METHODS AND STRATEGY

The general thermohaline circulation (Fig. 1) is at the origin of the wide range of oligotrophic conditions observed in the Mediterranean Sea. The “BOUM” cruise (Biogeochemistry from the Oligotrophic to the Ultra oligotrophic Mediterranean Sea) will focus on 2 transects East-West and North-South, and three selected stations (for process studies) sampled during 5 days (Fig. 2). They represent a unique context to understand at a regional scale, the link between biogenic biogeochemical cycles of C, N, P, Si, Fe and the planktonic community structures of highly contrasted oligotrophic marine waters. The two transects will include “core parameters” stations from the Rhone River mouth to the centre occidental Mediterranean Sea and from Gibraltar to the Levantine basin. They will be performed to describe the relevant biogeochemical parameters from surface to bottom in the whole Mediterranean Sea.

Figure 1: The schematic of the thermohaline circulation in the Mediterranean Sea with the major conveyor belt systems indicated by dashed lines with different colour. The yellow indicates the AW stream which is the surface manifestation of the zonal conveyor belt of the Mediterranean. The red indicates the mid-depth LIW recirculation branch of the zonal thermohaline circulation. The blue lines indicate the meridional cells induced by deep waters. LIW branching from the zonal conveyor belt connects meridional and zonal conveyor belts. (Pinardi & Masetti, 2000).

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1- General strategy at each station:

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1-1 Short duration stations

During transect to reach or between two long duration stations, 27 short “core parameters” stations of 3-h duration will be performed.

Map_Stations

Only one CTD profile with sampling from surface to bottom will be done for core parameter analyses (temperature, salinity, dissolved O2, dissolved CO2, alkalinity, inorganic nutrients, chlorophyll, particulate organic C, N and P (POC, PON, POP), particulate silica, dissolved organic C, N and P (DOC, DON, DOP). In &addition, two layers will be sampled (mixed layer, top of nutricline) for controlled in-board incubations. The distances between all short duration stations will be around 60 nmiles which corresponds to approximately 5 hours of ship time.

Niskin bottles (12 L) available for sampling:

 Deep cast O-bottom) : 23 Niskin bottles (mini PVM and Nitrate sensor occupies one bottle space)

BOUM_Parameters_Abreviations

CTD_SDst_cp

CTD_SDst_Bio(A)

CTD_SDst_Bio(B)

1-2 Long duration stations

To achieve our process study at each of the three long duration stations (1 in the central Western basin, 1 in the central Ionian basin and 1 in the central Levantin basin), a precise localisation and physical characterisation in low currents areas is needed. It will be performed by using all methodologies available at the present time. For each station, the successions of operations will be as follows:

1) Horizontal survey. We will consider satellite imagery (Taupier Letage, expert) and prevision from MERCATOR to define areas were a rapid survey (maximum 12h) using thermosalinograph and ADCP data will allow to find precisely the location of the sites with low potential advection. During this survey, only continuous sea surface sampling will be possible.

2) physical and chemical characterisation of the area The hydrological / chemical context will be appreciated by the exploration of 16 sub-stations in a 9 square miles geographic area around the position of the long duration station. In each sub station, profiles 0-500 m will be performed for CTD, O2, fluorescence, and nitrate (ISUS). Each of the 16 stations will be occupied for 30 minutes and separated by one hour maximum of ship time. Sampling by Niskin bottles will be possible only at the 4 angles during that survey.

CTD_LDst_Survey

3) 96-h process study The stations sampled during that period of time will be at the centre of the sampling grid before the drifting of the mooring line and near the drifting mooring the following days (Lagrangian strategy). The sequence of operations will be as follows:

Succession_of_Operations_at_Sea

Remark:

This time schedule will provide a continuous 93h of CTD cast every 3 hours (31 CTD cast).

Niskin bottles (12 L) available for sampling:
 CTD cast 0-500m Physics+Cycle: 23 Niskin bottles (1 bottle space for optical grape, miniPVM & Nitrate sensor)

CTD_LDst_Cycle_3h_J1	CTD_LDst_Cycle_3h_J2	CTD_LDst_Cycle_3h_J3J4
CTD_LDst_Cycle_6h_J1	CTD_LDst_Cycle_6h_J2	CTD_LDst_Cycle_6h_J3
CTD_LDst_Cycle_6h_J4	CTD_LDst_Cycle_9h_J1	CTD_LDst_Cycle_9h_J2
CTD_LDst_Cycle_9h_J3	CTD_LDst_Cycle_9h_J4	CTD_LDst_Cycle_12h_J1
CTD_LDst_Cycle_12h_J2	CTD_LDst_Cycle_12h_J3J4	CTD_LDst_Cycle_15h_J1
CTD_LDst_Cycle_15h_J2	CTD_LDst_Cycle_15h_J3	CTD_LDst_Cycle_15h_J4
CTD_LDst_Cycle_18h_J1	CTD_LDst_Cycle_18h_J2	CTD_LDst_Cycle_18h_J3
CTD_LDst_Cycle_18h_J4	CTD_LDst_Cycle_21h_J1	CTD_LDst_Cycle_21h_J2
CTD_LDst_Cycle_21h_J3	CTD_LDst_Cycle_21h_J4	CTD_LDst_Cycle_24h_J1
CTD_LDst_Cycle_24h_J2J3J4	.	.

 Deep cast: 23 Niskin bottles (1 bottle space for mini PVM & Nitrate sensor)

CTD_LDst_cp

• "production" cast: 23 Niskin bottles (1 bottle space for mini PVM & Nitrate sensor)

CTD_LDst_oxygen production

CTD_LDst_in situ production

• other sampling: 23 Niskin bottles (1 bottle space for mini PVM & Nitrate sensor)

CTD_LDst_Micr_Exp

CTD_LDst_AAG

4) 24h physical and chemical characterisation

In case of large physical variations occurs during the 4-5 days time survey, we will repeat the sampling grid at the end the measurements.

Three steps will be necessary in order to quantify the nutrients input by hydrological processes in the 3 contrasting locations.

1/Precise localisation of a site and estimation of the horizontal advection

 A few days before arriving on station: examination of SST and Sea color satellite images together with temperature and current fields from Mercator products in order to determine the zone to be studied.
 Arriving on the area of the site: perform a short horizontal survey to observe the SST, SSS and the currents. Choose the site location.
 Arriving on the site: Perform some vertical casts around the location to control the depths of the photic zone and of the pycnocline, and detail currents that will be analysed after the cruise to evaluate horizontal advection of each parameter.

2/ Estimation of the turbulent activity For each CTD profile, measure at 24 Hz each variable (T, S, O2, NO3) and calculate the vertical profile of the horizontal current (LADCP) at a vertical resolution of 2 m. The data treatment will give average profiles and TS anomalies with a high vertical resolution, the amplitudes of internal waves and eventually density inversions, which marks turbulent activity.

3/ Atmosphere ocean physical interactions Water, heat and moment surface fluxes, will be calculated with considering the bulk method in order to evaluate the atmospheric forcing on the mixed layer activity and its temporal variability.

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List of Parameters & Methods

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Hydrodynamics and optics :

Parameters	Method
Temperature, salinity	CTD SBE 911
Horizontal Current on vertical profile	LADCP 300 Khz (INSU) Rosette mounted
Horizontal Current	ADCP (75 KHz, 300 KHz Ship mounted)·
Kz	VMP5500
SST and SSS	Thermosalinograph SBE 21·
Wind speed and direction, Air temperature and humidity, solar radiation.	On board meteo station
PAR	Downwelling irradiance measured on the Deck by a TRIOS radiometer (350 nm to 850 nm every 3 nm)·
In water upwelling radiance and irradiance	Radiometer TRIOS (350 nm to 900 nm every 3 nm) mounted on a specific frame
Backscattering, attenuation, fluorescence, and CTD	Wetlabs optical package mounted on a specific frame·
Fo, Fm, Fv/Fm, sigma-PSII, tau-PSII, tau-PQ	Custom-built bench-top fast repetition rate fluorometer (FRRf)

Biogeochemistry :

Stocks

Parameters	Method
Inorganic nutrients concentration:  Nitrate, nitrite, phosphate, orthosilicic acid  Nitrate (ISUS in situ)  Ammonium  Dissolved iron concentration	-  Visible Spectrophotometry  UV spectrophotometry  Fluorescence  Chemiluminescence
TCO2	potentiometry
TA	potentiometry
Oxygen	- Winkler  SBE43 sensor
Dissolved Organic Matter: DOC, DON, DOP	Visible spectrophotometry after digestion
Particulate Matter:  COP,  NOP, POP, BSi and LSi  C, N, P, Mg, Fe, elemental analysis	-  CHN  Visible spectrophotometry after filtration and digestion  X-Ray Fluorescence spectroscopy
TOC analysis	High temperature catalytic oxidation
Carbohydrate analysis	Liquid chromatography High Pressure Anionic Exchange Chromatography / Pulsed Amperometric Detection (HPAEC-PAD)
Lipid analysis	Iatroscan Analyzer (Thin Layer Chromatography / Flame Ionisation Detection (TLC/FID)

Fluxes :

Parameters	Method
Primary production (carbon uptake)	RA incorporation ( 14C)
Nitrogen fixation	· Mass spectrometry, dual labelling 13C/15N procedure
Phosphate uptake (affinity, maximum uptake rate and Turnover time)	RA incorporation (33P)
Silicic acid uptake	RA incorporation (32Si)
NCP	Delta Oxygen
GCP	Delta Oxygen
DCR	Delta Oxygen
Ectoenzymatic activity (lipase, protease, aminopeptidase, phosphatases	Spectrofluorometry with substrate MUF- and MCA-fluorogenic substrates
Lipase activity	Radiolabelling with 3H-triolein
Turnover of DOP	Radiolabelling with 33P-ATP and 3H-ATP
Prokaryotic heterotrophic production	Radiolabelling with 3Hleucine and TCA extraction of the protein precipitate
Turnover of amino acids	Radiolabelling with 3H-amino acids

Biology :

Phytoplankton

Parameters	Method
Species richness of diazotrophs	Genetic comparison of nifH gene
Abundance of diazotrophs	Cell specific fluorescent probe hybridization (TSA-FISH technique)
Diatom abundance	Optical microscopy
Coccolithophore morphology	Polarization Optic Microscopy, Scanning Electron Microscopy
Coccoliths weight	SYRACO (Beaufort 2005)
Coccolithophore biodiversity	LSU rDNA clone libraries, after high-GC genomic extraction and PCR amplification with haptophyte specific primers.
Coccolithophore abundance	COD-FISH (Frada et al. 2006)
Pico-haptophyte diversity	LSU rDNA analyses

Protistan

Parameters	Method
Abundance and biomass	Optical and epifluorescence microscopy
Growth rates	Dialysis Bags for population estimates
Grazing rates and prey selectivity	Labelled preys

Mesozooplankton

Parameters	Method
Species richness	Taxonomical determination
Abundance and biomass of zooplankton	OPC: Optical plankton Counter, microscopic counts and dry weight
Fecal pellets production (bulk and individual production)
Respiration	O2 consumption, CO2 release
Excretion	Dissolved inorganic ammonium and phosphate meas.
Ingestion	Chlorophyll though gut fluorescence
Egg production

Upper meso- and macrozooplankton

Parameters	Method
Abundance and size spectra	Specific plankton nets and Zooscan
Taxonomic determination	Zooscan + experts (microscopy)
Diel migration	Plankton net + Underwater Video Profiler
Vertical structure of assemblages	Underwater Video Profiler

Picoplankton

Parameters	Method
Planktonic abundance (specific verification)	Flow cytometry
Microplankton characterization	Lugol fixed samples
Abundance of specific picoeucaryote groups  Prasinophyceae  Photosynthetic stramenopiles  Prymnesiophyceae  Alveolates group I and II	FISH (fluorescent in situ hybridization)

Bacteria (including AAP)

Parameters	Method
Phylogenetic diversity	FISH, SSCP, clone library, sequencing
Abundance of AAnPB	Flow cytometry, image analysis, epifluorescence and quantitative PCR
Molecular diversity of AAnPB	Cloning and sequencing pufM gene

Virus

Parameters	Method
Viral diversity	Pulsed-field gel electrophoresis and DGGE

Coupling Biogeochemistry and biology

Parameters	Method
Phosphate uptake at a group of species specific level	Flow cytometry (cell sorting) and RA incorporation (33P)
Flow cytometry (cell sorting) and RA incorporation (33P) in combination with MICRO-FISH
Si deposition rates at a species specific level	New fluorescent probe PDMPO and RA incorporation (32Si)
Specific incorporation or organic molecules by individual phylogenetic groups of prokaryotes	Micro-autoradiography coupled to FISH

Specific material

Parameters	Method
Marine snow vertical abundance	Underwater Video Profiler
Marine snow size spectra (integrated every 5m)	Underwater Video Profiler
Diel marine snow dynamics	Underwater Video Profiler
Vertical flux of calculated marine snow carbon	Underwater Video Profiler
Effect of hydrostatic pressure on marine prokaryotes	High Pressure Serial Sampler
Effect of hydrostatic pressure on degradation of sinking particles	Particles Sinking Simulator
O2 production	O2PA : oxygen productivity autosampler

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Previsional Schedule, Location of Stations

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Figure 2: General location of the Mediterranean Sea ecosystems under investigation in the sub-project (red dots) and short “core parameters” stations (blue dots).
 A: Alboran Sea,
 B: central Western basin,
 C: central Ionian basin,
 D: central Levantine basin) and transects of short stations.

Details concerning the sampling grid strategy (below) and position of the in situ mooring (yellow) deployed before the 96 h of process study.

Day	location	Operations
J1		Boarding
J2-J4		Transit Toulon - site B
	Localisation of site B
J5	Site B (1st stay)	28h Process study
J6-J9	From Site B to site C	Transect 1 (11 short duration stations)
J10	Site C	Sampling grid
J11-J14	Site C	96 h Process study
J15-J16	From site C to site B	Transit
	Localisation of site B
J17	Site B	Sampling grid
J18-J21	Site B	96 h process study
J22 –J26	Site B	Transect 2 (11short duration stations)
	Localisation of site A
J27	Site A	Sampling grid
J28-J31	Site A	96 h process study
J32-J34		Transect 3 (5 short duration stations)
J35		Deboarding

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Equipment

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Material that should be asked to the DT INSU:

The research vessel will be “L’Atalante” with all basic equipment for hydrological work (Hull-mounted ADCP :75 KHz, 300 KHz) .

Material

Equipment	Laboratories
ISUS Nitrate	Université Dalhousie/Satlantic Inc.
Mass Spectrometer	LOB
Flow cytometer (FACSAria) Cell sorting	LOBB
Flow cytometer (FACSCalibur)	LOBB
APO (Oxygen Productivity Auto Sampler)	LMGEM
Autoanalyser Bran&Luebbe (Nutrients)	LOBB
CHN LECO 900	LOV
OPC laser (Optical Particule Counter)	LOB
TCO2/TA	Université de Perpignan
pCO2 analysis system	Université de Perpignan
Radiometer TRIOS (350 nm à 850 nm)	ELICO
Radiometer TRIOS (350 nm à 900 nm pas 3 nm)	ELICO
Optical grapp Wetlabs	ELICO
PVM	LOV
Plankton et nanoplankton nets	LOV, LOB, SBR
Zooscan	LOV
HPLC	Woods Hole Oceanographic Institution
Fluorimeter et phyto-PAM	SBR
Fluorimeter (Fast Repetition Rate fluorometer)	Oregon State University USA
Hybridation oven	IRD, SBR, LMGEM
Microscopes inverses à Epifluorescence microscopes	LOB, ELICO
Microscopes à épifluoresence droits	LMGEM, LOBB, SBR
PCR	LMGEM, IRD, SBR
Material for cellular cultures	LMGEM, LOBB, SBR
Electrophorese	IRD, LOBB, LOV
Flow injection for dissolved iron analysis	LOV
8 GoFlo bottles	LOV
Teflon pump for surface sampling	LOV
TOC-V analyser	LOBB
Scintillation counting (at laboratory)	LOB, LMGEM, LOBB
Spectronic Spectrophotometer 401	LOB
Autoanalyser (Nutrients)	LOB
1 Spectrofluorimeter Kontron SFM 25	LMGEM
2 culture lab for phytoplankton	SBR
4 aluminium containers for temperature controlled incubations	LMGEM
2 centrifuge	LOB, SBR
1 micro-centrifuge	LMGEM
OPC	LOB
Systeme for liquid nitrogen production + containers	SBR

 LOB : Laboratoire d’Océanographie et de Biogéochimie
 LMGEM : Laboratoire de Microbiologie Géochimie et Ecologie Marines
 LOBB : Laboratoire d’Océanographie Biologique de Banyuls
 LOV : Laboratoire d’Océanographie de Villefranche
 ELICO : Ecosystèmes Littoraux et Côtiers, Wimereux
 IRD : Institut de Recherche pour le Développement (antenne COM)
 SBR : Station biologique de Roscoff

Equipment DT INSU	quantity
CTDOD SBE 911+ rosette 24 + 24 bottles (With special frame for min DIG-UVP)	1
Fluorimeter	1
Transmissiometer	1
PAR surface and in situ	1
pinger	1
Winch and kevlar cable	1
Oranos cable (650 m) + associated equipment	1
teflon messengers for GoFlo bottles
Container INSU	1
Container for iron chemistry (20 feet clean Container)	1
Driffting sediment traps. (3 traps+ argos + gonio)	1
LADCP 300 Khz (INSU) Rosette mounted	1
ADCP (75 KHz, 300 KHz Ship mounted)
In situ chemical profiler	1
Milli Q water system	1
Packard scintillation counter	1

Equipment IFREMER	quantity
Container for Radio isotopes utilisation (14C, 32Si, 33P, 3H-leucine)	1

Laboratory containers provided by french laboratories : Container station biologique Roscoff