Eva Izquierdo

Adrien Robin

Sean Keuleyan

Nicolas Lequeux

Emmanuel Lhuillier

Sandrine Ithurria
email: sandrine.ithurria@espci.fr

Strongly Confined HgTe 2D Nanoplatelets as Narrow Near-Infrared Emitters

Keywords: HgTe, nanoplatelets, near infrared, cation exchange, 2D

come

Introduction

The recent development of 2D semiconductor nanoplatelets (NPLs) 1 and quantum belts 2,3 has led to exceptional control of the optical features of cadmium chalcogenide compounds 4,5 due to the lack of inhomogeneous broadening (i.e., the ensemble spectrum is identical to the single particle emission). Their photoluminescence (PL) linewidths can be as narrow as 7 nm for an emission at 500 nm. Fine control of the thickness (the confined dimension) affords tuning of the emission throughout the visible range 5,6 . The bulk band gaps of cadmium chalcogenides prevent their use in the near infrared (IR). Therefore, narrower band gap materials need to be considered to cover this range of wavelengths. 2D colloidal lead chalcogenides have been grown using an oriented attachment of nanocrystals. 7,8 The non-uniform size of the nanocrystals, however, leads to an inhomogeneity in the NPL thickness. For the infrared range, great progress has been made with mercury chalcogenide colloidal quantum dots (CQDs) [9][10][11] . Nevertheless, they still suffer from a limited control of their size and shape, which leads to a broadening of their optical features. In the near IR, copper indium sulfide (CIS) and PbS have received the most attention. In CIS nanocrystals, the emission relies on trap states [12][13][14] , which are dependent on the stoichiometry, and results in extremely broad luminescence features (full width at half maximum (fwhm) ≈ 100 nm). Lead chalcogenide CQDs can be prepared with size polydispersity under 5%. Nonetheless, their PL linewidth remains relatively broad, especially for small particles emitting near 800 nm. 15 This appears to result from homogeneous broadening, 16 which leaves very little room for improvement of the synthesis. Alternative materials with better-controlled absorption and emission in the near IR are of the utmost interest.

Cation exchange [17][18][19][20] provides a way to prepare materials which cannot be directly synthesized. The process is now well established and is the most typical method used to obtain core-shell heterostructures on lead chalcogenides 21,22 . The method has also been applied to 2D NPLs by Bouet et al 23 to obtain zinc and lead based NPLs from cadmium based NPLs. However, the cation exchange fails to preserve the 2D shape of the thinnest NPLs.

Kuno and colleagues have shown cation exchange on the thinnest CdSe NPLs with copper and silver, but no further exchanges were performed 24 . Using cation exchange to prepare mercury chalcogenides is actually much more difficult because of the softness of the final material, which easily results in shape reconstruction. Nevertheless, mercury cation exchange has already been reported on CdTe CQDs to grow (HgCd)Te alloys [25][26][27][28][29] , and on CdSe wires and magic-sized clusters to give rise to (HgCd)Se 30,31 .

In this paper we describe a new procedure for completing mercury cation exchange to form HgTe and HgSe 2D NPLs. We use a bulky Hg precursor to slow down the reaction and obtain HgTe NPLs with exceptionally narrow near IR optical features. We attribute this to the atomically smooth 2D particles, which exhibit exceptionally high quantum confinement energies (≈ 1.4 eV). Similarly, the method is applied to obtain HgSe NPLs, which also present optical absorption in the near IR (≈ 800 nm). The integration of the HgTe NPLs into field effect transistors is also investigated and shows a switch from p-type CdTe to n-type HgTe NPLs. This work is paving the way for the development of a new generation of CQD based optoelectronic devices with better controlled optical features in the near IR, and this work might also find some applications in the design of colloidal topological insulators 32 .

Results and discussion

Cation exchange on CdTe with Hg 2+ to form narrow band gap HgCdTe has been proposed as a chemical path to push the absorption from the visible range to the infrared 25 . However, most of the reported methods do not lead to a complete exchange, which (i) limits the red shift of the optical feature or (ii) leads to severe inhomogeneous broadening due to the spatially inhomogeneous cation exchange 29 . To conduct a mercury cation exchange on CdTe NPLs, while preserving their 2D shape, we had to considerably slow down the reaction. Usually, copper and silver cation exchanges are performed in a homogeneous medium containing nanoparticles and a copper or silver salt. The nanoparticles are typically suspended in toluene and the salt is dissolved in methanol 18,33 . A mixture of the two solutions leads to a quick cation exchange. In these conditions, there is no control of the speed of reaction unless the salt is introduced by slow addition 24 . Using this method with a mercury salt leads to a fast exchange and a dissolution of the NPLs. This contrasts with the same reaction conducted with spherical nanocrystals, which leads to an alloy and heterostructures 34 . We therefore used bulky precursors to slow down the reaction (see Figure S1 in Supp. Info. for less bulky precursors).

3 monolayers (ML) thick CdTe NPLs (4 planes of cadmium and 3 planes of tellurium) were synthesized according to the procedure by Pedetti et al. 35 (see Figure S2 in Supp. Info.).

Despite their symmetric zinc blende structure, the CdTe NPLs are strongly anisotropic, with a thickness of 1.1 nm and a lateral dimension greater than 100 nm (see Figure S3). When the solution of NPLs was mixed with Hg(acetate)2 in oleylamine, a cation exchange occurred, with a typical time scale of 1 h. The exchange process was monitored by optical spectroscopy (see Figure 1a). The initial CdTe NPLs show two characteristic optical features in the visible spectrum, at 450 and 501 nm. These are attributed to the light hole and heavy hole to conduction band transitions 5 . During the cation exchange, these two peaks first disappear and slowly recover with a large red-shift in the near IR range at 735 and 882 nm. The Hg 2+ cation exchange with 3 ML CdSe NPLs occurs similarly, but at a lower rate than for the CdTe. The CdSe absorption features shift from 434 and 462 nm to 719 and 766 nm, as shown in Figure 1g. TEM images of HgTe NPLs in Figure 1c and 1d reveal that the NPL morphology and the zinc blende crystal structure are preserved after the cation exchange procedure (see Figure S3 and S4 in Supp. Info. for x-ray diffraction pattern and size distribution). The final NPLs are also observed to be homogeneous in TEM contrast. After several hours, the reaction saturates and no further shift of the exciton peak is observed, as shown in Figure 1a. We attribute this observation to the complete cation exchange to cadmium-free HgTe NPLs. In these NPLs, the confinement is limited to 1 dimension, the thickness of the NPLs, as the lateral dimensions are larger than the 40 nm, the Bohr radius in HgTe 36 . This finding is further confirmed by energy dispersive X ray spectroscopy (EDX), which does not show the presence of cadmium at the end of the reaction (see Figure S5 and S6 and Table S1 in Supp. Info.). Finally, we use Raman spectroscopy to confirm the complete transformation of the material (see Figure S9 in Supp.

Info.). Initially CdTe NPLs present one main feature at 166 cm -1 , which is attributed to the LO phonon mode of CdTe 37,38 . After cation exchange this mode completely disappears and we only observe peaks assigned to HgTe [39][40][41] at 94 cm -1 (ETO(Te) mode ), 123 cm -1 (A1(Te) mode

) and 142 cm -1 (ETO(Te) mode ) 31 .

In the preparation of HgSe, the shape of the NPLs is also conserved and accompanied by an unfolding of the NPLs 42,43 . This may come from a change of the surface ligands from oleic acid to oleylamine and additionally from the softness of the HgSe material. EDX analysis reveals a full exchange of the Cd to Hg (see Figure S7, S8 and Table S2 in Supp. Info.).

Absorption at short wavelength gives information on the amount and the composition of materials in solution 44 . Here, we can take the quantity of materials as constant because the particle shape and size does not change during the reaction. Figure 1b gives the optical density at 400 nm as a function of time during the cation exchange reaction. We observe a large increase in the optical density over the first ten minutes followed by a plateau. During this time, the CdTe absorption peaks also disappear. This suggests that the cation exchange happens in the first ten minutes. Annealing of the new HgTe NPLs and reorganization of the ligands takes a longer time, as indicated by the appearance and then narrowing of the absorption structure. In addition, the photoluminescence spectra in Figure S4 in the Supplementary Information show a shift of the emission maximum from 865 to 890 nm, with a reduction of the fwhm and an increase of its intensity. On the other hand, the Hg 2+ cation exchange performed on the CdSe NPLs takes much longer. After 2 hours of reaction, some CdSe NPLs remain in solution. The complete transition from CdSe to HgSe NPLs happens in approximately 3 hours.

The cation exchange is explained by Pearson's theory of hard and soft acids and bases (HSAB), in which soft acids will preferentially bind to soft bases, and hard acids to hard bases. Hg 2+ is a soft acid compared to Cd 2+ , and by choosing the right ligands and solvents, it is then possible to favor the cation exchange 20 . In general, carboxylic acids and amines can be considered hard bases. Thus, when a soft acid such as the Hg 2+ complexed with a hard base is introduced to a solution of CdTe nanocrystals, a cation exchange is favored. The Hg 2+ salt is dissolved in long chain amines, which are then miscible with a solution of nanocrystals in a non-polar solvent, such as hexane or octadecene, and form a homogeneous solution.

Hg(Acetate)2 was chosen as the Hg 2+ salt, as the acetate is a harder base than other anions such as Cl -in HgCl2. Cation exchange did occur when HgCl2 was used, but at a much slower rate (see Figure S10 in Supp. Info.).

Figure 2: (a.) Absorbance (dashed line) and photoluminescence (solid line) spectra of oleic acid capped CdTe NPLs (green), oleic acid capped HgTe NPL (OA -orange) and oleylamine capped HgTe NPLs (OLAm -red).(b.) Photoluminescence decay curve and TEM image (inset) of 3 ML oleic acid capped HgTe NPLs. All measurements are made in solution at room temperature.

In the HgTe NPLs, the confinement is so strong that we can use surface chemistry as a way to tune the optical features, while keeping the HgTe thickness unchanged. Using trioctylamine in place of oleylamine, we observe a blueshift of the transitions (see Figure 2a and Figure S11 in Supp. Info.). Trioctylamine is, however, not sufficient to stabilize the HgTe NPLs in solution.

As a result, additional ligands are necessary to recover a stable colloidal suspension after the cation exchange. The addition of a carboxylic acid, an amine or a thiol leads to a suspension of NPLs and the choice of ligand affects the absorption and emission spectra. With oleic acid, there is no shift of the absorption spectra compared to the NPLs in solution along with Hg(acetate)2 in trioctylamine. The fact that oleic acid and the acetate anion both interact with the NPLs through a carboxylate, combined with the lack of change in the absorbance spectrum after the addition of oleic acid, suggests that during the cation exchange, the NPLs are capped by acetate. Small angle diffraction shows a similar interparticle distance for the CdTe and HgTe NPLs. The difference is less than 1 Å, which is smaller than the 3.2 Å thickness of a ML (See Figure S3 in Supp. Info.). Addition of oleylamine leads to a red shift of the absorption spectrum to ~880 nm. Addition of thiols leads to a further red shift of ≈ 250 nm (see Figure 3). The strong mercury-thiol interaction favors the delocalization of the wavefunction into the ligands. Changes in the surface chemistry when oleic acid, oleylamine, and dodecanethiol are each added are also supported by IR spectroscopy (see Figure S12 in Supp. Info.).

Small angle diffraction shows a decrease in the interparticle spacing of approximately 1 nm when oleylamine is added after oleic acid. We attribute this change to a difference in packing density and interdigitation of the two ligands.

One of the most striking features of the HgTe NPLs is that their optical features are exceptionally narrow. The PL linewidth for ensemble measurement is 57 meV (less than 40 nm) for an emission peak at 887 nm (1.40 eV) for oleylamine capped NPLs (see Figure 2a).

For oleic acid capped NPLs, the PL signal is maximum at 828 nm (1.50 eV). Monodisperse PbS CQDs with similar peak emission wavelengths have a fwhm at least twice those observed here for HgTe NPLs. 15 In fact, the emission linewidth of a single PbS CQD is even broader (100 ± 30 meV). 16 This observation is even more striking because of the semimetal nature of bulk HgTe (i.e., zero bulk optical band gap). This means that the band gap energy is entirely a result of quantum confinement 44 . The fact that the fwhm remains narrow after the cation exchange reflects that no roughness is introduced in the confined direction.

The HgTe NPLs exhibit an emission quantum yield on the order of 10% with an average lifetime of 50 ns (Figure 2b and Figure S13 in Supp. Info). This lifetime is also much shorter than for lead chalcogenides [45][46][47] and CIS 13 CQDs, which have PL lifetimes in the µs range. This observation is consistent with the previous observation that for CdSe NPLs the PL emission lifetime was found to be shorter than for spherical particles of the same composition and band gap energy. 5 This cation exchange may also be applied to 2 ML CdTe NPLs, leading to HgTe NPLs with optical features in the visible and the near IR (Figure 3a). Again a dependence of the absorption peak wavelengths on the ligands is observed. With 2 ML HgTe NPLs, capped with oleic acid, the first excitation absorption peak appears at 568 nm. However, the oleylamine capping leads to a peak at 615 nm. Ligand exchange from oleylamine to dodecanethiol leads to a large red shift of more than 170 nm (Figure 3a). Two ML dodecanethiol capped HgTe NPLs have the first absorption peak at the same wavelength as 3 ML oleic acid capped HgTe NPLs. Sulfide has a strong affinity for mercury and the addition of sulfide on both surfaces of the 2 ML HgTe NPLs is apparently similar to the addition of a half ML on both sides. Subsequent ligand exchanges from oleic acid to oleylamine, then from oleylamine to dodecanethiol can be monitored by absorption spectroscopy, as shown for 3 ML HgTe NPLs in Figure 3b. Finally, we investigate how the transport properties of the NPLs are affected by the cation exchange. To efficiently gate the system, we use ion gel gating, which has several advantages such as (i) large tunability of the carrier density 48 , (ii) in-air measurements and (iii) low bias (< 3 V) operation 49 . We first conduct a solution phase ligand exchange to promote S 2- capping 50 to increase the inter-NPL coupling. Then, we use LiClO4 in polyethylene glycol as the ion gel electrolyte (see part 8 of Supp. Info. for details). The initial CdTe contains only ptype II-VI semiconductor nanocrystals. 49 After cation exchange, the HgTe NPLs behave as ntype semiconductors (see Figure 4 and Figure S14 in Supp. Info.) and the conductance is typically a factor of 50 higher than for a CdTe NPL film of similar thickness. In narrow band gap materials, such as HgTe, the carrier density n is thermally activated and therefore follows an Arrhenius behavior, 𝑛 ∝ 𝑒𝑥𝑝(-𝐸 𝑎 𝑘 𝑏 𝑇 ⁄ ). In intrinsic materials, the activation energy is half the band gap. During the cation exchange process, the band gap decreases by 800 meV, which is responsible for the observed rise in the conductance. For similar surface chemistry and gating, HgTe CQDs were previously observed to be ambipolar 49,51 . Here we use S 2-as the capping ligand, which tends to form an HgS surface layer. Moreover, HgS CQDs were recently reported to be stably n-doped. 52 We thus believe that the formation of this n-type surface layer is responsible for the observed switch in the majority carrier during the cation exchange.

Conclusions

We report the synthesis of 2D HgTe NPLs via a cation exchange procedure starting with CdTe NPLs and using a mercury amine complex. We show that the cation exchange is complete (i.e., the final material is Cd-free) and has exceptionally narrow near IR optical features. The fwhm can be as small as 57 meV for ensemble emission centered near 890 nm.

Because of the extreme confinement energy in this system, we show that surface chemistry can be used to tune the band gap, which reflects the partial delocalization of the wavefunction into the ligand shell. Finally, we build an electrolytic transistor and show that the conductivity switches from p-type in CdTe to n-type in HgTe.

In addition to the bright and fast near IR luminescence of this material, we believe these results are a promising first step to building 2D colloidal topological insulators 53,54 . In particular, theory predicts HgTe QD based honeycomb lattice structures would have a Dirac cone and non-trivial band gap, but this remains unconfirmed due to the difficulties in preparing such materials. 55

Figure 1 :

 1 Figure 1: Absorbance spectra of the CdTe→HgTe (a.) and CdSe→HgSe (g.) NPLs at different times during the cation exchange reaction. The dilution remains the same for all spectra, which are offset for clarity. (b.) Absorbance at 400 nm as a function of time from Figure 1a. (c.) and (d.) Transmission electron microscopy (TEM) images of the final 3 ML HgTe NPLs (e.) TEM image of 3 ML HgSe NPLs (f.) TEM image of 3 ML CdSe NPLs.

Figure 3 :

 3 Figure 3: Absorption spectra of the oleic acid (orange), OLAm (red) and dodecanethiol (black) capped (a.) 2 ML and (b.) 3 ML HgTe NPLs. (c.) Absorption spectra during ligand exchange from oleate (yellow) to oleylamine (red) and then to dodecanethiol (black) on 3 ML HgTe NPLs. Intermediate species are in orange and in brown.

Figure 4 :

 4 Figure 4: Drain and gate current as a function of gate voltage for an electrolyte-gated thin film of CdTe (green) and HgTe (brown) NPLs. Measurements are made at room temperature. The gate bias is stepped in 1 mV increments. The drain-source bias is 0.5 V for CdTe and 10 mV for HgTe.

ACKNOWLEDGEMENTS

We thank Thomas Pons for fruitful discussions. We thank the SBPC (Réseau des Salles Blanches Paris Centre) for Raman Spectroscopy. We thank the Agence National de la Recherche for funding through grants Nanodose and H2DH. This work has been supported by the Region Ile-de-France in the framework of DIM Nano-K. This work was supported by French state funds managed by the ANR within the Investissements d'Avenir programme under reference ANR-11-IDEX-0004-02, and more specifically within the framework of the Cluster of Excellence MATISSE.

SUPPORTING INFORMATION AVAILABLE

Details of CdTe and CdSe NPLS synthesis; details of cation exchange with different mercury precursors; absorption and emission spectroscopies of HgTe NPLS; Energy Dispersive X-ray spectroscopy (EDS), Raman spectroscopy, X-ray diffraction patterns of NPLs; lifetime measurements of HgTe NPLs; Infrared spectroscopy of HgTe NPLS capped with different ligands; Ligand exchange and fabrication of field effect transistors. This information is available free of charge on the ACS Publications website http://pubs.acs.org


Dr Marc Bouvy
email: marc.bouvy@ird.fr

Patrice Got

Isabelle Domaizon

Marc Pagano

Christophe Leboulanger

Corinne Bouvier

Claire Carré

Cécile Roques

Christine Dupuy

Plankton communities of the five "Iles Eparses" (Southwestern Indian Ocean) considered to be pristine ecosystems

Keywords: microorganisms, distribution, enrichment experiment, nutrient limitation, islands, Mozambique Channel microorganisms, distribution, enrichment experiment, nutrient limitation, islands, 28 Mozambique Channel

Coral reef environments are generally recognized as being the most threatened of fragile marine ecosystems. They are highly susceptible to stress and disturbance, especially to anthropogenic pressure. However, it is extremely difficult to distinguish the effects of climate change from other forcing factors, mainly because it is difficult to study ecosystems that are isolated from human pressure. The five Iles Eparses (Scattered Islands) are located in the Western Indian Ocean (WIO) and form the 5th district of the French Southern and Antarctic Lands (TAAF). They are considered to be pristine ecosystems as they are occupied only by a small group of military personnel. This study described the plankton assemblages for the first time, by determining the abundances of microbial (virus, bacteria, heterotrophic protists and phytoplankton) and metazooplankton communities in various lagoon and ocean sites around each island. The Europa lagoon has extensive, productive mangrove forests. Their particular structure and functioning, with the highest nutrient concentrations (nitrogen forms, dissolved organic carbon), explain bacterial production and growth rates that are higher than those reported for the other islands. The marine ecosystem of Tromelin, which lies outside the Mozambique Channel, had the lowest biological productivity, with the lowest nutrient concentrations and bacterial growth rates. Comparison with a sampling station in Mayotte lagoon, considered to be the reference for an anthropized island, showed that the patterns of microbial assemblages in the Iles Eparses were different from those found in Mayotte lagoon.

H ighlights:

-The microbial and zooplankton assemblages in the five Iles Eparses were studied -Bacterial growth rates and grazing rates by HNF were determined for various sites -Bacterial production and growth rates were higher in lagoons with mangroves Highlights (for review)

Introduction 55

The conservation and sustainable use of marine resources are priority goals in a growing 56 number of national and international policy agendas [START_REF] Spalding | 792 Marine ecoregions of the world: a bioregionalization of coastal and shelf areas[END_REF]. Biogeographic 57 classification is essential for developing ecologically representative systems in protected areas 58 to protect a complete range of biodiversity genes, species, populations and communities 59 throughout the trophic chain. A recent report analyzed the effects of anthropogenic activities 60 in the oceans worldwide, focusing on stressors that can be evaluated at global scale (Halpern 61 et al., 2008). According to these authors, all the oceanic ecosystems analyzed (coral reefs, 62 mangroves, seagrass meadows, seamounts, rocky reefs, soft shallow areas, continental shelf 63 areas, slope areas, pelagic waters and the deep sea) may be affected by anthropogenic 64 activities to varying degrees. Special attention should be paid to the smallest living marine 65 organisms as marine microbes provide essential ecosystems functions and are threatened by 66 pollution [START_REF] Nogales | Anthropogenic 727 perturbations in marine microbial communities[END_REF]. It is now recognized that marine microbial components are 67 associated with the channeling of a large proportion of carbon fluxes through the microbial 68 food web [START_REF] Estrada | 637 The Mediterranean Sea: its history and present challenges[END_REF]. Where there is eutrophication or pollution pressure, the 69 composition and structure of microbial communities are basic indicators of the ecosystem 70 status, providing information on the type of factors regulating the dynamics of these 71 communities [START_REF] Fuhrman | Marine viruses and their biogeochemical and ecological effects[END_REF][START_REF] Suttle | Viruses in the sea[END_REF]. 72

Coastal marine habitats at the land-sea interface are threatened by human activities at sea and 73 on shore, and are, therefore, among the most heavily degraded (Costanza et al., 1997). Coral 74 reef environments are generally recognized as being among the most threatened of the fragile 75 marine ecosystems, and are highly susceptible to stress and disturbance, especially to 76 anthropogenic pressure [START_REF] Mellin | Natural and anthropogenic influences on the diversity structure of reef fish 712 communities in the Tuamotu Archipelago (French Polynesia)[END_REF] and climate change [START_REF] Miller | Coral disease following massive bleaching in 2005 causes 60 % decline in 720 coral cover on reef in the US Virgin Islands[END_REF]. Studies 77 of coral reef systems to understand the causes, effects and consequences of direct 78 anthropogenic pressure are generally conducted in anthropized zones. However, it is 79 extremely difficult to distinguish the effects of climate change from other forcing factors, 80 mainly because it is difficult to study ecosystems that are isolated from human pressure. 81 82

The five Iles Eparses (Scattered Islands) are located in the Western Indian Ocean (WIO) and 83 form the 5 th district of the French Southern and Antarctic Lands (TAAF). Several studies of 84 these remote ecosystems were performed recently, which established ecological baselines for 85 pristine coral reef systems. However, most previous studies have dealt with reef fish 86 inventories [START_REF] Chabanet | Coral reef fish communities of Mayotte (western Indian Ocean) two years 610 after the impact of the 1998 bleaching event[END_REF]Quod et al., 2004;Chabanet and Durville, 2005) following the 87 extensive coral bleaching event in 1998, and only a few studies have considered the 88 biodiversity, structure and functioning of the marine microbial communities in these islands 89 (for example, the nutrient status was studied by [START_REF] Riaux-Gobin | 756 Nutrient status in coral reefs of the Îles Eparses (Scattered Islands): comparison to nearby 757 reefs subject to higher anthropogenic influences (Mozambique Channel and Mascarenes, 758 Indian Ocean)[END_REF]. The structure of 90 the plankton foodweb in these ecosystems considered to be pristine ecosystems, must be 91 understood in order to protect them from future human threats. The lack of "reference 92 environments" raises serious problems: (1) limited knowledge about the diversity and 93 functions of microbial communities from unaffected coastal sites, (2) lack of knowledge on 94 the sensitivity of reference communities (e.g. bacteria, phyto-and zooplankton) to local 95 environmental forcing (nutrient inputs, organic and mineral pollution, etc). 96

As part of the international 2011-a survey was carried out from 97

April 5 to 23, 2011 by several scientific teams aboard the N/O Marion Dufresne II, the TAAF 98 supply vessel, to collect data on many aspects of terrestrial and marine biodiversity in the Iles 99

Eparses. This study focused on plankton microorganisms, based on samples taken from 100 various lagoon and ocean stations in each of the five islands (see Table 1), as well as from a 101 station in Mayotte lagoon, which is considered be a typical anthropized island in the 102 geographic zone. 103

The overall aim of the project was to determine whether there were differences between such 104 105 compartments owing to environmental conditions in each island ecosystem. This preliminary 106 study characterized the plankton assemblages by (1) determining the abundances of microbial 107 communities (virus, bacteria, protists and phytoplankton) and metazooplankton in the lagoon 108 and ocean sites around each island, and (2) exploring relationships between environmental 109 conditions and biological components at regional scale. The Iles Eparses (Scattered Islands) are small coral reef islands located in the Indian Ocean 116 close to Madagascar, and became the 5th district of the French Southern and Antarctic Lands 117 (TAAF) in February 2007. Four of these islands lie in the Mozambique Channel, west of 118 Madagascar (from south to north: Europa, Bassas da India, Juan de Nova and Glorieuses) and 119 the fifth (Tromelin island) lies about 450 kilometers east of Madagascar (Figure 1). The 120 sas da India have 121 a meteorological station. Each island except Bassas da India, has an airstrip more than 122 1,000 m long for use by small groups of military personnel. 123

Samples were taken during a single period ( S 124 from 16 stations. There was one ocean station 125 and two or three stations (depending on the lagoon area) for each island. Samples were taken 126 at a depth of 0.5 m using a Niskin bottle, transferred immediately to acid-washed 127 polyethylene bottles and kept in the dark at in situ temperatures until processed in the 128 laboratory on board within 2 hours. A total of 25 parameters were measured to characterize 129 the physical, chemical and biological environment of the water column. Metazooplankton was 130 collected using a WP2 plankton net (80 µm mesh-size) equipped with a Hydrodata flowmeter. 131

The net was hauled vertically from near bottom to surface at ocean and deep lagoon stations, 132

and horizontally (at a depth of 0.5 m) at shallow stations. After collection, metazooplankton 133 samples were rapidly preserved in a 4% buffered formaldehyde solution. 134 135

Physical and chemical parameters 136

At each sampling station, a CTD profiler (YSI 600 XLM) was deployed to record 137 temperature, depth, pH and dissolved oxygen profiles. Dissolved organic carbon (DOC) 138 analyses were performed on 30 ml subsamples collected in pre-combusted (450°C overnight) 139 glass vials, preserved with 35 µl of 85% phosphoric acid. Samples were stored in the dark 140 until analysis using a Shimadzu TOC VCPH analyzer [START_REF] Rochelle-Newall | Phytoplankton-764 bacterioplankton coupling in a subtropical South Pacfic coral reef lagoon[END_REF]. 141

Chlorophyll concentrations were determined by fluorometry after filtration onto Whatman 142 GF/F fiberglass filters and direct extraction using methanol [START_REF] Yentsch | A method for the determination of phytoplankton, 843 chlorophyll and phaeophytin by fluorescence[END_REF]. 143

Samples for measuring dissolved nutrients (SiO 4, NH 4 -N, NO 3 -N, NO 2 -N, PO 4 -P) were 144 145 Strickland and Parsons (1972). Sampled water was filtered through a pre-combusted GF/F 146 Whatman filters to determine particulate organic nitrogen (PON) and particulate organic 147 carbon (POC), using a nitrogen carbon analyzer after acid decarbonation. 148 149

Biological parameters 150

For bacterial and viral abundances, samples were fixed with prefiltered (0.02 µm) buffered 151 formaldehyde (2% final concentration), stored in liquid nitrogen (-196°C) and analyzed on 152 return to Montpellier University. Total bacterial cells (TB) were enumerated by flow 153 cytometry using the method described by [START_REF] Marie | Enumeration and cell cycle analysis of 704 natural populations of marine picoplankton by flow cytometry using the nucleic acid stain 705 SYBR Green I[END_REF]. One milliliter of fixed 154 subsamples was incubated with SYBR Green I (Molecular Probes, Eugene, OR, USA) at a 155 final concentration of 1/375 for 15 min at 4°C in the dark. For each subsample, three replicate 156 counts were performed using a FACSCalibur flow cytometer (Becton Dickinson, San Jose, 157 CA, USA) equipped with an air-cooled argon laser (488 nm, 15 mW). Stained bacterial cells, 158 excited at 488 nm, were enumerated according to their right angle light scatter (RALS) and 159 green fluorescence (FL1) was measured using a 530/30 nm filter. These cell parameters were 160 recorded on a four-decade logarithmic scale mapped onto 1024 channels. Fluorescent beads 161

(1-2 µm, Polysciences, Warrington, PA, USA) were systematically added to each sample. 162

Standardized RALS and FL1 values (cell RALS and FL1 divided by 1 µm beads RALS and 163 FL1 respectively) were used to estimate the relative size and nucleic acid content of bacterial 164 cells respectively [START_REF] Troussellier | Flow cytometric discrimination 813 of bacterial populations in seawater based on SYTO 13 staining of nucleic acids[END_REF]. List-mode files were analyzed using BD 165

Cellquest Pro software. The number of virus-like particles (VLPs) contained in triplicates of 166 50-200 µl samples was determined after particles had been retained on 0.02 µm pore-size 167 membranes (Anodisc) and stained with SYBR Gold (Patel et al., 2007). On each slide, VLPs 168 were counted in 15-20 fields using an epifluorescence microscope, with final numbers giving 169 a precision of <10% at 95% confidence limit. Culturable heterotrophic bacteria (CB) were 170 counted by plating 100μL of serial dilutions of each sample on marine agar plates (Marine 171

Agar;

in situ room temperature (close to 27C°), 172 colony forming units (CFU) were counted (it was established that the counts did not increase 173 for longer incubation periods). 174 Bacterial Production (BP) was estimated from the DNA synthesis rates measured by (3H-175 methyl) thymidine (3H-TdR) incorporation using the microcentrifuge method (Smith and 176 Azam, 1992). On board, a sample aliquot (1.4 ml) was added to a sterile polystyrene snap cap 177 tube containing a final saturating concentration of 20 nM of 3 H-TdR (specific activity 53 Ci 178 mmol -1 , Amersham). Triplicate live samples and a control were run for each assay. Killed 179 controls were prepared by adding 70 µl of 100% of trichloroacetic acid (TCA) 15 min before 180 the addition of 3 H-TdR. Bacterial growth was measured in the dark at in situ temperature for a 181 short incubation time (no longer than 1 h). Incorporation was terminated by adding 70 µl of 182 100% TCA. Samples were stored for at least 2 h at 4°C and then centrifuged at 14000 g for 183 15 min. The precipitates were rinsed three times with 5% TCA and once with 70% ethanol 184 and were resuspended in 1.5 ml of liquid scintillation cocktail (Ultima Gold LLT, Perkin 185 Elmer) before determining the radioactivity using a liquid scintillation counter (Beckman LS 186

6500). 187

Pico and nano-phytoplankton samples were fixed with paraformaldehyde (2% final 188 concentration), and counted using a Facs Aria Flow cytometer (Becton Dickinson, San Jose, 189 CA, USA) equipped with an HeNe air-cooled laser (633 nm, 20 mW). Cells excited at 633 nm 190 were detected and enumerated according to their FALS and RALS properties and their orange 191 fluorescence (576/26 nm) and red fluorescence (> 650 nm) from phycoerythrin and 192 chlorophyll pigments, respectively. Fluorescent beads (1-2µm for pico and 2-6-10-20µm for 193 nano) were systematically added to each sample. List-mode files were analyzed using BD 194 FACSDiva software. To enumerate heterotrophic (HNF) and pigmented nanoflagellates 195 (PNF), water samples were fixed with paraformaldehyde (2% final concentration) and stored 196 at 4°C for 24 h. Twenty-five millilitres of preserved water samples were then stained with 197 DAPI (final concentration, 15 mg ml -1 ) for 15 min, filtered onto a black Nuclepore filter (0.8 198 mm pore size), stored at -20°C, and counted using an epifluorescence microscope (Nikon 199 Eclipse TE200) with UV excitation (Boenigk et al., 2004, modified). Pigmented 200 nanoflagellates were discriminated by blue and green fluorescence under UV excitation. 201

For microzooplankton (ciliates) and microphytoplankton (dinoflagellates, diatoms) 202 abundances, water samples (500 mL) were concentrated by gravity filtration onto a 5µm 203 porosity filter (polycarbonate membrane, Nuclepore), and fixed with alcalin lugol iodine (2% 204 final concentration). The remaining 50 mL was then stored at 4°C in the dark until analysis in 205 the laboratory. Microorganisms were enumerated in an Utermöhl settling chamber (Hydro-206 Bios combined plate chamber) using a reverse microscope (Zeiss Axiovert, magnification 207 x400) for ciliates, and an inverted microscope (Olympus IX70), equipped with digital camera 208 (Moticam MoticamPro) for phytoplankton. 209

Metazooplankton were counted under a stereomicroscope (Olympus SZX200), using a 210 Dolfuss counting chamber and were identified as described by [START_REF] Tregouboff | Manuel de planctonologie méditerranéenne[END_REF]Rose (1957), 211 and[START_REF] Conway | 619 Guide to the coastal and surface zooplankton of the South-Western Indian Ocean[END_REF]. The abundance of the various taxa was discriminated by group, 212 genus or species. 213 214 2.4. Bacterial growth rates and grazing rates by HN F 215 Water samples were taken at 0.5 m depth at the various stations for each island. Two bioassay 216 series were performed to compare the responses of the bacterial community to inorganic 217 All Whirlpaks were incubated for 24 hours on board in a tank filled with a seawater circuit to 224 ensure that the water temperature remained fairly constant during the experiments. 225

Subsamples were removed at time zero (t0) and after 24 h (t24) incubation to enumerate the 226 bacterial abundance (see above). The bacterioplankton growth rates in each triplicate were 227 calcula 24 lnN0)/t, where t was the incubation time and N24 and N0 were the 228 bacterial concentrations at the end and at the beginning of incubation. The grazing rates were 229 calculated as the difference between net growth rate 230 231 232

Data processing 233

The differences between sites for all parameters were tested using the non parametric Mann-234 Whitney U-test. Differences were considered as significant at p<0.05 (Sigma Stat version 3.5). 235

The relationships between environmental parameters and biological variables were studied 236 using multivariate analysis, with data from the 16 sampling stations. Principal component 237 analysis (centered PCA) was performed for each of two data sets: an Environmental System 238 based on 8 parameters, and a Biological System based on 12 variables. The results of the two 239 analyses were combined by co-inertia analysis, which allows two tables with a different 240 number of variables to be compared (Dolédec and Chessel, 1994). Two sets of factor scores 241 were obtained for the sampling points: scores of the rows seen by the environmental 242 parameters , and scores of the rows seen by the biological variables . The significance of the 243 co-inertia analysis was tested after randomizing the results, by a repeated random permutation 244 of the rows of both tables, and a comparison of these results obtained with a standard PCA. 245

The resulting distribution of 2000 replicated matches of the two arrays gave an estimated 246 significance of p < 0.001 for the difference from the original value. Cluster classification of 247 observation scores from the first factorial planes of the co-248 aggregation) was performed to classify the 16 stations. All this data processing was 249 performed using ADE-4 software [START_REF] Thioulouse | ADE 4 : a multivariate analysis 804 and graphical display software[END_REF]. 250 251

Results

252

Environmental parameters 253

The surface temperature ranged from 26.9°C (Europa lagoon) to 30.1°C (Juan de Nova 254 lagoon) with mean values similar for the lagoon and ocean stations (Table 1). There was little 255 variation in salinity, with average values of 35.34 and 35.05, for lagoon and ocean sites. There 256 was little variation in pH with values ranging from 7.95 to 8.02 (average of 7.98 and 8.01 in 257 lagoon and ocean sites, respectively). The dissolved oxygen concentrations ranged between 258 4.27 mg l -1 at Glorieuses lagoon (G3) to 9.43 mg l -1 at Europa lagoon (E1), except for Bassas 259 da India lagoon where a high concentration was reported (B1: 10.96 mg l -1 ) (Table 1). 260

Low dissolved nutrient concentrations were found, for all nutrients, without significant 261 differences between the sites (p>0.05; Table 2). Dissolved phosphorus concentrations were 262 below the detectable limits (threshold 0.010 µM). Silicate concentrations did not vary 263 significantly (p>0.05) between the stations, with an average of 2.41 and 1.90 µM for ocean 264 and lagoon sites, respectively. Ammonium concentrations accounted for the larger 265 contribution of nitrogen forms in ocean sites with an average of 0.16 µM (Table 2). The 266 highest concentrations of nitrite and nitrate were reported in the Europa lagoon (0.19 µM). 267

The mean values of DOC concentrations averaged 103 µM and 96.4 µM in ocean and lagoon 268 sites respectively, with the highest concentrations for the Europa stations. Particulate organic 269 nitrogen averaged 24.1 and 34.5 µg l -1 in ocean and lagoon sites, respectively, while 270 particulate organic carbon averaged 171.5 and 182.8 µg l -1 . There were higher particulate 271 organic nitrogen forms in the Europa stations, especially in the lagoon. The particulate 272 organic C/N averaged 7.3 and 5.4 in ocean and lagoon sites, respectively, with the highest 273 values in Europa and Bassas da India ocean sites. 274 275

Biological variables 276

The culturable bacteria (CB) density varied from 330 to 60,000 CFU ml -1 (colony forming 277 unit) with lower values reported in Europa sites (Table 3). The bacterial abundance averages 278 were similar (9 x 10 5 ml -1 for ocean sites and 12 x 10 5 ml -1 for lagoon sites). The highest 279 abundances of total bacteria (TB) were found in Europa lagoon, and in the Mayotte lagoon 280 which was considered to be a polluted site (not part of the Iles Eparses). The highest values 281 for bacterial production, (BP) were found in the Europa lagoon (close to 400 pmole l -1 h -1 ) 282 whereas the mean of the ocean sites was only 27 pmole l -1 h -1 . Virus like particle (VLP) 283 concentrations were significantly different (mean of 2.86 x 10 7 ml -1 for the ocean sites and a 284 mean of 15.46 x 10 7 ml -1 for the lagoon sites (t-test; p<0.006), with the highest values 285 reported in Juan de Nova lagoon (stations J1, J2 and J3). 286 and lagoon sites (mean of 21 x 10 3 ml -1 and 23 x 10 3 ml -1 , respectively) whereas 290 picoeukaryotes abundances were significantly higher in lagoon sites, especially in the Europa 291 lagoon (t-test; p<0.05). There were very few nanophytoplankton cells (Nano) and pigmented 292 nanoflagellates (PNF), especially in ocean sites (mean of 1.06 and 1.60 x 10 2 ml -1 , 293 respectively). Microphytoplankton densities were higher in lagoon sites (mean of 11,715 cells 294 l -1 ) than in ocean sites (mean of 2,190 cells l -1 ) with the highest abundances found in the 295 Europa and Juan de Nova lagoon stations (Table 3). The mean chlorophyll-a concentrations, 296 considered here as a proxy of total phytoplankton biomass, were generally low and 297 statistically different (t-test; p<0.05) between the type of ecosystem (0.181 µg l -1 in ocean and 298 0.507 µg l -1 in lagoon sites). 299

The heterotrophic nanoflagellates (HNF) abundances were low and similar in both types of 300 site. The ciliate community was identified as two groups, with aloricate abundances higher 301 than loricate abundances, especially in lagoon sites (no loricate organisms were found in Juan 302 de Nova and Tromelin ocean sites) (data not shown). Ciliate abundances were very low for all 303 the stations (Table 3). Metazooplankton numbers varied from 607 (G1, Glorieuses lagoon) to 304 6008 ind.m -3 (Mayotte lagoon) with similar means in ocean and lagoon sites (1824 and 2583 305 ind.m -3 in ocean sites and lagoon sites, respectively). Copepods were the most abundant in all 306 sites (>75% abundance) except for Bassas de India (39%) where meroplankton larvae were 307 dominant (45%) (data not shown). 308 309

Bacterial growth rates and HN F grazing rates 310

The 24 h bioassay experiments were conducted for each island enumerating the bacteria for 311 each treatment (presence or absence of predators, with or without enrichment). The results 312 showed that the nanoflagellate abundance did not increase during incubation in the various 313 assays. Bacterial growth rates in the presence (gross rate; µ+g) or absence (net rate; µ) of 314 predators, and grazing rates of bacteria by predators (g) in the different nutrient conditions 315 (with or without added nutrients) are given in Table 4. With no added nutrients (control), 316 bacterial growth rates were higher for the Europa lagoon (E2: 4.25 d -1 ) and Bassas lagoon 317 (B1: 4.17 d -1 ), and values were lower for ocean sites (from 0.48 d -1 in GO to 2.73 d -1 in BO). 318

Grazing rates by predators were generally lower than growth rates (Figure 2) and net bacterial 319 growth rates (µ) in the absence of predators were greater than in the presence of predators 320 (µ+g) (Table 4). With enrichment, net growth rates (µ) were higher in both ocean and lagoon 321 sites than without enrichment. With the exception of the Tromelin ocean station (TO), growth 322 rates were clearly higher with enrichment but there was no close correspondence between 323 growth and grazing rates values (Figure 2). Net growth of the bacterial communities from 324 ocean stations (BO, EO, JO, GO) and Mayotte was clearly higher with enrichment and 325 absence of predators (values of µ close to 5 d -1 ) 326 327

Co-inertia analysis 328

The two PCAs on environmental and biological variables were performed on all data sets (16 329 stations; 8 environmental parameters; 12 biological variables). The first two eigenvalues of 330 the co-inertia analysis accounted for 90.9% of the total variability (Figures 3 and4), 331

indicating that the analysis focused on the first 2 axes. The values of the projected variables of 332 the Environmental and Biological tables on the axes (F1, F2) of the co-inertia analysis (Iner E 333 and Iner B) were close to the values of projected variables on the same axes of the standard 334 (PCA) analysis (Var E and Var B) (Table 5). The factorial plane of the co-inertia for the 335 Biological table explained 88.9% of the variance, and the factorial plane for the 336 Environmental table explained 87.9% of the variance. The co-inertia analysis, therefore, 337 demonstrated a co-structure between the 2 data sets. Figures 4 and5 represent the factorial 338 planes of the variables (columns) and stations (lines) in the Environmental and the Biological 339 System, respectively. 340

In the Environmental System (Figure 3), the first axis (F1) showed an opposition between the 341 342 aggregation recognized 4 groups of stations. The first group ENV-1 comprised the Europa 343 lagoon stations (E1, E2 and E3) and had significantly higher concentrations of organic 344 nutrient (PON, POC and DOC), whereas the second group ENV-2 (JNO, EO and BO) was 345 more associated with ammonium (NH 4 ). The third group (JN3, G1) was in opposition with 346 the two first groups without specific information, whereas the fourth group (ENV-4) was 347 characterized by low organic concentrations, high inorganic concentrations (NO 3 , NO 2 ), and 348 high silicate concentrations. Consequently, the factorial plane defined by the first 2 axes in 349 this environmental system distinguished stations according to nutrient richness (Axis F1). 350

In the Biological System (Figure 4), the F1 axis showed the association of the bacterial 351 variables (TB, BP) and the phytoplankton variables (CHLO, NANO, PNF), opposed to 352 cyanobacteria concentrations (CYANO) and culturable bacteria (CB). The factorial plane 353 highlighted the neutral position of metazooplankton (ZPK), ciliates (CIL) and heterotrophic 354 355 the scores of stations identified 3 main groups. Group BIO-1 comprised the Europa lagoon 356 stations as for the Environmental system (see above), with higher values of total bacterial 357 abundance (TB) and production (BP) linked to high autrophic algal biomass (CHLO, PNF, 358 NANO). The second group BIO-2 comprised all ocean stations and two lagoon stations (JN1 359 and G3), and had significantly higher concentrations of picocyanobacteria (CYANO) with the 360 genera Prochlorococcus and Synechococcus, and culturable bacteria (CB). In the third group 361 BIO-3, Bassas and Mayotte lagoons were in a neutral position, characterized by higher 362 concentrations of metazooplankton, ciliates and heterotrophic nanoflagellates (ZPK, CIL, 363 HNF). Thus, the mean values of biological characteristics in the 3 groups of stations varied 364 according to biological richness (Axis F1). 365

The correlation between the new environmental and biological ordination of the stations, 366 reflecting the degree of association between the Environmental and Biological systems, was 367 highly significant, with R-values of 0.828 and 0.677 for the 2 factorial planes (Axis F1 368

Environment/Axis F1 Biology; Axis F2 Environment/Axis F2 Biology). There was a good fit 369 for all these results between the 2 new ordination sets. 370 371 4. Discusssion 372 4.1. Regional context 373

In the Western Indian Ocean (WIO), the Mozambique Channel is dominated by southward 374 migrating eddy trains with large anticyclones of 300 km in diameter (Quarty and Srokosz, 375 2004). Many studies conducted in this zone were based on mesoscale features with physical 376 and biological approaches demonstrating the importance of the frontal areas and the cyclonic 377 and anticyclonic eddies (Ménard et al., 2014;Debourges-Dhaussy et al., 2014). Various 378 studies have shown the importance of eddies for increasing nutrient concentrations and 379 primary production, attracting zooplankton and top-predators around these zones 380 [START_REF] Weimerskirch | Foraging strategy 833 of a top predator in tropical waters: great frigate birds in the Mozambique Channel[END_REF]. However, it is now recognized that coral reefs are one of the 381 coastal ecosystems most threatened by climate change and anthropogenic pressures (Hoegh-382 Guldberg et al., 2007;[START_REF] Sandin | 775 Baselines and degradation of coral reefs in the Northern line Islands[END_REF]. The WIO, therefore, provides a unique 383 environmental gradient for examining the interactive effects of environmental variation, 384 climate change, connectivity, resilience and adaptation of coral communities (McClanagan et 385 al., 2014). However, little attention has been paid to microbial communities in coral reefs, and 386 their role in the nutrition of reef organisms, or in the transfer of dissolved organic matter from 387 microorganisms to predators [START_REF] Dinsdale | 628 Microbial ecology of four coral atolls in the northern line islands[END_REF]. So far as we are aware, there are very 388 few microbial studies of the coral reef lagoons located in the WIO, with only one study of the 389 nutrient status of the Iles Eparses [START_REF] Riaux-Gobin | 756 Nutrient status in coral reefs of the Îles Eparses (Scattered Islands): comparison to nearby 757 reefs subject to higher anthropogenic influences (Mozambique Channel and Mascarenes, 758 Indian Ocean)[END_REF] and a few studies on microbial 390 ecology in Mayotte lagoon [START_REF] Vacelet | Influence of freshwater and 821 terrigenous material on nutrients, bacteria and phytoplankton in a high island lagoon: Mayotte, 822 Comoro Archipelago, Indian Ocean[END_REF][START_REF] Gourbesville | Coastal environment assessment procedure for 659 sustainable wastewater management in tropical islands: the Mayotte example[END_REF]391 Houlbrèque et al., 2006). The coral reef lagoons in Madagascar are very different (e.g. Great 392

Reef of Toliara). However, very few studies of aquatic microorganism dynamics have been 393

conducted in this zone, except for the phytoplankton components [START_REF] Vasseur | Main changes in the 828 last 30-40 years on coral reefs and coastal areas induced mostly by human pressure: the 829 Tuléar region example (SW Madagascar, Indian Ocean)[END_REF] and 394 recently for bacterial and viral interactions [START_REF] Bouvy | Importance of predation and viral lysis for bacterial mortality western Indian coral 603 reef ecosystem (Toliara, Madagascar)[END_REF]. This study should, therefore, 395 provide information about the relationships between environmental conditions and the main 396 plankton compartments of the Iles Eparses, considered here to be pristine systems at a 397 regional scale. 398 399 4.2. Nutrient context and plankton structure 400

The water temperature, salinity and pH were uniform, with no stratification along the water 401 column at any of the lagoon or ocean stations. The Mozambique Channel is subject to 402 anticyclonic and cyclonic mesoscale eddies throughout the year with a southward water 403 migration [START_REF] Schouten | Eddies and 778 variability in the Mozambique Channel[END_REF]. [START_REF] Roberts | Interaction of dipole eddies with the western 761 continental slope of the Mozambique Chanel[END_REF] reported that there was no significant 404 difference in surface temperature in April along the Channel. This was confirmed by our 405 results obtained in April 2011 with surface temperatures varying between 28.1°C and 29.8°C. 406

On average, nutrient concentrations were low, similar to those reported in oceanic zones, with 407 undetectable levels of orthophosphate. Most of the WIO is nutrient-limited (especially 408 phosphates) with little or no upwelling along most of the East African coast (Porter et al., 409 2013) and prevailing downwelling [START_REF] Mcclanahan | water[END_REF]. However, mesoscale eddies can 410 occasionally increase nutrient concentration and primary production (Weimerskirch et al., 411 2004). There is very little human activity in the WIO region, especially in the Iles Eparses 412 and nutrient concentrations in mangroves are mainly derived from the release of nutrients 413 and organic matter from mangrove litter during leaching and decomposition. This study 414 produced the first measurements of virioplankton and protozoan concentrations in the WIO. 415

Virioplankton concentrations were similar to those found in Pacific near-shore oceanic coral 416 reefs [START_REF] Seymour | Spatial dynamics of virus like 781 particles and heterotrophic bacteria within a shallow coral reef system[END_REF]Pattern et al., 2011;Bouvy et al., 2012a). Virioplankton 417 abundances (min-max, 1.4 34.4 x 10 7 VLPs ml -1 ) were within the usual range (10 7 10 8 ml-418 1) for temperate productive systems [START_REF] Weinbauer | Ecology of prokaryotic viruses[END_REF]. VLP concentrations were 419 significantly higher in the lagoon than in the ocean sites, confirming that viral abundances 420 tend to be greater in productive, nutrient rich environments [START_REF] Weinbauer | Distribution of viruses and dissolved DNA 839 along a coastal trophic gradient in the northern Adriatic sea[END_REF]. The 421 bacterial densities were similar to those occurring in near-shore through to oceanic coral reefs 422 [START_REF] Seymour | Spatial dynamics of virus like 781 particles and heterotrophic bacteria within a shallow coral reef system[END_REF][START_REF] Dinsdale | 628 Microbial ecology of four coral atolls in the northern line islands[END_REF]Patten et al., 2008). The highest chlorophyll 423 values were observed in the lagoons (especially Europa) with an average of 0.507 µg l -1 , 424 while significantly lower values, close to concentrations recorded in South Pacific coral reef 425 lagoon, were found in ocean sites [START_REF] Rochelle-Newall | Phytoplankton-764 bacterioplankton coupling in a subtropical South Pacfic coral reef lagoon[END_REF]. These results were 426 confirmed by the statistical difference in algal concentrations between the two types of 427 systems. The phytoplankton composition, dominated by picoeucaryotes (Pico) and 428 picocyanobacteria (Cyano), was in the range reported by surveys in tropical lagoons (Ferrier-429 Pages and Furla, 2001;Houlbréque et al., 2006;Bouvy et al., 2012b) and in oceanic waters 430 [START_REF] Flombaum | 652 Present and futre global distribtions of the marine Cyanobacteria Prochlorococcus and 653 Synecochoccus[END_REF]. Our results confirmed the importance of picoplankton (defined here 431 as cells <3µm), primarily found in oligotrophic areas of the oceans, where they contribute to 432 nutrient remineralization [START_REF] Azam | The 587 ecological role of bacteria in the sea[END_REF] and may account for up to 95% of primary 433 production [START_REF] Raven | Small is beautiful: the picoplankton[END_REF]. On average, picocyanobacteria and picoeukaryotes were 434 significantly less abundant in oceanic waters than in the lagoons. However, their 435 concentrations were lower in Europa mangrove waters as they are believed to be outcompeted 436 by other phytoplankton in high-nutrient waters [START_REF] Partensky | Prochlorococcus, a marine photosynthetic 734 prokaryote of global significance[END_REF]. 437

It is now accepted that nanoflagellates are commonly the most abundant group of coastal 438 marine protists in nanoplankton communities (70-80% < 5 µm), with a count of heterotrophic 439 nanoflagellates (HNF) and pigmented nanoflagellates (PNF) of between 10 2 and 10 3 cells ml -1 440 [START_REF] Massana | Eukaryotic picoplankton in surface oceans[END_REF]. There were relatively low concentrations of both groups in the Iles Eparses, 441 with no significant differences between lagoon and ocean sites. The low abundances of 442 nanoflagellates were also in the range reported for the Mayotte reef (Houlbréque et al. 2006) 443 and in the Great Reef of Toliara, Madagascar [START_REF] Bouvy | Importance of predation and viral lysis for bacterial mortality western Indian coral 603 reef ecosystem (Toliara, Madagascar)[END_REF]. 444

Overall, aloricate ciliates were more abundant than loricate ciliates in the Iles Eparses, 445 confirming that oligotrophic marine waters generally have significantly more aloricate ciliates 446 than tintinnids [START_REF] Quevedo | The protistan microzooplankton 747 community in the oligotrophic northeastern Atlantic: large and mesoscale patterns[END_REF]. There was no significant difference in total ciliate 447 concentrations between the lagoon and ocean ecosystems, with abundances generally lower 448 than those found in other coastal localities [START_REF] Jyothibabu | The 684 response of microzooplankton (20 200 mm) to coastal upwelling and summer stratification in 685 the southeastern Arabian Sea[END_REF]Liu et al 2012 in the 449 southeastern Arabian Sea and the Bay of Bengal, respectively). Recently, Fournier et al. 450 (2012) reported abundances of ciliates in French Polynesian Atoll lagoons (with a mean of 451 740 cell l -1 ) that were significantly larger than those reported in Iles Eparses lagoons (mean of 452 44 cell l -1 ). These low concentrations of heterotrophic protists may significantly affect the 453 flow of organic matter in the microbial food web in the Iles Eparses ecosystems. 454

The mean total metazooplankton abundance in the Iles Eparses (0.6 to 6 10 3 ind m -3 ) was 455 lower than those found in a French Polynesian Atoll (between 5 x 10 3 ind m -3 and 23 x 10 3 ind 456 m -3 ; [START_REF] Pagano | Metazooplankton communities in the Ahe atoll lagoon (Tuamotu Archipelago, French 731 Polynesia); spatio-tempral variations and trophic relationships[END_REF] and in the coral reef lagoon of New Caledonia (mean of 3.5 x 10 5 ind 457 m -3 ; [START_REF] Carassou | Spatial and temporal distribution 606 of zooplankton related to the environmental conditions in the coral reef lagoon of New 607 Caledonian Southern Pacific[END_REF]. There was no significant difference between the two types of 458 ecosystem (mean of 1.8 x10 3 and 2.5 x 10 3 ind m -3 , respectively in ocean and lagoon sites). 459

Our results showed that copepod nauplii (copepodids) and adult copepods accounted for a 460 large proportion of the total density of zooplankton (close to 75%). This proportion is lower 461 in atoll lagoons of the Tuamotu archipelago (French Polynesia), where copepods account for 462 nearly 35% of the total zooplankton abundance whereas meroplankton account for up to 74%, 463 owing to the importance of pearl farming (Le Borgne et al., 1989;[START_REF] Carassou | Spatial and temporal distribution 606 of zooplankton related to the environmental conditions in the coral reef lagoon of New 607 Caledonian Southern Pacific[END_REF]464 Pagano et al., 2012). 465 466 4.3. Influence of nutrient and predator levels (HN F) on the bacterial growth rates and 467 grazing rates 468

The enrichment experiments were performed in nutrient conditions comparable to those 469 observed in some of the Iles Eparses in 2009 [START_REF] Riaux-Gobin | 756 Nutrient status in coral reefs of the Îles Eparses (Scattered Islands): comparison to nearby 757 reefs subject to higher anthropogenic influences (Mozambique Channel and Mascarenes, 758 Indian Ocean)[END_REF], and also to those 470 reported in Ahe atoll lagoon (Bouvy et al. 2012b) and in the Great Reef of Toliara (Bouvy et 471 al. 2015). The study also measured bacterial growth rates and bacterial grazing rates by 472 nanoflagellates, as it is now commonly accepted that nanoflagellates are the most important 473 grazers on bacteria in most environments (e.g. Sanders et al. 1992;[START_REF] Tsai | 817 Importance of bacterivory by pigmented and heterotrophic nanoflagellates during the warm 818 season in a subtropical western Pacific coastal ecosystem[END_REF]. Our 474 results showed a large range of bacterial growth rates with different patterns in lagoonal and 475 oceanic conditions. Bacterial growth rates were higher when the nanoflagellate concentration 476 was reduced by 99%, confirming that nanoflagellates are the main bacterial predators (see 477 above). Overall, all bacterial growth rates (µ, table 4) were relatively high in the upper range 478 of data reported in other ocean ecosystems [START_REF] Tsai | 817 Importance of bacterivory by pigmented and heterotrophic nanoflagellates during the warm 818 season in a subtropical western Pacific coastal ecosystem[END_REF]Bouvy et al., 2012b;Bouvy et 479 al., 2015). The highest growth rates were reported in lagoon ecosystems with 4.25 d -1 in 480 Europa, which also had the highest bacterial production and dissolved and particulate organic 481 matter. Microbial communities are adapted to intertidal zones and are subject to highly 482 variable salinity, floating, light and temperature conditions, which give rise to the high 483 diversity that characterizes mangrove ecosystems [START_REF] Feller | 641 Biocomplexity in mangrove ecosystems[END_REF]. In the other 484 environments studied, the growth rates of bacterial communities can be expected to be limited 485 by dissolved inorganic and organic matter, given that mineral limitation of growth rates is 486 widespread in various ecosystems including atoll systems (e.g. Torreton et al., 2000;Bouvy et 487 al., 2012b). Our results showed that bacterial growth rates were higher with added nutrients 488 for each station, especially for the ocean stations at Juan de Nova (JO) and Europa (EO), 489

indicating that nutrient limitation is a major factor for bacterial communities in the conditions 490 encountered in the Iles Eparses. Furthermore, a high ratio between heterotrophic bacteria and 491 picoautotrophic cells was reported at all stations (H/A; mean of 20.1), especially in the 492 Europa mangroves, suggesting that these microbial communities can act as CO 2 sources 493 [START_REF] Duarte | The CO 2 balance of unproductive aquatic ecosystems[END_REF]. The greater heterotrophic effect (such as external eutrophication) 494 could, therefore, be related to changes in the relative contribution of the various microbial 495 components, or to an increase of nutrient concentrations. 496 Prey abundance and water temperature are usually considered to be among the most important 497 factors regulating the grazing activity of nanoflagellates [START_REF] Choi | The dynamic nature of protistan ingestion response to prey abundance[END_REF]. Our experiments 498 showed that grazing rates by nanoflagellates increased with increasing bacterial growth rates, 499

indicating that bacterial and nanoflagellate dynamics were closely coupled at certain lagoon 500 stations (J2, E2 and B1). In the tropical conditions encountered in the Iles Eparses, water 501 temperature is certainly not the main factor controlling bacterial and nanoflagellate activities. 502 Moreover, our study did not consider alternative sources of bacterial mortality, such as lysis 503 by viruses. Very few studies have been carried out on viruses in atoll lagoons (Seymour et al., 504 2005;[START_REF] Dinsdale | 628 Microbial ecology of four coral atolls in the northern line islands[END_REF][START_REF] Patten | Uptake of picophytoplankton, 737 bacterioplankton and virioplankton by a fringing coral reef community (Ningaloo reef, 738 Australia)[END_REF]Bouvy et al., 2012a), and very little is known 505 about the response of tropical bacterioplankton to the presence of viruses. 506 507

Relationships between biological components and trophic variables 508

Multivariate analysis demonstrated clear differences between nutrient levels and biological 509 productivity in the Iles Eparses. Co-510 defined a group with the three stations sampled in Europa lagoon, characterized by high 511 nutrient levels associated with high bacterial dynamics. In the Environmental system, 512 particulate organic carbon (POC), particulate nitrogen (PON) and dissolved organic carbon 513 (DOC) were the parameters that had the strongest correlation with the Europa lagoon (ENV-514 1), confirming the role of mangroves as a provider of nutrients and organic matter for the 515 biological compartments [START_REF] Saenger | Mangrove ecology, silviculture and conservation[END_REF]. Microbial communities, especially bacteria (TB 516 and BP), are, therefore, fundamental in these sites (BIO-1) for the mineralization of organic 517 matter and its transformation into nutrients, as confirmed by the correlation found in the 518 Biological system. The release of ammonium (NH 4 ) into the overlying water following high 519 rates of remineralization in the mangrove sediment may support the high bacterioplankton 520 activities in the Europa lagoon, as demonstrated by Miyajima et al. (2001). Pigmented 521 nanoflagellates (PNF) and nanophytoplankton (NANO) were strongly linked to bacterial 522 abundances in the Europa lagoon, suggesting a high trophic coupling between bacteria and 523 nanoplankton. Co-inertia analysis also revealed a group (ENV-2) with three southern ocean 524 stations (JNO, EO, BO), characterized by higher nutrient richness than the other ocean 525 stations (TO and GO). However, all the ocean stations were grouped into one group (BIO-2) 526 for the biological variables reflecting the large contrast in terms of functioning between 527 lagoon and ocean ecosystems. The ocean stations were characterized by higher concentrations 528 of nitrate and nitrite, and by picocyanobacteria (PICO) which played a major role in ocean 529 ecosystems. These data confirmed that these pico-autotrophic cells can represent a substantial 530 fraction of marine primary production [START_REF] Flombaum | 652 Present and futre global distribtions of the marine Cyanobacteria Prochlorococcus and 653 Synecochoccus[END_REF]. Our study also confirmed that 531 picocyanobacteria were outcompeted by other phytoplankton in high nutrient waters as 532 mangroves [START_REF] Partensky | Prochlorococcus, a marine photosynthetic 734 prokaryote of global significance[END_REF]. 533

Finally, Mayotte lagoon has a particular biological composition which differs from the Iles 534

Eparses. It has higher concentrations of heterotrophic groups (HNF, CIL and ZPK) associated 535 with high microalgae abundance (ALG), suggesting an omnivorous network with a 536 dominance of the herbivorous food chain, as observed in coastal upwelling systems (e.g. 537 Vargas and Gonzales, 2004). As reported by [START_REF] Gourbesville | Coastal environment assessment procedure for 659 sustainable wastewater management in tropical islands: the Mayotte example[END_REF], Mayotte 538 lagoon is subject to organic and inorganic pollution from sewage, which probably stimulates 539 primary production and the microheterotrophic pathway and their transfer to metazooplankton 540 (mainly copepods and appendicularians; data not shown). 541

Two of the Iles Eparses had very different ecological functioning: 542 -The Europa lagoons with large and productive mangrove forests are clearly 543 distinguished with higher nutrient concentrations (nitrogen forms, dissolved organic 544 carbon) than those reported in other islands. Mangroves are transitional coastal 545 ecosystems in tropical and sub-tropical regions with biologically important, productive 546 ecosystems [START_REF] Kathiresan | Biodiversity of Mangrove Ecosystems[END_REF]. These intense nutrient pressures influence the 547 structure and dynamics of the microbial compartments. The high bacterial production and 548 growth rates are linked to the presence of mangroves, owing to the high concentrations of 549 dissolved and particulate organic matter (POC, PON, DOC; Table 2) that are released into the 550 lagoon waters driven by tides and freshwater flow [START_REF] Silveira | 785 Influence of salinity on bacterioplankton communities from the Brazilian Rain forest to the 786 coastal Atlantic ocean[END_REF]. 551 -The marine ecosystem of Tromelin had the lowest biological productivity, with the 552 lowest nutrient concentrations and bacterial growth rates. Bacterial communities appeared to 553 be adapted to the oligotrophic conditions as enrichment had no significant effect on the 554 growth rates. This may be because Tromelin is outside the Mozambique Channel, and so the 555 marine system is not subject to the frontal areas and cyclonic and anticyclonic eddies 556 characterizing the Mozambique Channel. 557

In conclusion, this survey provided the first regional study giving a preliminary insight into 558 the spatial distribution of the plankton communities in the Iles Eparses. The coral reef lagoons 559 off the Iles Eparses are particularly well preserved, with low nutrient levels, while those in 560

Mayotte can be considered to be an anthopized, polluted ecosystem with a predominance of 561 herbivore networks. The Europa lagoon had productive bacterial and pigmented 562 nanoflagellate communities related to the presence of mangroves. Picocyanobacteria were the 563 main component found at the oligotrophic ocean stations (low phosphate concentrations). The 564 Iles Eparses can, therefore, be considered to be pristine ecosystems and should be protected 565 from human pressure and pollution. Ecological changes generally take place over the long 566 term and each new generation of scientists and the general public alike fail to recognize the 567 changes that have taken place gradually over time [START_REF] Katikiro | Perceptions on the shifting baseline among coastal fishers of Tanga, 680 Northeast Tanzania[END_REF]. Although there is a lack 568 of ecological data on this area of the West Indian Ocean, identifying changes occurring in the 569 ecosystem over time will show how the environmental baseline (established by this study) has 570 shifted to such an extent that it no longer includes species that were found in the past. In this 571 context, data mining is necessary and should focus essentially on the genetic diversity and 572 functioning of microbial communities in the Iles Eparses, for which there have been very few 573 Units ml -1 10 5 ml -1 pmole l -1 h -1 10 7 ml -1 10 3 ml -1 10 3 ml -1 10 2 ml -1 10 2 ml -1 10 2 ml -1 µg l

-

  filtered water) and with 1% of predators 218 --filtered water). The samples were 219 homogenized and distributed equally into 2 series of 3 x 100 ml Whirl-Pak® polyethylene 220 sterile bags. At time zero (t0 4 221 NaNO 3 NaH 2 PO 4 222 combination. All assays were performed in triplicate with a total of 6 Whirlpaks per station. 223

Figure 1 :Figure 2 :Figure 3 :

 123 Figure 2: Growth rates of bacteria (day -1 ) versus grazing rates of bacteria by predators (day -1 ) 853 in the different lagoon and oceanic sites from experimental procedures, with nutrient added 854 (black square) and in absence of nutrient addition (Control: gray circle). Standard deviation is 855 reported from triplicates analysis. Abbreviation of the 10 stations: TO: Tromelin ocean; GO: 856 Glorieuses ocean; JO: Juan de Nova ocean; EO: Europa ocean; BO: Bassas da india ocean; 857 G2: Glorieuses lagoon; J2: Juan de Nova lagoon; ML: Mayotte lagoon; E2: Europa lagoon; 858 B1: Bassas da India lagoon. 859 860 Figure 3: Co-inertia analysis with the position of the environmental parameters on the F1 x F2 861plane. Position of the sites were linked to environmental parameters co-inertia weights, with 862 -1; ENV-2, ENV-3 and ENV-4). See 863 abbreviations in Table1for the sites. Abbreviations of parameters: PON: particulate organic 864

Figure

  Figure 1 : Bouvy et al.

Table 1 :

 1 Table 1 for the sites. Abbreviations of parameters: PON: particulate organic 864 List of stations studied in the five Iles Eparses and in Mayotte island in West Indian Ocean in April 2011. Sampling data, sitecode, geographical coordinates and some physical parameters (temperature, salinity, dissolved oxygen amount and pH) are reported as average along the water column (undet : not determined).

	865	carbon; POC: particulate organic nitrogen; C/N: ratio of particulate organic compounds;
	866	DOC: dissolved organic carbon; NH 4 : ammonia; NO 3 : nitrate; NO 2 : nitrite; Si: silicate.
	867
	868	Figure 4: Co-inertia analysis with the position of the biological variables on the F1 x F2 plane.
	869	Position of the sites was linked to biological variables co-inertia weights, with identification
	870	O-1; BIO-2 and BIO-3). See abbreviations in Table 1
	871	for the sites. Abbreviations for variables: TB: total bacteria abundance; BP: bacterial
	872	production; CB: culturable bacteria; VLP: virus like particles; CHLO: chlorophyll-a; NANO:
	873	nanophytoplankton; PICO: picoeukaryotes; CYANO: picocyanobacteria; PNF: pigmented
	874	nanoflagellates; ALG: microalgal density; HNF: heterotrophic nanoflagellates; CIL: total
	875	ciliates (loricates and aloricates); ZPK: metazooplankton

Table

Table 2 :

 2 Mean of the chemical parameters of the 16 studied sites (lagoon and oceanic stations) located in the Iles Eparses and in Mayotte lagoon. PO4 : dissolved phosphorus ; SI(OH)4 : silicates ; NO2 : nitrite ; NO3 : nitrate ; NH4 : ammonia ; PON : particulate organic nitrogen ; POC : particulate organic carbon ; C/N : ratio between particulate organic forms ; DOC dissolved organic carbon. (nd: non detectable).

	Parameters	Code	PO 4	SI(OH) 4 NO 2 NO 3 NH 4 PON	POC	C/N	DOC
	Units		µM	µM	µM µM µM µM	µM	µM
	EUROPA
	ocean	EO	nd	1.94	0.04 0.12 0.16 21.70 222.4 10.25 136.3
	lagon	E1	nd	1.59	0.05 0.23 0.49 54.55 250.2 4.59 149.8
	lagon	E2	nd	1.51	0.04 0.21 0.07 47.94 311.4 6.50 142.5
	lagon	E3	nd	1.57	0.04 0.12 0.37 47.11 260.9 5.54 168.5
	BASSAS DA INDIA
	ocean	BO	nd	2.90	0.05 0.07 0.08 22.32 238.3 10.54 95.2
	lagon	B1	nd	2.34	0.07 0.20 0.25 26.66 143.2 5.37 79.6
	JUAN DE NOVA
	ocean	JO	nd	2.04	0.07 0.05 0.40 31.41 171.9 5.47 104.6
	lagon	J1	0.06	2.40	0.11 0.10 0.15 31.88 161.1 5.05 87.8
	lagon	J2	nd	1.65	0.04 0.05 0.07 27.89 138.5 4.97 78.1
	lagon	J3	nd	1.26	0.03 0.05 0.04 36.13 188.4 5.21 77.2
	MAYOTTE
	lagon	ML	nd	2.79	0.06 0.26 0.13 32.59 121.2 3.72 73.4
	GLORIEUSES
	ocean	GO	nd	nd	nd	nd 0.04 27.28 119.9 4.39 91.9
	lagon	G1	nd	1.43	0.05 0.02 0.01 38.85 208.4 5.36 71.5
	lagon	G2	nd	1.83	0.02 0.03 0.01 24.80 140.3 5.66 71.1
	lagon	G3	nd	2.15	0.02 0.12 0.06 17.85 127.1 7.12 77.1
	TROMELIN
	ocean	TO	nd	2.73	0.04 0.05 0.13 17.98 107.9 6.00 87.1
	Mean of ocean sites	nd	2.41	0.05 0.07 0.16 24.1 171.5 7.3 103.0
	Mean of lagoon sites	nd	1.90	0.05 0.14 0.15 34.5 182.8 5.4 96.4

Table 3 :

 3 Mean of the biological variables of the 16 studied sites (ocean and lagoon) located in the Iles Eparses and in Mayotte lagoon. Concentrations of each variable are reported: CB : culturable bacteria ; TB : total bacterial ; BP : bacterial production ; VLP : virus like particles ; Pico : picoeukaryotes : Cyano : picocyanobacteria ; Nano : nanophytoplankton ; HNF : heterotrophic nanoflagellates ; PNF : pigmented nanoflagellates ; Chloro : chlorophyll--a ; ZpK : metazooplankton.

	Parameters	Code	C B	T B	BP	V L P	Pico	C yano Nano H N F	PN F	C hloro M icroalga C iliates	Zp K

Table 4 :

 4 Bacterial growth rates in presence (µ+g) or absence (µ) of HNF (respectively in 100% and 1% fraction), and grazing rates of bacteria by predators (g) in the different experimental conditions (control and with nutrient added; see Material and methods).

	-1	l -1	l -1	m 3

Table 5 :

 5 Comparison of inertia from the 2 data sets based on Environmental (E) and Biological (B) variables resulting from the co--inertia analysis. Two co--inertia axes (F1 and F2) are selected. Var E and Var B: inertia of each table projected on the co--inertia axes. Iner E and Iner B: maximal projected inertia of each table. Covar: covariance of the 2 sets of coordinates projected on the co--inertia axes. R--value represents the correlation between the 2 new sets of coordinates resulting from the co--inertia analysis

	Axis	Var E Var B Iner E Iner B Covar R--value
	F1	2.621 4.137 2.908 4.747 2.706	0.828
	F2	1.504 2.561 2.146 2.692 1.330	0.677

picoeukaryotes (Pico) and the picocyanobacteria (Cyano) mainly Synechococcus and

Prochlorococcus. Picoeukaryotes and picocyanobacteria abundances were similar in ocean