1. Flux studies in contrasting environments (obj. 2) The role of heterotrophy (bact. microzoo)

2. What is the impact of natural iron fertilization - On the structure of the microbial food web - On the functioning of the microbial food web - On the fate of primary production And How the magnitude of carbon fluxes (grazing, mineralization) is affected by iron availability ? We will focus on two axes to differentiate heterotrophic responses - Direct vs indirect effects on heterotrophic bacteria, DOC utilization and respiration - Cascade effect on the trophic web Specific objectives Quantification of the carbon flux exported – Obj. 2.2-

3. ciliates copepods heterotrophic bacteria picophytoplankton nanophytoplankton microphytoplankton DOC P, N nanoflagellates Silicates CO2 O2 CO2 Gross Community production GCP and Dark Community Respiration DCR ? IRON ? ? ? ? ? ? ? ? ?? ? ?? ? ?? ? ?? ?

4. ciliates copepods heterotrophic bacteria picophytoplankton nanophytoplankton microphytoplankton DOC P, N + iron nanoflagellates Silicates CO2 O2 CO2 Gross Community production GCP and Dark Community Respiration DCR Direct effect of IRON on heterotrophs 1)Are bacteria Fe limited? Is there changes in bacterial biomass, ectoenzymatic activities, production, respiration ?

5. ciliates copepods heterotrophic bacteria picophytoplankton nanophytoplankton microphytoplankton DOC P, N + Fe nanoflagellates Silicates CO2 O2 CO2 Gross Community production GCP and Dark Community Respiration DCR Indirect effects of IRON on microbial food web 1. Is the response of the microbial food web a cascade effect from Phytoplankton stimulation? What is the fate of the phytoplankton ? Phytoplankton growth  phytoplankton grazing rates, abundance of predators, relations between predators? ? ? grazing ? sinking ?

6. ciliates copepods heterotrophic bacteria picophytoplankton nanophytoplankton microphytoplankton DOC nutrients nanoflagellates Silicates CO2 O2 CO2 Gross Community production GCP and Dark Community Respiration DCR Heterotrophy and remineralisation Indirect effects of IRON on microbial food web: 2. Do bacteria benefit from the carbon derived from Fe stimulated primary production? Does the Fe fertilization influence - the production and respiration of bacterioplankton and consequently the BGE ? - the factors limiting bacterial activity (Fe vs DOC)

7. Heterotrophic and phototrophic nanoflagellates - epifluorescence microscopy - size classes - biovolumes - carbon equivalents Ciliates - formol/lugol fixation - Sedimentation and counting on inverted microscope equipped for fluorescence - size classes / taxonomy + with flow cytometry data (pico autotrophs, heterotrophic bacteria) and the microphytoplankton mesozooplancton stocks Tools for studying biomasses 1. Structure of the food web in terms of stocks

8. Bacterial production 3H-leucine incorporation into proteins, with micro-centrifuge technique Gross community production and Dark community respiration : 24h variations of O2 in Winkler flasks, in situ-simulated conditions (running water bathes and screens) Bacterial ectoenzymatic activity Hydrolysis of fluorogenic substrates (aminopeptidase, glucosidase) Grazing fluxes Use of fluorescent labelled preys Fluorescent labelled bacteria for bacterial grazing by flagellates Fluorescent labelled algae for grazing of nanophytoplankton by ciliates. Tools for studying fluxes 2. Fluxes

9. FLS (fluorescently labelled Synechococcus) Synechococcus analog FLS FLA (fluorescently labelled algae, Rublee & Gallegos 1989) Nanophytoplankton analog FLA Nanochloropsis sp. (2-4 μm) Grazing of pico and nano autotrophs by ciliates Tools for studying fluxes

10. M2 D6 D5 D4 D3 D2 D1 A5 Where do we sample ?  across gradients Vertical profiles (euphotic zone – 0-200m) Kerguelen Plateau A5 Open Sea D6 The transect Plateau – Open Sea 5 stations D1 to D5 Sampling strategy

11. In situ Profiles : standing stocks and BP, O2/CO2 fluxes Surface layer : grazing, growth of heterotrophs We need : - to sample at the same time of the day every profile -to coincide with PP (14C) rosette, nutrients, DOC profile, flow cytometry, bacterial taxonomy, FISH Volumes necessary : BP, stocks (HNAN/PNAN, ciliates) : 750 ml O2/CO2 fluxes : Grazing bact, nanophyto (surface only) : 2 litres Growth (cil, flag, surface only): 10 lt

12. on-board experiments Process studies: Effect of Iron limitation on microbial food webs OBEX 1 : microb comm. growth, on-board experiments Response of the microbial food web Parameters to follow - BP (all time points) - HNAN/PNAN, ciliates stocks (T0h, T final) - grazing fluxes ( T0h, Tfinal) OBEX 4, OBEX 3 < 0,8 µm mesocosms in the dark? Direct iron effect on bacteria - BP - O2 consumption  BGE (bacterial growth efficiency) - Other Collaborations?

13. Which material which person in charge - Scintillation counter : Brest ? (Stéphane, Bernard ?) - Microcentrifuge (Urania ?, Markus ?) - Spectrofluorometer : possibly that desembarked after DYNAPROC ? - One Millipore filtration apparatus (France, LMGEM) - One Millipore filtration apparatus (Urania MREN ? Markus LOV ?) - Inverted flux system (membranes 142 mm) (France, LMGEM) - Refrigerated incubators ? Do we need on board ?