was verified before inoculation in dinoflagellates cultures and in control cultures using the MNB assay according to [START_REF] Gallacher | Evidence for production of paralytic shellfish toxins by bacteria associated with Alexandrium spp. (Dinophyta) in culture[END_REF]. Dinoflagellates from control and experimental cultures at stationary phase (Day 34) were harvested by centrifugation (5 min, 2000 × g). Harvested cells (approximately 10 6 cells) were resuspended in 1 ml of 0.05 M acetic acid before storage at -20°C. Following thawing of samples, cells were disrupted by the addition of 25% (w/v) glass beads (150 to 212 mm diameter, Sigma) and vortex mixed for 5 min. Microscopic examination of cell debris confirmed complete cell disruption. Samples were centrifuged (5 min, 2000 × g) prior to filtration (Ultrafree centrifugal units, 10 000 NMWL [nominal molecular weight limit], Millipore). HPLC analysis of PST was performed according to the method of [START_REF] Franco | Separation of paralytic shellfish toxins by reversed phase high performance liquid chromatography, with postcolumn reaction and fluorimetric detection[END_REF] with amendments detailed in [START_REF] Smith | Biotransformation of paralytic shellfish toxins (PST) by bacteria isolated from bivalve molluscs[END_REF].

RESULTS AND DISCUSSION

Dinoflagellates and bacterial growth

Dinoflagellates growth did not seem to be much affected by any of the bacterial combinations tested (Fig. 2A). A lag period was observed only in cultures inoculated with bacteria but the stationary phase was reached at the same time with the same cell densities in all cultures. During the first 15 d of the experiment, dinoflagellates' influence on bacterial growth was very limited (Fig. 2B-D) and bacteria were able to grow in dinoflagellate-free f/2 seawater medium almost as well as (Fig. 2B), and sometimes slightly better than (Fig. 2D), when algal cells were present. This suggests that initial bacterial growth was supported by organic matter present in the natural seawater used to prepare the medium but not by organic matter derived from algal cells. In contrast, after Day 15, bacterial growth was strongly affected by the presence of algal cells (Fig. 2B-D). After Day 15, bacteria either disappeared (407-2) or remained at relatively low concentrations (253-11 and 253-11 plus 407-2) in control cultures, whereas after Day 20, bacterial cell densities sharply increased in all 3 cultures containing dinoflagellates (Fig. 2B-D). At this stage, stationary growing dinoflagellates probably stimulated bacterial growth through the production of organic exudates. In cultures where both bacteria were introduced either with or without dinoflagellates, the behavior of each bacterial strain roughly resembled the behavior of the corresponding strain inoculated, respectively, alone in f/2 medium or alone in a culture of Alexandrium tamarense (Fig. 2D). Thus, both bacterial isolates behaved like many marine 252 Fig. 1. Epifluorescence microscopy image from a culture containing both Roseobacter strain 253-11 (open arrows) and Alteromonas strain 407-2 (solid arrows) together with Alexandrium tamarense. Cells were sampled at Day 13 and were hybridized with the probe AMAC137R detected using fluorescein tyramide and then with the probe EUB 338R detected with coumarin-tyramide. All bacteria were labeled by the probe EUB 338R (blue color), whereas strain 407-2 was specifically labeled by the probe AMAC137R (green color) bacteria that rely on organic material released by algae [START_REF] Cole | Interactions between bacteria and algae in aquatic ecosystems[END_REF]. However, in the literature, the effect of bacteria on the growth of algal species and, conversely, the effect of algae on bacteria, are extremely variable. Cases have been observed of stimulation, but also of inhibition by bacteria on algae (including harmful algal species), or conversely by algae on bacteria as well as cases where none of the organisms showed any response [START_REF] Cole | Interactions between bacteria and algae in aquatic ecosystems[END_REF][START_REF] Doucette | Interaction between bacteria and harmful algae: a review[END_REF]. Culture or environmental conditions may also modulate these interactions. Indeed, independent experiments conducted on A. tamarense and the bacteria 253-11 and 407-2 showed that when larger algal inocula were used, bacteria enhanced algal growth (E. Smith unpubl. data).

Dynamics of bacterial attachment

Both bacterial strains (Roseobacter strain 253-11 and Alteromonas strain 407-2) were observed attached to the cell surface of morphologically intact dinoflagellates throughout the growth of the dinoflagellate cultures (Fig. 3). Although highly variable among coexisting dinoflagellate cells, the number of attached bacteria per algal cell clearly increased as cultures aged (Figs. 3 & 4). Bacterial load was rather low in all cases during initial growth (<10 bacteria per dinoflagellate until Day 10) but could reach 16, 43 and 80 attached bacteria (average numbers 9, 16 and 30) at Day 34 in the cultures containing 407-2, 253-11 and 407-2 together with 253-11, respectively (Fig. 4). The percentage of attached bacterial cells relative to total bacterial cells was maximal when dinoflagellates entered stationary phase. At Day 34, 5% of the 253-11 bacteria and 16% of the 407-2 bacteria were found attached when those bacteria were inoculated separately in dinoflagellates cultures. When both bacteria were inoculated together, a maximum of 18% of the 253-11 and 52% of the 407-2 were found attached to dinoflagellate cells. Overall, 407-2 exhibited a more pronounced potential for attachment. This strain also had a tendency to aggregate in clumps (data not shown) while most of the 253-11 bacteria remained free living.

There was no clear relationship between the number of attached bacteria per algal cell and the total number of free bacteria or the number of free bacteria available per dinoflagellate cell (Fig. 4). This shows that bacterial colonization of algal cells is not only constrained by the probability of encounter between both organisms. However, during dinoflagellates' stationary phase, the sharp increase observed in free bacterial cell counts (Fig. 2) was also observed for attached bacteria (Fig. 4). This suggests that the physiological state of dinoflagellates was important for the attachment of those potentially toxic bacteria and that dinoflagellates in stationary phase stimulated both bacterial growth and attachment. A similar pattern of bacterial attachment has already been observed when non-toxic bacteria were introduced to diatoms, chlorophytes or other non-toxic dinoflagellate cultures (Bell & Mitchell 1972[START_REF] Kogure | Bacterial attachment to phytoplankton in sea water[END_REF], Albright et al. 1986). In this respect, the general behavior of these toxic or potentially toxic bacteria towards phytoplankton cells was similar to the behavior of other marine bacteria. Ageing phytoplankton cells probably release increasing amounts of exudates through porous membranes and cell walls [START_REF] Fogg | The ecological significance of extracellular products of phytoplankton photosynthesis[END_REF]. The physical associations and kinetics of the association could result from chemotactic responses of the bacteria to algal exudates (Bell & Mitchell 1972, Albright et al. 1986) and/or from an increased bacterial potential for attachment (through the production of exopolymers for example).

Attachment is probably beneficial for bacterial growth since these algal exudates represent sources of organic carbon for bacteria [START_REF] Paerl | Specific associations of the bluegreen algae Anabaena and Aphanizomenon with bacteria in freshwater blooms[END_REF], Azam et al. 1995).

Toxicity tests

Both the Alteromonas strain 407-2 and Roseobacter strain 253-11 are capable of SCB toxin production and the chemical nature of the toxin produced by 407-2 was determined as PST by capillary electrophoresismass spectrometry (Gallacher et al. 1997, Gallacher & Smith unpubl. data). However, only 407-2 produced SCB toxins when tested using the MNB assay prior to this study (data not shown), while strain 253-11 did not consistently demonstrate toxicity. In our study, although bacterial attachment was clearly occurring, addition of the 2 bacteria either individually or in combination did not induce any detectable PST production (detection limit for gonyautoxins 1, 2, 3 and 4, saxitoxin and neosaxitoxin on the column were respectively 0.28, 0.33, 0.08, 0.12, 0.57 and 3.77 ng) in the stationary dinoflagellate cultures. Previous experiments involving inoculation of toxigenic or non-toxigenic bacteria into axenic toxic dinoflagellate cultures suggested that bacteria enhanced toxin production via attachment: indeed, inoculation of viable bacteria considered to be toxigenic into an axenic culture of the toxic dinoflagellate from which they were isolated (Alexandrium lusitanicum) enhanced toxin production, whereas addition of bacteria in dialysis tubing (which prevented attachment) was inefficient [START_REF] Doucette | Algal bacterial interactions: can they determine the PSP-related toxicity of dinoflagellates?[END_REF]. Similarly, experiments conducted on a toxic non-axenic strain of the benthic dinoflagellate Ostreopsis lenticu- laris showed that marked increases in the proportion of non-toxic bacteria directly associated with the surfaces of algal cells were correlated with the development of peak dinoflagellate toxicity [START_REF] Tosteson | Associated bacterial flora, growth, and toxicity of cultured benthic dinoflagellates Ostreopsis lenticularis and Gamberdiscus toxicus[END_REF]).

In conclusion, we have shown by a direct approach that toxigenic bacteria are able to attach to Alexandrium tamarense in culture and that this mainly occurs when the culture enters stationary growth phase. However, attachment of those bacteria does not necessarily induce toxin production in A. tamarense. This does not support the hypothesis that toxic bacteria could simply control toxin production by attachment to particle surfaces such as eukaryotic organisms (Doucette et al. 1998). Attachment may still be crucial to induce and/or modulate toxin production by bacteria and/or dinoflagellates in the environment. Other factors such as nutrient availability [START_REF] Kodama | Possible links between bacteria and toxin production in algal blooms[END_REF][START_REF] Doucette | Characterization of bacteria associated with different isolates of Alexandrium tamarense[END_REF] or strain-to-strain specificity [START_REF] Imam | Specificity of marine microbial surface interactions[END_REF] may also be important. In this context, our study shows that the use of a combination of sophisticated microscopy techniques with nucleic acid probes should permit deep insights, at the cellular level, into specific physical interactions between phytoplankton and bacteria. Given the important ecological implications of those interactions, for example on population dynamics or toxin production, such studies are clearly justified.

  KEY WORDS: Attached bacteria • Dinoflagellate • Alexandrium • Bacteria-phytoplankton interactions • In situ hybridization • Tyramide signal amplification • Confocal micoscopy Resale or republication not permitted without written consent of the publisher

  -AMAC-0137-a-A-18 Alteromonas genus T G T T A T C C C C C T C G C A A A Brinkmeyer et al. (2000) ROSEO536R S-*-ROSEO-0536-a-A-18 Roseobacter clade C A A C G C T A A C C C C C T C C G Brinkmeyer et al. (2000) ROSEOC536R S-*-ROSEOC-0536-a-A-18 Competitor for ROSEO536R C A A C G C T A T C C C C C T C C G Brinkmeyer et al. (2000) EUB 338R Eubacteria G C T G C C T C C C G T A G G A G T Amann et al. (1990)*Oligonucleotide Probe Database(Alm et al. 1996)

Fig. 2 .

 2 Fig. 2. (A) Changes in Alexandrium tamarense cell densities (D, in culture together with the bacterial strain 253-11; m, in culture together with the bacterial strain 407-2; j, in culture together with both the bacterial strains 253-11 and 407-2; s, in the control axenic culture). Changes in bacterial cell densities: (B) free-living bacterial strain 253-11, respectively, with (D) and without (s) dinoflagellates; (C) free-living bacterial strain 407-2, respectively, with (m) or without (n) dinoflagellates; (D) both bacterial strains 253-11 and 407-2, with or without dinoflagellates (same symbols as for B and C)

Fig. 3 .

 3 Fig. 3. Confocal laser scanning microscopy images obtained by the superposition of twenty 0.7 µm thick optical sections taken through Alexandrium tamarense and its associated microflora. The samples shown were collected in the culture containing both the Alteromonas (407-2) and Roseobacter (253-11) bacterial strains during lag (Day 4; A,D), exponential (Day 13; B,E) and stationary (Day 26; C,F) phases of dinoflagellates growth. Hybridizations were performed, respectively, with the probes AMAC137R to detect specifically the bacterial strain 407-2 (A,B,C) and ROSEO536R to detect specifically the bacterial strain 253-11 (D,E,F). Detection used tyramide-fluorescein. Dinoflagellates autofluorescence (e.g. open arrow in F) is very faint, whereas bacteria are very brightly stained (e.g. solid arrow in F). Scale bars = 10 µm

  Fig. 4. Changes in the number of free and attached bacteria per dinoflagellate cell. For attached bacteria, each symbol represents a data point for a single dinoflagellate cell and data obtained for each dinoflagellate analyzed were plotted. Experimental culture containing (A) dinoflagellates and Roseobacter strain 253-11: free (d) and attached (s) 253-11; (B) dinoflagellates and Alteromonas strain 407-2: free (m) and (n) attached 407-2; (C) dinoflagellates and both strains 407-2 and 253-11: free (d) and attached (s) 253-11 and free (m) and (n) attached 407-2

251 Table 1 .

 2511 Oligonucleotide probes used in this study. All oligonucleotide probes are targeted to the 16S rRNA molecules

Acknowledgements. We thank Susan Gallacher for providing us with the bacterial strains, Jean-François Lennon for assistance with CLSM and Florence Le Gall for assistance with dinoflagellate cultures. This study was supported by the European Grant FAIR No. CT96-1558.