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Objective 2

lundi 12 mai 2014


The second objective is to study how production, mineralisation and export of organic matter depends on the N2 fixation process in contrasting oligotrophic areas: 3 long duration (LD: 7 days) stations. We will quantify the flow of material (biogenic elements) through each ecosystem and particularly focus on the role of N2 fixation in the production and fate of organic C. The fate of newly fixed N by diazotrophs in the ocean has been poorly studied so far, despite the fact that N2 fixation provides the major external source of N for the global ocean. In most studies, the input of N brought to surface waters by N2 fixation is converted using Redfield ratios in order to ‘theoretically’ evaluate the C production potentially sustained by this source of N. It is not clear whether this N fuels photo-autrotrophic phytoplankton and can be transferred along the trophic chain and rapidly exported out from the euphotic zone, and/or if it fuels heterotrophic growth and thereby contributes to remineralisation in surface waters. In addition, the plankton diversity and its evolution after the development of a diazotroph bloom (classical food web versus microbial loop) have been very poorly studied, mainly due to the difficulty of observing and monitoring a bloom in the natural environment.

Objective 2.1. Physical and biogeochemical characterization of contrasting oligotrophic environments

We will sample 3 water masses along the trophic gradient characterized by different populations of N2-fixing organisms. The real added value will be the coupling between physical and biogeochemical strategies. We expect to locate the first LD station (A) (see Document 2) in an area dominated by autotrophic diazotrophs Trichodesmium spp. and/or UCYN-B, which appear often spatially associated in the SW Pacific (Moisander et al., 2010). The previously developed algorithms TRICHOSAT (Dupouy et al., 2011) and PHYSAT (Alvain et al. 2005; 2008; the Trichodesmium algorithm is currently being developed in the framework of the SAVALEFER project, PI: C. Menkès) will help to locate a Trichodesmium spp. bloom from space. We expect to locate the second LD station (B) in an area associated with high abundances of UCYN-A, probably in the transition zone between the SW Pacific and the gyre as shown by Halm et al. (2011). Because it will not be possible to identify it from space, we will use diazotroph abundance, diversity and gene expression assessed onboard in near real time time using qPCR at each 18 SD stations to decide which one will be used for a process study (LD station). The Third LD station (C) will be located in the low N2 fixation region (gyre), where heterotrophic diazotrophs dominate (Bonnet et al., 2008; Halm et al., 2011).
The physical dynamics of the regions around the 3 LD stations will be first characterized in terms of large scale general circulation through the analysis of satellite (SST, Ocean color, Altimetry) and operational model data. These will be automatically retrieved and processed (to derive Eulerian and Lagrangian diagnostics such as the Okubo-Weiss parameter and Lagrangian Coherent Structures), and then transmitted onboard by a dedicated server on land. These data will be analysed in near realtime both on board and on land in order to optimize the sampling strategy according to the local physical and ecological characteristics. This approach has already been successfully implemented by the proposed team and employed during several previous cruises: LATEX (Nencioli et al. 2011), KEOPS2 (d’Ovidio et al., 2012) and STRASSE. Local meso and submesoscale dynamics within each region (eddy, filaments and frontal processes and their impact on regulating vertical fluxes and structuring the ecological community) will be first characterized through multisensor MVP 200 mapping at the beginning and at the end of each survey. This will allow to accurately identify the homogeneus and coherent water masses which will be the focus of each LD station. For this purpose the MVP 200 will represent an extremely useful instrument to sample a large zone in a short time to preserve synopticity. If not available, a more classical strategy with numerous CTD casts will be undertaken as during the BOUM cruise (Moutin et al., 2012). A glider mapping within a smaller region around the station will integrate the initial and final LD mappings. This will allow to investigate the impact of high-frequency small-scale physical phenomena on biomasses and biogeochemical fluxes. A recently improved version of the Lagrangian navigation software developed during the LATEX project (Doglioli et al., 2013) will be adopted in order to take into account the translation of the studied water masses, as well as their rotation and deformation.
3 Bio-Argo floats, specifically equipped with low level phycoerythrin sensors will be deployed at each LD station. In addition to conventional sensors for monitoring physical and biogeochemical parameters, they will allow to characterize Trichodesmium spp. blooms in the SW Pacific ocean during our cruise and on a longer time scale.

Specific questions: How do physical processes structure the planktonic community? How accurately are phytoplanktonic communities, and specially N2-fixing organisms, represented by remote sensing data?

Objective 2.2. Measurements of nutrient concentrations and/or availabilities in the photic zone, and identification and quantification of the major external fluxes.

Nutrient availability (N, P, Si, Fe) in the photic zone controls primary production of organic matter, and thus exerts a strong influence on the species composition of the food web. It is dependent on several input, recycling and export fluxes. Nutrient concentrations in the upper layer represent the equilibrium state between all those fluxes and is the first indicator of nutrient availability. Nevertheless, as nutrient concentrations are very low in marine oligotrophic habitats, these measurements represent a major observational challenge. Nanomolar methods developed by the proposed team will be used for nutrient concentrations measurements when necessary (Rimmelin & Moutin, 2005, Rimmelin et al., 2007, Pascoa et al., 2012).
New nutrient inputs in the upper mixed layer include fluxes induced by ocean dynamics (meso- and submesoscale pumping; vertical turbulent mixing), interior sources (N2 fixation) and surface fluxes (atmospheric deposition). The dynamics of meso- and submesoscale structures is often associated with strong vertical motion that can induce intense upwelling of deeper, nutrient-rich waters into the euphotic zone (Levy et al., 2012). The impact of those structures in regulating local nutrient availability and primary production will be investigated through the observations described in section 2.1.
Vertical turbulent mixing is a key process driving physical and biogeochemical fluxes between the mixed layer and the stratified ocean (Cuypers et al 2012) and can strongly impact primary production. While mixing in the mixed layer is mostly driven by direct atmospheric forcing, mixing in the stratified water column is mainly driven by internal wave breaking. Internal waves can be generated from inertial currents at the surface or tidal forcing at the bottom. The study area encompasses a large variety of environments regarding the internal waves (Internal tides notably vary from strong to negligible over the cruise area). As well we expect significant mixing resulting from instabilities of the strong currents of the western part. Therefore the measurements would provide a unique data set characterizing turbulent mixing in a poorly sampled area and under a variety of internal wave dynamical regimes and different current structures. Microstructure measurements that resolve the dissipative scales will be performed using a full ocean depth un-tethered profiling system, “VMP6000” (or a SCAMP that samples the first 100m). This profiler, that can profile down to 6000 m is equipped with microsensors for temperature and shear that enable two independent accurate estimates of the eddy diffusion coefficient Kz. These two estimates will be used to test the relevance of the different indirect methods based on classical CTD and ADCP measurements. This validation will allow to estimate Kz also when microstructure measurements are not performed. The same strategy has been used by the proposed team duing the BOUM cruise (Cuypers et al., 2012).
In addition to the input of nutrients by turbulent mixing, we will quantify the N input through biological N2 fixation, as it may be a major source in the SW Pacific. Gross and net N2 fixation rates will be measured at every station (SD and LD) between 0 and 2000 m depth using both the acetylene reduction method (Capone, 1993) and the 15N2 labelling procedure, incubation (24 h) and mass spectrometry analyses (MIO Marseille) as described in Montoya et al., (1996) and Bonnet et al., (2011). The15N2 enriched seawater method (Mohr et al., 2010; Wilson et al., 2012; Grosskopf et al., 2012) will be used during the cruise and several method inter-comparisons will be performed between the 15N2 bubble method and the 15N2 seawater enriched method, both performed under trace metal and DOM-clean (Bonnet et al., 2009) and non-trace metal clean and DOM-clean procedures.
In order to better understand the relationship between (photo)-heterotrophic diazotrophs and DOM, we will perform DOM characterization through FT-ICR MS (which allows the detection of individual DOM compounds) simultaneously to N2 fixation measurement and diazotrophs diversity.
Aeolian dust transport represents, on a global scale, the dominant source of iron, an essential micronutrient for phytoplankton growth, to the ocean (Jickells et al., 2005). Some of the melanesian archipelagos crossed during the transect (Vanuatu, Fiji) exhibit active volcanoes, which have been seen (in the North Pacific) to provide sources of very soluble and bioavailable trace metals such as iron (Olgun et al., 2011) and potentially N and P. Consequently, dust inputs could stimulate in situ biological productivity in oligotrophic ecosystems and alter community composition, distribution of nutrients and finally the net sequestration of atmospheric CO2. We will quantify the input of nutrients by the atmosphere and study the influence of dust deposits on the food web and their potential impact on N2 fixation, using microcosm experiments.

Specific questions: Is N2 fixation a major source of new N for primary production in the photic layer? Do direct measurements of eddy diffusion coefficients reasonably agree with previous estimations? What could be the role of dust?

Objective 2.3. Organic matter production and food web structure

Because of light/energy requirements, marine organic matter is almost entirely produced in the upper photic zone of the ocean. Variable primary production fuelled by physical processes acting over a wide range of scales interacts with predation to define the species composition of planktonic populations (Gargett & Marra, 2002). The basic principle of the conceptual food web structure, which has not been critically challenged since its original description (Johannes, 1965; Thingstad and Rassoulzadegan, 1999), is to consider two trophic strategies. Osmotrophy refering to organisms that feed by taking up dissolved nutrients and phagotrophy refering to organisms that feed by eating particulate matter. Osmotrophs include both heterotrophic bacteria and autotrophic phytoplankton, while the predatory food chain includes protozoa, mesozooplankton and higher predators (all heterotrophs).

Biogeochemical fluxes in relation to osmotrophic production
We will measure fluxes of biogenic elements (including C and O2) and determine parameters that will help to represent these fluxes (P vs I parameters, Ks, Vmax, affinity constants…). Classical approaches using stable (15N, 13C) and unstable (14C, 33P, 32Si) isotopes for measuring biogeochemical fluxes will be used for bulk measurements, along with new techniques at the single-cell level, which address by which species (or group of species) a specific element has been taken up. New methods concerning both dominant species separation as cell sorting by flow cytometry developed by the proposed team (Duhamel et al., 2009, Gregori et al., 2011; Talarmin et al., 2011) and low detection limits of chemical analyses will allow us to define species-specific uptake parameters. The contribution of mixotrophy and photoheterotrophy to the biogeochemical fluxes of C and N will be studied together with the environmental factors that control mixotrophy and symbiosis by microbial eukaryotes. The methods proposed will allow us to significantly improve our understanding of the factors controlling the distribution and activity of different groups of plankton functional types and to link their phylogenetic and functional diversity.
The coupling between phytoplankton assemblages and production will be studied in the surface mixed layer by measuring simultaneously and at a high frequency both the net community production (Oxygen/Argon method) and the pico, nano- and microphytoplancton abundances by flow cytometry (cytosense).
Regarding N2-fixing organisms, recent major development in SIMS (Secondary Ion Mass Spectrometry) instrumentation (NanoSIMS 50TM) now allows for the measurement of isotopic ratios with a spatial resolution better than 100 nm (Guerquin-Kern et al., 2005; Slodzian et al., 1992). The capability of such instrument to measure isotopic composition makes it suitable to study environmental microbiology at the single-cell level (Lechene et al., 2007). Most of these recently discovered N2-fixing micro-organisms are uncultured and many unresolved questions remain about their physiology and their importance in term of input of new N in the ocean. The nanoSIMS technology associated with cell sorting (Bonnet et al., In Rev.) or FISH (HISM-SIMS, Thompson et al., 2012, Krupke et al., 2013) will be applied to assess the contribution of every diazotrophic group to overall fluxes, and determine organic/inorganic nutrients controlling their activity. We will focus on the recently described UCYN (Moisander et al. 2010, Thompson et al. 2012) group whose role in C and N cycling is not well understood thanks to recent methods developments realized by the proposed team (see references above).
The silicification process in marine diatoms will be investigated using the PDMPO labelling technique (Leblanc & Hutchins, 2005).

Phagotroph production (secondary production)
The planktonic food web structure influences the fate of primary production in the ocean and has consequences for the CO2 transfer process. It is generally understood that when small eukaryotes or prokaryotes dominate the microbial community, the grazers of picoplankton are small protozoa which do not produce rapidly sinking faecal pellets. Despite their omnipresence and their pivotal role in the energy flow in marine waters on a global scale, the physiological ecology of these organisms is poorly understood. Several grazing steps are necessary to enable primary production to be incorporated into the upper trophic levels. Therefore, most of the C fixed by phytoplankton is respired and remineralised by the microbial community in the surface mixed layer and there is little or no net uptake of CO2 from the atmosphere to the sea. In contrast, when large phytoplankton cells dominate, they can either sink to depth or be grazed by copepods and other mesozooplankton which produce rapidly sinking faecal pellets. However, Mesozooplankton, i.e. copepods, have recently been coupled to lower trophic levels in the eastern Mediterranean Sea (Thingstad et al. 2005) and their contribution to the C cycle of oligotrophic areas needs to be reconsidered. The upper meso- and macrozooplankton, largely under-sampled by classical techniques, may also significantly contribute to the vertical transport of organic matter.
An apparently high contribution of diazotroph N to zooplankton directly east of New Caledonia was recently evidenced (Hunt et al., in press), supporting similar findings in diazotroph rich areas of the Atlantic (Hauss et al. 2013). It is possible to trace the diazotroph signature through the macrozooplankton to the micronekton component of the food web. These are the first detailed stable isotope measurements of the pelagic community presented for the region. We will now increase the geographic coverage of zooplankton and stable isotope sampling across the SW Pacific diazotroph gradient, specifically to investigate the relative contributions of diazotrophs to food web N and the their role in structuring community composition. We will apply bulk stable isotope analysis in combination with amino acid specific N isotope analysis (CSIA). The latter method provides the added resolving power of direct estimate of source N isotope signature for any organism measured (McClelland et al. 2003). A key question remains what are the pathways that fixed N takes to reach primary consumers (microzooplankton and zooplankton grazers) and upwards. In part we can contribute to answering this question by detailed species level stable isotope analysis of all lower trophic level community components. Additional insights will be gained by experimental studies using labelled N tracers (Loick-Wilde et al. 2012).

Biogeochemical process from optical measurements
Integrated measurements of bio-optical properties and pigments will be made with instruments measuring hyperspectral radiometry in the UV-Visible domain, with UV-VIS Trios spectroradiometers (Murakami and Dupouy, 2013). Two consecutive profiles of downward irradiance [Ed(Z,λ)] will be conducted at solar noon with a MicroPro free-fall profiler (Satlantic) that is equipped with pressure, temperature and tilt sensors and OCR-504 downward irradiance sensors for the UVR-B (305 nm), UVR-A (325, 340 and 380 nm) and PAR (412, 443, 490 and 565 nm) spectral domains, according to the protocol given in Tedetti et al. (2007) and Para et al. (2013). Surface (atmospheric) irradiance [Es(λ)], which is equivalent to the downward irradiance just above the sea surface [Ed(0+,λ)], will be continously measured in the same channels from the ship deck with other OCR-504 downward irradiance sensors (surface reference). These measurements will be made to account for the variations in cloud conditions that occurred during measurement. Backscattering at the satellite channels will be obtained with the Hydroscat-6 (Dupouy et al., 2008; 2010; Loisel et al., 2011). Hyperspectral light absorption of particles and CDOM and FDOM characterization (with the identification of markers of bloom degradation in the surface, above and below the euphotic depth, etc...) in relation with pigment concentrations (Coble, 1996; Tedetti et al., 2011; 2012). Additionally, the characterization of optical properties of dominant species (Dupouy et al., 2008) encountered along the transect will be realized. In addition, inorganic aerosol quantification and atmospheric radiance measurements will be continuously done along the transect, especially around the Vanuatu archipelago. Simulations reveal that in Northern Atlantic atmosphere, dusts are able to induce a significant decrease of PP due to the attenuation of light by about 15-25% for dust optical depth (DOD) larger than 0.6-0.7 (at 550 nm).

Specific questions: What are the characteristics of the dominant species concerning photosynthesis, N2 fixation, nutrient uptake, osmotrophy and phagotrophy?

Objective 2.4. Organic matter mineralization and food web structure

The study of organic matter degradation will focus on the relationship between mineralization and bacterial diversity. Hence, examining the relationship between diversity and functionality within the bacterial community (ectoenzymatic activity, uptake of specific organic compounds representative of a chemical family) is a major challenge for understanding the impact of prokaryotic heterotrophic processes on mineralization of organic matter along the water column. We will also study the factors controlling bacterial production and the consequences of such controls on heterotrophic activity, and the hydrolysis rate of Dissolved Organic Phosphate and nitrogen.
The wide geographical and vertical (in the oceanic water column) distribution of heterotrophic diazotrophs suggests they are bound to have a key role in global N2 fixation. However, failure to isolate and maintain these organisms in culture limits our knowledge on their activity and nutritional needs (Zehr, 2011). While the genetic information of these organisms reveals their dependence on organic matter (Riemann et al., 2010; Tripp et al., 2010), this relationship has not been studied so far. We will perform microcosm experiments at LD stations to test whether different sources of DOM (C-enriched, N-enriched or P-enriched commercially available sources, and natural in situ DOM) trigger N2 fixation and/or the expression of nifH by different groups of heterotrophic diazotrophs? Additional nutrient amendment experiments will be performed using 13C- and 15N-labelled organic molecule and the HISH-SIMS (Thompson et al., 2012) method in order to identify which diazotrophs assimilate which organic molecule and in which quantity.
Another question concerns the possible relationship between TEP production/concentration and heterotrophic diazotrophs. Do TEP/marine aggregates provide hot spots for heterotrophic diazotrophs especially in the mesopelagic/aphotic depths as suggested by Rahav et al. (2013)?

Specific questions: What are the changes in prokaryotic heterotrophic activity and community composition in relation with horizontal (west-east) and vertical (surface to depth) nutrient gradients and the composition/lability of the dissolved organic matter?

Objectif 2.5. Organic matter export (particulate and dissolved matter)

The structure of pelagic ecosystems influences the size and type of downward flux of biogenic C in the sea (Legendre and Rassoulzadegan, 1996). This vertical flux is composed mainly of large particles such as fecal pellets, hard parts of zooplankton, amorphous aggregates, marine snow (Fowler and Knauer, 1986; Silver and Gowing, 1991), and senescent diatoms, particularly in the aftermath of blooms (Billet et al., 1983). Fecal pellets can sink quickly: 20-900 m d-1 for copepods (Lorenzen, 1983; Welschmeyer and Lorenzen, 1985), and up to 2700 m.d-1 for large gelatinous zooplankton (Bruland and Silver, 1981; Madin and Purcell, 1992). High sinking velocities can lead to the efficient export of biogenic C. The downward flux of biogenic C can therefore be largely dominated by C of zooplankton origin (Thibault et al., 1999). We will use the Underwater Vision Profiler (UVP; Picheral et al. 2010) to assess vertical POC flux from particle size distribution (PSD) (Guidi et al. 2008) and vertical distribution of zooplankton (Stemmann et al., 2008) that are directly linked to the vertical flux. We will also complement the zooplankton distribution by an indirect estimate of zooplankton vertical profiles through acoustics from the ship borne S-ADCP and Lowered ADCP on stations as well as from ER 60 (200kHz) echosounder. In addition, using images from the UVP, we will describe vertical, horizontal, and temporal evolution of the Trichodesmium spp. colonies similar to what has been done recently in the North Pacific (Guidi et al. 2012). Trichodesmium spp. colonies will be spatially and temporally correlated to vertical particle flux and their potential contribution will be estimated.
We will study the C/N/P/Si stoichiometry of settling particulate matter because despite its biogeochemical relevance, there are few direct measurements available (Geider & La Roche, 2002). We will also examine the role of larger particulate matter responsible for the marine snow rarely observed in oligotrophic areas and quantify instantaneous DOC export.
It has been recently reported that bacteria living within colonies of the N2-fixing cyanobacterium Trichodesmium spp. use cell-cell signaling to regulate the degradation of organic phosphorus compounds (Van Mooy et al., 2012). In a separate study, it has been found that bacteria living on sinking particles may also use cell-cell signaling to regulate organic matter degradation (Hmelo et al., 2011). Finally, rates of N2-fixation were also found to be affected by cell-cell signaling (Van Mooy and Dyhrman, unpublished). We will combine these three findings and ask the next question: How does cell-cell signaling via acylated homoserine lactone (AHL) and autoinducer-2 (AI-2) molecules in senescent Trichodesmium spp. colonies affect the export of new organic N in sinking particles?

Specific questions: What is the ratio of particulate vs dissolved organic matter export? What is the role of Trichodesmium spp., and of cell-cell signalling in the export?

Objective 2.6. Fate of N2 fixation

Although direct evidence of trophic transfer from diazotrophs through planktonic food webs is lacking, studies based on natural abundance of 15N in suspended particles and zooplankton suggest that N from diazotrophy would support a part of primary and secondary production in the subtropical Atlantic Ocean (Montoya et al., 2002; Landrum et al., 2011). In addition, a low delta 15N signature of exported material at the time series station HOT (Karl et al., 1997; 2002; Casciotti et al., 2008), and of nitrate accumulating in the upper thermocline of the Sargasso Sea (Knapp et al., 2008) would indicate that recently fixed N is transferred out of the euphotic zone. As fragments of Trichodesmium spp. are rarely recovered in sediment traps (Walsby, 1992) and are mostly ungrazed (O’Neil, 1999), transfer of fixed N is probably done through the dissolved pool (Mulholland, 2007). Indeed, Trichodesmium spp. has been seen in the natural environment and in cultures to release up to 50-80% of recently fixed N2 as dissolved organic N (DON) and ammonium (NH4+) (Capone et al., 1994; Mulholland & Bernhardt, 2005) potentially available for surrounding planktonic communities. Alternatively, Brandes et al., (1998) suggested that some material derived from N2 fixation can be remineralized in surface waters, lightening the isotopic nitrate signal, which may be propagated along the trophic chain.
This lack of knowledge is essentially due to the lack of techniques which allow us to trace the N transfer through the different compartments of the food web. We propose here to overcome these difficulties by using a combination of powerful techniques previously used by the proposed team in the framework of the VAHINE project (INSU-LEFE – ANR JCJC PI: S. Bonnet), including: 1/ high-resolution nanometer scale secondary ion mass spectrometry (NanoSIMS) coupled to 2/ flow cytometry cell sorting and 3/ 15N2 isotopic labelling (Bonnet et al., In Rev.).
Previous studies have shown that increased TEP production by Trichodesmium was coupled with caspase activation and programmed Cell Death (PCD, Berman-Frank et al., 2004, 2007). The concentration of TEP will be correlated to PCD of diazotrophs using diagnostic genetic markers developed by I. Berman-Frank (Univ. Bar Ilan, Israel) at LD stations.
In order to quantify the part of export production that has been sustained by N2 fixation, a δ15N budget will be performed at LD stations, based on 15N natural abundance (collaboration with A. Knapp, Florida State University, see attached letter). δ 15NO3, δ 15DON will be measured along the vertical at LD stations. Using the PN sinking flux and PN sinking δ15N, we will construct a 1-D (vertical) isotope budget, where the water mass at LD stations is treated as a single box. We will assume that there are two sources of new N to the surface waters, the first of which being total dissolved N (TDN) (TDN = NO3-+NO2-+DON) mixed up from subsurface waters, using the δ15N of TDN from the base of the seasonal mixed layer as the δ15N for this source term. The other source of new N will be assumed to be N from N2 fixation, with a δ15N of -1‰ (Hoering and Ford, 1960; Minagawa and Wada, 1986; Carpenter et al., 1997). First we will construct a δ15N budget assuming that the TDN supplied from the subsurface is the only important source of new N, and assume that the two dominant export terms are represented by 1) the gradient in