Pierre E Galand
email: pierre.galand@obs-banyuls.fr

Sabrina Lucas

Sonja K Fagervold

Erwan Peru

Audrey M Pruski

Gilles Vétion

Christine Dupuy

Katell Guizien

Disturbance increases microbial community diversity and production in marine sediments

Disturbance strongly impacts patterns of community diversity, yet the shape of the diversitydisturbance relationship remains a matter of debate. The topic has been of interest in theoretical ecology for decades as it has practical implications for the understanding of ecosystem services in nature. One of these processes is the remineralization of organic matter by microorganisms in coastal marine sediments, which are periodically impacted by disturbances across the sediment-water interface. Here we set up an experiment to test the hypothesis that disturbance impacts microbial diversity and function during the anaerobic degradation of organic matter in coastal sediments. We show that during the first 3 weeks of the experiment, disturbance increased both microbial production, derived from the increase in microbial abundance, and diversity of the active fraction of the community. Both community diversity and phylogenetic diversity increased, which suggests that disturbance promoted the cohabitation of ecologically different microorganisms. Metagenome analysis also showed that disturbance increased the relative abundance of genes diagnostic of metabolism associated with the sequential anaerobic degradation of organic matter. However, community composition was not impacted in a systematic way and changed over time. In nature, we can hypothesize that moderate storm disturbances, which impact coastal sediments, promote diverse and productive communities. These events, rather than altering the decomposition of organic matter, may increase the substrate turnover and, ultimately, remineralization rates.

Introduction

One of the key features of microbial communities in most natural ecosystems is that they harbor a tremendous diversity [START_REF] Keller | Tapping into microbial diversity[END_REF]. Community diversity is affected by a number of biotic interactions but also by abiotic factors such the surrounding environmental conditions. Environmental conditions change with time and can fluctuate dramatically when impacted by episodic disturbances. A disturbance can be defined as "any relatively discrete event in time that disrupts ecosystem, community, or population structure and changes resources, substrate availability, or the physical environment" [START_REF] Pickett | The ecology of natural disturbance and patch dynamics[END_REF]. The response of communities to disturbance has been a central interest in ecology studies for decades. More specifically, investigations have aimed to resolve how disturbances in an ecosystem influence the diversity of the communities [START_REF] Sousa | The role of disturbance in natural communities[END_REF]. In this respect, the intermediate disturbance hypothesis (IDH) [START_REF] Grime | Competitive exclusion in herbaceous vegetation[END_REF][START_REF] Connell | Diversity in tropical rain forests and coral reefs[END_REF] suggests that the relationship between diversity and disturbance can be represented graphically as a unimodal distribution. Community diversity is low when disturbance levels are low and increases with disturbance frequency or strength. If disturbance persists, however, diversity decreases again.

Nevertheless, recent studies have refuted the idea of a positive relationship between disturbance and community diversity. A survey of empirical studies reported that nonsignificant relationships were the most common [START_REF] Mackey | The diversity-disturbance relationship: is it generally strong and peaked?[END_REF], and a recent paper proposed, based on both empirical and theoretical grounds, that the IDH should be abandoned [START_REF] Fox | The intermediate disturbance hypothesis should be abandoned[END_REF]. For microorganisms, soil experiments showed that diversity declined with increasing disturbance frequencies, [START_REF] Kim | Changes in Soil Bacterial Community Structure with Increasing Disturbance Frequency[END_REF] and a negative diversitydisturbance relationship was also observed in natural marine sediments [START_REF] Boer | Time-and sediment depth-related variations in bacterial diversity and community structure in subtidal sands[END_REF].

Recently, it has been emphasized to incorporate genetic diversity into disturbancediversity studies because the topic remains a major knowledge gap [START_REF] Banks | How does ecological disturbance influence genetic diversity?[END_REF].

Despite the recognition of the importance of genetic diversity in ecology [START_REF] Hughes | Ecological consequences of genetic diversity[END_REF], its role as a driver for microbial diversity and function remains poorly understood. Overall, data on the IDH for microorganisms remains scarce because studies have mainly focused on the impact of disturbance on community composition, rather than diversity [START_REF] Allison | Resistance, resilience, and redundancy in microbial communities[END_REF][START_REF] Berga | Effects of disturbance intensity and frequency on bacterial community composition and function[END_REF][START_REF] Yeo | Coastal bacterioplankton community dynamics in response to a natural disturbance[END_REF]. Communities are hypothesized to be resistant or resilient to disturbance when they are not definitely altered in their composition [START_REF] Allison | Resistance, resilience, and redundancy in microbial communities[END_REF].

Episodic disturbances, which are thought to have an important role in maintaining diversity, impact many of the ecosystems of the Earth. Coastal sediments are regularly exposed to disturbances due to increases in turbulence that result from the dissipation of energetic flows in the benthic boundary layer. These turbulences occur with different strengths and at different temporal scales. Tidal flow generates periodic increases of turbulence above the sediment with moderate intensity, whereas waves cause larger but more sporadic turbulences, which lead to sediment resuspension. Turbulence intensity in the benthic boundary layer controls diffusive fluxes across the sediment-water interface; as a consequence, turbulence intensity fluctuations disturb the stratification of dissolved compounds in the sediments and may affect organic matter degradation [START_REF] Arzayus | Organic matter degradation in sediments of the York River estuary: Effects of biological vs. physical mixing[END_REF]. Organic matter degradation is an essential service provided by heterotrophic microorganisms [START_REF] Ducklow | Microbial services: challenges for microbial ecologists in a changing world[END_REF]. This is particularly true in estuarine sediments and deltaic systems, where most of the terrestrial organic matter delivered by rivers is deposited before being remineralized or buried [START_REF] Hedges | What happens to terrestrial organic matter in the ocean?[END_REF][START_REF] Burdige | Preservation of organic matter in marine sediments: controls, mechanisms, and an imbalance in sediment organic carbon budgets?[END_REF]. Understanding the fate of terrestrial or marine organic matter exported down to sediments may help gain greater insight into the cycling of carbon on a global scale and its possible alteration by climate change and anthropogenic perturbations [START_REF] Regnier | Anthropogenic perturbation of the carbon fluxes from land to ocean[END_REF][START_REF] Reichstein | Climate extremes and the carbon cycle[END_REF]. Degradation or remineralization of the complex mixture of molecules composing sediment organic matter is mediated by the combined activity of an array of both aerobic and anaerobic microorganisms with different metabolic potential. Although aerobic processes are more effective, the anoxic degradation rates are often similar to the oxic rates in surface sediments [START_REF] Arndt | Quantifying the degradation of organic matter in marine sediments: A review and synthesis[END_REF].

The identity of the main players in the terminal degradation process and the structure of the communities involved are not always well known. The composition of the microbial communities can for instance be correlated to the origin and lability of organic matter [START_REF] Fagervold | River organic matter shapes microbial communities in the sediment of the Rhône prodelta[END_REF], the geochemical composition of the sediments [START_REF] Jorgensen | Correlating microbial community profiles with geochemical data in highly stratified sediments from the Arctic Mid-Ocean Ridge[END_REF] and pigment concentrations [START_REF] Bienhold | The energy-diversity relationship of complex bacterial communities in Arctic deep-sea sediments[END_REF]. The impact of disturbance, which may disrupt the commonly observed vertical zonation of marine sediments, remains poorly studied.

Here, we test the hypothesis that disturbance impacts microbial community diversity in coastal sediments during organic matter mineralization. We conducted an experiment in which sediment cores were regularly exposed to enhanced turbulence at the sediment-water interface, without sediment resuspension, mimicking the effect of moderate swell events.

Sediments were enriched with plant-derived detritus to simulate the sedimentation of labile terrestrial organic matter. Organic matter degradation was monitored during the course of the experiment and so was the concentration of oxygen. Microbial abundance was measured as a way to derive production. Bacterial and archaeal communities were described before and after each disturbance event at two sediment depths by targeting both 16S rRNA genes and 16S rRNA transcripts to identify the active fraction of the communities. We also constructed metagenomes to test whether disturbance impacted potential sedimentary metabolic pathways and to verify whether possible changes in community taxonomic composition were correlated with changes in functional gene composition.

Materials and methods

Experimental setup

A total of 36 sediment cores enriched with duckweed (Lemna sp.) were placed in a water tank incubated in the dark at 16 °C (see Supporting information for details). The sediment cores were exposed to disturbance in the form of a spatially uniform diffusive turbulence generated by an oscillating grid that controlled the diffusive fluxes across the sediment-water interface (Fig. S1, [START_REF] Lucas | Oscillating grid mesocosm for studying oxygen dynamics under controlled unsteady turbulence[END_REF]). Turbulence frequency and strength were chosen to simulate the disturbance generated by moderate swell events in the infralittoral area. Overall, the experiment included four disturbance events preceded by calmperiods. Twenty-four-hour disturbance events (turbulent velocity=6.15 cm s -1 ) were separated by calm-intervals of 10 days (turbulence < 1.18 cm s -1 ). The entire experiment lasted for 5 weeks. The low turbulence in the calm-periods corresponded to turbulent conditions in the benthic boundary layer during usual wind-driven circulation. The disturbance events did not cause any sediment resuspension.

Sampling was conducted before and after each disturbance event. A total of 8 different time points (T0-T7) were sampled (4 before disturbance and 4 after disturbance). For each sampling, 3 cores were removed from the water tank. The cores were sliced and sediment samples were taken from two layers: 0-5 mm (surface) and 5-10 mm (deep). Organic matter concentrations and microbial abundances were measured on triplicate cores, and oxygen, fatty acids and microbial molecular analysis were conducted on 1 of the 3 cores.

Fatty acids, organic matter, oxygen and bacterial abundance

Labile OM compounds, namely total carbohydrates, was assessed using colorimetric methods according to previously published protocols [START_REF] Dubois | Colorimetric Method for determination of sugars and related substances[END_REF][START_REF] Barnes | Estimation of lipids in marine animals and tissues: detailed investigation of the sulphophosphovanilun method for 'total'lipids[END_REF][START_REF] Bradford | A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[END_REF]. Total fatty acids were extracted by direct acid transmethylation and analyzed using a Saturn 2100T iontrap GC-MS instrument (VARIAN, Les Ulis, France) equipped with a fused-silica-capillary column (Factor Four, VF-Waxms, 30mX0.25mm ID, 0.25 mm film thickness, VARIAN). Details of the extraction procedure and analytical conditions are given in [START_REF] Bourgeois | Distribution and lability of land-derived organic matter in the surface sediments of the Rhône prodelta and the adjacent shelf (Mediterranean Sea, France): a multi proxy study[END_REF] [START_REF] Bourgeois | Distribution and lability of land-derived organic matter in the surface sediments of the Rhône prodelta and the adjacent shelf (Mediterranean Sea, France): a multi proxy study[END_REF]. Oxygen concentrations were profiled at a 200 µm vertical spatial resolution every 15 min with a Unisense O 2 Clark-type microsensor (Unisense, Aarhus, Denmark) from 20 mm above to 10 mm below the sediment-water interface. Time series of the average O 2 concentration in the sediment layers (0-5 mm, 5-10 mm) were calculated from these profiles.

Prokaryotic abundance was measured by flow cytometry. Five milliliters of sediment was fixed with 0.2 µm filtered formaldehyde solution (vol/vol, 2% final concentration), and cells were separated from the sediment and homogenized according to the protocol described by [START_REF] Lavergne | An efficient and rapid method for the enumeration of heterotrophic prokaryotes in coastal sediments by flow cytometry[END_REF]. One milliliter of sediment was diluted sequentially to 1/2000 with 0.01 M sodium pyrophosphate (NaPp) and was mixed by vortexing. Samples were then incubated at +4 °C for 30 min before sonication (60 W for 30 s). Finally, an aliquot of the sample was stained with SYBR Green I (1:10,000) for 15 min in the dark and was analyzed by flow cytometry as previously described [START_REF] Lavergne | An efficient and rapid method for the enumeration of heterotrophic prokaryotes in coastal sediments by flow cytometry[END_REF]. Bacterial production was derived from the increase in bacterial abundance and was expressed as the number of cells per mL of sediment and per day. Production was calculated for each time point as:

ProductionT 2 =(ln(AbT 2 )-ln(AbT 1 ))/(T 2 -T 1 ), where Ab is the prokaryotic abundance and T is the time.

Nucleic acid extraction, RT-PCR and sequencing.

DNA and RNA were co-extracted using the RNA PowerSoil Total RNA Isolation Kit and the DNA Elution Accessory Kit (MoBio, Carlsbad, CA, USA) from 1g of frozen sediment according to the manufacturer's protocol. RNA samples were DNAse treated using the RNase-Free DNase Set (Qiagen, Valencia, CA, USA) and concentrated with the RNeasy MinElute Cleanup Kit (Qiagen). The RNA were reverse-transcribed with random primers using the SuperScript III Reverse Transcriptase kit (Invitrogen, Carlsbad, CA, USA) and tested for the presence of contaminating genomic DNA by performing PCR before the reverse transcription.

Portions of the 16S rRNA genes were amplified with the bacterial primer pair 27F (5'-AGRGTTTGATCMTGGCTCAG-3') and 519R (5'-GTNTTACNGCGGCKGCTG-3'), and archaeal primers 349F (5'-GYGCASCAGKCGMGAAW-3') and 806r (5′ - For the metagenomes, libraries for shotgun sequencing were prepared using Nextera DNA Sample Preparation Kit (Illumina, San Diego, CA, USA) with 50 ng of DNA from each sample. Library insert size was determined by Experion Automated Electrophoresis Station (Bio-Rad, Hercules, CA, USA). The insert size of the libraries ranged from 300 to 850 bp (average 500bp). Pooled libraries (12pM) were loaded to a 600 Cycles v3 Reagent cartridge (Illumina) and the sequencing was performed on Miseq (Illumina) by a commercial laboratory (Research and Testing Laboratory, Lubbock, TX, USA). Metagenomes are archived in MG-RAST under accession numbers 4612993.3, 4612994.3, 4612995.3, 4612996.3 and 4612997.3.

GGACTACVSGGGTATCTAAT-3′). A 30 cycle PCR with the

Sequence analysis

All reads that had a mismatch with the 16S rRNA primers, contained ambiguous nucleotides (N) or were longer than 390 after the forward primer, were removed. Then, reads that had ≥ 3% of bases with Phred values < 27 (0.2% per-base error probability) were removed. These steps are recommended to ensure that when clustering at 97%, the influence of erroneous reads is minimized [START_REF] Huse | Ironing out the wrinkles in the rare biosphere through improved OTU clustering[END_REF]. Sequences were clustered at a 97% threshold using Uclust [START_REF] Edgar | Search and clustering orders of magnitude faster than BLAST[END_REF]. Representative sequences of each OTU were classified by comparison with Blast to the Greengenes database [START_REF] Desantis | Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB[END_REF]. Sequence analyses were conducted with Pyrotagger [START_REF] Kunin | PyroTagger: A fast, accurate pipeline for analysis of rRNA amplicon pyrosequence data[END_REF]. To compare bacterial communities for diversity analysis, all samples were randomly re-sampled to the size of the sample containing the fewest sequences (n=950 for Bacteria and n=424 for Archaea) using Daisy Chopper [START_REF] Gilbert | The seasonal structure of microbial communities in the Western English Channel[END_REF].

For metagenomes, sequence ends were cut to remove adapters. Sequences were considered low quality when >5 bases had a phred score <15 and were subsequently removed.

FastqJoin was used to merge paired end reads when possible with a minimum overlap setting of 8 bp and a maximum difference of 10%. Both paired and unpaired sequences were kept for automated annotation in MG-RAST using Hierarchical Classification subsystems against the KEGG Orthology database. Annotations were conducted with a maximum e-value cutoff of 1e -10 , a minimum 60% identity cutoff and 15 amino acids as the minimum alignment length cutoff. Identified microbial gene families (specified by KEGG orthology groups) were grouped into metabolic pathways (Table S1). Metagenome reads were normalized by evenly resampling at random to 1,254,500 annotated reads per sample to allow comparison.

Data analysis

Bacterial diversity was estimated for both the RNA and DNA fractions by calculating the Shannon diversity index (H') and by the standardized effect size (SES) [START_REF] Kembel | Picante: R tools for integrating phylogenies and ecology[END_REF], which is a standardized measure of phylogenetic diversity. Phylogenetic diversity was computed using the Picante package [START_REF] Kembel | Picante: R tools for integrating phylogenies and ecology[END_REF] in R. To calculate SES, 300-bplong representative sequences for each OTU were aligned using MUSCLE [START_REF] Edgar | MUSCLE: a multiple sequence alignment method with reduced time and space complexity[END_REF]) and the alignment was then cleaned to remove nonoverlapping sequence regions. A phylogenetic tree was constructed using FASTTREE [START_REF] Price | FastTree 2-approximately maximumlikelihood trees for large alignments[END_REF]. The observed phylogenetic diversity was compared to the average phylogenetic diversity in a randomly generated community (null model) and divided by the standard deviation of phylogenetic distances in the null model [START_REF] Webb | Phylocom: software for the analysis of phylogenetic community structure and trait evolution[END_REF]. The null model randomizes community data matrix with the independent swap algorithm maintaining species occurrence frequency and sample species richness [START_REF] Kembel | Disentangling niche and neutral influences on community assembly: assessing the performance of community phylogenetic structure tests[END_REF]. SES is equivalent to -1 times the Nearest Relative Index (NRI) [START_REF] Webb | Phylogenies and Community Ecology[END_REF]). Faith's PD [START_REF] Faith | Conservation evaluation and phylogenetic diversity[END_REF] is the most common measure of phylogenetic diversity, but because the number of taxa in a sample affects PD and because the number of taxa varied significantly between our samples, we chose to use SES instead. Positive SES values indicate greater phylogenetic distance among co-occurring species than expected by chance, whereas negative values indicate small phylogenetic distance.

The Bray Curtis similarity index was computed to compare the community and functional gene composition between samples and to conduct cluster analysis. The effects of disturbance and time on community composition were tested with a permutational multivariate analysis of variance using distance matrices as implemented in the adonis function in the Vegan package (v.2.3-0) in the R software (v.3.1.2). To identify OTUs that had a significant difference in abundance between groups we used an ANOVA with a Tukey-Kramer post-hoc test as implemented in STAMP [START_REF] Parks | STAMP: statistical analysis of taxonomic and functional profiles[END_REF].

Results

Dynamics of oxygen, carbohydrates and fatty acid concentrations

Each of the four disturbance events, which lasted for 24 h (T0-T1, T2-T3, T4-T5, and T6-T7), oxygenated the water above the sediment, resulting in O 2 saturation. Oxygen concentrations in the overlying water decreased at the beginning of each interleaved calm-interval (T1-T2, T3-T4, and T5-T6) (Fig. 1a). Oxygen concentrations in the sediment did not follow the same trend as in water: the concentration increased after the first disturbance and remained low at 0.20 mg L -1 on average in the upper layer of the sediments during the following calm interval (T1-T2) (Fig. 1a). After the second disturbance event (T2-T3), the oxygen concentrations in the upper part of the sediment increased with a peak of 0.60 mg L -1 three days after the end of the disturbance, and then decreased to 0.40 mg L -1 at the end of the calm interval (T3-T4) (Fig. 1a). The third disturbance (T4-T5) immediately increased oxygen concentrations up to 1.50 mg L -1 in the upper sediment layer, but the concentrations rapidly dropped to lower levels than during the first and second calm intervals (Fig. 1). The last disturbance (T6-T7) increased the oxygen concentration in the sediment to 0.50 mg L -1 .

In summary, turbulent disturbance oxygenated the mesocosm water, thereby increasing oxygen flux to the sediment. However, the average concentrations in the sediment varied during the course of the experiment, and the 0-5 mm surficial sedimentary layer remained anoxic on average (<0.6 mg L -1 ), with the exception of the first hours after the T4-T5 disturbance, during which the sediment was in hypoxia. The deeper part of the sediment (5-10 mm) was always anoxic (data not shown).

The average carbohydrate concentration was 8.1 µg g -1 in the surface sediment layer at the beginning of the experiment and decreased to 5.4 µg g -1 at T2 (Fig. 1b). The concentrations remained low, with values varying from 4.5 to 5.6 µg g -1 of dry sediment.

Among the fatty acids quantified by GC-MS, four were characteristic of the fatty acid signature of the duckweed used to enriched the sediments (C16:0, C18:1ω9 cis, C18:2ω6 cis and C18:3ω3) and accounted for 77% of the fatty acids in the sediments at T0. The concentrations of these biomarkers decreased strongly from T0 to T3 and remained at low concentrations after that (Fig. 1b), which indicates that the decrease in carbohydrate concentrations reflected the degradation of duckweed detritus in the surface sediment layers.

Prokaryote production and community diversity

Bacterial community diversity, represented by the Shannon index (H), varied during the course of the experiment in both the surface and deep layers (Fig. 2a) and for both the RNA and DNA fractions (Fig. S2). For the RNA fraction, the surface and deep sediment bacterial communities had similar patterns (Fig. 2a). Bacterial community diversity increased sharply after each of the 3 first disturbance events and remained stable or even decreased during calm periods (Fig. 2a). For the DNA fraction, the surface and deep layers had similar diversity dynamics patterns, with the exception of the T2-T3 disturbance event (Fig. S2), but the patterns differed from the RNA fraction. The first disturbance resulted in a sharp decrease in bacterial diversity (Fig. S2). Then, increases were observed with turbulence, and there was a decrease at the end of the experiment.

Similar to community diversity, the phylogenetic diversity for the RNA fraction, measured as SES, increased in surface sediments after each of the first three disturbance events (Fig. 2b) and decreased after T5. Moreover, the SES values were negative at T0, T1 and T2 for both sediment depths (Fig. 2b), positive in the T3 to T5 samples, and then negative again in the T6 and T7 samples. The deep sediment SES patterns were similar to the surface patterns. All SES values were significant against a null model, with the exception of the surface sample at T1.

In surface sediments (0-5 mm), the prokaryote production, expressed as the number of cells per mL of sediment per day, increased after each disturbance event and decreased during calm intervals (Fig. 2c). In deep sediments (5-10 mm), we observed an opposite trend, with a decrease in prokaryote production after each turbulence events (Fig. 2c).

Effect of time and disturbance on community composition

We calculated the Bray Curtis similarity index between each possible sample pair to compare community composition similarity with time. In both surface and deep sediment samples, the Bray Curtis values were higher when samples were separated by shorter time intervals (Fig. 3a), and the similarity decreased as the time separating communities increased (ANOVA, F 1,26 =22.76, p<0.001), which indicated that turbulence (samples separated by 1 day) had less effect on community composition compared to time.

We further tested the effect of disturbance and time with a permutational multivariate analysis of variance using distance matrices (adonis statistics). Disturbance did not impact the community composition at the RNA level (F 1,8 =0.04, p= 0.73), whereas time was a significant factor impacting the community composition (F=0.36, p= 0.001). There was no difference in community composition between depths (F=0.046, p= 0.65).

Community succession

The composition of microbial communities was compared between samples with a hierarchical cluster analysis based on the Bray Curtis index. Active bacteria communities (RNA) were separated into 3 main clusters (Fig. 4a). One contained samples taken at T0, T1 and T2, one contained samples taken at T3, T4 and T5 and the last contained samples from T6 and T7. At T0, T1, T2, T6 and T7, surface and deep sediment communities grouped together, showing similar community composition between depths. At T3 and T5, the deep and surface communities were separated, suggesting a stronger stratification of the communities with depth (Fig. 4a). Pre-and post-disturbance samples grouped together, indicating that disturbance events had a lower impact on the composition of the active communities than calm periods, despite the increase of community diversity. The second disturbance event represented an exception because it induced a major change in community composition (Fig. 4a). The T5 and T6 communities were separated in two different clusters, which indicated that the third calm interval had a strong impact on the active bacterial community composition.

At the phylum/class level, the first cluster of active bacteria (RNA fraction) (T0, T1, and T2) contained a majority of Gammaproteobacteria, Fusobacteria and Firmicutes sequences (Fig. 5). In the second cluster (T3, T4, and T5), the proportion of Deltaproteobacteria and Bacteroidetes sequences increased. The third cluster (T6 and T7) was composed mostly of Firmicutes (> 70% of the sequences). The composition of the bacterial communities described with DNA was similar to that observed at the RNA level, but there were more Bacteroidetes sequences and fewer Deltaproteobacteria.

At the OTU level, the first cluster (T0, T1, and T2) had a higher abundance of sequences belonging to OTU 1 and OTU 1657 (Fig. 4a, Fig. S3), which were similar to sequences from Propionigenium maris (Fusobacteria) (98% similarity), an anaerobic strain found in sediments that can ferment glucose and succinate primarily to propionate [START_REF] Watson | Reductively debrominating strains of Propionigenium maris from burrows of bromophenol-producing marine infauna[END_REF]. Other specific OTUs included OTU 80, 98% similar to the sediment sequences belonging to the order Clostridiales (Firmicutes), and OTU 27, identified as Vibrio kanaloae (Gammaproteobacteria) (99% similarity), which can ferment different compounds as the sole carbon source [START_REF] Thompson | Vibrio kanaloae sp. nov., Vibrio pomeroyi sp. nov. and Vibrio chagasii sp. nov., from sea water and marine animals[END_REF]. The second cluster (T3, T4, and T5) had more sequences belonging to OTU 239, OTU 943, OTU 137 and OTU 84 (Fig. 4a, Fig. S3). All belonged to the order Desulfobacterales (Deltaproteobacteria), a group of sulfate-reducing bacteria. The third cluster (T6 and T7) was characterized by the presence of OTU 476, OTU 302, OTU 8 and OTU 2, all belonging to Clostridiales (Firmicutes), an order with many fermenting bacteria.

For Archaea, active communities (RNA fraction) were separated in 2 main clusters (Fig. 4b). The first contained samples from T0 and T1, and the second cluster grouped the other samples, with the exception of the deep T2 sample. At the phylum/class level, the T0-T1 cluster contained mostly Thaumarchaeota (>85% of the sequences), whereas the second cluster (T2-T6) contained mostly Methanomicrobia sequences (Fig. S4). At the DNA level, the Methanomicrobia were less abundant, and there were more sequences from the SAGMEG-1 and C2 group, as well as Methanobacteria.

At the OTU level, the T0-T1 cluster had a higher abundance of sequences belonging to OTU 54, OTU 3 and OTU 4 (Fig. S5), which were 99%, 98% and 97% similar to the Candidatus Nitrosopumilus koreensis AR1 (Thaumarchaeota), respectively [START_REF] Park | Draft genome sequence of an ammonia-oxidizing archaeon,"Candidatus Nitrosopumilus koreensis" AR1, from marine sediment[END_REF].

The second cluster (T2-T6) was characterized by OTU 8, which was 99% similar to the methanogen Methanococcoides methylutens (Methanomicrobia), and OTU 13 and OTU 711, which were 99% and 97% similar, respectively, to Methanolobus oregonensis (Methanomicrobia). All belong to the family Methanosarcinaceae, a group known for performing all pathways of methanogenesis.

Functional genes dynamics

We constructed metagenomes for surface sediment samples T2, T3, T4, T5 and T6 and obtained a total of ca. 66.8 million sequences after quality check (Table S2). The Bray-Curtis index, calculated from the abundance of annotated sequences found in the functional genes categories, showed that there was no correlation between the time separating two samples and the functional gene composition of the samples (ANOVA, F=0.03, p=0.85) (Fig. 3b).

Within the annotated metagenome sequences, we identified the level 2 gene categories that varied the most between samples (Fig. S6). The number of sequences associated with membrane transport was highest at T2 and then decreased to similar lower levels. Energy metabolisms showed an opposite trend. There were fewer sequences at T2, and after an increase at T3, the number of sequences did not change significantly. Sequences for genes involved in cell motility were abundant in samples T2 and T4 and then decreased at T5 and again at T6. Cell growth and death increased after each turbulence event.

We then specifically targeted some metabolic pathways present in sediments that may be impacted by disturbances. Genes typical for methanogenesis, sulfate reduction, nitrate reduction, aerobic respiration, fermentation and nitrification were identified (Table S1). The genes that had the highest number of sequences were chosen to monitor the pathway dynamics in the sediments (Table S1). All of the gene sequences increased in abundance after the first turbulence event, with the exception of genes involved in fermentation (Fig. 6). After the second turbulence event, genes for sulfate reduction and aerobic respiration showed large increases (Fig. 6a), whereas genes for nitrate reduction and nitrification showed small increases (Fig. 6b). The number of sequences associated with sulfate reduction and aerobic respiration increased overall over time during the course of the experiment, whereas the number of sequences for genes associated with methanogenesis, nitrification and nitrate reduction decreased after the first turbulence event (Fig. 6).

To compare the metagenome and amplicon results, we counted the number of 16S rRNA amplicon sequences identified as methanogens (belonging to the classes Methanobacteria, Methanococci and Methanomicrobia) and sulfate reducers (order Desulfobacterales). For methanogens, the DNA and RNA counts showed similar patterns, which indicated that the microbes that were present were also active. The disturbance events resulted in an increase of the number of methanogen 16S rRNA sequences. For sulfate reducers, the DNA and RNA patterns were also similar (Fig. S7). The second and third disturbance events resulted in an increase in the number of sequences in a pattern similar to that observed for metagenomic functional genes (Fig. 6).

Discussion

We showed that disturbance increased the diversity of marine sediment bacterial communities during the first three weeks of incubation in a mesocosm experiment. Our finding can be interpreted in the context of the intermediate disturbance hypothesis, IDH [START_REF] Grime | Competitive exclusion in herbaceous vegetation[END_REF][START_REF] Connell | Diversity in tropical rain forests and coral reefs[END_REF], which predicts that disturbance will increase community diversity up to a certain level of disturbance strength or frequency, after which diversity will decrease. The increase in diversity is hypothesized to originate from the creation of niches because disturbances contribute to environmental heterogeneity. These niches are colonized by new species and the effect of competitive dominance is reduced [START_REF] Violle | Experimental demonstration of the importance of competition under disturbance[END_REF]. If disturbance is too frequent, extinction will occur, only the few species able to cope with an unstable environment will grow, and diversity will decrease. In our experiment, diversity increased until T5 (3 weeks) and then started to decrease. The last disturbance event did not augment diversity, and we propose that we observed the decrease predicted by the intermediate disturbance hypothesis. The IDH has been challenged recently, however, and the shape of the diversity-disturbance relationship remains a matter of discussion [START_REF] Mackey | The diversity-disturbance relationship: is it generally strong and peaked?[END_REF][START_REF] Hughes | Reciprocal relationships and potential feedbacks between biodiversity and disturbance[END_REF][START_REF] Fox | The intermediate disturbance hypothesis should be abandoned[END_REF]. This is the first report of a positive disturbancediversity relationship for benthic microorganisms. The hypothesis was tested previously with in situ data from marine sediment, where depth was used as a proxy for disturbance [START_REF] Boer | Time-and sediment depth-related variations in bacterial diversity and community structure in subtidal sands[END_REF], but only a weak relationship was observed. A number of studies have been conducted on soil microorganisms, in which no relationship [START_REF] Fierer | Influence of drying-rewetting frequency on soil bacterial community structure[END_REF][START_REF] Wakelin | A single application of Cu to field soil has long-term effects on bacterial community structure, diversity, and soil processes[END_REF][START_REF] Deng | Pyrene effects on methanotroph community and methane oxidation rate, tested by dose-response experiment and resistance and resilience experiment[END_REF] or only a negative impact of disturbance has been reported [START_REF] Kim | Changes in Soil Bacterial Community Structure with Increasing Disturbance Frequency[END_REF]. Here, the positive relationship was observed both at the beginning of the experiment, when the nutrient load was high, and later, when the organic carbohydrate concentrations were significantly lower. This is an important point because community diversity may be directly influenced by the resources available in the ecosystem. It has been shown, for instance, that high nutrient availability may reduce the number of species [START_REF] Torsvik | Prokaryotic diversity--magnitude, dynamics, and controlling factors[END_REF] and that the decline in energy over time may impact the level of diversity [START_REF] Scholes | The combined effects of energy and disturbance on species richness in protist microcosms[END_REF]. Our observation of diversity increasing independent of nutrient availability argues for a direct effect of disturbance on diversity. It should, however, be noted that the availability of electron acceptors is also very important in determining sediment community composition. The differences in cell numbers observed between surface and deep layer may reflect differences in oxygen availability between depths.

Interestingly, phylogenetic diversity was also positively impacted by disturbance, which indicates that disturbance promoted the co-occurrence of phylogenetically distinct species. Bacteria with distant common ancestors are more likely to be ecologically different.

Thus, the presence of distantly related species after disturbance supports the notion that diversity increased because additional niches were created and occupied by ecologically different species, which reduced competitive exclusion. Inversely, the low values measured toward the end of the experiment indicate phylogenetic clustering, which can be interpreted as communities structured by environmental filtering [START_REF] Webb | Phylogenies and Community Ecology[END_REF]. The environment selects a subset of ecologically similar taxa able to dominate under the given environmental conditions.

We also showed that in surface sediments, diversity bursts were accompanied by an increase in prokaryote production. The finding of stimulated production is supported by the observation of a relative increase, after each disturbance event, of genes that belonged to the "cell growth and death" category, which may indicate that cells were actively growing. The link between production and diversity is a key topic in ecology [START_REF] Naeem | Biodiversity, ecosystem functioning and human wellbeing. An ecological and economic perspective[END_REF][START_REF] Cardinale | Biodiversity loss and its impact on humanity[END_REF] and a positive relationship is usually observed for community diversity [START_REF] Gillman | The influence of productivity on the species richness of plants: a critical assessment[END_REF][START_REF] Cardinale | Biodiversity loss and its impact on humanity[END_REF]. For marine microbes, contrasting results show a negative relationship for bacterial richness [START_REF] Reinthaler | Relationship between bacterioplankton richness, respiration, and production in the southern North Sea[END_REF][START_REF] Obernosterer | Linkage between bacterial carbon processing and the structure of the active bacterial community at a coastal site in the NW Mediterranean Sea[END_REF] but a positive relationship between production and the phylogenetic diversity of active bacteria [START_REF] Galand | Microbial productivity is associated with phylogenetic distance in surface marine waters[END_REF]. High-diversity communities may use resources more efficiently through positive interactions and/or niche partitioning (complementary effect); alternatively, more diverse communities are more likely to contain highly productive organisms (sampling effect) [START_REF] Loreau | Partitioning selection and complementarity in biodiversity experiments[END_REF]. In our experiment, the relationship did not hold in the deeper layer of the sediments, where production decreased with disturbance. Negative relationships between production and diversity have been shown for bacteria under controlled experimental settings [START_REF] Horn | Hydrogenotrophic methanogenesis by moderately acid-tolerant methanogens of a methane-emitting acidic peat[END_REF][START_REF] Becker | Increasing antagonistic interactions cause bacterial communities to collapse at high diversity[END_REF], suggesting that other factors may constrain the relationship. We also found that prokaryote production decreased during calm intervals. Whereas increased microbial abundance has been detected at intermediate disturbance level in soil [START_REF] Kim | Changes in Soil Bacterial Community Structure with Increasing Disturbance Frequency[END_REF], our data suggest that high production was not maintained after disturbance events, or that only a few of the sediment species could maintain growth as time passed.

We identified specific groups of active microorganisms that increased in relative gene abundance when the microbial biomass increased. Methanogens and sulfate reducers both increased in sequence abundance with disturbance and may be contributors to the overall biomass bursts. The question remains as to why these groups were stimulated and others, such as fermenters, were inhibited. We can speculate that disturbance made fermentation end products more available and that they were then used by methanogens and sulfate reducers, which both represent the final step of anaerobic organic matter remineralization.

Although we observed changes in sequence abundance for some groups, our experiment showed that disturbance did not have a systematic impact on the composition of active communities, which indicates that the communities overall changed according to the time of incubation. The presence of a temporally dynamic community implies that communities will not recover to their initial composition after disturbance. In such a dynamic ecosystem, resilience can probably not be observed. Our results suggest that for defining a general response to disturbance in terms of community composition [START_REF] Allison | Resistance, resilience, and redundancy in microbial communities[END_REF] an ecosystem has to be stable or in a steady state. However, stability, which results from a combination of biotic and abiotic factors, is never or rarely observed in coastal sediments impacted by seasonal variations in physical forcing and nutrient loads.

We identified three main stages during our incubation that corresponded to different community compositions. The first period was dominated by sequences affiliated with potential fermenting bacteria and with archaea identified as ammonia oxidizers. During the second stage, we detected more sequences belonging to sulfate reducing bacteria and methanogenic archaea. For the last stage, methanogens were still present, and sequences associated with potential fermenters, different from the first ones, appeared. We hypothesize that the general pattern of changing communities over time followed the transformation of organic matter contained in the sediments.

The overall potential microbial metabolism did not change as much as the composition of the communities during the course of the incubation. The first stage was the most dissimilar, with relatively fewer genes associated with energy metabolism but more membrane transport, whereas cell motility decreased toward the end of the incubation.

However, the comparison of the gene function composition did not show significant changes with time, which indicated that there was a certain level of functional redundancy within the communities. Assemblies that appear different at the 16S rRNA gene level may actually contain similar metabolic functions. These functions would be maintained during the course of the experiment, but the identity of the microorganisms bearing these functions would change with the evolving environmental conditions. More studies are needed to resolve the question of microbial functional redundancy because our observation remains limited to one type of experimental microbial ecosystem.

Conclusion

We showed that disturbance had a positive effect on sediment microbial communities by increasing their production and both their community and phylogenetic diversity during the first 3 weeks of the experiment. This result implies that disturbance stimulated the overall community growth and promoted the development of many phylogenetically different microorganisms. These diverse communities occupy a wide range of niches and consume a variety of substrates produced during the sequential anaerobic degradation of organic matter.

In nature, we can hypothesize that the moderate storm disturbances that regularly impact coastal sediments also promote diverse communities. These events, rather than disturbing the decomposition of organic matter, probably increase the substrate turnover and, ultimately, remineralization rates.

Ackowledgments

This work was funded by the French National Program for Continental and Coastal Ecosphere

  HotStarTaq Plus Master Mix Kit (Qiagen, Valencia, CA) was conducted under the following conditions: 94 o C for 3 minutes, followed by 28 cycles of 94 o C for 30 seconds, 53 o C (bacteria) or 50 o C (archaea) for 40 seconds and 72 o C for 1 minute, after which a final elongation step at 72 o C for 5 minutes was performed. Pyrosequencing was conducted by a commercial laboratory (Research and Testing Laboratory, Lubbock, TX, USA) on a Roche 454 FLX (Brandford, CT, USA) using commercially prepared Titanium reagents. Sequences have been deposited to the GenBank Sequence Read archive under number SAMN04126915-SAMN04126946.

Fig. 1 .

 1 Fig. 1. Oxygen concentrations in the water (crosses) and sediments (triangles) along the

Fig. 2 .

 2 Fig. 2. Changes in the RNA-based community diversity (a) and phylogenetic diversity (b),

Fig. 3 .

 3 Fig. 3. Differences in the taxonomic community composition between samples (a) and

Fig. 4 .

 4 Fig. 4. Hierarchical clustering of the composition of the bacterial (a) and archaeal (b)

Fig. 5 .

 5 Fig. 5. Taxonomic affiliation and proportion of the bacterial 16S rRNA sequences in

Fig. 6 .

 6 Fig. 6. Variation in functional gene sequence numbers expressed as z-scores during the

Author Contributions:

SL, KG designed the experiments, PEG, SL, AP, EP, SF, GV, CD, KG carried out the work, SL, PEG, KG interpreted the results, PEG wrote the manuscript that was revised and improved by all the coauthors

Conflict of Interest Statement:

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Laurent Diologent

Gérard Bolbach

Cristian Focsa

Michael Ziskind
email: michael.ziskind@univ-lille1.fr

Prof Isabelle Fournier
email: isabelle.fournier@univ-lille1.fr

Laser induced post-desolvation of MALDI clusters

Keywords: Matrix Assisted Laser Desorption/Ionization, mechanism, cluster, desolvation

teaching and research institutions in France or abroad, or from public or private research centers.

Introduction

Matrix-Assisted Laser Desorption Ionization (MALDI) has revolutionized the field of biomolecules analysis thanks to the pioneering work of Hillenkamp and Karas [START_REF] Karas | Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons[END_REF][START_REF] Karas | Matrix-Assisted Laser Desorption of Non-Volatile Compounds[END_REF]. Although very high analytical performances have now been reached, fundamental mechanisms governing the MALDI process are still not fully understood. One illustration is the very different charge distribution shown between MALDI and ElectroSpray Ionization (ESI) [START_REF] Fenn | Electrospray ionization for mass spectrometry of large biomolecules[END_REF] despite very similar performances in terms of both sensitivity and molecular weight/polarity of the accessible analytes. It is then clear that a better understanding of these mechanisms is necessary when aiming at optimizing the MALDI source.

Several models have been proposed to explain the features observed in the MALDI spectra. Certain models have focused on the desorption/ablation event [START_REF] Dreisewerd | The desorption process in MALDI[END_REF], others have addressed in more detail the ionization stage [START_REF] Knochenmuss | Ion formation mechanisms in UV-MALDI[END_REF][START_REF] Knochenmuss | A quantitative model of ultraviolet matrix-assisted laser desorption/ionization including analyte ion generation[END_REF][START_REF] Knochenmuss | MALDI ionization: the role of in-plume processes[END_REF], while some have considered the desorption/ionization event as a continuous integrated process [START_REF] Chen | Near-ultraviolet-induced matrix-assisted laser desorption/ionization as a function of wavelength[END_REF][START_REF] Karas | Ion formation in MALDI: the cluster ionization mechanism[END_REF][START_REF] Beavis | Phenomenological models for matrix assisted laser desorption ion yields near the threshold fluence[END_REF]. Currently, the model on which instruments are designed is based on the hypothesis that gas phase molecules, including ions produced in MALDI, are individualized and separated from each other. However, because the analyte is diluted in matrix crystals during cocrystallization and is further ejected together with the surrounding matrix molecules, a significant number of analyte-matrix clusters might be present in the plume. Fundamental studies [START_REF] Fournier | Delayed extraction experiments using a repulsing potential before ion extraction: evidence of non covalent clusters as ion precursor in UV matrix assisted laser desorption/ionization. Part II-Dynamic effects with α cyano 4 hydroxycinnamic acid matrix[END_REF][START_REF] Fournier | Delayed extraction experiments using a repulsive potential before ion extraction: evidence of clusters as ion precursors in UV-MALDI. Part I: dynamical effects with the matrix 2, 5-dihydroxybenzoic acid[END_REF][START_REF] Karas | Ionization in matrix-assisted laser desorption/ionization: singly charged molecular ions are the lucky survivors[END_REF][START_REF] Sachon | Protein desolvation in UV matrix-assisted laser desorption/ionization (MALDI)[END_REF][START_REF] Jaskolla | Compelling evidence for Lucky Survivor and gas phase protonation: the unified MALDI analyte protonation mechanism[END_REF] and numerical simulations [START_REF] Zhigilei | Molecular dynamics simulations of matrix-assisted laser desorption-connections to experiment[END_REF][START_REF] Zhigilei | Molecular dynamics model for laser ablation and desorption of organic solids[END_REF][START_REF] Zhigilei | Microscopic mechanisms of laser ablation of organic solids in the thermal and stress confinement irradiation regimes[END_REF][START_REF] Zhigilei | Dynamics of the plume formation and parameters of the ejected clusters in shortpulse laser ablation[END_REF][START_REF] Zhigilei | Computer simulations of laser ablation of molecular substrates[END_REF][START_REF] Knochenmuss | Molecular dynamics model of ultraviolet matrix-assisted laser desorption/ionization including ionization processes[END_REF] have gradually converged towards a comprehensive MALDI model including cluster formation. Numerical simulations have demonstrated the transition from a thermal vaporization regime, characterized by the exclusive ejection of neutrals, to a phase explosion regime with emission of charged compounds and clusters when laser fluence is increased. These results are supported by a series of experiments demonstrating the existence of these clusters using repulsive fields in the source during the delayed extraction [START_REF] Fournier | Delayed extraction experiments using a repulsing potential before ion extraction: evidence of non covalent clusters as ion precursor in UV matrix assisted laser desorption/ionization. Part II-Dynamic effects with α cyano 4 hydroxycinnamic acid matrix[END_REF][START_REF] Fournier | Delayed extraction experiments using a repulsive potential before ion extraction: evidence of clusters as ion precursors in UV-MALDI. Part I: dynamical effects with the matrix 2, 5-dihydroxybenzoic acid[END_REF]. If cluster formation has become a model accepted by the community, its impact on the ionization mechanisms is still under discussion [START_REF] Knochenmuss | Ion formation mechanisms in UV-MALDI[END_REF][START_REF] Karas | Ionization in matrix-assisted laser desorption/ionization: singly charged molecular ions are the lucky survivors[END_REF].

According to the simulations, clusters are already present in the early desorption phase (<1ns after the laser shot) where they have a broad size distribution. The main problem raised by their presence is that some (possibly many) analyte ions are trapped in these complexes and are being solvated by matrix molecules (by analogy with the mechanisms associated with ESI). Various experiments have shown that the electric field applied to transfer the ions in the mass analyzer (e.g. ion traps IT, Orbitrap, Ion Cyclotron Resonance ICR or Time-of-Flight TOF) contributes to the desolvation of these complexes, which nevertheless remains incomplete. Studies using atmospheric pressure MALDI (AP-MALDI) sources [START_REF] Verentchikov | Collisional cooling and ion formation processes in orthogonal MALDI at intermediate gas pressure[END_REF] have demonstrated that clusters can be stabilized by AP relaxing collisions. Relaxation is then sufficient to allow for the observation of analyte molecules (M) clustered with matrix molecules (m), resulting in the presence of [M + nm] + peaks in the mass spectra.

In fact, stabilized clusters were shown to desolvate inside the heated capillary of the instrument under AP-MALDI conditions, making possible the observation of multiply charged ions and thus reinforcing the similarity between MALDI and ESI models [START_REF] Inutan | Commercial intermediate pressure MALDI ion mobility spectrometry mass spectrometer capable of producing highly charged laserspray ionization ions[END_REF][START_REF] Trimpin | Laserspray ionization, a new atmospheric pressure MALDI method for producing highly charged gas-phase ions of peptides and proteins directly from solid solutions[END_REF][START_REF] Mcewen | Laserspray ionization on a commercial atmospheric pressure-MALDI mass spectrometer ion source: selecting singly or multiply charged ions[END_REF]. However, for high-vacuum MALDI, which is still the most sensitive configuration, the experiments have demonstrated that collisions neither effectively stabilize the clusters nor provide their full desolvation. This phenomenon remains a major problem for the quality of the spectra. The naked analyte ions, formed before the ion extraction, will be detected at a time fully defined together by the ejection parameters and the analytical conditions (geometry of the instrument, applied voltages, delayed extraction...). In contrast, there is no direct correlation between the time-of-flight and the m/z for clusters which are not fully desolvated before their extraction but continue to desolvate during their acceleration. Clusters with a broad size distribution and long desolvation time appear as unresolved signals and contribute to increase the ("chemical") background noise in MALDI-TOF spectra. This phenomenon has been described by several authors. In particular, the work of Krutchinsky and Chait [START_REF] Krutchinsky | On the nature of the chemical noise in MALDI mass spectra[END_REF] has pointed out the existence of matrix ions or clusters in the background "noise" when performing MS/MS studies. Specifically, the authors demonstrated using a MALDI-IT instrument the presence of clusters for almost all selected mass-to-charge (m/z) values and with a lifetime significantly higher than that expected in MALDI-TOF. Sachon et al. [START_REF] Sachon | Protein desolvation in UV matrix-assisted laser desorption/ionization (MALDI)[END_REF] studied the desolvation of clusters in MALDI-TOF linear mode by varying the beam deflection voltage. These experiments highlighted, for two different matrices, the existence of a slow component compatible with the hypothesis of desolvation occurring in the source. The same authors showed that the analyte ion was the main fragment released when portions of "chemical background noise" above the analyte m/z value were selected for subsequent MS/MS studies. All these studies have demonstrated the limitation of a gradual and partial cluster desolvation during analysis, which results ultimately in a reduced spectral quality due to a concurrent increase in background noise and decrease in analyte signal, especially for high-mass analytes such as proteins. Therefore, achieving full cluster desolvation before their transfer into the analyzer (especially during the delayed extraction occurring before the acceleration for conventional MALDI-TOF systems) should improve spectral quality. More specifically, spectral sensitivity should be increased because of the rise in analyte signal intensity accompanied by the reduction in background noise.

The aims of this study are twofold: first is to develop and test a system that achieves more efficient cluster desolvation during the MALDI process, and second is to observe the effects of such a postdesolvation on MALDI signals. Taking into account the prominent role of MALDI-TOF systems for analytical purposes, we have developed a system based on a commercial instrument. To achieve effective cluster desolvation on a short time scale (shorter than the extraction delay, i.e. few hundred nanoseconds at most) we use a second pulsed laser beam which intercepts the expanding plume perpendicularly in the vicinity of the sample plate surface. The effect of various experimental parameters on the analyte, matrix and cluster signals has been investigated.

Material &Methods

Chemicals

HPLC grade water, ACN and TFA were purchased from BioSolve (Valkenswaard, The Netherlands).

Standard calibration peptides Bradykinin (Mw = 1060.2 u.) and ACTH 18-39 (Mw=2465.7 u.), as well as the -cyano 4-hydroxycinnamic acid matrix (HCCA, Mw=189.2 u.) were purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France) and used without further purification.

Sample preparation

Peptides were dissolved in water to prepare solutions at 5. 10 -6 M for both analytes. The HCCA matrix solution was prepared just before the experiments by dissolving 10 mg/mL of HCCA in 0.1% TFA in water:ACN (7:3, v/v). The sample was prepared according to the dried droplet method. Briefly, 1µL of matrix solution and 1 µL of analyte were mixed onto the MALDI sample plate. The sample was then left at room temperature and normal pressure conditions until co-crystallization of the matrix/analyte system.

MALDI-TOF MS instrument

The laser post-desolvation setup was installed on a commercial MALDI-TOF (Voyager Elite system upgraded to STR, PerSeptive Biosystems, Framingham, MA). The instrument is equipped with a delay extraction system and a single stage reflectron, and can be operated both in linear and reflectron modes. The laser used on this instrument is a pulsed N2 laser (=337 nm) with a 3 ns pulse width at a repetition rate of 3 Hz. Two adjacent stages are used for delayed extraction-ion acceleration in the instrument [START_REF] Juhasz | On the initial velocity of ions generated by matrix-assisted laser desorption ionization and its effect on the calibration of delayed extraction time-of-flight mass spectra[END_REF]. The N2 laser is focused at 45° incidence, with an irradiation spot of roughly 200 µm size on the sample plate. A neutral density filter placed on the laser beam path outside the chamber insures the control of the delivered laser energy. The maximum output laser energy is 200 µJ/pulse, this is decreased to 5-15 µJ/pulse using the filter for MALDI experiments. Inside the vacuum chamber the sample plate is inserted in a support mounted on an automatic (x, y) plate allowing sample motion under the N2 laser beam. A camera (placed at 45° with respect to the TOF axis) is used to monitor the sample during experiments.

Experiments are performed by working just above the laser energy threshold allowing analyte ion observation. Conventional MALDI MS spectra of the standard peptides are recorded using both the delayed extraction and reflectron in positive mode. During the delay time td, the first (sample plate) and second electrodes are fixed to 23 kV. After td, the first electrode is switched to a total acceleration of 25 kV and the second electrode is kept to 23 kV. td is adjusted in the range 0-200 ns to provide the highest spectral resolution for the used analyte. It must be noticed that a supplementary electronic delay of 180 ns must be taken into account. Signal at the detector is monitored during the acquisition using a 500 MHz digitizing oscilloscope (2 ns resolution, Tektronix TD520). Recorded spectra are the average of a fixed number of individual spectra (500 laser shots) obtained while moving regularly the sample under the laser beam to achieve good statistics. Recorded spectra are then processed using the Data Explorer v5.0 software.

Setup of the desolvation laser

A second laser was added on the commercial MALDI-TOF instrument for the purpose of matrix/analyte cluster desolvation. The laser has to be chosen so that it induces the dissociation of matrix clusters and avoids gas phase post-ionization in the meantime [START_REF] Rohlfing | Investigation of the desorption process in UV matrix-assisted laser desorption/ionization with a liquid 3-nitrobenzyl alcohol matrix by photoacoustic analysis, fast-flash imaging, and UV-laser postionization †[END_REF][START_REF] Soltwisch | Mass spectrometry imaging with laser-induced postionization[END_REF]. Accordingly, we used a ns pulsed Nd:YAG laser emitting at 1064 nm (Brillant B, Quantel). Taking into account the ionization potential of the irradiated species (typically 8-9 eV), eight photons are generally required to ionize them, which is highly unlikely in the fluence range used (~320-3200 J/m², corresponding to 1-10 mJ/pulse). The second laser was implemented on the side of the instrument allowing the laser beam to enter the source chamber through a newly mounted quartz window. Beam alignment was obtained using two 45° mirrors and beam collimation to a 2 mm diameter insured via a telescope setup (Figure 1A). The optical path was set so that the desolvation laser beam enters the source chamber between the sample and the first acceleration grid, since during the delayed extraction time td the ions are not accelerated (electric field null in the first extraction stage of the source). Ideally, the desolvation laser beam should intercept the MALDI plume in the earlier stage of the desorption/ionization process, just above and parallel to the sample surface and before ions are accelerated. If we consider an average initial axial velocity of 500 m/s for HCCA matrix [START_REF] Juhasz | On the initial velocity of ions generated by matrix-assisted laser desorption ionization and its effect on the calibration of delayed extraction time-of-flight mass spectra[END_REF][START_REF] Glückmann | The initial ion velocity and its dependence on matrix, analyte and preparation method in ultraviolet matrix assisted laser desorption/ionization[END_REF][START_REF] Zhou | Kinetic energy measurements of molecular ions ejected into an electric field by matrix assisted laser desorption[END_REF][START_REF] Beavis | Velocity distributions of intact high mass polypeptide molecule ions produced by matrix assisted laser desorption[END_REF], the plume material will travel an average of 90 µm for 180 ns delayed extraction time and 500 µm if the delay is increased to 1000 ns. Therefore, it is crucial that the laser beam intercepts the plume as close as possible to the sample surface. However, the fact that the second laser entrance window and the sample plate are not in a perfect line of sight, along with the short length of the first acceleration stage (2.8 mm) require the laser beam to be slightly tilted (Figure 1B). In this configuration, the laser beam hits the edge of the sample plate. A careful alignment of the 1064 nm laser beam makes sure that the laser hits only the stainless-steel plate and not the MALDI sample. In addition, such irradiation of the stainless-steel plate shows no effect on our studies, since no signal is detected in the mass spectrometer as long as the 337 nm MALDI laser is turned off. Synchronization of the MALDI (337 nm) and desolvation (1064 nm) laser pulses was realized through a 4-channel digital delay pulse generator (Stanford Research System DG 535) controlling the flash lamp (T0), the N2 laser irradiation pulse (T0+t), and the Q-Switch of the Nd:YAG laser (T0+t+t').

A frequency divider was added to decrease the N2 laser repetition rate for its synchronization with the Nd:YAG laser, which also decreases the MS instrument duty cycle. The synchronization sequence is presented on Figure 2. During the experiments, 10-100 ns N2 -Nd:YAG time delays (t') were studied since t'<td. In order to provide enough time and space for the MALDI plume to develop while enabling a good interaction between the MALDI plume and the post-desolvation laser, we operated with td =280 ns.

Results

The properties (density, composition, dimension, temperature,...) of the MALDI plume evolve spatially and temporally. It is therefore very important to trigger the desolvation laser in such a way that the interaction between the plume and the laser pulse is optimized to intercept the largest possible part of the plume and promote desolvation. We thus first investigated the delay between the laser pulse promoting the MALDI process and the laser set up for the desolvation experiments. Figure 3 displays the evolution of the analyte signal [M+H] + with the delay between the two laser pulses. A clear increase in the intensity of the protonated ions of both Bradykinin (Figure 3A) and ACTH 18-39 (Figure 3B) peptides is evidenced from t'=20 ns up to 100 ns. For both peptides, over a 2-fold increase in signal intensity is observed. This demonstrates a beneficial interaction between the desolvation laser beam and the plume. Assuming an average plume initial velocity of about 500 m/s and about 1000 m/s for the plume front, and neglecting the time required to establish this velocity, one can roughly estimate that at t'=20 ns the front plume is localized only 20 µm away from the surface of the irradiated sample at the farthest, and the "center" of the plume at about 10 µm. For t'=100 ns the plume front is expected to be located 100 µm above the sample surface and the plume "center" at about 50 µm. Since the plume velocity is only weak at the beginning of the expansion and increases to reach a plateau [START_REF] Spengler | On the formation of initial ion velocities in matrix-assisted laser desorption ionization: Virtual desorption time as an additional parameter describing ion ejection dynamics[END_REF], herein above are the maximum distances that we can expect. These distances are very small compared to the diameter of the desolvation beam (2 mm). Thus, for t'=20 ns, we assume that only the front of the plume interacts with the Nd:YAG laser beam. One can therefore expect that the interaction increases for longer times, which is actually observed in the spectra recorded for a delay between the two lasers of t'=80 and 100 ns. The evolution of the signals corresponding to the [m+H] + and [2m+H] + matrix ions contrasts with that of the analytes (Figure 4): the intensity of the matrix peaks varies little over the studied delay range (or in absence of the desolvation beam). More specifically, the [m+H] + matrix ion shows a slight decrease of intensity until a 40 ns time delay between the two lasers is reached and the [2m+H] + protonated dimer ion is roughly constant over the time range studied. Therefore, the matrix ions show only little effect of the desolvation beam, at least for the selected fluence of 3200 J/m². This demonstrates that efficient conditions of interaction between the desolvation laser and the MALDI plume can be obtained.

To deepen our understanding of the observed mechanism, the effect of the desolvation laser fluence Fd was studied for different delays between the triggering of the two lasers. Figure 5 shows the evolution of the [M+H] + ion signal of Bradykinin in the mass spectra for three laser fluences and for a delay of 80 ns between the two laser pulses. A steady increase in the signal intensity of this ion up to Fd 3200 J/m² (highest fluence used in this study) is observed. Importantly, it must be noted that the increase in peak intensity is not accompanied by a deterioration of the spectral resolution. The average intensities of the Bradykinin peaks obtained at different fluences and delays are plotted in Figure 6A. A clear increase in signal intensity is observed above Fd1000 J/m², which strengthens for the highest delay between the two laser i.e. for 80 and 100 ns. Specifically, at Fd3200 J/m² and for the shortest delays (t'=20 and 50 ns) the peak recorded is about twice as intense as the peak recorded at zero fluence, i.e. in the absence of the desolvation laser pulse. For the longest delays t'=80 and 100 ns, the peak grows to being about 3 times higher than that recorded at zero fluence. A similar trend is observed for the ACTH 18-39 as shown in Figure 6B. Similarly to Bradykinin, an increase of the ion signal is observed when increasing the fluence of the desolvation laser. Again the intensity of the peak is maximum at the highest desolvation fluence i.e. Fd 3200 J/m². At this fluence, a 2fold increase (with respect to the signal recorded without post-desolvation) is observed (depending on the delay t' between the lasers), which is slightly lower than the increase recorded for Bradykinin.

This might indicate that desolvation is more difficult for higher molecular weight, ACTH 18-39 molecular weight being more than twice that of Bradykinin. This is well in line with studies demonstrating the increase of cluster size with analyte molecular weight [START_REF] Sachon | Protein desolvation in UV matrix-assisted laser desorption/ionization (MALDI)[END_REF]. The evolution of the intensity of the peaks corresponding to the [m+H] + and [2m+H] + signals of matrix ions is again radically different (Figure 7). For both ions a 2-fold decrease is first observed up to Fd=1500 J/m². Above this value, the matrix signal is again increasing. Again the [m+H] + matrix ion seems more affected by the post-desolvation process than [2m+H] + .

Discussion

For all experimental conditions investigated in this work a similar desolvation trend is observed. A significant increase in analyte signal intensity is observed above Fd=1000 J/m² for all delays between the two lasers. Matrix ions show a less marked dependence on the desolvation laser fluence, slightly decreasing at low fluence and leveling at higher fluences. The clear effect of the desolvation laser on the analyte ion signals is in line with a cluster model for MALDI. Indeed, most of the observations can be explained in a model of cluster desolvation during the field free expansion of the MALDI plume. At the maximum desolvation fluence used in this study (3200 J/m 2 ), a 2 to 3-fold increase in intensity is observed for the analyte signal whereas the peak width remains strictly unchanged. This suggests the absence of metastable fragmentation in the field-free region. This is in favor of an effective cluster desolvation while preserving the analyte from fragmentation. MALDI clusters are expected to be composed of an analyte surrounded by a large number of matrix molecules. Previous studies [START_REF] Fournier | Delayed extraction experiments using a repulsing potential before ion extraction: evidence of non covalent clusters as ion precursor in UV matrix assisted laser desorption/ionization. Part II-Dynamic effects with α cyano 4 hydroxycinnamic acid matrix[END_REF][START_REF] Fournier | Delayed extraction experiments using a repulsive potential before ion extraction: evidence of clusters as ion precursors in UV-MALDI. Part I: dynamical effects with the matrix 2, 5-dihydroxybenzoic acid[END_REF][START_REF] Sachon | Protein desolvation in UV matrix-assisted laser desorption/ionization (MALDI)[END_REF][START_REF] Lai | Sinapinic acid clusters distribution from monomer to mega Dalton's region in MALDI process[END_REF] suggest that the number of matrix molecules forming the clusters increases with the molecular weight of the analyte. This might explain the lower signal intensity increase observed for ACTH18-39 (Mw=2465.7 u.) compared to Bradykinin (Mw=1060.2 u.) even when using a desolvation fluence of 3200 J/m². A larger number of matrix molecules would require more laser energy to be efficiently removed from the cluster.

In addition, no significant increase in intensity for matrix ions and no new ion fragments are observed in the spectra. This concurs with no significant analyte fragmentation during plume expansion before ion extraction. In contrast, the slight decrease of the matrix [m+H] + ion signal at low fluence could be associated with fragmentation processes of a small proportion of the matrix ions. However, at higher fluences, for which an increase in the analyte signal is observed, the matrix signal tends to go back to its initial level. This trend supports a desolvation model releasing free analytes but also matrix ions. In this view, the desolvation could progressively compensate for matrix fragmentation and thus explain the change in behavior observed for the matrix signal, which first decreases before increasing.

We could also hypothesize, since we do not observe any features in the spectra supporting a fragmentation process, that desolvation might prevent reneutralization of the analyte ions inside the clusters, which would be in agreement with the lucky survivor model [START_REF] Karas | Ionization in matrix-assisted laser desorption/ionization: singly charged molecular ions are the lucky survivors[END_REF][START_REF] Sachon | Protein desolvation in UV matrix-assisted laser desorption/ionization (MALDI)[END_REF][START_REF] Jaskolla | Compelling evidence for Lucky Survivor and gas phase protonation: the unified MALDI analyte protonation mechanism[END_REF].

According to the desolvation model, natural cluster desolvation and cluster collisions within the plume with neutrals result in partial to possibly complete cluster desolvation. The change in cluster sizes during ion acceleration, especially for m/z above that of the analyte, should yield in the end a signal that is diluted in the background spectra (the so-called chemical noise), and should herein increase the chemical background associated with the analyte signal. Consequently, the use of a postdesolvation laser should reduce the chemical noise. Yet, after careful examination of the spectra, notably the m/z region above the analyte signals, we could not evidence any particular change in the background. Therefore, if the desolvation process increases in our experiments, it is likely that this process is far from being complete for all clusters. It is, however, possible that the decrease of the background noise might be observed over a range of m/z higher than that recorded [START_REF] Sachon | Protein desolvation in UV matrix-assisted laser desorption/ionization (MALDI)[END_REF] or is too weak to be evidenced. Finally, the increase of the analyte signal by fast desolvation after desorption/ionization could also indicate that a higher number of analytes are ionized in the early beginning of the desorption and would be reneutralized within the clusters. This would be in line with simulations and experiments showing that laser irradiation results in the ejection of a mixture of individual molecules and molecular aggregates of different sizes [START_REF] Knochenmuss | Molecular dynamics simulations of MALDI: laser fluence and pulse width dependence of plume characteristics and consequences for matrix and analyte ionization[END_REF]. The aggregates require further desolvation to release naked analyte ions. This also indicates that fast desolvation of clusters is favorable to achieve a better sensitivity. This hypothesis is supported by the increased sensitivity reached (at the expense of lower mass resolution) in the continuous extraction mode compared to the delayed extraction, where in the former configuration clusters are immediately accelerated and tend to desolvate through collisions in the dense plume.

Conclusion & Outlook

These preliminary studies show that MALDI signals are affected by the use of a post-desolvation IR laser. The results demonstrate a beneficial effect of early cluster desolvation on the analyte signal, which brings about a 2-fold increase in intensity using a fluence of 3200 J/m² at 1064 nm. Regarding the matrix, signal evolution with increasing laser fluence suggests that desolvation competes with fragmentation. There would be then a threshold, under which fragmentation dominates and induces the decrease of the matrix peaks in the mass spectrum, and above which cluster desolvation compensates for matrix ion fragmentation. The analyte signals seem not to be affected by the fragmentation processes, probably because of their lower concentration in the plume and the concomitant buffer effect played by matrix molecules. Therefore, we have shown that the release of analyte ions during cluster desolvation significantly improves the sensitivity of the MALDI technique.

This scenario has to be confirmed by increasing the fluence range tested, and by testing the desolvation effect on heavier analytes such as proteins (e.g. Bovine Serum Albumin). Studies involving other laser sources (different wavelengths, pulse durations etc.) could also be carried out in order to amplify the desolvation effect observed or to improve basic understanding of the mechanisms involved. Generally, desolvation requires an energy transfer to the irradiated clusters. This transfer is more effective if the clusters (or the matrix monomer) present an absorption band which coincides with the desolvation laser wavelength. The experiments described here were conducted with a laser which does not satisfy this requirement. Desolvation is then achieved via a purely thermal process whose performance is directly related to the laser fluence employed. A natural extension of this work would be to replicate these experiments with more suitable wavelengths, e.g. in the 2.9 -3.4 µm spectral range that is in coincidence with the intense O-H, C-H or N-H stretching vibrational bands present in most of the commonly used matrices. This range of wavelength is reachable using a tunable

Figure 1 :

 1 Figure 1: Schematic representation of the setup realized (top views) for cluster desolvation adding a second pulsed laser (Nd:YAG pulsed, 1064 nm). Representation of (A) the laser setup and (B) the laser beam path inside the ion source chamber.

Figure 2 :

 2 Figure 2: Synchronization time sequence and apparatuses used for the pulsed laser with respect to the MALDI laser and the delay extraction system equipping the instrument.

Figure 3 :

 3 Figure 3: Evolution of the signal intensity of the [M+H] + ion of the peptides (A) Bradykinin and (B) ACTH 18-39 with respect to the delay t' between the MALDI laser and the desolvation laser. The fluence of the desolvation laser is set to 3200 J/m².

Figure 4 :

 4 Figure 4: Evolution of the signal intensity of the matrix (A) [m+H] + and (B) [2m+H] + ion with respect to the delay t' between the MALDI laser and the desolvation laser. The fluence of the desolvation laser is set to 3200 J/m².

Figure 5 :

 5 Figure 5: MALDI mass spectra of Bradykinin recorded for 0, 400, 1600, 3200 J/m 2 desolvation fluences. The delay between the MALDI laser and desolvation laser pulses is set to 80 ns.

Figure 6 :

 6 Figure 6: Evolution of the signal intensity of the [M+H] + ion of the peptides (A) Bradykinin and (B) ACTH (18-39) with respect to the desolvation laser fluence for different delays between the MALDI laser and the desolvation laser

Figure 7 :

 7 Figure 7: Evolution of the signal intensity of the matrix (A) [m+H] + and (B) [2m+H]+ ion with respect to the desolvation laser energy for different delays between the MALDI laser and the desolvation laser.

Figure 3 Figure 7

 37 Figure 3

Acknowledgements

Supported by grants from Ministère de l'Enseignement Supérieur et de la Recherche via Institut Universitaire de France (Prof. I. Fournier) and ANR Programme Blanc 2008 ANR-08-BLAN-0026 Fun-MALDI (Dr. G. Bolbach and Prof. I. Fournier).