Gérald Grégori
email: gerald.gregori@mio.osupytheas.fr

Michel Denis

Sergio Sgorbati

Sandra Citterio

Resolution of Viable and Membrane-Compromised Free Bacteria in Aquatic Environments by Flow Cytometry

Keywords:

In aquatic environments, free heterotrophic bacteria play an extremely important role due to their high biomass, wide panel of metabolisms, and ubiquity, as well as the toxicity of certain species. This unit presents a nucleic-acid doublestaining protocol (NADS) for flow cytometry that can distinguish fractions of viable, damaged, or membrane-compromised cells within the free-bacterial community. The NADS protocol is based on the simultaneous utilization of two nucleic acid stains-membrane-permeant SYBR Green and membraneimpermeant propidium iodide (PI). The efficiency of the double staining on fresh samples is magnified by the FRET from SYBR Green to PI when both are bound to the nucleic acids. Full quenching of SYBR Green fluorescence by PI identifies cells with a compromised membrane, partial quenching indicates cells with a slightly damaged membrane, and lack of quenching characterizes cells with an intact membrane. Samples do not require any pretreatment and this protocol can be performed almost anywhere.

Cellular viability is a key parameter in the study of aquatic microbial assemblages and their relationships with the natural environment because it allows one to correlate total activities measured by a global method with the only fraction of active cells responsible for these activities [START_REF] Gasol | Significance of size and nucleic acid content heterogeneity as measured by flow cytometry in natural planktonic bacteria[END_REF]. Membrane integrity is one of the main criteria generally accepted to characterize a living cell and to distinguish it from a dead or damaged one. Membranes isolate the cell from the exterior medium, regulate ion or molecule exchanges between them, and are indispensable to the establishment of the proton electrochemical potential difference driving ATP synthesis at the bioenergetic-membrane level during oxidative phosphorylation [START_REF] Mitchell | Coupling of phosphorylation to electron and hydrogen transfer by chemiosmotic type mechanism[END_REF][START_REF] Nicholls | Bioenergetics. An Introduction to the Chemiosmotic Theory[END_REF].

In aquatic environments, free heterotrophic bacteria play an important role because of their high biomass, wide panel of metabolisms, and ubiquity, as well as the toxicity of certain species [START_REF] Porter | Fluorescent probes and flow cytometry new insights into environmental bacteriology[END_REF]. They are extremely small with relatively low amounts of cell constituents (Swan et al., 2013).

Bacteria are responsible for most of the mineralization of organic matter in bodies of water, and the determination of their abundance and biomass are basic parameters in related investigations [START_REF] Kirchman | High turnover rates of dissolved organic carbon during a spring phytoplankton bloom[END_REF][START_REF] Pomeroy | Significance of microorganisms in carbon and energy flow in marine ecosystems[END_REF][START_REF] Robertson | Determination of bacterial biomass from flow cytometric measurements of forward light scatter intensity[END_REF]. Although determination of the total abundance of bacteria is routinely performed [START_REF] Hobbie | Use of nuclepore filters for counting bacteria by fluorescence microscopy[END_REF][START_REF] Kogure | A tentative direct microscopic method for counting living marine bacteria[END_REF][START_REF] Klug | Direct measurements of natural planktonic bacterial community viability by flow cytometry[END_REF], quantification of the viable fraction is not easily achieved. This unit presents a nucleic-acid double-staining protocol (NADS) for flow cytometry that can distinguish fractions of viable, damaged, or membranecompromised cells within the marine or freshwater free-bacterial community, while distinguishing them from debris. Flow cytometry is particularly suited for analysis of aquatic bacteria because it is able to perform rapid multiparametric analysis on individual cells at cell numbers representative of natural environments [START_REF] Trousselier | Recent applications of flow cytometry in aquatic microbial ecology[END_REF].

Since heterotrophic bacteria do not contain natural photosynthetic pigments and are not autofluorescent, staining must be performed to discriminate and enumerate these bacteria by flow cytometry [START_REF] Marie | Enumeration of phytoplankton, bacteria, and viruses in marine samples[END_REF]. Some external cell structures may also prevent the fluorochromes from reaching their cellular target [START_REF] Gasol | Using flow cytometry for counting natural planktonic bacteria and understand the structure of planktonic bacterial communities[END_REF]. The NADS protocol is based on the simultaneous utilization of two nucleic acid stains-SYBR Green and PI. SYBR Green (I or II) is a membranepermeant dye [START_REF] Haugland | Handbook of Fluorescent Probes and Research Chemicals[END_REF], whereas PI is membrane impermeant [START_REF] Jones | An improved method to determine cell viability by simultaneous staining with fluorescein diacetatepropidium iodide[END_REF][START_REF] López-Amorós | Assessment of E. coli and Salmonella viability and starvation by confocal laser microscopy and flow cytometry using Rhodamine 123, DiBAC4(3), propidium iodide and CTC[END_REF][START_REF] Sgorbati | Characterization of number, DNA content, viability and cell size of bacteria from natural environments using DAPI PI dual staining and flow cytometry[END_REF][START_REF] Williams | Distinguishing between living and nonliving bacteria evolution of the vital stain propidium iodide and its combined use with molecular probes in aquatic samples[END_REF]. Both are readily excited by the blue line from an argon laser or an arc lamp, the excitation sources generally found in benchtop analytical flow cytometers. The efficiency of the double staining is magnified by the FRET from SYBR Green to PI when both are bound to the nucleic acids [START_REF] Barbesti | Evaluating the flow cytometric nucleic acid double staining protocol (NADS) in realistic planktonic bacterial death situations[END_REF], where the green fluorescence of SYBR Green is quenched by PI (for more details on FRET, refer to [START_REF] Szöllősi | Principles of resonance energy transfer[END_REF]). According to the FRET phenomenon, full quenching of SYBR Green fluorescence by PI will identify cells with compromised membranes fully permeable to PI, which will result in cells with red-only fluorescence. Partial quenching will indicate cells with a slightly damaged membrane that allows a small amount of PI to penetrate, which will result in cells with both red and green fluorescence. Lack of quenching will characterize cells with an intact membrane stained only by SYBR Green and are identified as viable by their green-only fluorescence. Figure 1 displays an example of this cluster resolution for an E. coli culture sample. This approach, originally described by [START_REF] Barbesti | Evaluating the flow cytometric nucleic acid double staining protocol (NADS) in realistic planktonic bacterial death situations[END_REF] for cultured bacteria, has been adapted to bacteria from fresh and marine waters [START_REF] Grégori | Resolution of viable and membranecompromised bacteria in freshwater and marine waters based on analytical flow cytometry and nucleic acid double staining[END_REF]. For marine samples, SYBR Green II is preferred over SYBR Green I because it shows higher fluorescence intensity [START_REF] Lebaron | Comparison of blue nucleic acid dyes for flow cytometric enumeration of bacteria in aquatic systems[END_REF].

Samples do not require any pretreatment and this protocol can be performed either on board a ship or in the laboratory. Samples must not be fixed to keep cells alive. The different clusters of bacteria-i.e., intact membranes (viable), damaged membranes, and compromised membranes-present in natural marine samples are discriminated on the basis of the green and red fluorescence of the cells after staining. Analogous fluorescence quenching could be achieved by using Hoechst dyes over SYBR Green [START_REF] Lebaron | Comparison of blue nucleic acid dyes for flow cytometric enumeration of bacteria in aquatic systems[END_REF][START_REF] Nebe-Von Caron | Viability assessment of bacteria in mixed populations using flow cytometry[END_REF]; however, those fluorochromes require ultraviolet excitation, which, in general, necessitates the use of more costly flow cytometers.

Materials

Natural fresh-or seawater samples SYBR Green I (fresh water) or II (seawater) working solution (see recipe) Fresh water or seawater (filtered through a 0.2-μm filter) Cells killed by paraformaldehyde fixation, heat, or ozone treatment Freshly harvested cells: freshly harvested natural sample with >95% viability or sample from a bacterial culture in exponential growth phase 1 mg/ml propidium iodide (PI, Molecular Probes), store ࣘ1 month at 4°C in the dark 10% bleach: dilute standard bleach (5% sodium hypochlorite) 1:10 in water 70% ethanol Sheath fluid: distilled water passed through a 0.2-μm filter 100-μm filters 12 × 75-mm tubes, sterile Vortexer Flow cytometer with 488-nm argon laser or arc lamp and filters: 525 ± 15-nm (green) fluorescence and >650-nm (red) fluorescence NOTE: All water, including fresh water or seawater used to dilute samples and used to prepare solutions, should be passed through a 0.2-μm filter prior to use.

Stain cells

1. Filter natural fresh-or seawater samples through a 100-μm filter to avoid clogging flow cytometer.

2a. For natural freshwater samples: Add 20 μl SYBR Green I working solution to 1.980 ml freshwater sample in a sterile 12 × 75-mm tube and vortex. In the same manner, also stain three controls:

i. 1.980 ml water (dye background levels) ii. Cells killed by paraformaldehyde fixation, heat, or ozone treatment (negative control) iii. Freshly harvested cells (positive control) 2b. For natural seawater samples: Add 20 μl SYBR Green II working solution to 1.980 ml seawater sample in a sterile 12 × 75-mm tube and vortex. In the same manner, also stain three controls:

i. 1.980 ml seawater (dye background levels)

ii. Cells killed by paraformaldehyde fixation, heat, or ozone treatment (negative control) iii. Freshly harvested cells (positive control).

The original 10,000× concentration of dye has been reduced to 1× for both types of sample.

3. Add 20 μl of 1 mg/ml PI (10 μg/ml final) and vortex. 4. Incubate 30 min at room temperature in the dark.

Calibrate flow cytometer

5. Run the water-only sample on a flow cytometer to localize the background noise due to the dyes and adjust the detection threshold to it.

6. Resolve viable, membrane-damaged, and membrane-compromised bacteria by defining three quadrants from the background noise limits as follows:

a. Run the negative (dead) control with all parameters set on logarithmic scale. b. Adjust the PMT voltages as necessary to ensure that red-fluorescent cells (>620 nm) are to the right-hand side of the display, and that their green fluorescence (515 to 530 nm) is as weak as possible.

Position the vertical axis of the quadrant at the limit between noise and dead cells (Fig. 1). Typical settings on a flow cytometer used in aquatic sciences, the FACSCalibur (BD biosciences), are SS = 430, green PMT = 450, and red PMT = 540 (Gasol et al., 2016).

Another advantage of the NADS protocol is that it can readily be used on most flow cytometers. Most flow cytometers collect:

i. Green fluorescence (GF) with a band-pass filter centered at 530 nm. This channel is sometimes labeled as FITC(fluorescein isothiocyanate), or FL1. ii. Orange fluorescence collected at 585 nm. This channel is sometimes labeled PE(phycoerythrin), or FL2. iii. Red fluorescence centered at ß650 nm. This channel is sometimes refered as PerCP (peridininchlorophyll protein complex), or FL3.

Instruments where detectors are by default labeled FL1, FL2, FL3, etc., follow, in most cases, the sequence green, orange, and red fluorescence according to their wavelengths (from shortest to longest). However, it is recommended to customize the names of the detectors to better fit the experimental set-up with the names of the fluorescent molecules responsible for the fluorescences. This allows data interpretation and comparison (with other instruments) much easier.

7. Run the positive (live) control. If necessary, adjust the PMT voltages to ensure that the green-fluorescent cells (525 nm) are to the upper-left side of the display and that their red fluorescence signal is as weak as possible. Position the horizontal axis of the quadrant at the limit between noise and viable cells (Fig. 1).

Analyze and resolve viable, membrane-damaged, and membrane-compromised bacteria

8. Analyze bacterial samples on the flow cytometer using the settings determined by the calibration steps.

9. Record the percentages, numbers, or abundances of viable (green only; upperleft quadrant), membrane-damaged (green and red; upper-right quadrant), and membrane-compromised (red only; bottom-right quadrant) bacteria (Fig. 1). If the sample is too concentrated, dilute with fresh water or seawater as appropriate.

10. Clean cytometer by running successive volumes of 10% bleach, 70% ethanol, and sheath fluid until <1 events/sec are detectable.

REAGENTS AND SOLUTIONS

SYBR Green I or II working solution

In a 1.5-ml microcentrifuge tube, dilute 10 μl SYBR Green I or 100 μl SYBR Green II (Molecular Probes) in 990 or 900 μl water, respectively. Store ࣘ1 month at 4°C in the dark.

membrane bacteria, respectively. On the other hand, green-only fluorescing bacteria represent the fraction of viable cells with preserved membrane integrity (Fig. 2).

Since the initial publication of this unit, several studies (>120) have used the NADS method in several cases where bacterioplankton is faced with either typical laboratory death-causing factors or natural mortality. It has been proven that its performance can be applied not only in surface samples, but also along vertical profiles, down to the mesopelagic zone [START_REF] Barbesti | Evaluating the flow cytometric nucleic acid double staining protocol (NADS) in realistic planktonic bacterial death situations[END_REF].

Based on the literature already published, the NADS protocol can be considered a robust tracer of natural heterotrophic prokaryote mortality (by evidence of damaged membranes) and can provide information on its temporal and spatial variations.

Critical Parameters

Flow cytometer calibration

It is always innocuous to keep the data rate <1000 particles/sec [START_REF] Gasol | Using flow cytometry for counting natural planktonic bacteria and understand the structure of planktonic bacterial communities[END_REF]. Analyze the sample, adjusting the flow rate and/or the cell concentration (by dilution) to avoid coincidence and saturation of the flow cytometer. Typically, the authors run samples for 60 sec at a flow rate of 10 μl/min and maintain a data rate <1000/sec by diluting samples if necessary. If this frequency is not exceeded, it is recommended to dilute sample to 2×, 5×, or 10× with filtered sample water to avoid any damage to cells. It is not necessary to add additional dye solution. Once the stains have linked to the nucleic acids of the cells prior to sample dilution, the molecules remain in the cell. Only unbound molecules (i.e., still in suspension) will dilute with the filtered water added to the sample.

of 9 COMMENTARY Background Information

To better understand the function of the microbial ecosystem, it is necessary to investigate the different activities of the microorganisms at the cellular level. Usually, bulk activity measurements refer to total counts and imply the involvement of all cells. This is not correct for natural samples because total counts may include ghost, dead, or damaged cells [START_REF] Gasol | Significance of size and nucleic acid content heterogeneity as measured by flow cytometry in natural planktonic bacteria[END_REF][START_REF] Zweifel | Total counts of marine bacteria include a large fraction of non-nucleoid-containing bacteria (ghosts)[END_REF]. It is therefore crucial to quantify the amounts of both viable and dead cells. The nucleic-acid double-staining (NADS) protocol developed by Barbesti and co-workers (Barbesti et al., 2000) for cultured bacteria in fresh water, and was adapted to natural samples from fresh and marine environments [START_REF] Grégori | Resolution of viable and membranecompromised bacteria in freshwater and marine waters based on analytical flow cytometry and nucleic acid double staining[END_REF], was developed with this goal in mind. This protocol specifically addresses membrane integrity, which is a widely accepted criterion for viability [START_REF] Joux | Use of fluorescent probes to assess physiological functions of bacteria at single-cell level[END_REF][START_REF] Nebe-Von Caron | Viability assessment of bacteria in mixed populations using flow cytometry[END_REF]. The general cell physiological condition can be addressed thanks to the membrane [START_REF] Tyndall | Application of flow cytometry to detection and characterization of Legionella spp[END_REF]. The loss of membrane integrity results in the collapse of cellular energetics and active transports, quickly leading to cell death [START_REF] Nebe-Von Caron | Viability assessment of bacteria in mixed populations using flow cytometry[END_REF]. The principle of the NADS protocol described in this unit is based on the excitation energy transfer from an excited donor (SYBR Green) to an acceptor molecule (PI). This fluorescence resonance energy transfer (FRET) occurs when the distance between nucleic acidbound molecules (PI and SYBR Green) is <70 ˚A because the emission spectrum of SYBR Green (maximum emission at 520 nm) covers the absorption spectrum of PI (maximum absorption at 535 nm). Thus, on the one hand, red-only fluorescing bacteria and cells emitting both green and red fluorescence represent the fractions of dead and damaged Quadrant A includes green-fluorescent cells stained by SYBR Green only and identified as viable because membrane integrity prevents uptake of PI. Quadrant B contains green-plus redfluorescent cells stained by both SYBR Green and PI and identified as membrane-damaged cells because they enable various amounts of PI to penetrate the cell and bind to nucleic acid, inducing a corresponding increase of red fluorescence and decrease of green fluorescence, depending on the extent of FRET from SYBR Green to PI. Quadrant D includes red-fluorescent cells, which are identified as dead because the total quenching of SYBR Green fluorescence by PI reveals the presence of highly compromised membranes. Background noise is confined to quadrant C. Note that with the NADS protocol it is possible to distinguish the two E. coli cultures by their relative nucleic-acid content.

Sample double staining

The NADS protocol is based on a FRET mechanism between PI and SYBR Green I or II; therefore, it is important to simultaneouldy double stain each sample. [START_REF] Barbesti | Evaluating the flow cytometric nucleic acid double staining protocol (NADS) in realistic planktonic bacterial death situations[END_REF] reported that incubation with a single dye first would end up with results difficult to interpret.

Threshold limit

Set the discriminator to green and red fluorescence if possible (e.g., Cytoron Absolute Count flow cytometer) or on the FS or SS signals. Adjust the threshold to the background noise to improve the signal-tonoise ratio to better resolve the different clusters of bacteria and avoid instrument saturation.

Preserving sample

Determination of bacterial viability with the NADS protocol is performed only on fresh natural samples. The samples must not be fixed, but must be analyzed as soon as possible after collection. Because the analyses generally must be done in the laboratory, the authors suggest keeping samples in the dark and cold (4ºC) during transport to preserve freshness.

Troubleshooting

Optimal dye concentrations

In natural samples containing insufficient concentrations of bacteria (i.e., <0.5 × 10 6 cells/ml), the background fluorescence of SYBR Green dyes and PI can be high enough to affect signal quality. To resolve this problem, it is necessary to adapt the concentrations of the fluorescent probes to that of bacteria by decreasing the added amount of dye working solution. For example, a final PI concentration of 1 μg/ml and a final dilution of 1/100,000 (v/v) for the SYBR Green I commercial solution are optimal for freshwater samples with <0.5 × 10 6 bacteria/ml. For more concentrated samples (up to 20 × 10 6 bacteria/ml), 10 μg PI/ml and a final dilution of 1/10,000 (v/v) of SYBR Green I or 1/1000 (v/v) of brane permeability with respect to the fluorescent probes in use. Addressing membrane integrity, the NADS protocol provides resolution of bacteria into viable (active or inactive), membrane-damaged, and membranecompromised groups, but other complementary approaches are needed to independently demonstrate the presence of specific activity or metabolism, as well as the capacity of a cell to undergo division [START_REF] Lloyd | Vigor, vitality and viability of microorganisms[END_REF]1995;[START_REF] Mcfeters | Physiological assessment of bacteria using fluorochromes[END_REF][START_REF] Porter | Fluorescent probes and flow cytometry new insights into environmental bacteriology[END_REF][START_REF] Grégori | Resolution of viable and membranecompromised bacteria in freshwater and marine waters based on analytical flow cytometry and nucleic acid double staining[END_REF].

Time Considerations

The NADS protocol can be achieved in <1 hr and yields both quantitative (i.e., cell abundance) and qualitative (i.e., viable, membrane-damaged, and membranecompromised bacteria; nucleic acid content) information. This protocol is compatible with routine sampling and analyses and with the high-frequency sampling required to perform spatial or temporal monitoring of bacteria in aquatic environments. High-frequency sampling is not feasible with other methods that are much more time-consuming than flow cytometry, such as epifluorescence microscopy and acridine-orange or DAPI staining on membrane filters [START_REF] Hobbie | Use of nuclepore filters for counting bacteria by fluorescence microscopy[END_REF][START_REF] Zimmerman | A new method for fluorescence staining of bacterial populations on membrane filters[END_REF].

SYBR Green II have found to be best for bacterial cultures. [START_REF] Barbesti | Evaluating the flow cytometric nucleic acid double staining protocol (NADS) in realistic planktonic bacterial death situations[END_REF] suggested that the ideal set of concentrations for PI and SYBR Green may vary depending upon the amount and severity of the mortality induced on the bacteria. In this study, it was reported that concentrations in the range of 5 to 20 μg/ml of propidium iodide, equivalent to a 10× concentration of SYBR green I, would be optimal for detecting simultaneously "live" (green-only cells) and "dead" (red-only cells) bacteria.

This showed that working with a commercial kit (such as the BacLight kit), where dyes are present in predefined concentrations, is less efficient than combining two separate dyes, with concentrations adapted by the user to fit the sample conditions.

Instrument sterility

The 0.2-μm filtered distilled water used as the sheath fluid ensures the sterility of the flow cytometer and minimizes background particlescatter noise. Nevertheless, it is also important to clean the flow cytometer when the analyses are finished in order to remove residual bacteria or dye molecules from the fluidic system. The authors accomplish this by running the following solutions through the flow cytometer: (1) 10% bleach solution, (2) 70% ethanol solution, and (3) 0.2-μm filtered distilled water. The flow cytometer is considered clean when the detection rate is <1 event/sec.

Anticipated Results

The NADS protocol is an assay based on analytical flow cytometry that allows one to distinguish viable from membrane-damaged and membrane-compromised bacteria and to sort out noise and debris. Viable cells include both active (e.g., measurable metabolic activity) and inactive states (e.g., lack of enzyme activity in nutrient-depleted environments; [START_REF] Nebe-Von Caron | Viability assessment of bacteria in mixed populations using flow cytometry[END_REF]. This protocol should not be considered a strict live/dead assay, but rather as a proxy whose efficiency depends on the characteristics (cell type, physiologic state, and so on) of the cells occurring in natural samples where several populations in different physiologic and cytostructural terms can be simultaneously encountered. This large heterogeneity of bacteria in aquatic samples, where most of the species are still unknown, is the main limitation to the NADS assay as well as to other protocols using fluorochromes. Some unknown species may not be consistent with observations made thus far regarding bacterial mem-

Figure 1

 1 Figure1Determination of the A, B, and D quadrants used to resolve viable, membranedamaged, and membrane-compromised bacteria (dead cells), respectively, on a green-versus red-fluorescence histogram. On the left, positioning of the vertical limit between red-fluorescing dead bacteria and the background noise of an ozone-treated sample. On the right, positioning of the horizontal limit between green-fluorescing viable bacteria and background noise in an untreated sample containing mainly viable bacteria.

Figure 2

 2 Figure 2 Application of the NADS protocol to a sample containing two different E. coli cultures. Flow cytometric analysis yields this typical cytogram characterizing cells by their fluorescence. Quadrant A includes green-fluorescent cells stained by SYBR Green only and identified as viable because membrane integrity prevents uptake of PI. Quadrant B contains green-plus redfluorescent cells stained by both SYBR Green and PI and identified as membrane-damaged cells because they enable various amounts of PI to penetrate the cell and bind to nucleic acid, inducing a corresponding increase of red fluorescence and decrease of green fluorescence, depending on the extent of FRET from SYBR Green to PI. Quadrant D includes red-fluorescent cells, which are identified as dead because the total quenching of SYBR Green fluorescence by PI reveals the presence of highly compromised membranes. Background noise is confined to quadrant C. Note that with the NADS protocol it is possible to distinguish the two E. coli cultures by their relative nucleic-acid content.

Acknowledgements

The authors wish to acknowledge the support of the French-Italian Integrated Action Program "GALILEE" and of the Council of the Provence-Alpes-Côte d'Azur Region. The authors would also like to acknowledge Sam Duncan, who provided E. coli cultures.

Key References

 [START_REF] Barbesti | Evaluating the flow cytometric nucleic acid double staining protocol (NADS) in realistic planktonic bacterial death situations[END_REF]. See above. The original presentation of the NADS protocol. [START_REF] Grégori | Resolution of viable and membranecompromised bacteria in freshwater and marine waters based on analytical flow cytometry and nucleic acid double staining[END_REF] See above. This article describes the adaptation of the NADS protocol described by [START_REF] Barbesti | Evaluating the flow cytometric nucleic acid double staining protocol (NADS) in realistic planktonic bacterial death situations[END_REF] to natural fresh and marine water samples.

Internet Resources

https://www.probes.com/servlets/pis This Web site displays technical bulletins on Molecular Probes fluorochromes with useful storage and handling information and suggestions for their use.


Emilio Garcia-Robledo

Cory C Padilla

Montserrat Aldunate

Frank J Stewart

Osvaldo Ulloa

Aurélien Paulmier

Gérald Grégori

Niels Peter Revsbech

Gerald Gregori

Cryptic oxygen cycling in anoxic marine zones

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Oxygen availability drives changes in microbial diversity and biogeochemical cycling between the aerobic surface layer and the anaerobic core in nitrite-rich anoxic marine zones (AMZs), which constitute huge oxygen-depleted regions in the tropical oceans. The current paradigm is that primary production and nitrification within the oxic surface layer fuel anaerobic processes in the anoxic core of AMZs, where 30-50% of global marine nitrogen loss takes place. Here we demonstrate that oxygenic photosynthesis in the secondary chlorophyll maximum (SCM) releases significant amounts of O 2 to the otherwise anoxic environment. The SCM, commonly found within AMZs, was dominated by the picocyanobacteria Prochlorococcus spp. Free O 2 levels in this layer were, however, undetectable by conventional techniques, reflecting a tight coupling between O 2 production and consumption by aerobic processes under apparent anoxic conditions. Transcriptomic analysis of the microbial community in the seemingly anoxic SCM revealed the enhanced expression of genes for aerobic processes, such as nitrite oxidation. The rates of gross O 2 production and carbon fixation in the SCM were found to be similar to those reported for nitrite oxidation, as well as for anaerobic dissimilatory nitrate reduction and sulfate reduction, suggesting a significant effect of local oxygenic photosynthesis on Pacific AMZ biogeochemical cycling.

Prochlorococcus | oxygen minimum zone | secondary chlorophyll maximum | metatranscriptomics | aerobic metabolism I n coastal zones of the eastern tropical Pacific Ocean, the up- ward transportation of nutrient-rich waters results in relatively Nitrite is also produced and consumed in the aerobic nitrification pathway involving the two-step process of aerobic ammonia and nitrite oxidation [START_REF] Dalsgaard | Anammox and denitrification in the oxygen minimum zone of the eastern South Pacific[END_REF][START_REF] Babbin | Organic matter stoichiometry, flux, and oxygen control nitrogen loss in the ocean[END_REF]. Despite the absence of measureable O 2 in the core of eastern Pacific AMZs, biomolecular evidence (DNA, RNA, and proteins) indicates the presence of aerobic microbial processes. The expression of genes encoding for nitrification and other O 2 -dependent microbial metabolisms, potentially including heterotrophic respiration, have been found well below the oxycline [START_REF] Stewart | Microbial metatranscriptomics in a permanent marine oxygen minimum zone[END_REF][START_REF] Kalvelage | Aerobic microbial respiration in oceanic oxygen minimum zones[END_REF], raising the question of how aerobic processes could persist under apparent anoxia.

In the three oceanic AMZs of the Arabian Sea, ETNP, and ETSP, dense populations of phototrophs have been observed at the base of the photic zone but below the oxycline that separates oxic from anoxic waters [START_REF] Lavin | Novel lineages of Prochlorococcus thrive within the oxygen minimum zone of the eastern tropical South Pacific[END_REF][START_REF] Goericke | A novel niche for Prochlorococcus sp in low-light suboxic environments in the Arabian Sea and the Eastern Tropical North Pacific[END_REF][START_REF] Cepeda-Morales | Effect of the oxygen minimum zone on the second chlorophyll maximum in the Eastern Tropical Pacific off Mexico[END_REF]. This deep secondary chlorophyll maximum (SCM) is mainly composed of novel, yet uncultivated, lineages of the cyanobacterium Prochlorococcus [START_REF] Lavin | Novel lineages of Prochlorococcus thrive within the oxygen minimum zone of the eastern tropical South Pacific[END_REF], with chlorophyll concentrations that can equal that of the primary chlorophyll peak near the surface [START_REF] Cepeda-Morales | Effect of the oxygen minimum zone on the second chlorophyll maximum in the Eastern Tropical Pacific off Mexico[END_REF]. The presence of this large population of putative oxygenic phototrophs has suggested a mechanism by which aerobic metabolism can be maintained in a zone where in situ measurements indicate anoxic conditions [START_REF] Ulloa | Microbial oceanography of anoxic oxygen minimum zones[END_REF]. Although an active photosynthetic community produces and releases oxygen to the environment, coupled O 2 consumption by an aerobic microbial community may keep seawater O 2 concentration at very low and possibly subnanomolar levels, thereby resulting in a cryptic O 2 cycle. The existence of such a cryptic Significance Anoxic marine zones (AMZs) create expansive habitats for microbes whose anaerobic metabolisms help drive global nutrient cycles, for example, by removing nitrogen from the oceans by producing N 2 gas. AMZ cycles may also be shaped by oxygen intrusion from outside the AMZ, creating opportunities for aerobic microbial metabolisms. Here we show that aerobic processes in AMZs are linked to oxygen production within the anoxic zone. Oxygen is produced during daytime in a layer of photosynthetic cyanobacteria near the top of the AMZ and then rapidly consumed by aerobic processes without accumulating. Oxygen turnover and carbon fixation rates are comparable to those of microbial N 2 production, suggesting an important role for internal oxygen cycling in AMZ transformations of matter and energy.

high primary productivity at surface depths. Sinking of organic matter produced by surface production coupled with sluggish circulation leads to the formation of oxygen-deficient water masses at intermediate depths below the mixed layer. Due to strong stratification, these oxygen minimum zones (OMZs) extend far offshore over vast swaths of the eastern Pacific. In these regions, oxygen availability plays a major role in structuring organism distributions and biogeochemical processes in the pelagic ocean [START_REF] Paulmier | Oxygen minimum zones (OMZs) in the modern ocean[END_REF].

Recently developed sensor techniques [START_REF] Revsbech | Construction of STOX oxygen sensors and their application for determination of O 2 concentrations in oxygen minimum zones[END_REF] show that in much of the OMZ water column, from about 30-100 m to about 800 m, O 2 concentrations fall below sensor-specific detection limits of down to 3 nmol•L -1 (3•10 -9 moles per liter) [START_REF] Thamdrup | Widespread functional anoxia in the oxygen minimum zone of the Eastern South Pacific[END_REF][START_REF] Tiano | Oxygen distribution and aerobic respiration in the north and south eastern tropical Pacific oxygen minimum zones[END_REF]. OMZs in the eastern tropical North and South Pacific (ETNP and ETSP, respectively) and in the Arabian Sea are subject to such intense O 2 depletion and therefore have been redefined as anoxic marine zones (AMZs) [START_REF] Ulloa | Microbial oceanography of anoxic oxygen minimum zones[END_REF]. In other oceanic OMZs, including in the Bay of Bengal and northeast Pacific, oxygen concentrations may decrease to a few micromolar, but total O 2 depletion occurs only occasionally [START_REF] Bristow | N2 production rates limited by nitrite availability in the Bay of Bengal oxygen minimum zone[END_REF]. AMZs are often distinguished from more oxygenreplete OMZs by the accumulation of nitrite, which is typically most pronounced when O 2 falls below the nanomolar detection limit [START_REF] Ulloa | Microbial oceanography of anoxic oxygen minimum zones[END_REF][START_REF] Bristow | N2 production rates limited by nitrite availability in the Bay of Bengal oxygen minimum zone[END_REF][START_REF] Canfield | A cryptic sulfur cycle in oxygen-minimum-zone waters off the Chilean coast[END_REF][START_REF] Ganesh | Size-fraction partitioning of community gene transcription and nitrogen metabolism in a marine oxygen minimum zone[END_REF]. Nitrite is a key substrate in microbial N 2 and N 2 O production by either denitrification or anaerobic ammonium oxidation (anammox), which together in AMZs mediate 30-50% (9) of the marine recycling of inorganic nitrogen compounds (nitrate, nitrite, and ammonium) to atmospheric N 2 .

oxygen cycle has been suggested by biomolecular evidence [START_REF] Stewart | Microbial metatranscriptomics in a permanent marine oxygen minimum zone[END_REF] but has not yet been demonstrated.

In this study we used a combination of high-resolution oxygen profiling, metabolic rate measurements, and community mRNA sequencing to explore the potential for oxygen cycling in the SCMs of the ETNP off Mexico and the ETSP off Peru. Our results show that the photosynthetic community of the SCM produces significant amounts of O 2 , sufficient to maintain an aerobic community in an otherwise anoxic environment. Rates of O 2 production and carbon fixation in the SCM in both ETNP and ETSP AMZs are comparable to previously measured rates of aerobic processes like nitrite and ammonium oxidation [START_REF] Ganesh | Size-fraction partitioning of community gene transcription and nitrogen metabolism in a marine oxygen minimum zone[END_REF][START_REF] Bristow | Ammonium and nitrite oxidation at nanomolar oxygen concentrations in oxygen minimum zone waters[END_REF], as well as anaerobic AMZ processes like denitrification, anammox, and sulfate reduction [START_REF] Canfield | A cryptic sulfur cycle in oxygen-minimum-zone waters off the Chilean coast[END_REF][START_REF] Ganesh | Size-fraction partitioning of community gene transcription and nitrogen metabolism in a marine oxygen minimum zone[END_REF]. Although the measured metabolic rates exhibit large spatial and temporal variability, our data collectively suggest a significant effect of local photosynthesis on the biogeochemical cycling in Pacific Ocean AMZs.

Results and Discussion

Sampling in both the ETNP and ETSP revealed a typical AMZ O 2 distribution in the upper 200 m of the water column. Oxygen concentrations in the 0-35 m surface layer in the ETNP were stable at ∼200 μmol•kg -1 , before declining along a clearly defined oxycline from 35-45 to 60-80 m, and then falling below the detection limit of the switchable trace amount oxygen (STOX) sensors (few nanometers) at 80-100 m (Fig. 1B). In the ETSP AMZ off Peru, O 2 concentrations and the depth of the oxycline were more variable and clearly influenced by proximity to the shore, with anoxic depths beginning at ∼30 m at the coastal station but at ∼70 m for the more oceanic station (Fig. 1H). In both the ETNP and ETSP, the chlorophyll concentration below the primary maximum decreased in parallel with O 2 concentration, reaching a minimum before complete O 2 depletion and then increasing again to form an SCM in which 90% of the phototrophs were Prochlorococcus (Fig. 1 and Table S1). Although the upper region of the SCM was consistently located near the oxic-anoxic interface, maximum in vivo fluorescence and Prochlorococcus abundance were usually localized within the anoxic zone a few meters below. Low O 2 concentrations (<500 nmol•L -1 ) were occasionally found inside the SCM (Table S2), suggesting intrusion of overlying oxygenated waters or in situ O 2 production and accumulation.

Oxygenic Photosynthesis and Carbon Fixation in the SCM. Shipboard experiments using water from the SCM incubated under trace O 2 conditions revealed that O 2 concentration with time differed substantially between dark-and light-incubated samples (Fig. 2 A and D). Net community production (NCP), corresponding to the slope of the O 2 concentration curves and hence the balance between O 2 production and consumption, gradually increased to more positive values with increasing irradiance. NCP was also variable between stations, reflecting the spatial and temporal variability of the metabolic activity in terms of photosynthesis and respiration rates (Fig. 2 B andE). At several stations, net consumption of O 2 occurred at all applied irradiances, although a clear decrease in consumption rate was always measured with increasing light intensities. At other stations, a net increase in O 2 was measured when the samples were exposed to an irradiance of only 10 μmol photons•m -2 •s -1 . The observed maximum irradiance in situ was, however, only in the range of 2-5 μmol photons•m -2 •s -1 at most stations (see examples in Fig. S1); at such low light levels, net O 2 consumption was always observed. for the SCM of the Arabian Sea [START_REF] Johnson | Energetics and growth kinetics of a deep Prochlorococcus spp. population in the Arabian Sea[END_REF], whereas the ETSP SCM values were more similar to those from the laboratory cultures. Although it is not yet possible to directly quantify in situ O 2 transformations in the AMZ, in situ GCP rates can be estimated based on water column chlorophyll concentrations and light conditions (Fig. S1). The light intensity at the SCM was variable and almost always substantially below 10 μmol photons•m -2 •s -1 . Under such conditions, O 2 production rates are lower than potential respiration rates (R*), and the O 2 produced is immediately consumed by the microbial community, resulting in a cryptic O 2 cycle in the seemingly anoxic environment of the SCM (Fig. 1). However, at some stations the irradiance in the SCM was similar or close to the E k . The occasional detection of low O 2 concentrations in the SCM (4, 8) (Table S2) may thus be explained by photosynthetic activity in the SCM increasing O 2 concentrations to measurable levels. Such daily changes are difficult to measure by discrete sampling, but recurrent measurements in the same water mass might reveal hourly and daily changes in the SCM.

Oxygen Production Coupling with Aerobic Microbial Processes. Even if undetectable, O 2 production in the SCM may support important (micro)aerobic metabolisms. To explore this prediction, we looked for signatures of such aerobic metabolism in available metatranscriptomes along the AMZ depth gradient in the ETNP during two cruises in 2014 and 2013, focusing on station T6 where the SCM was well developed and for which the metatranscriptome dataset was most comprehensive. Transcripts encoding terminal oxidases, including both LATO and HATO (Table S4), were detected at all depths (Fig. 3 and Fig. S2), including deep within the AMZ, where the transcript pool was dominated by sequences affiliated with diverse Gammaproteobacteria and Alphaproteobacteria (Fig. S3). The presence of oxidase transcripts within anoxic marine waters has been reported previously [START_REF] Kalvelage | Aerobic microbial respiration in oceanic oxygen minimum zones[END_REF] and may reflect constitutive expression by groups at high abundance in the AMZ core, potentially to capitalize quickly on O 2 if it becomes available [START_REF] Tsementzi | SAR11 bacteria linked to ocean anoxia and nitrogen loss[END_REF]. The relative abundance of both LATO and HATO transcripts exhibits a local peak within the SCM compared with depths immediately above (base of oxycline) and below the SCM (Fig. 3 and Fig. S2). Similar trends were observed at stations T4 and T10, although limited sampling affected our ability to fully resolve oxidase distributions immediately above the SCM at these sites (Fig. S2). Together, these data provide evidence of a local peak in O 2 scavenging within the SCM.

Oxygen produced in the SCM may also be consumed through key steps of the OMZ nitrogen cycle. Comparatively high rates of autotrophic nitrification (ammonia and nitrite oxidation) are known to occur close to the oxic-anoxic boundary of AMZs [START_REF] Dalsgaard | Anammox and denitrification in the oxygen minimum zone of the eastern South Pacific[END_REF]. Here transcripts affiliated with ammonia oxidizing bacteria Tracking O 2 consumption during our experiments allowed for estimates of aerobic respiration rates. O 2 consumption curves were linear down to about 50 nmol•L -1 during dark incubations (Fig. 2 A andD). Aerobic respiration by prokaryotes is generally driven by two classes of terminal oxidases: low-affinity terminal oxidases (LATO) with a half saturation constant (K m ) of about 200 nmol O 2 •L -1 and high-affinity terminal oxidases (HATO) with K m values of 3-8 nmol O 2 •L -1 [START_REF] Morris | Shallow breathing: Bacterial life at low O(2)[END_REF]. Marine bacteria possessing HATO can decrease apparent K m values of aerobic respiration down to less than 10 nmol O 2 •L -1 [START_REF] Gong | Respiratory kinetics of marine bacteria exposed to decreasing oxygen concentrations[END_REF], and a linear O 2 decrease may thus be expected down to about 50 nmol O 2 •L -1 . Therefore, O 2 consumption rates (referred to as respiration for simplicity) obtained at concentrations >50 nmol•L -1 represent potential respiration rates (R*) because they were measured above the threshold of O 2 limitation. The estimated R* rates were significantly higher in the ETSP compared with the ETNP (Fig. 2 and Table S2), consistent with a higher microbial and particle abundance measured in the ETSP (Fig. 1 and Tables S1 andS2).

3.17nM h -1 1.33nM h -1 -0.37nM h -1 -3.
Experiments under the unique, almost anoxic conditions, of AMZs have not been performed in previous measurements of photosynthetic activity in the SCM [START_REF] Johnson | Energetics and growth kinetics of a deep Prochlorococcus spp. population in the Arabian Sea[END_REF]. We conducted our experiments at O 2 levels below those sporadically detected by in situ measurements (up to 500 nmol•L -1 ) but far above the K m values for HATO. In this range, we can assume that gross community production of O 2 (GCP-O 2 ) can be calculated as the sum of NCP and R*. We also validated these production calculations by simultaneously measuring the incorporation of inorganic carbon (using 13 Cor 14 C) into biomass [gross community carbon production (GCP-C)], as has been done previously to quantify Prochlorococcus carbon fixation [START_REF] Johnson | Energetics and growth kinetics of a deep Prochlorococcus spp. population in the Arabian Sea[END_REF][START_REF] Moore | Photophysiology of the marine cyanobacterium Prochlorococcus: Ecotypic differences among cultured isolates[END_REF]. Both GCP-O 2 and GCP-C followed a classical photosynthesis-irradiance curve (Fig. 2 C and F), with maximum (GCP max ) values above saturating light intensities (E k ) of 10.5 ± 2.0 and 21.4 ± 9.3 μmol photons•m -2 •s -1 (0.5 and 1% of the incident light) for the ETNP and ETSP, respectively. The low E k values reflect adaptation to the dim light environment, being similar to values found for the SCM community in the Arabian Sea [START_REF] Johnson | Energetics and growth kinetics of a deep Prochlorococcus spp. population in the Arabian Sea[END_REF] or in Prochlorococcus cultures [START_REF] Moore | Photophysiology of the marine cyanobacterium Prochlorococcus: Ecotypic differences among cultured isolates[END_REF]. Above E k , mean GCP max -O 2 values in the ETNP and ETSP were 16.6 ± 9.1 and 52.5

± 30.4 nmol O 2 •L -1 •h -1
, respectively, and were generally consistent with maximum GCP-C rates (GCP max -C: 8.1 ± 11.2 and 44.4 ± 30.3 nmol C•L -1 •h -1 in the ETNP and ETSP, respectively) (mean values of all stations ± SD in Table S3 and model in Fig. 2 C and F). The parameters describing the photosynthesis characteristics of the SCM community (maximum gross production rates, photosynthetic efficiency, and E k ) were similar to the values previously found for the SCM community of the Arabian Sea and the characterization of several Prochlorococcus isolates from the Pacific Ocean [START_REF] Johnson | Energetics and growth kinetics of a deep Prochlorococcus spp. population in the Arabian Sea[END_REF][START_REF] Moore | Photophysiology of the marine cyanobacterium Prochlorococcus: Ecotypic differences among cultured isolates[END_REF]. The values found for the ETNP were similar to those found the SCM was calculated by integrating the GCP profiles (Fig. S1) over a diel cycle, using measured (ETSP) or estimated (ETNP) scalar irradiance profiles (Fig. S4 and Table 1). Higher chlorophyll and estimated irradiance values at the SCM in the ETNP off Mexico resulted in higher mean production rates (0.83/0.39 mmol

O 2 /C•m -2 •d -1 ) compared with the ETSP off Peru (0.32/0.31 mmol O 2 /C•m -2 •d -1
). Although in situ light attenuation profiles were used for the ETNP, cloud coverage and other local factors reducing the incident light could not be included in the calculations, (AOB) and ammonia oxidizing archaea (AOA), notably those encoding the ammonia monooxgynease (amo) enzyme catalyzing aerobic ammonia oxidation, peaked in the upper part of the oxycline and declined in abundance into the core of the ETNP AMZ (Fig. 3 and Fig. S2). In contrast, transcripts of nitrite oxidizing bacteria (NOB), primarily those of the marine NOB genus Nitrospina, spiked within the SCM, coinciding in most cases with a local enrichment in transcripts encoding nitrite oxidoreductase (nxr) (Fig. 3 and Fig. S2). A prior study showed that potential nitrite oxidation rates at station T6 in the ETNP peaked in the anoxic SCM at 10.8 nmol N•L -1 •h -1 , a rate approximately double that of the maximal O 2 respiration rate measured in this study (Table S3). Taking the stoichiometry of nitrite oxidation into account, we can infer that most of the measured O 2 consumption (R*) could be due to nitrite oxidation. The nitrite oxidation rates reported at the same stations were measured at low O 2 concentrations (<80 nmol•L -1 ), and we assume that the conditions were similar to our incubations, suggesting that the previous nitrite oxidation and our present R* rates are directly comparable. The balance between heterotrophic and nitrite oxidizer O 2 consumption may, however, vary as a function of the actual O 2 concentration in the 0 to ∼100 nmol•L -1 range that we measured in the ETNP SCM (Table S2). The cooccurrence of elevated Nitrospina transcription and nitrite oxidation rates in the SCM suggests that NOB is fueled by local O 2 production.

Implications for Oxygen Minimum Zones. The results of this study indicate that the SCM is a significant source of O 2 for both nitrite and organic matter oxidation, as well as a source of fixed carbon. Total productivity in terms of O 2 released and C fixed in community) were analyzed by staining the cells with SYBR Green (Molecular Probes) as described previously [START_REF] Lebaron | Comparison of blue nucleic acid dyes for flow cytometric enumeration of bacteria in aquatic systems[END_REF][START_REF] Grégori | Resolution of viable and membranecompromised free bacteria in aquatic environments by flow cytometry[END_REF].

Oxygen Production and Carbon Fixation Measurements. Water samples from the SCM (summarized in Table S1) were collected using Niskin bottles or a PPS. To minimize the O 2 leaking from the polymers of the Niskin bottles, the water was transferred to a 20-L glass bottle previously purged with N 2 gas as soon as the rosette was on deck. If the samples were collected using the PPS, the 20-L glass bottle purged with N 2 gas was filled directly from the outlet of the PPS. A certain O 2 contamination (1-5 μmol•L -1 ) during the sampling procedure could not be avoided, and the seawater was therefore immediately degassed in the 20-L bottle by bubbling with N 2 + 0.05% CO 2 . A STOX sensor was inserted inside the bottle to determine when anoxia was approached (<100 nmol O 2 •L -1 ). After adjusting the O 2 concentration to 100-400 nmol•L -1 , samples were siphoned to custom made incubation vessels (n = 12-16) (Fig. S5) [START_REF] Tiano | A new highly sensitive method to assess respiration rates and kinetics of natural planktonic communities by use of the switchable trace oxygen sensor and reduced oxygen concentrations[END_REF][START_REF] Garcia-Robledo | Determination of respiration rates in water with sub-micromolar oxygen concentrations[END_REF], containing either STOX sensors (ETSP) or a combination of STOX and optode sensors with a measuring range of 0-1,000 nmol•L -1 (32, 33) (ETNP). Each vessel was placed inside a light incubation tube immersed in a constant temperature water bath, enabling maintenance of in situ temperature (14-15 °C) and quantification of very low O 2 transformation rates. The light incubation tubes consisted of a black PVC tube with white LEDs (LF06S-W3F-850; OSRAM) installed along the whole periphery of the tube and with a custom-built waterproof magnetic stirrer fitted at the bottom. The LEDs were covered with a blue filter (131 Marine Blue filter; LEE Filters) to simulate the in situ light spectrum. Oxygen concentrations (Fig. 2) throughout the incubation period (8-12 h) were measured in treatments spanning a range of bluish light intensities slightly above maximum in situ levels (10, 20, and 40 μmol photons•m -2 •s -1 ) and in darkness (n = 3-4, per treatment). Rates of oxygen consumption or production (here named NCP) were obtained by linear regression of the oxygen evolution during the incubations. GCP rates were calculated by subtracting the mean respiration value (NCP rate measured in darkness) from the NCP rates measured at different irradiances.

Rates of carbon incorporation were measured simultaneously during the incubations for oxygen measurements using stable (ETNP) or radioactive (ETSP) isotopes. Incubations amended with Na 14 C-HCO 3 (450 μCi/L final concentration) were done in parallel incubation bottles of only 110 mL (but otherwise similar to the one described in Fig. S5) following the procedure described by Telling et al. [START_REF] Telling | Measuring rates of gross photosynthesis and net community production in cryoconite holes: A comparison of field methods[END_REF]. Incubations amended with Na 13 C-HCO 3 (0.27 mM 13 C final concentration) were done in the same incubation bottles used for O 2 measurements. Incorporation of 14 C was measured by counting on a Perking Elmer Tri-Carb 2900 TR scintillation counter, whereas the 13 C incorporation was analyzed in an Elemental Analyzer (Thermo Elemental Analyzer Flash EA 1112 HT) coupled to an Isotope Ratio Mass Spectrometer (Delta V; Thermo Scientific). The 13 C enrichment in the produced organic carbon was calculated as the difference between the amounts of 13 C in the sample minus the natural 13 C abundance measured on blank filters. Decays per minute values ( 14 C incubations) and 13 C incorporation were converted to 12 C uptake values or GCP (nmol C•L -1 •h -1 ) rates using the formula described in Telling et al. [START_REF] Telling | Measuring rates of gross photosynthesis and net community production in cryoconite holes: A comparison of field methods[END_REF].

Rates Modeling and Upscaling of Processes. The photosynthesis-irradiance model described by Jassby and Platt [START_REF] Jassby | Mathematical formulation of the relationship between photosynthesis and light for phytoplankton[END_REF] was fitted to the measured GCP (nmol•L -1 •h -1 ) rates for both O 2 production and C assimilation (Fig. 2), being

GCP = GCP max × tanhðα × E=GCP max Þ, where GCP max (nmol•L -1 •h -1
) is the maximum gross community production rate, reached at saturating irradiances, tanh is the hyperbolic tangent,

α [(nmol•L -1 •h -1 )(μmol photons•m -2 •s -1 ) -1
] is an index of the photosynthetic efficiency, and E (μmol photons•m -2 •s -1 ) is the spherical irradiance.

The obtained parameters were normalized by the chlorophyll concentration and used to estimate the in situ O 2 production and C fixation using the light and chlorophyll profiles measured in the SCM by the fluorescence and photosynthetic active radiation (PAR) sensors connected to the CTD (ETSP cruise) or the PPS (ETNP cruise). During the ETSP cruise off Peru, casts were consistently repeated every 3-4 h, and thus, the light profiles from the CTD were used in our calculations. During the ETNP cruise, light profiles measured with the PPS during daytime were normalized to the incident irradiance at the surface. The light attenuation profiles were assumed to be constant at each station, and the incident irradiance was used to calculate the change in light profile during the day. The values of incident irradiance were taken from the closest National Radiation station located in San Diego (National Solar Radiation Database, National Oceanic and Atmospheric Administration, United States).

and therefore, the production values should be taken as maximum values. Productivity was also highly variable among sites (0.43-1.70/0.15-0.95, and 0.03-0.91/0.02-0.87 mmol O 2 /C•m -2 •d -1 for ETNP and ETSP, respectively), reflecting the heterogeneous spatial distribution of the SCM (Fig. 1 and Tables S1 andS2).

Although primary production in surface waters largely exceeds these values [START_REF] Kalvelage | Nitrogen cycling driven by organic matter export in the South Pacific oxygen minimum zone[END_REF], the vast majority of surface production is remineralized before reaching the AMZ core. Indeed, the range of particulate organic carbon supply to the AMZ is 0.83-7.81 mmol C•m -2 •d -1 [START_REF] Babbin | Organic matter stoichiometry, flux, and oxygen control nitrogen loss in the ocean[END_REF][START_REF] Devol | Role of the oxygen-deficient zone in transfer of organic carbon to the deep ocean[END_REF] in the ETNP or 1.52-14.70 mmol C•m -2 •d -1 in the ETSP [START_REF] Escribano | Biological and chemical consequences of the 1997-1998 El Niño in the Chilean coastal upwelling system: A synthesis[END_REF]. This wide range highlights the variability in export rates in these regions. Nonetheless, comparing these estimations with our data, the carbon production in the SCM could provide 5-47% and 2-20% of the organic matter supplied to the anoxic waters of the ETNP and ETSP, respectively, where part of it is then mineralized by dissimilatory nitrate reduction to nitrite and denitrification [START_REF] Ganesh | Size-fraction partitioning of community gene transcription and nitrogen metabolism in a marine oxygen minimum zone[END_REF][START_REF] Babbin | Organic matter stoichiometry, flux, and oxygen control nitrogen loss in the ocean[END_REF][START_REF] Kalvelage | Nitrogen cycling driven by organic matter export in the South Pacific oxygen minimum zone[END_REF][START_REF] Ward | Oceans. How nitrogen is lost[END_REF]. Nitrate respiration to nitrite appears as the dominant mineralization step in the ETNP [START_REF] Ganesh | Size-fraction partitioning of community gene transcription and nitrogen metabolism in a marine oxygen minimum zone[END_REF], and mineralization rates of about 1 mmol C•m -2 •d -1 can be calculated from published data [START_REF] Canfield | A cryptic sulfur cycle in oxygen-minimum-zone waters off the Chilean coast[END_REF][START_REF] Kalvelage | Nitrogen cycling driven by organic matter export in the South Pacific oxygen minimum zone[END_REF]. These rates are close to the C fixation rate in the SCM, highlighting the relevance of the SCM in OMZ metabolism.

Global warming is expected to result in shoaling of the OMZ oxycline and overall expansion of OMZ volumes [START_REF] Gilly | Oceanographic and biological effects of shoaling of the oxygen minimum zone[END_REF]. Mesoscale physical processes such as local upwelling and anticyclonic eddies that shoal the oxic-anoxic boundary have been shown to enhance the development of SCMs [START_REF] Goericke | A novel niche for Prochlorococcus sp in low-light suboxic environments in the Arabian Sea and the Eastern Tropical North Pacific[END_REF][START_REF] Cepeda-Morales | Effect of the oxygen minimum zone on the second chlorophyll maximum in the Eastern Tropical Pacific off Mexico[END_REF]. Oxycline shoaling increases the light intensities in the anoxic cores of the AMZs, thereby potentially stimulating the photosynthetic community. The effects of these changes on microbial communities and microbial biogeochemical cycling in AMZs are difficult to predict, although significant changes in carbon, nitrogen, and sulfur cycling are expected [START_REF] Gilly | Oceanographic and biological effects of shoaling of the oxygen minimum zone[END_REF]. Our data show a significant carbon supply to the anoxic core of the Pacific AMZs by SCM photosynthetic activity, and it is likely that the situation is similar in the Arabian Sea. Although we did not measure nitrogen transformation processes, the nitrifying community was also enriched at the SCM, potentially reflecting elevated metabolic rates. A shoaling of the AMZ coupled with increases in irradiance and SCM photosynthetic activity would increase the carbon and daytime oxygen supply to the upper part of the AMZ. Shoaling of the AMZ due to global warming could thus lead to more extensive areas with high rates of SCM biological activity, with the diel oxic/anoxic cycles of these SCMs influencing marine productivity and coupled global nitrogen cycling.

Materials and Methods

Sampling Sites and in Situ Measurements. The two main oxygen minimum zones of the ETSP and ETNP were investigated during two cruises during 2014: the Activities of Research Dedicated to the Minimum of Oxygen in the Eastern Pacific (AMOP) cruise on the R/V L'Atalante to the ETSP off Peru during late January and February 2014 and the Oxygen Minimum Zone Microbial Biogeochemistry Expedition 2 (OMZoMBiE2) cruise on the R/V New Horizon to the ETNP region off Mexico during May-June 2014. Profiles of physical and chemical variables were obtained with a Seabird SBE-911 CTD system, equipped with a SBE 43 oxygen sensor and a Seapoint Chlorophyll Fluorimeter (R/V New Horizon) or a Chelsea Aqua 3 fluorimeter (R/V L'Atalante). CTD sensors were calibrated according to the manufacturer. The fluorometers used for the determination of chlorophyll were calibrated using pure chlorophyll solutions in 90% acetone (from 0.1 to 100 μg/L). In the ETNP, a pump profiling system (PPS) was also used for water collection. High-resolution O 2 profiling was performed during the CTD and PPS casts during the ETNP cruise. A highresolution STOX (2, 28) was used to measure O 2 concentration at nanomolar levels as described previously [START_REF] Revsbech | Construction of STOX oxygen sensors and their application for determination of O 2 concentrations in oxygen minimum zones[END_REF][START_REF] Tiano | Oxygen distribution and aerobic respiration in the north and south eastern tropical Pacific oxygen minimum zones[END_REF].

Flow Cytometry Analysis. Samples for cell counts were taken at several depths from the rosette (ETNP and ETSP) and the PPS (ETNP), fixed with glutaraldehyde and stored at -80 °C until analysis. Cell abundance was determined by flow cytometry using a FACSCalibur flow cytometer (Beckton Dickinson). Prochlorococcus, Synechococcus, and other autofluorescent cells (identified as picoeukaryotes) were counted in untreated samples, whereas autofluorescent plus nonautofluorescent cells (bacteria + archaea, referred as total microbial Metatranscriptome Analysis. Community cDNA sequencing was used to characterize microbial gene transcription in biomass (retained on 0.22-μm filters) from a subset of AMZ samples at the ETNP region off Mexico (Tables S4 andS5). These included samples collected during the OMZoMBiE2 cruise (2014) and a subset of samples previously reported by Padilla et al. [START_REF] Padilla | NC10 bacteria in marine oxygen minimum zones[END_REF]. Seawater from discrete depths spanning the oxic zone, SCM, lower oxycline, upper AMZ, and AMZ core was collected using Niskin bottles or the PPS. The sampling, preservation, RNA extraction, and sequencing were done following the procedure described by Padilla et al. [START_REF] Padilla | NC10 bacteria in marine oxygen minimum zones[END_REF]. Barcoded sequences were demultiplexed, and low-quality reads (Phred score < 25) were removed. Paired-end sequences were merged using custom scripts incorporating the FASTX toolkit (hannonlab.cshl.edu/fastx_toolkit/index.html) and USEARCH algorithm, with criteria of minimum 10% overlap and 95% nucleotide identity within the overlapping region. Ribosomal RNA (rRNA) transcripts were identified with riboPicker (37) and removed from the analysis. Merged nonrRNA sequences were queried via DIAMOND using sensitive search parameters [START_REF] Buchfink | Fast and sensitive protein alignment using DIAMOND[END_REF] against the National Center for Biotechnology Information (NCBI)-nr database (November 2013). DIAMOND-identified protein-coding transcripts were assigned a functional annotation based Kyoto Encyclopedia of Genes and Genomes (KEGG) orthology (KO) identifiers (39) using Metagenome Analyzer 5 (MEGAN5) [START_REF] Huson | Integrative analysis of environmental sequences using MEGAN4[END_REF], with taxonomic classification assigned using the lowest common ancestor (LCA) algorithm in MEGAN5 based on the NCBI taxonomy. Counts per KO were normalized to the total number of protein coding transcripts classified within bacteria and archaea (i.e., prokaryotes). Transcripts encoding LATO and HATO, nxr, and amo (all subunits) were identified by the KO identifiers listed in Table S4.

NOB abundances were determined by taxonomic LCA assignment according to NCBI taxonomy of DIAMOND-identified mRNA transcripts normalized to the total number of prokaryotic mRNA sequences. Taxonomic affiliation of both LATO and HATO were also assigned according to NCBI taxonomy via the LCA algorithm in MEGAN5. Fig. S4. Depth integration of in situ gross oxygen production and carbon fixation rates. Three stations off Mexico (T4, F4, T7; Upper) and off Peru (am_04, am_25, and am_28; Lower) were selected as examples. Modeled in situ gross oxygen production and carbon fixation rate profiles were integrated with depth to calculate the aerial contribution of the SCM. We used only GCP rates from the SCM, being delimited as the water layer extending from the minimum fluorescence value between the primary and secondary chlorophyll maxima to the bottom of the photic layer. Light at the depth where the maximum fluorescence value was measured is also shown as reference. The abundance of Prochlorococcus sp. (Prochl.), Synechococcus sp. (Syn.), photosynthetic pico-eukaryotes (P.euk.), and total microbial community (T.Microb.) are expressed as cells per mL. The percentage of Prochlorococcus sp. in the phototrophic microbial community (% photot.) and in the total microbial community (% total) were also calculated. Mean chlorophyll values (Chl.) from in situ CTD measurements were added for comparison. Stn, stations in the ETNP; Total, number of sequences post trimming, merging, and quality control; Read length, average read length (bp) of merged and quality controlled sequences; nonrRNA, number of nonrRNA reads; DIAMOND, number of sequences with DIAMOND matches (bit score >50) to protein-coding sequences in the NCBI-nr database; MEGAN bacteria, number of reads assigned to the bacteria node using the lowest common ancestor (LCA) algorithm in MEGAN5; MEGAN archaea, number of reads assigned to the archaea node using the LCA algorithm in MEGAN5; MEGAN Prok, the sum of bacteria and archaeaassigned reads by the LCA algorithm; and Accession, NCBI accession number or BioProject ID. *Samples collected in 2013 published in Ganesh et al. [START_REF] Ganesh | Size-fraction partitioning of community gene transcription and nitrogen metabolism in a marine oxygen minimum zone[END_REF]. † Samples collected in 2014 published in Padilla et al. [START_REF] Jassby | Mathematical formulation of the relationship between photosynthesis and light for phytoplankton[END_REF].

•L -1 •h -1 GCP max , nmol•L -1 •h -1 α, (nmol•L -1 •h -1 ) (μmol photons•m -2 •s -1 ) -1 O 2 C O 2 C OMZoMBiE2 (

Fig. 1 .

 1 Fig. 1. Maps with sampled stations and main characteristics of the upper part of the (A-F) ETNP AMZ and (G-L) ETSP AMZ. Stations off Mexico (A) and Peru (G) where the SCM was found and sampled. (B and H) Dissolved oxygen profiles, based on SBE43 and STOX sensors (zooming in at low STOX O 2 values in B or corrected SBE O 2 in H). (C and I) Profiles of chlorophyll concentration inferred from in vivo fluorescence. (D and J) Prochlorococcus abundance. (E and K) Total microalgae (Prochlorococcus, Synechococcus, and picoeukaryotes) and (F and L) total microbial community (Total Microb. com.) abundance measured by flow cytometry.

FFig. 2 .

 2 Fig. 2. Oxygen production and carbon fixation during incubations of samples from the SCM off (A-C) Mexico and (D-F) Peru. (A and D) Evolution of O 2 concentration during incubation of SCM samples exposed to a range of scalar irradiances (0-40 μmol photons•m -2 •s -1 ). (B and E) Net community production (NCP) rates versus scalar irradiance. (C and F) Gross community production (GCP): O 2 -GCP was measured as the net O 2 production, and C-GCP was measured by the incorporation of 13 C (at ETNP) or 14 C (ETSP). Data were fitted to a photosynthesis-irradiance model to calculate maximum rates (GCP max ) and the initial slope of the curve (α), an index of the photosynthetic efficiency at low light (values in the main text). Error bars represent the SE.

Fig. 3 .

 3 Fig. 3. Water column dissolved oxygen (O 2 ), chlorophyll concentrations (Chl.), and microbial transcript abundances at station T6 in the ETNP in (A-D) 2013 and (E-H) 2014. (A and E) O 2 based on SBE and STOX sensor measurement and chlorophyll inferred from in vivo fluorescence. (B and F) Abundances of transcripts encoding LATO and HATO, amo, and nxr, as a percentage of total prokaryotic mRNA. (C and G) Taxonomic classification of total mRNA affiliated with NOB and (D and H) AOB (Nitrosomonas) and AOA (Thaumarchaeota), as a percentage of total prokaryotic mRNA.

Fig. S2 .

 S2 Fig. S2. Water column dissolved oxygen (O 2 ) and chlorophyll concentrations and microbial transcript abundances at stations (A-D) T4 and (E-H) T10 in the ETNP in 2014. (A and E) O 2 , based on SBE and STOX senor measurement, and chlorophyll inferred from in vivo fluorescence. (B and F) Abundances of transcripts encoding LATO and HATO, amo, and nxr, as a percentage of total prokaryotic mRNA. (C and G) Taxonomic classification of total mRNA affiliated with NOB and (D and H) AOB and AOA, as a percentage of total prokaryotic mRNA.

Fig. S3 .

 S3 Fig. S3. Taxonomic identities of terminal oxidases. Taxonomic assignment of (Upper) HATO and (Lower) LATO at stations T4, T6, and T10 off Mexico in 2014 and T6 in 2013 as determined via lowest common ancestor binning in MEGAN5. Abundances are presented as the percentage of total prokaryotic mRNA from each sample.

Fig. S5 .

 S5 Fig.S5. Experimental setup for measurement of O 2 production/consumption. Glass bottle (labeled as 1) with a volume of 1160 mL. A series of modifications were done to a standard Schott-Duran glass bottle including (i) the insertion of a long open glass tube (internal diameter 2.5 mm) for pressure compensation and for the injection of calibration solution or tracers; (ii) the addition of a short 8.1-mm (inner diameter) glass tube for insertion of the STOX sensor (labeled as 2) or a solid 8-mm glass bar if STOX sensors were not used; (iii) a modification of the bottle neck to accommodate a ground glass stopper (internal diameter 12 mm) so that the inner part of the bottle could be accessed; and (iv) insertion of a sensing dot for optode-based measurements that was glued onto the inner surface of the bottle, whereas the reading device (LUMOS) was positioned on the outside, held by a PVC frame glued to the bottle. STOX sensors (labeled as 2) were connected to a picoammeter (labeled as 3) equipped with an external battery and a switch for the periodic polarization of the front guard of the sensor. The signal from the picoammeter was fed into an A/D converter connected to a laptop (labeled as 4). The LUMOS (labeled as 5) device was directly connected to the laptop. A magnetic stirrer (labeled as 6) and a controller (labeled as 7) ensured homogeneous mixing of the water in the bottle by a glass coated magnet. A white LED system surrounded the bottle (labeled as 8) to ensure a homogeneous light distribution. The light intensity was modulated by an external controller (labeled as 9), and the light spectrum of the SCM was simulated by coating the LEDs with a Marine Blue filter (LEE Filters). The were installed within a nontransparent PVC tube. The incubation bottles and associated lighting and stirring equipment were immersed into a water bath held at the in situ temperature (14 °C).

Table 1 .

 1 Depth-integrated oxygen production and carbon fixation rates

		GCP, mmol•m -2 •d -1
	Station	O 2	C
	ETNP-Mexico
	T4	0.43	0.15
	F4	1.70	0.19
	T7	0.48	0.13
	T9	0.97	0.93
	T10	0.59	0.55
		0.83 ± 0.53	0.39 ± 0.35
	ETSP-Peru
	am_04	0.03	0.02
	am_25	0.03	0.03
	am_28	0.91	0.87
		0.32 ± 0.51	0.31 ± 0.49

Table S1 .

 S1 Flow cytometry analysis of the SCM microbial community

				Prochl., 10 4	Syn., 10 4	P.euk., 10 4	T.Microb., 10 5
	Cruise	Station Depth, m	cells per mL	cells per mL	cells per mL	cells per mL Prochl., % photot. Prochl., % total Chl., μg•L -1
	OMZoMBiE2 (ETNP)	T4	100	5.06	0.29	0.001	3.56	94.5	12.4	0.55
		T7	80	2.99	0.66	0.000	3.95	81.8	6.9	0.15
		T9	90	6.97	0.74	0.009	4.76	90.3	12.6	0.67
		T10	110	5.99	0.45	0.002	8.67	93.0	6.4	0.52
		T3	115	1.76	0.08	0.001	2.12	95.4	7.6	0.20
		F12	85	5.56	0.43	0.000	6.49	92.8	7.8	0.43
	Mean		97	4.72	0.44	0.002	4.92	91.3	9.0	0.42
	SD		14	1.96	0.24	0.003	2.33	5.0	2.8	0.21
	AMOP'14 (ETSP)	am_4	50	13.80	0.30	0.006	3.49	97.8	28.1	0.13
		am_25	30	21.97	3.70	0.035	19.02	85.5	10.2	0.16
		am_28	60	18.96	3.06	0.012	14.66	86.0	11.2	0.21
	Mean		47	18.25	2.35	0.018	12.39	89.8	16.5	0.16
	SD		15	4.13	1.81	0.015	8.01	7.0	10.1	0.04

Table S2 .

 S2 Descriptive data and CTD measurements from within the SCM at sampled stations in both the ETNP and ETSP

						Beam	Chlorophyll,
	Cruise	Station Date (2014) Offshore (K m ) Depth, m	transmission, %	μg•L -1	O 2 , μmol•kg -1 T, °C Density (σ θ ), kg•m -3
	OMZoMBiE2 (ETNP)	T4	15/05	140	100	88.92	0.54	0.002	14.2	26.0
		F4	18/05	150	100	88.93	0.54	0.013	14.1	25.8
		T7	21/05	70	95	89.10	0.38	0.439	15.6	25.6
		T7	21/05	70	90	89.04	0.24	-	15.1	25.8
		T9	22/05	125	90	-	0.67	0.002	14.3	26.0
		T10	24/05	190	100	88.61	0.64	0.000	14.6	25.9
		T3	27/05	230	110	89.49	0.25	0.314	14.1	26.0
		T3	27/05	230	105	88.94	0.44	0.102	14.5	25.9
		F13	01/06	280	90	88.62	0.75	0.006	14.4	25.9
	Mean				98	88.96	0.49	0.112	14.6	25.9
	SD				7	0.28	0.18	0.170	0.5	0.1
	AMOP'14 (ETSP)	am_4	31/01	97	46	80.73	0.21	0.16	14.7	26.3
		am_25	14/02	30	45	79.68	0.30	0.07	14.7	25.8
		am_25	15/02	30	53	79.82	0.30	0.22	14.6	26.2
		am_28	17/02	125	70	80.25	0.20	0.04	13.6	26.2
		am_28	19/02	125	70	80.95	0.12	68.56	13.6	26.2
	Mean				57	80.29	0.23	0.123	14.3	26.2
	SD				12	0.55	0.08	0.083	0.6	0.2

Table S3 .

 S3 Summary of metabolic rates calculated from oxygen and carbon incorporation data

				R*, nmol
	Cruise	Station	Depth, m	O 2

Table S4 .

 S4 KEGG orthology identifiers used to screen metatranscriptomic datasets for terminal oxidase and nitrification marker genes

	Table S5. ETNP sequencing statistics
	Year Stn Depth, m	Total	Read length, bp nonrRNA DIAMOND MEGAN bacteria MEGAN archaea MEGAN Prok	Accession
	2013	6	30	1,521,531	203	1,053,980	108,873	62,934	5,476	68,410	PRJNA263621*
			85	1,364,985	201	294,080	175,178	115,127	4,935	120,062	PRJNA263621*
			100	1,024,593	214	218,551	158,677	115,316	1,922	117,238	PRJNA263621*
			125	1,494,459	204	377,156	256,113	174,678	4,559	179,237	PRJNA263621*
			300	712,020	212	159,091	110,937	78,842	2,389	81,231	PRJNA263621*
	2014	4	60	2,302,732	197	417,033	232,758	126,802	6,986	133,788	PRJNA305951
			100	2,520,169	199	805,179	359,336	258,010	15,993	274,003	PRJNA305951
			150	1,291,622	208	310,389	183,424	128,402	6,995	135,397	PRJNA305951
			200	1,212,126	205	253,637	155,219	110,453	5,938	116,391	PRJNA305951
		6	75	4,786,146	164	957,802	298,788	187,702	63,517	251,219	PRJNA277357 †
			80	1,407,348	183	591,754	165,806	80,047	41,583	121,630	PRJNA305951
			100	1,601,640	183	611,712	192,294	133,385	13,799	147,184	PRJNA305951
			150	1,452,296	202	359,133	169,271	113,485	14,564	128,049	PRJNA305951
			200	2,325,341	177	482,694	210,303	158,319	5,971	164,290	PRJNA305951
			300	18,781,558	185	7,710,908 1,645,992	1,540,161	51,789	1,591,950	PRJNA277357 †
	10	100	2,149,661	192	439,535	106,713	62,268	3,823	66,091	PRJNA277357 †
			150	4,523,472	166	1,276,310	495,416	416,236	14,970	431,206	PRJNA277357 †
			300	1,335,682	194	439,535	226,427	187,523	6,458	193,981	PRJNA277357 †
			ETNP)	T4		100		2.6	13.3	2.4	1.26	0.2
				F4		100		5.7	8.8	1.1	0.75	0.1
				T7		95		5.3	8.0	2.6	0.61	0.2
				T7		90		6.9	10.0	0.0	0.76	0.0
				T9		90		3.2	35.5	30.3	3.19	3.1
				T10		100		7.1	19.4	9.2	1.77	0.8
				T3		110	17.4	16.8	3.1	1.32	0.4
				T3		105	11.4	-	-	1.60	-
				F13		90		8.2	21.3	-	1.89	-
	Mean					98		7.5	16.6	6.9	1.46	0.69
	SD					7		4.5	9.1	10.7	0.80	1.09
	AMOP'14 (ETSP)	am_4	46	32.6	32.5	28.5	2.4	1.9
				am_25	45	20.1	32.1	33.1	1.61	1.02
				am_25	53	22.8	105.0	98.3	3.49	3.25
				am_28	70	25.7	51.9	35.2	2.11	2.01
				am_28	70	63.2	41.0	27.1	4.08	2.28
	Mean					57	32.9	52.5	44.4	2.74	2.09
	SD					12	17.6	30.4	30.3	1.02	0.80
			Function		Protein	Abbreviation		KEGG orthology identifier (KO)
			Terminal oxidase Cytochrome C	LATO	K02277, K02276, K02274, K15408, K02275, K02258, K02259
			Terminal oxidase cbb3		HATO		K00404, K00405, K15862, K00407, K00406
			Terminal oxidase bd		HATO			K00425, K00246
			Nitrification	Nitrite oxidoreducatase	nxr			K00370, K00371
			Nitrification	Ammonia monooxygenase	amo		K10944, K10945, K10946

ACKNOWLEDGMENTS. We thank P. Sørensen and L. B. Pedersen for the fabrication of sensors and conscientious and continuous technical support. We are grateful for the support from P. Lehner, S. Borisov, and I. Klimant in enabling high-resolution optode measurements. We also thank the captains and crews of the R/V New Horizon and L'Atalante. We additionally thank J. C. Kondrup for making the glass bottles and G. Alarcón, C. Venegas, M. Soto, C. Henry, M. Dugenne, D. Lefevre, A. Franco-Garcia, J. Grelet, O. Depretz-De-Gesincourt, and A. Barani for operational and experimental support. We are also grateful to H. Maske and G. Rodriguez for the logistical support dealing with radioactive compounds, as well as K. B. Oest and J. Pedersen for the support and analysis of isotopes samples. We thank M. Altabet, B. Dewitte, C. Maes, and V. Garçon. This work was supported by the European Research Council Grant 267233 and European Union Seventh Framework Programme Project 614141; the National Science Foundation 1151698 and 1558916 (to F.J.S.); the Sloan Foundation RC944 (to F.J.S.); the Chilean National Commission for Scientific and Technological Research 1130784 (to O.U.) and a graduate fellowship (to M.A.); the Millennium Science Initiative IC120019 (to O.U.); and Activity of Research Dedicated to the Minimum of Oxygen in the Eastern Pacific project supported by Institut de Recherche pour le Développement, CNRS/Institut National des Sciences de l'Univers, and Laboratoire d'Etudes en Géophysique et Océanographie Spatiales.

Supporting Information

Garcia-Robledo et al. [START_REF] Dalsgaard | Anammox and denitrification in the oxygen minimum zone of the eastern South Pacific[END_REF].1073/pnas.1619844114 Fig. S1. PAR, chlorophyll, and in situ gross oxygen production and carbon fixation rates in the SCM estimated from onboard experimental data for O 2 metabolism and C fixation. GCP rates were calculated only at the SCM, considered as the water layer extending from the minimum fluorescence value between the peaks to the bottom of the photic layer. An oceanic station with a deeper SCM (T4) off Mexico (ETNP, Upper) and a coastal station with a shallow SCM (am_4) off Peru (ETSP, Lower). Note the different scales.