Laure Coudrat

Majdi Rouis

Hamdi Jaafar

Elvis Attiogbe

Thierry Gélat

Tarak Driss

Emotional pictures impact repetitive sprint ability test on cycle ergometre Interaction between emotion and power output

Keywords: power output, repetitive sprint ability test, affective load, emotion

This study investigated the interaction between emotional eliciting pictures and power output during a repetitive supra-maximal task on a cycle ergometre.

Twelve male participants (mean (+SD) age, height and weight: 28.58±3.23 years, 1.78±0.05 m and 82.41±13.29 kg) performed five repeated sprint tests on a cycle ergometre in front of neutral, pleasant or unpleasant pictures. For each sprint, mechanical (peak power and work), physiological (heart rate) and perceptual (affective load) indices were analysed. Affective load was calculated from the ratings of perceived exertion and reflected the amount of pleasant and unpleasant responses experienced during exercise. The results showed that peak power, work and heart rate values were significantly lower (P < 0.05) for unpleasant pictures (9.18+0.20 W.kg -1 ; 47.69+1.08 J.kg -1 ; 152+4 bpm) compared to pleasant ones (9.50+0.20 W.kg - 1 ; 50.11+0.11 J.kg -1 ; 156+3 bpm). Furthermore, the affective load was found to be similar between the pleasant and unpleasant sessions.

All together, these results suggested that the ability to produce maximal power output depended on whether the emotional context was pleasant or unpleasant. The fact that the power output was lower in the unpleasant vs pleasant session could reflect a regulatory process aimed at maintaining a similar level of affective load between both sessions.

Introduction

Sports performance depends not only on the ability to produce a maximal or nearmaximal effort but also on the ability to repeat it. These skills could be measured by cycling laboratory tests such as the repetitive sprint ability (i.e. RSA) test [START_REF] Bishop | The validity of a repeated sprint ability test[END_REF]. Performance in such a test, estimated from the power output, depends on several factors such as recovery patterns [START_REF] Billaut | Effect of different recovery patterns on repeatedsprint ability and neuromuscular responses[END_REF], previous fatiguing [START_REF] Mendez-Villanueva | Physical fitness and performance. Fatigue responses during repeated sprints matched for initial mechanical output[END_REF], or time of the day (Racinais, Connes, Bishop, Blonc, & Hue, 2005). For example, in this latter study, the peak power developed in the first sprint was higher in the evening than in the morning. Otherwise, it has been recently suggested that power output was also affected by the knowledge of sprint number [START_REF] Billaut | Influence of knowledge of sprint number on pacing during repeated-sprint exercise[END_REF]. These findings demonstrated the existence of a pacing strategy managing the total energy expenditure during exercise in order to ensure that the exercise bout can be completed with the maximal performance and without a catastrophic biological failure (St clair [START_REF] St Clair Gibson | Evidence for complex system integration and dynamic neural regulation of skeletal muscle recruitment during exercise in humans[END_REF]Noakes, St Clair Gibson, & Lambert, 2005). From the central governor model (CGM) [START_REF] Noakes | From catastrophe to complexity: a novel model of integrative central neural regulation of effort and fatigue during exercise in humans: summary and conclusions[END_REF], this pacing strategy is thought to be controlled by the brain and selected according to internal (e.g. from peripheral physiological systems) and external (e.g. environmental conditions) information [START_REF] St Clair Gibson | Evidence for complex system integration and dynamic neural regulation of skeletal muscle recruitment during exercise in humans[END_REF][START_REF] St Clair Gibson | The role of information processing between the brain and peripheral physiological systems in pacing and perception of effort[END_REF][START_REF] Noakes | From catastrophe to complexity: a novel model of integrative central neural regulation of effort and fatigue during exercise in humans: summary and conclusions[END_REF]. Among this information, the conscious sensory perception of fatigue may have a crucial role [START_REF] Baron | The role of emotions on pacing strategies and performance in middle and long duration sport events[END_REF][START_REF] St Clair Gibson | Evidence for complex system integration and dynamic neural regulation of skeletal muscle recruitment during exercise in humans[END_REF].

Interestingly, based on the hypothesis that rating of perceived exertion (RPE) has an affective component [START_REF] Baden | Effect of anticipation during unknown or unexpected exercise duration on rating of perceived exertion, affect, and physiological function[END_REF][START_REF] St Clair Gibson | The role of information processing between the brain and peripheral physiological systems in pacing and perception of effort[END_REF], Baron and colleagues (Baron et al., 2011) recently proposed an affective loading scale computed on the basis of RPE. Affective loading is here defined as the difference between negative and positive affect arising from the exercise intensity and represent internal information that contributes to the regulation of pacing strategy. In this framework, the authors suggested that the more positive the affective responses during exercise, the greater the desire to maintain or to increase the exercise intensity. In contrast, the more negative the affective responses, the lesser this desire, resulting in a performance decrease. The role of this emotion-motivational process has been studied under conditions where changes in emotional state stemmed from the exercise intensity and not from external sensory information [START_REF] Smith | Physical activity does not disturb the measurement of startle and corrugator responses during affective picture viewing[END_REF][START_REF] Tian | Attentional bias to emotional stimuli is altered during moderate-but not high-intensity exercise[END_REF]. For example, affective valence of the experienced exercise was less pleasant during a high intensity (i.e. 105% of ventilatory threshold) compared to a low intensity (i.e. 85% of ventilatory threshold) cycling bout, suggesting that affective responses were less pleasant and the desire to maintain the exercise was lower during a high intensity exercise [START_REF] Kilpatrick | Affective responses to exercise are dependent on intensity rather than total work[END_REF]. From another point of view, other studies showed that unpleasant emotions such as anxiety and anger might be used for enhancing performance, from emotion regulation strategies [START_REF] Lane | The BASES expert statement on emotion regulation in sport[END_REF].

Furthermore, it has been hypothesized that emotional motivation, such that involved by appetitive and defensive motivational systems [START_REF] Lang | Motivated attention: affect, activation, and action[END_REF], was activated by pleasant and unpleasant external sensory information (e.g. emotional pictures), respectively, contrasting with emotional motivation arising from effort intensity. In this framework, many studies showed that emotional information could have some beneficial or deleterious effects on many goal-directed movements such as those involved in button press tasks [START_REF] Pereira | Sustained and transient modulation of performance induced by emotional picture viewing[END_REF], arm movement [START_REF] Chen | Consequences of automatic evaluation: immediate behavioral predispositions to approach or avoid the stimulus[END_REF]Coombes, Cauraugh, & Janelle, 2006) and gait initiation (Gélat, Coudrat, & Le Pellec, 2011;[START_REF] Stins | Organization of voluntary stepping in response to emotion-inducing pictures[END_REF]. In these studies, the impact of emotion on motor performance, measured in terms of reaction time, accuracy and amplitude of motor responses, usually depended on the congruency between the direction of the considered movement (i.e. approach, avoidance) and the valence of stimuli. To the extent that supra-maximal exercises are often produced without any approach-avoidance direction, one study appears to be particularly interesting as emotional effects were analysed on a non-directional arm movement such as a square-tracing task (Coombes, Janelle, & Duley, 2005). In Coombes et al.'s study, an extended exposure to unpleasant pictures (i.e. 8 s) decreased the movement accuracy (i.e. greater error). This result illustrated a deleterious effect of unpleasant stimuli on performance, resulting from the defensive motivational system activation.

To our knowledge, while sports competitions are performed in varied emotional contexts, no study has investigated the role of external emotional information during supra-maximal exercise. The potential role of emotional information to change power output during supra-maximal exercise might have strong implications on training.

The aim of the present study was to explore the effect of continued emotional information on a non-directional movement: a repetitive supra-maximal task on a cycle ergometre. Emotional context was modulated by a display of neutral (i.e. control condition), pleasant and unpleasant pictures, traditionally used to induce emotion. Based on Baron's model and the results of Coombes, we hypothesized that peak power and work should be greater in a pleasant context, compared to an unpleasant one.

Methods

Participants

Twelve active and healthy men practicing recreational sporting activities for about 4-5h a week (jogging, soccer) volunteered for this study. None of them were cyclists.

Their mean (± SD) age, height and weight were 28.58±3.23 years, 1.78±0.05 m, and 82.41±13.29 kg, respectively. Each participant signed an informed consent before the experimental procedures. The study complied with the Helsinki Declaration for human experimentation and was approved by the Institutional Review Board of the University.

Experimental design

Three days after experienced a familiarization session, participants completed three test sessions in a randomized order: a neutral, a pleasant and an unpleasant one (2 days apart). Thus, 6 possible combinations were performed: neutral-pleasantunpleasant, neutral-unpleasant-pleasant, pleasant-neutral-unpleasant, pleasantunpleasant-neutral, unpleasant-neutral-pleasant and unpleasant-pleasant-neutral, with 2 participants per combination. Each session consisted of a warm-up and a RSA cycle test [START_REF] Bishop | The validity of a repeated sprint ability test[END_REF].

Warm-up

The standardized warm-up consisted of 5 min of cycling at 80 W, followed by 3 min of rest and then a 10-s maximal sprint test on cycle ergometre. Upon completion of the 10-s test, participants rested for 5 min before performing the RSA cycle test.

Importantly, participants were required to gaze horizontally during the 10-s maximal sprint test. This instruction served as training the participants to keep their gaze horizontally during the subsequent RSA cycle test, for which a projected picture had to be viewed during each 6-second sprint. The participants were informed that if their head got down during the RSA test, then the trial would be discarded.

RSA cycle test

Each RSA cycle test consisted of 5 x 6-s sprints separated each by 24-s of passive recovery period on a cycle ergometre (Monark 894E, Stockholm, Sweden). Load was set at 60 g.kg -1 of body mass on the basis of previous results showing that a load between 50 and 75 g. kg -1 of body mass is suitable for the determination of maximal power in active adults (Doré et al., 2000).

Participants were seated on the cycle ergometre placed at a distance of 2.50 m in front of a white wall of the laboratory on which visual stimuli was presented. The temperature was 22°C.

At trial onset, adjustments to saddle height, foot position on pedals, and upper body position were made according to each subject's satisfaction. Toe clips and heel straps were used to prevent the feet from slipping. The optimal riding position was maintained identical throughout the study. The start and end of each sprint was performed according to the onset and the extinction of a single visual stimulus, respectively. Participants were instructed (1) to assume the ready position while awaiting the start visual signal, (2) to pedal as soon as and as fast as possible after stimulus onset, (3) to perform an all-out effort throughout the duration of the stimulus presentation and (4) to look at the stimulus the entire time it was on the wall, i.e. do not get the head down during the 6-s sprint test.

Stimuli presentation

SuperLab Pro v.2 was used to control the visual stimuli presentation of each sprint which began with a 4-s preparatory period, characterized by the display of a fixation cross, followed by a 6-s visual stimulus (95 cm × 160 cm). During the familiarization session, the visual stimulus was a white square. During the three test sessions, it was a neutral, pleasant or unpleasant image. Thus, 15 images were selected from the International Affective Picture System (IAPS) according to their normative ratings for male participants [START_REF] Lang | International affective picture system (IAPS): affective ratings of pictures and instruction manual[END_REF]. These images represented neutral people for the neutral session, sports images for the pleasant session and mutilation for the unpleasant session.

According to the IAPS model, valence of each image category was chosen by the experimenter as significantly different from each other (P < 0.05). Sports images (6.9) were rated as more pleasant than neutral people images (5.1), which were rated as more pleasant than mutilation ones (2.2). However, while the arousal of sports and mutilation pictures were not significantly different (P = 0.480), neutral people images were rated as less arousing (P < 0.0001) than both sport and mutilation images (neutral people: 3.2, sport: 6.1, mutilation: 5.8). IAPS image numbers were: i) neutral, 2102, 2191, 2383, 2513, 2570; ii) pleasant, 8060, 8090, 8200, 8210, 8260; iii) unpleasant, 3015, 3051, 3062, 3068, 3100. Immediately following the completion of all sessions, a computerized 9-point version of the self-assessment manikin (SAM) [START_REF] Lang | International affective picture system (IAPS): affective ratings of pictures and instruction manual[END_REF] was used to obtain subjective ratings of valence and arousal of all images viewed during the sessions. In this system, ratings of valence are indicated by five graphical representations of facial expressions ranging from a severe frown (most unpleasant) to a broad smile (most pleasant). For the arousal, the manikin varies from a state of low to high agitation. Participants may select any of the five figures, or boxes in between.

Variables

Mechanical indices

The Monark cycle ergometer interfaced with a microcomputer was used to collect the data and to calculate the power and the work performed during each sprint.

The peak power output (P Peak ) elicited during each sprint was calculated from the relationship between the peak pedal velocity and the braking force as described by Vandewalle and colleagues (Vandewalle, Peres, Heller, & Monod, 1985). The mechanical work (W) performed during each sprint was calculated by the integral of the power vs. time curve.

Two decrement values were calculated by considering all sprints: the decrement in peak power output (PP dec ) and the decrement in work done (W dec ) using the following equations: [START_REF] Bishop | The validity of a repeated sprint ability test[END_REF]Fitzsimmons, Dawson, & Ward, 1993)

Physiological and perceptual responses to exercise

The heart rate (HR) was continuously measured during each trial using a heart rate monitor with 1-second frequency (Polar RS400, Polar Electro Oy, Kempele, Finland) strapped to the chest wall directly above the heart. The peak of heart rate was obtained for each sprint.

For each 6-s sprint, participants were asked to rate their subjective perceived exertion (RPE) using the 6-20 point Borg scale [START_REF] Borg | Psychophysical bases of perceived exertion[END_REF]. The end of each 6-s sprint corresponded to the offset of a picture. At this time, the RPE scale appeared on the wall and participants had to give one number from 6 to 20 after each 6-s sprint.

According to Baron and colleagues [START_REF] Baron | The role of emotions on pacing strategies and performance in middle and long duration sport events[END_REF] and considering affect as bipolar, RPE scores allowed to calculate the affective load defined as the difference between the coexisting negative and positive affective responses associated to the exercise. As RPE score indicates a physical discomfort, it directly reflects the amount of unpleasant affective (UA) responses, which cannot be lower than 6 (very very light) and greater than 20 (very very hard). Pleasant affective (PA) responses could be thus deduced from the amount of UA responses. For instance, if the RPE score is equal to 6, then PA responses will be equal to 14. In this case, affective load computed as the difference between UA and PA responses, will be equal to -8. At the opposite, if the RPE score is equal to 20, then PA responses will be equal to 0. In this case, affective load will be equal to 20. A negative affective load score indicates the dominance of pleasant affective responses whereas a positive affective load score indicates the dominance of unpleasant ones.

Emotional bias scores

In order to assess the positive or negative effect of emotional pictures on performance, two indexes were created for each dependent variable: a pleasant and an unpleasant effect bias scores. They were calculated as the value associated to the pleasant or unpleasant context, respectively, minus the value associated to the neutral one (Fox, 2002;[START_REF] Naugle | Emotional influences on locomotor behavior[END_REF]. A positive score indicates greater values during an emotional relative to a neutral context while a negative score indicates reduced values during the emotional relative to a neutral context. The bias scores served as the bases for all statistical analyses.

Statistical analysis

Analyses were performed using Statistica 7.1 software for windows (StatSoft, France). Data distribution normality was confirmed with the Kolmogorov-Smirnov distance test. Bias scores for PP eak , W, HR and affective load were analysed using a two-way (emotional pictures: pleasant, unpleasant) x five (sprints) analysis of variance (ANOVA) with repeated measures on the two factors. PP dec and W dec bias scores were analysed using a one-way repeated-measures ANOVA (emotional pictures: pleasant, unpleasant).

The difference between the ratings of self-reported valence and arousal of each image category was analysed using one-way repeated-measures ANOVA (image categories: neutral, pleasant and unpleasant).

Post hoc analyses were conducted using the Bonferroni test. For all analyses, the probability was set at P < 0.05. Effect sizes were calculated as partial eta-squared to estimate the meaningfulness of significant findings.

Results

Pictures ratings and affective load bias score

The one-way ANOVA performed on the ratings of self-reported valence (SAM procedure) revealed a main effect of image category (F 2,22 = 156.45, P < 0.0001, = 0.93). Post hoc test showed that valence for pleasant image (6.3+0.4) was significantly higher (P = 0.01) than neutral images (5.3+0.2); the latter being significantly higher (P < 0.0001) than unpleasant ones (1.2+0.1).

The one-way ANOVA performed on the ratings of self-reported arousal also revealed a main effect of image category (F 2,22 = 93.81, P < 0.0001, = 0.89).

In contrast with normative arousal ratings (IAPS), post hoc test demonstrated that pleasant images (4.5+0.5) were rated as less arousing (P < 0.0001) than unpleasant ones (7.8+0.3). Neutral images (2.3+0.4) were rated as less arousing (P < 0.0001) than pleasant and unpleasant images.

Regarding the affective load bias score, the two-way ANOVA revealed no main effect of image (P = 0.088) despite a trend indicating that the affective load value was lower for pleasant pictures (0.43+0.50), compared to unpleasant ones (1.73+0.58) (Table 1). No main effect of sprint and no interaction were found on this variable (P > 0.05).

Mechanical and physiological bias scores

Table 1 presents summary data for all these dependent variables.

**** Table 1 near here ****

The two-way ANOVA (image x sprint) showed a significant main effect of image for the P Peak (F 1,11 = 21.71, P < 0.001, = 0.66), W (F 1,11 = 56.02, P < 0.0001, = 0.84), and HR (F 1,11 = 6.71, P = 0.025, = 0.38). Exposure to pleasant pictures led to significantly greater peak power (Figure 1a), work (Figure 1b) and heart rate (Figure 2c) following picture onset, as compared to unpleasant ones. However, the two-way ANOVA (image x sprint) showed no significant main effect of sprint for P Peak , W and HR. There was a significant interaction (sprint x image) effect for P Peak (F 4,44 = 6.52, P < 0.001, = 0.

Discussion

According to our hypothesis, greater peak power and work were obtained in the pleasant context, compared to the unpleasant ones. Specifically, peak power and work bias scores were positive for pleasant trials and negative for unpleasant ones (Figure 1). These results demonstrated that emotional pictures affected power output during a supra-maximal task depending on whether their affective valence was pleasant or unpleasant. Whereas pleasant pictures increased performance, unpleasant ones impaired it. In line with Baron et al.'s model, the performance improvement observed in pleasant trials accounted for their associated positive affective responses, which tended to increase the motivation to produce greater exercise intensity. In contrast, negative affects experienced during unpleasant trials tended to reduce the motivation to produce a high exercise intensity, which resulted in a decrease in performance. Indeed, in agreement with normative valence ratings [START_REF] Lang | International affective picture system (IAPS): affective ratings of pictures and instruction manual[END_REF], SAM results indicated that, in comparison with neutral people pictures, sport and mutilation ones induced pleasant and unpleasant emotion, respectively. Thus, emotional pictures took an active part in modifying the environmental affective content, likely explaining why power output during repeated sprints was affected.

In the present study, emotional impact on power output could be explained by appetitive and defensive motivational systems activated by the viewing of pleasant and unpleasant pictures, respectively [START_REF] Lang | Motivated attention: affect, activation, and action[END_REF]. Indeed, these motivational systems were considered to mediate action in order to promote individual survivals [START_REF] Lang | Motivated attention: affect, activation, and action[END_REF][START_REF] Bradley | Emotion and motivation I: defensive and appetitive reactions in picture processing[END_REF]. A large body of the literature demonstrated that motivational system activations gave rise to physiological changes, mediated by the somatic and autonomic nervous system [START_REF] Bradley | Emotion and motivation I: defensive and appetitive reactions in picture processing[END_REF][START_REF] Bradley | A multi-process account of startle modulation during affective perception[END_REF]. For example, while sitting or standing, the passive viewing of highly unpleasant pictures (e.g. mutilation) led to a significantly larger cardiac deceleration compared to the view of pleasant or neutral pictures [START_REF] Azevedo | A freezing-like posture to pictures of mutilation[END_REF][START_REF] Bradley | Natural selective attention: orienting and emotion[END_REF]. According to these previous studies, heart rate was lower in front of unpleasant pictures, compared to pleasant ones. This physiological change is reminiscent of the ''fear bradycardia'' and reflects a common adaptive defensive behaviour like the behavioural phenomenon of "freezing" when a potential threat is imminent [START_REF] Bradley | Natural selective attention: orienting and emotion[END_REF]Fanselow, 1994).

Thus, the freezing-like behaviour associated with highly unpleasant pictures could explain the lower power output observed during unpleasant trials, compared to pleasant ones, as this behaviour was not compatible with the task requirements, i.e.

supra-maximal effort generating over a 6-s sprint.

Previous studies showed a link between exercise intensity and affective responses [START_REF] Kilpatrick | Affective responses to exercise are dependent on intensity rather than total work[END_REF][START_REF] Smith | Physical activity does not disturb the measurement of startle and corrugator responses during affective picture viewing[END_REF][START_REF] Tian | Attentional bias to emotional stimuli is altered during moderate-but not high-intensity exercise[END_REF].

While pleasant affective responses dominate during moderate exercise intensity, they decreased during high intensity exercises for the benefit of unpleasant responses [START_REF] Kilpatrick | Affective responses to exercise are dependent on intensity rather than total work[END_REF][START_REF] Tian | Attentional bias to emotional stimuli is altered during moderate-but not high-intensity exercise[END_REF]. In the present study, the results of affective load scores were consistent with these previous findings. Affective load scores were positive from the second sprint regardless the image category (Table 1),

indicating the dominance of unpleasant responses during the supra-maximal task.

However, although affective load in the unpleasant context was greater than in the pleasant one, this difference was not significant. This lack of significance could stem from the fact that peak power and work were significantly lower in the unpleasant context, compared to the pleasant one. If peak power and work would have been similar between the pleasant and unpleasant sessions, affective load should have been higher in the unpleasant context, because of higher exercise intensity. The fact that participants produced an exercise intensity lower in the unpleasant context than in the pleasant one may be interpreted as the effect of a regulatory process to experience the exercise as less unpleasant, enabling thus the affective load score to not increase. Accordingly, if this regulatory process were reliable, it would be then relevant during training sessions to use a pleasant context to be able to produce high exercise intensity.

Moreover, emotional effects on performance were observed for the only two first sprints since emotional peak power and work bias scores were not significantly different from each other, from the third sprint to the end of the exercise. These results suggested that emotional effects present at the beginning of the exercise were only transient. During RSA tests, fatigue manifests as a decrease in peak power or work, which generally occurs after the first sprint [START_REF] Racinais | Morning versus evening power output and repeated-sprint ability[END_REF]Girard, Mendez-Villanueva, & Bishop, 2011). Even if emotional pictures affected the ability to repeat sprints, as power output was also affected during the second cycling bout, it could be assumed that the fatigue generated immediately after the first sprint has interfered with image effect.

Power and work decrement values were greater in pleasant than in unpleasant trials. This result was in agreement with previous studies [START_REF] Racinais | Morning versus evening power output and repeated-sprint ability[END_REF][START_REF] Billaut | Influence of knowledge of sprint number on pacing during repeated-sprint exercise[END_REF][START_REF] Racinais | Maximal power, but not fatigability, is greater during repeated sprints performed in the afternoon[END_REF] according to which decrement values would depend on the highest peak power or work values. The greater these values were, the greater were the decrement values. In our study, the highest peak power and work were obtained for pleasant trial, for which decrement values were the greatest. Thus, the greater decrement values observed during pleasant did not necessarily mean a greater fatigability but could simply be the consequence of a higher peak power or work [START_REF] Racinais | Maximal power, but not fatigability, is greater during repeated sprints performed in the afternoon[END_REF].

Conclusion

The results of the present study showed that emotional pictures affected power output during a supra-maximal repetitive sprint test when emotional context was changed. Peak power and work were higher for pleasant pictures compared to unpleasant ones. As exercise intensity has been shown to impact affect during exercise, the lower exercise intensity in the unpleasant session compared to the pleasant session might be viewed as the effect of a regulatory process aimed at making less unpleasant the exercise. If this regulatory process were relevant, the ability of participants to produce maximal power output would thus depend on whether the emotional context is pleasant or unpleasant. These findings highlighted that emotional context should be taken into account in training and competition in which maximal power output is required.

Contributorship statement: each author contributed to the conception and design, analysis and interpretation of data, the drafting of the article or revisiting it critically for important intellectual content and approved the final version to be published.

  37) and W (F 4,44 = 5.05, P < 0.01, = 0.31). Post hoc tests revealed that the image effect was only observed during the two first sprints (P < 0.001) (Figure 2 a,b). The one-way ANOVA revealed a significant effect of image for PP dec (F 1,11 = 10.03, P < 0.01, = 0.48) and W dec (F 1,11 = 5.49, P = 0.039, = 0.33), which were greater during exposure to pleasant (8.07 + 0.71 % and 7.69 + 0.64 %) images (Figure 1c,d), compared to unpleasant (5.99 + 0.74 % and 5.97 + 0.93 %) ones. **** Figure 1 near here **** **** Figure 2 near here ****

Figure 1 .

 1 Figure 1. Mean (and standard error) bias scores for (a) P Peak ,(b) W, (c) PP dec and (d) W dec across trials. * Significant (p<0.05) difference between emotional bias scores.

Figure 2 .

 2 Figure 2. Mean (and standard error) bias scores across the picture categories and sprints for (a) P Peak , (b) W, (c) HR and (d) AL. * Significant (p<0.05) difference between emotional bias scores. Negative bias score indicates a lower values for the emotional trial (pleasant or unpleasant) compared to the neutral trial.

Table 1 .

 1 Coombes, S.A., Cauraugh, J.H., & Janelle, C.M. (2006). Emotion and movement: Trends of peak power, heart rate and affective load during the five sprints and according to the neutral, pleasant and unpleasant pictures. Values are mean + SD.P: significance level for the difference between emotional bias scores; ***: P < 0.001, **: P < 0.01, *: P < 0.05, NS: not significant.

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Ibtissem Doghri
email: ibtissem.doghri@univ-lr.fr

Johann Lavaud
email: johann.lavaud@takuvik.ulaval.ca

Alain Dufour
email: alain.dufour@univ-ubs.fr

Alexis Bazire
email: alexis.bazire@univ-ubs.fr

Isabelle Lanneluc
email: isabelle.lanneluc@univ-lr.fr

Sophie Sablé
email: sophie.sable@univ-lr.fr

Cell-bound exopolysaccharides from an axenic culture of the intertidal mudflat Navicula phyllepta diatom affect biofilm formation by benthic bacteria

Keywords:

At low tide, intertidal mudflat biofilms cover large surfaces and are mainly responsible for the high productivity of these marine areas. In the European Atlantic coast, such biofilms are mainly composed of diatoms, especially Navicula phyllepta, bacteria, and microbial extracellular polymeric substances (EPS). To better understand interactions occurring between microorganisms, we first axenized a N. phyllepta culture with a new and simple protocol. Colloidal and bound EPS secreted by diatom cells during the exponential growth and the stationary phase were then harvested, and we tested their effects on the in vitro formation of biofilms by three marine bacteria. The latter had been isolated from a French Atlantic intertidal mudflat and were previously selected for their strong in vitro biofilmforming ability. They belong to the Flavobacterium, Roseobacter, and Shewanella genera. Navicula phyllepta-bound EPS synthesized during the stationary phase specifically inhibited the biofilm formation by the Flavobacterium sp. strain, whereas they stimulated biofilm development by the two other strains. The EPS acted in all cases during the first stages of the biofilm establishment. Saccharidic molecules were found to be responsible for these activities. This is the first report on marine bacterial antibiofilm saccharides of microalgal origin. This work points out the complexity of the benthic natural biofilms with specific microalgae/bacteria interactions and underlines the possibility to use axenic diatoms as a source of bioactive compounds.

Introduction

AQ2

Intertidal mudflats are among the most productive marine areas [START_REF] Admiraal | The seasonal succession patterns of diatom species on an intertidal mudflat-an experimental analysis[END_REF], and they may provide up to 50 % of the primary production of estuaries, in particular due to the formation of photosynthetic biofilms at their e.Proofing http://eproofing.springer.com/journals/printpage.php?token=nuGKxB4... surface [START_REF] Underwood | Primary production by phytoplankton and microphytobenthos in estuaries[END_REF]. By providing shelter and food, mudflats especially serve as pantry and nursery for many animal species of commercial interest, including fishes [START_REF] Beck | The identification, conservation and management of estuarine and marine nurseries for fish and invertebrates[END_REF].

One of the peculiarities of intertidal mudflat biofilms lies in their transient character. During daily tidal emersion periods, autotrophic motile microalgae (microphytobenthos) move upward through the sediment to the surface to access the light they need for photosynthesis [START_REF] Consalvey | The ups and downs of life in a benthic biofilm: migration of benthic diatoms[END_REF] ). When microalgae and heterotrophic prokaryotes associate at the surface, they secrete a matrix of mucilaginous extracellular polymeric substances (EPS) and form a biofilm surrounding the sediment particles [START_REF] Decho | Microbial biofilms in intertidal systems: an overview[END_REF][START_REF] Consalvey | The ups and downs of life in a benthic biofilm: migration of benthic diatoms[END_REF]. During immersion and night, microalgae migrate downward into deeper sediment layers [START_REF] Consalvey | The ups and downs of life in a benthic biofilm: migration of benthic diatoms[END_REF] ), part of the biofilm biomass is resuspended into the water column, and the temporary biofilm disappears [START_REF] Colijn | Light absorption in the waters of the ems-Dollard estuary and its consequences for the growth of phytoplankton and microphytobenthos[END_REF][START_REF] Serôdio | Nondestructive tracing of migratory rhythms of intertidal benthic microalgae using in vivo chlorophyll a fluorescence[END_REF].

This transient biofilm is thus mainly composed of microalgae [START_REF] Admiraal | The seasonal succession patterns of diatom species on an intertidal mudflat-an experimental analysis[END_REF][START_REF] Underwood | Primary production by phytoplankton and microphytobenthos in estuaries[END_REF], prokaryotes (mainly bacteria) [START_REF] Van Duyl | Consequences of diatom mat erosion for carbohydrate concentrations and heterotrophic bacterial activities in intertidal sediments of the ems-Dollard estuary[END_REF], and a variety of EPS [START_REF] Underwood | The importance of extracellular carbohydrate production by marine epipelic diatoms[END_REF] constituting complex assemblages of polysaccharides, proteins, glycoproteins, uronic acids, lipids, and many other compounds [START_REF] Underwood | Environmental effects on exopolymer production by marine benthic diatoms: dynamic, changes in composition, and pathways of production[END_REF][START_REF] Pierre | Biochemical composition and changes of extracellular polysaccharides (ECPS) produced during microphytobenthic biofilm development (Marennes-Oléron, France)[END_REF]. The EPS matrix is believed to limit sediment desiccation by maintaining high pore water content, to stabilize sediment layers, and to protect microorganisms against exogenous stresses [START_REF] Flemming | The biofilm matrix[END_REF][START_REF] Orvain | Tidal and seasonal effects on the short-term temporal patterns of bacteria, microphytobenthos and exopolymers in natural intertidal biofilms (Brouage, France)[END_REF]. Motile epipelic diatoms secrete into the surrounding sediment 30 to 60 % of photoassimilated carbon as EPS [START_REF] Underwood | The measurement of microbial carbohydrate exopolymers from intertidal sediments[END_REF]Middelburg et al. 2000 ;[START_REF] Smith | The production of extracellular carbohydrates by estuarine benthic diatoms: the effects of growth phase and light and dark treatment[END_REF]. In European intertidal mudflats, epipelic diatoms dominate the microphytobenthic community, representing up to 97 % of the biomass (de Jonge and [START_REF] De Jonge | Dynamics of microphytobenthos biomass in the ems estuary[END_REF][START_REF] Méléder | Spatio-temporal changes in microphytobenthos structure analysed by pigment composition in a macrotidal flat (Bourgneuf Bay, France)[END_REF] ). In the French Atlantic coast, the dominant species is Navicula phyllepta [START_REF] Haubois | Spatio-temporal structure of the epipelic diatom assemblage from an intertidal mudflat in Marennes-Oléron Bay, France[END_REF]. Whereas bacteria are also very abundant, with generally 10 cells per mL of sediment [START_REF] Pascal | Seasonal variation in consumption of benthic bacteria bymeio-and macrofauna in an intertidal mudflat[END_REF], their diversity and activity remain largely uncharacterized [START_REF] Gontang | Phylogenetic diversity of gram-positive bacteria cultured from marine sediments[END_REF]. The bacterial communities depend on the depth of sediment layers, and Gammaproteobacteria might be highly predominant (almost 90 %) in surface layers [START_REF] Urakawa | Characterization of depth-related population variation in microbial communities of a coastal marine sediment using 16S rDNA-based approaches and quinone profiling[END_REF].

Within mudflat biofilms, multiple interactions such as synergy, competition, or defense occur between microalgae and prokaryotes. Although these interactions are recognized as essential to the biogeochemistry of marine ecosystems [START_REF] Amin | Interactions between diatoms and bacteria[END_REF], their study is limited. There is a strong correlation between diatom and bacterial abundances [START_REF] Cammen | Annual bacterial production in relation to benthic microalgal production and sediment oxygen uptake in an intertidal sandflat and an intertidal mudflat[END_REF][START_REF] Van Duyl | Tidal coupling between carbohydrate concentrations and bacterial activities in diatominhabited intertidal mudflats[END_REF][START_REF] Hamels | Uncoupling of bacterial production and flagellate grazing in aquatic sediments: a case study from an intertidal flat[END_REF]. The presence of bacteria closely associated to diatom cells (referred to as "satellite" bacteria by [START_REF] Schäfer | Genetic diversity of "satellite" bacteria present in cultures of marine diatoms[END_REF] seems essential for some diatom strains to their optimal growth in nature as well as in laboratory cultures [START_REF] Bruckner | Bacteria associated with benthic diatoms from Lake Constance: phylogeny and influences on diatom growth and secretion of extracellular polymeric substances[END_REF][START_REF] Bruckner | Growth and release of extracellular organic compounds by benthic diatoms depend on interactions with bacteria[END_REF][START_REF] Amin | Interactions between diatoms and bacteria[END_REF]. Diatom cell aggregation is also enhanced in the presence of bacteria [START_REF] Bruckner | Bacteria associated with benthic diatoms from Lake Constance: phylogeny and influences on diatom growth and secretion of extracellular polymeric substances[END_REF][START_REF] Lubarsky | The stabilisation potential of individual and mixed assemblages of natural bacteria and microalgae[END_REF][START_REF] Gärdes | Diatom-associated bacteria are required for aggregation of Thalassiosira weissflogii[END_REF]. [START_REF] Bruckner | Bacteria associated with benthic diatoms from Lake Constance: phylogeny and influences on diatom growth and secretion of extracellular polymeric substances[END_REF] and [START_REF] Windler | Biofilm and capsule formation of the diatom Achnanthidium minutissimum are affected by a bacterium[END_REF] showed that bacteria can stimulate diatom biofilm formation, as biofilm formation was directly induced by the cell-free spent medium of bacteria, making modification of the diatom metabolism most likely. It has been shown that the carbon photosynthetically assimilated by diatoms is transferred into bacteria in few hours, indicating a rapid use of labile carbon sources, likely including EPS (Middelburg et al. 2000 ;[START_REF] Goto | Biodegradation of photosynthetically produced extracellular organic carbon from intertidal benthic algae[END_REF][START_REF] Cook | Effect of nutrient availability on carbon and nitrogen incorporation and flows through benthic algae and bacteria in near-shore sandy sediment[END_REF][START_REF] Bellinger | Significance of diatom-derived polymers in carbon flowdynamics within estuarine biofilms determined through isotopic enrichment[END_REF] ). To remain within the "phycosphere" where their growth is stimulated by algal EPS [START_REF] Bell | Chemotactic and growth responses of marine bacteria to algal extracellular products[END_REF], bacteria often need to attach to the surface of diatom cells. Mechanisms underlying attachment remain unclear, but they likely involve extracellular molecules such as polysaccharides and/or proteins that are also released by bacteria themselves [START_REF] Amin | Interactions between diatoms and bacteria[END_REF]. EPS are thus crucial intermediaries between bacteria and diatoms. However, interactions between diatoms and bacteria are not always beneficial: Mmany diatoms have defense mechanisms against unwanted and/or algicidal bacteria [START_REF] Amin | Interactions between diatoms and bacteria[END_REF]. They secrete fatty acids, esters, and polyunsaturated aldehydes that can act as antibacterial compounds and influence the bacterial community structure [START_REF] Lebeau | Diatom cultivation and biotechnologically relevant products. II. Current and putative products[END_REF][START_REF] Ribalet | Differential effect of three polyunsaturated aldehydes on marine bacterial isolates[END_REF]. However, some diatom-associated bacteria were shown to be resistant to bioactive polyunsaturated aldehydes, suggesting that satellite bacteria may have developed resistance to toxic molecules released by diatoms [START_REF] Ribalet | Differential effect of three polyunsaturated aldehydes on marine bacterial isolates[END_REF] ). To our knowledge, nothing is known about the effect of molecules excreted by diatoms in their immediate surroundings on the bacterial biofilm formation.

We previously selected three bacterial strains from intertidal mudflat biofilms of the French Atlantic coast that were able to attach to different surfaces and build biofilms with specific 3D structures [START_REF] Doghri | Marine bacteria from the French Atlantic coast displaying high forming-biofilm abilities and different biofilm 3D architectures[END_REF]. These strains are appropriate models for deciphering the colonization of surfaces and the interactions between microorganisms in marine biofilms. Such knowledge is essential to the better understanding of biofouling which causes various and e.Proofing http://eproofing.springer.com/journals/printpage.php?token=nuGKxB4... costly damages to maritime human activities and industries. Here, we investigated the specific effects of diatom organic EPS on the formation of bacterial biofilms. A N. phyllepta isolate was selected since this species is one of the most abundant in temperate intertidal mudflats. To avoid the presence of EPS secreted by satellite bacteria, we axenized N. phyllepta prior to examine the effects of different EPS fractions on the bacterial models.

Materials and methods

Diatom and bacterial strain isolation and culture

Benthic diatom Navicula phyllepta Kützing (Culture Collection Yerseke, CCY9804) cells were grown in batch cultures at 20 °C in sterile artificial F/2 seawater medium (Tropic Marin salt, Guillard and Ryther 1962 ) with a light intensity of 75 µmol photons m s and a 16-h light/8-h dark photoperiod (white fluorescent tubes, L58W/840, Osram, Germany). Diatom cultures were started with a concentration of 0.3 µg chlorophyll a (Chl a) mL using a Chl a spectrophotometric quantification [START_REF] Jeffrey | New spectrophotometric equations for determining chlorophylls a, b, c and c in higher plants, algae and natural phytoplankton[END_REF]. Specific growth rates, µ (day ), were calculated from regression of the natural logarithm of the Chl a (mg mL ) during their exponential growth phase. During its growth, N. phyllepta forms a biofilm in the flask bottom that can be visually observed.

Benthic bacteria

We used strains Flavobacterium sp. II2003, Roseobacter sp. IV3009, and Shewanella sp. IV3014. They were isolated from the intertidal temperate mudflat biofilm of the Marennes-Oléron Bay (French Atlantic coast) and selected for their ability to form biofilms on different surfaces, under static and dynamic conditions [START_REF] Doghri | Marine bacteria from the French Atlantic coast displaying high forming-biofilm abilities and different biofilm 3D architectures[END_REF]. Strains were conserved as frozen stocks with 25 % glycerol at -80 °C. For all tests, the bacteria were grown at 22 °C in Zobell broth (Bio-Rad pastone 4 g L ; Bio-Rad yeast extract 1 g L ; sea salts Sigma 30 g L ) supplemented with agar (12 g L Biokar) for solid medium.

Axenization of N. phyllepta

Different axenization strategies were investigated, combining mechanical cell separation and antibiotic treatments (Fig. 1 and Table 1 ). Based on [START_REF] Shishlyannikov | A procedure for establishing an axenic culture of the diatom Synedra acus subsp. radians (Kütz.) Skabibitsch from Lake Baikal[END_REF], two pretreatments of the diatom suspensions were tested and compared with a non-pretreated control (Fig. 1 , "Physicochemical pretreatments"). The suspensions, pretreated with or without Triton X-100, were filtered through a 5-µm polycarbonate membrane (Millipore,

-2 -1 -1 -1 -1 -1 -1 -1
-1

e.Proofing http://eproofing.springer.com/journals/printpage.php?token=nuGKxB4... USA) put on a Whatman membrane by using a manual filtration unit (Sartorius, Germany). Diatom cells were then washed with artificial seawater and transferred to F/2 medium. Different antibiotics, alone or in combination (Table 1 ), were added to the pretreated or the control suspensions, and diatom cells were grown as described above, with soft shaking at regular time intervals (Fig. 1 , "Antibiotic treatments"). The final concentrations of the tested antibiotics are described in Table 1 . Diatom cells were then harvested by filtration, rapidly washed with culture medium, and resuspended in F/2 medium. The degree of axenization of diatom cell suspensions was then immediately checked by (i) bacterial counting with epifluorescence microscopy after 4′6-diamidino-2-phenylindole (DAPI) staining and (ii) counting the number of colony forming units (CFU-culturable bacteria) after plating diatom cell suspensions on Zobell agar (Fig. 1 , "Checking"). For the microscope counting, diatom cell samples were fixed in formaldehyde (final concentration 2 %) and diluted in di-sodium pyrophosphate 10 mM with 0.1 % of Tween 80 in order to enhance detachment of bacteria from the diatoms [START_REF] Lavergne | An efficient and rapid method for the enumeration of heterotrophic prokaryotes in coastal sediments by flow cytometry[END_REF]. After incubation for 30 min at 4 °C, samples were stained with DAPI (1 mg L ) for 15 min and filtered through a 0.2-µm polycarbonate membrane (Nuclepore Track-Etch Membrane, Whatman Schleicher & Schuell, USA). The membranes were mounted on a glass slide in a non-fluorescent oil drop, and bacterial cells were counted with an epifluorescence microscope (Leica DMRB, mercury steam light, magnification ×1000, filter UV A Leica) on an average of five fields of 0.0415 mm each. Extraction of EPS from axenic cultures of N. phyllepta

Fig. 1

The detailed protocol was adapted from [START_REF] Takahashi | Optimising extraction of extracellular polymeric substances (EPS) from benthic diatoms: comparison of the efficiency of six EPS extraction methods[END_REF] and is summarized in Fig. 2 . The axenic N. phyllepta suspension was cultured during 6 days (D , middle of the exponential phase, 1.16 µg Chl a mL ) and 12 days (D , stationary phase, 1.24 µg Chl a mL ) in order to collect the colloidal and bound EPS. D and D samples were centrifuged to harvest the supernatants containing the colloidal EPS fraction. To extract EPS bound to the diatom cells, the pellets were resuspended in F/2 medium containing activated cation exchange resin (Dowex Marathon C Na , Sigma-Aldrich, USA) and left under gentle shaking. After centrifugation, both colloidal and bound EPS fractions were filtered (Millipore PVDF filters, USA) and lyophilized. The dried fractions were resuspended in 1 mL ultra-pure water.

The salt excess was removed by dialysis (molecular cutoff of 1000 Da, Spectrum Labs, USA) against ultra-pure water, and EPS samples were then stored at -80 °C until further use.

Fig. 2

Protocol for the extraction of EPS from axenic N. phyllepta cultures

Antibacterial assay

Antibacterial (i.e., bactericidal or bacteriostatic) activity of EPS was tested by using the agar well diffusion assay previously described by [START_REF] Sablé | Antibacterial activity evaluation of microcin J25 against diarrheagenic Escherichia coli[END_REF]. Sterile glass rings (4-mm inside diameter) were placed on solid nutrient plates (15 mL of Zobell agar inoculated with 10 of the target bacteria) and filled with 30 µL of the EPS 20× samples to be tested. The plates were incubated at 22 °C for 48 h to allow bacterial growth and EPS diffusion. The presence of a halo around the glass cylinder indicates an inhibition of bacterial growth if the halo is clear (without cell growth) or eventually a stimulation if the halo is denser than the remaining plate (with cell growth).

Effect of the diatom EPS on bacterial biofilm formation

Microtiter plate assay: static conditions According to [START_REF] Pitts | A microtiter-plate screening method for biofilm disinfection and removal[END_REF], the EPS fractions were mixed to bacterial suspensions during the adhesion step. After overnight growth (Zobell broth, shaking at 150 rpm, 22 °C) and harvesting by centrifugation (7000×g, 10 min), bacteria were resuspended in artificial seawater (for the control) or in EPS 20× fractions to a final optical density at 600 nm (OD ) of 0.25. Of the resulting suspensions (10 CFU mL ), 150 µL was then loaded per well of a 96-well microtiter plate (MICROTEST 96, Falcon, USA). After a bacterial attachment step of 2 h at 22 °C, the wells were gently washed three times with artificial seawater, and 150 µL Zobell medium were added to each well. After incubation at 22 °C for 24 h, the bacterial biofilms were washed three times with artificial seawater, stained with a 0.8 % crystal violet solution for 20 min, and rinsed with ultra-pure water until the wash liquid was clear (10 times on average). Crystal violet was then eluted from attached bacteria with 96 % ethanol (150 µL well ), and the absorbance was measured with a plate reader at 595 nm (FLUOstar Omega, BMG Labtech, Germany) to quantify the biofilms.

Flow cell assay: dynamic conditions Bacterial biofilms were grown on glass slides in continuous culture three-channel flow cells (channel dimensions 1 by 4 by 40 mm, Technical University of Denmark Systems Biology, Denmark) as described by [START_REF] Pamp | Insight into the microbial multicellular lifestyle via flow-cell technology and confocal microscopy[END_REF]. Flow cells were inoculated with 24-h old bacterial cultures diluted in artificial seawater (for the control) or in EPS 20× fractions to a final OD of 0.1. Bacteria were allowed to attach to the glass slide during 2 h at 22 °C without medium flow. Channels were washed to remove non-attached bacteria by applying a flow of artificial seawater for 15 min at a rate of 2 mL h , and biofilm growth was allowed for 24 h at 22 °C under a constant flow (2 mL h ) of Zobell medium. The biofilms were then stained with 5 µM Syto 61 Red and observed by confocal laser scanning microscopy (CLSM) using a TCS-SP2 system (Leica Microsystems, Germany). The biofilm stacks were analyzed with the COMSTAT software (developed in MATLAB, [START_REF] Heydorn | Quantification of biofilm structures by the novel computer program COMSTAT[END_REF] to estimate the maximal and the average thicknesses (µm) as well as the biovolume (µm µm ) of each biofilm.

Biochemical assays and characterization of the diatom active EPS fraction

Proteins assays were carried out with a bicinchoninic acid protein assay kit (Sigma-Aldrich, USA), using bovine serum albumin as a standard [START_REF] Smith | 43 of protein using bicinchoninic acid[END_REF]. The sugar content was determined using the phenol-sulfuric acid assay, using glucose as standard [START_REF] Dubois | Colorimetric method for determination of sugars and related substances[END_REF] different treatments were performed to digest proteins, lipids, nucleic acids, and saccharides. Proteinase K or Pronase E was added to a final concentration of 1 mg mL , and the reaction was incubated for 1 h at 37 °C. Lipase acrylic resin form was used at a final concentration of 2 mg mL , and the reaction was incubated for 48 h under shaking at 37 °C. DNaseI (100 µg mL ) or RNaseA (25 µg mL ) was added for 12 h at 37 °C. NaIO was used to hydrolyze saccharides by cleaving the C-C bonds and by oxidizing the carbon of vicinal hydroxyl groups [START_REF] Mack | The intercellular adhesin involved in biofilm accumulation of Staphylococcus epidermidis is a linear beta-1,6-linked glucosaminoglycan: purification and structural analysis[END_REF][START_REF] Bendaoud | Broad-spectrum biofilm inhibition by Kingella kingae exopolysaccharide[END_REF]. We improved the classical treatment (20 mM NaIO , incubation for 12 h at 37 °C) by adding a neutralization step of NaIO with ethylene glycol (1:100) [START_REF] Babor | Periodate oxidation of saccharides. III. Comparison of the methods for determining the consumption of sodium periodate and the amount of formic acid formed[END_REF] for 2 h at 37 °C followed by a final dialysis step (1000 Da). Each treated EPS fraction was mixed to bacterial suspension during the adhesion step, and a microtiter plate assay was performed as previously described.

Statistical analyses

All values presented in the " Results " part are the average of three independent experiments. In order to analyze differences between a sample and the corresponding control, Student's t tests were performed. ANOVA tests gave the same results.

Results

Axenization of the N. phyllepta diatom from associated bacteria

The uni-algal N. phyllepta cultures were non-axenic, and the initial suspension (0.3 µg Chl a mL ) contained in average about 10 bacteria mL (Table 2 ). In order to test the effect of EPS specifically secreted by N. phyllepta and to avoid interference with EPS synthesized by the bacteria associated with diatom cells, it was crucial to remove bacteria present in these cultures. Different antibiotic treatments as well as pretreatments potentially increasing the effectiveness of antibiotics were then investigated (Fig. 1 ).

While the detergent treatment associated with the filtration reduced the bacterial load by 40 % (Table 2 ), it considerably affected the viability of the diatom cells (Chl a in cultures decreased by more than 50 %) and the ability of N. phyllepta to form a biofilm (visual observation of a brown layer of diatom cells in the flask bottom). The filtration without detergent treatment reduced the bacterial contamination by up to 26 % ( and tetracycline [Tc]) were tested, alone or in combination. The treatments including Ap, Tc, or Ipm were quickly discarded owing to the modification of the visual aspect (i.e., pigmentation), the ability to form a biofilm, and the growth rate of the diatom cultures. The results obtained with the most promising antibiotics are shown in Table 2 . Cip and Sm treatments reduced the bacterial contamination by 94 % according to microscope counting and by 60 and 73 % according to agar medium plating, respectively. Km treatment also reduced significantly the bacterial contamination, in particular the bacteria growing on agar medium. Nevertheless, the strongest effect was exerted by the Km + Sm mix which completely removed bacterial contamination.

Table 2

Enumeration of bacteria associated with N. phyllepta before and after physico-chemical and antibiotic treatments

Microscope counting Counting by plating on agar

Initial algal suspension without treatment

1.1 × 10 ± 1.4 × 10 1.9 x 10 ± 3. To summarize the selected protocol of axenization, the algal suspension was first filtered, treated with Km and Sm for 18 h, again filtered, and transferred in new culture medium. The growth rate of the obtained axenic diatom cultures was ∼1.2 day , similar to untreated diatom cultures (data not shown).

Effects of the N. phyllepta EPS fractions on bacterial biofilm formation

In order to observe biological effects, N. phyllepta EPS were concentrated.

Based on the EPS protein and saccharide contents, a 20× concentration was chosen, to reach concentrations slightly higher than what was observed in the intertidal mudflats where N. phyllepta is the major diatom [START_REF] Pierre | Biochemical composition and changes of extracellular polysaccharides (ECPS) produced during microphytobenthic biofilm development (Marennes-Oléron, France)[END_REF].

Antibacterial assays, performed with the agar well diffusion techniques, did not exhibit any clear halo characteristic of inhibition of bacterial growth. These experiments then showed that no EPS fractions from diatom cultures had a bactericidal or bacteriostatic activity against the three tested marine bacterial strains: Flavobacterium sp. II2003, Roseobacter sp. IV3009, and Shewanella sp. IV3014. Moreover, no stimulation of the bacterial growth (halo denser than the remaining plate) was observed around the deposit of EPS fractions.

We then examined whether EPS fractions affected biofilm formation. In static conditions (on polystyrene surface, microtiter plate assay, quantification of biofilms by spectrophotometry), the D colloidal and bound EPS fractions (Fig. 3a ), as well as the D colloidal EPS fraction (Fig. 3b ), did not show any significant activity on bacterial biofilm formation. On the contrary, the D bound EPS fraction showed significant effects: Ccompared with the not treated control biofilms, the biofilms treated with this EPS fraction were reduced by more than 40 % for Flavobacterium sp. II2003 (t = -13.63; p < 0.01) and were increased by more than 30 % for Roseobacter sp. IV3009 (t = 6.97; p < 0.05) and Shewanella sp. IV3014 (t = 11.99, p < 0.01) (Fig. 3b ). In a second set of experiments, the activity of the D bound EPS fraction on bacterial biofilm formation was further tested by growing the biofilms under dynamic conditions (on glass surface, flow cell assay, observation of biofilms by confocal laser scanning microscopy). The main aim of this set of experiments was to confirm EPS effects obtained under static conditions. The antibiofilm activity against Flavobacterium sp. II2003 was also observed in the second conditions. Compared with the untreated sample, the treated biofilm was very thin: -76 % of the biovolume (t = -6.82, p < 0.01), -47 % of the average thickness (t = -5.89, p < 0.01), and -60 % of the maximal thickness of the biofilm (t = -13.20, p < 0.001), and it lost its mushroom-like 3D structures (Fig. 4a ). Under dynamic conditions, the Roseobacter sp. IV3009 biofilm seemed to be still stimulated compared with the untreated sample, but this positive effect of D bound EPS was not significant anymore (t = 1.41, p = 0.24 for the biovolume, t = 1.78, p = 0.25 for the maximum thickness, and t = 2.28, p = 0.09 for the average thickness) (Fig. 4b ). However, the stimulating effect of D bound EPS was maintained for Shewanella sp. IV3014 (Fig. 4c ): Aan increased biovolume (+40 %, with t = 3.59, p < 0.05) and increased average thickness (+43 %, with t = 4.64, p < 0.01) and maximal thickness (+37 %, with t = 3.62, p < 0.05) were observed, without modification of the hairy architecture compared with the control biofilm. The results obtained under static and dynamic conditions were thus consistent for Flavobacterium sp. II2003 and Shewanella sp. IV3014.

Fig. 3

Effect of EPS fractions from axenic N. phyllepta cultures on bacterial biofilm formation in static conditions.

Biochemical characterization of the N. phyllepta active EPS fraction

The carbohydrate and protein contents of all EPS fractions were quantified (Table 3 ), in order to compare the active and inactive fractions. It is noticeable that carbohydrate and protein quantities were higher in D than in D fractions. The measure of the chlorophyll content of the cultures allowed us to evaluate the number of diatoms and then to show that the quantities of carbohydrates and proteins per cell were higher in the D fractions than in the D fraction. Moreover, the carbohydrate content was higher than the protein content in all fractions, and the active fraction (D bound EPS) was [START_REF] Mack | The intercellular adhesin involved in biofilm accumulation of Staphylococcus epidermidis is a linear beta-1,6-linked glucosaminoglycan: purification and structural analysis[END_REF][START_REF] Jiang | Antibiofilm activity of an exopolysaccharide from marine bacterium Vibrio sp[END_REF]. These results indicated that the active compounds of the D bound EPS fraction were saccharidic molecules.

Table 3 Determination of protein and carbohydrate contents of the N. phyllepta EPS 20× fractions and estimation of these contents per diatom cell Proteins (mg L ) 36.3 ± 2.9 45.9 ± 2.3 114.0 ± 8.7 151.0 ± 5.4

Proteins (mg cell ) 6.2 × 10 7.9 × 10 1.8 × 10 2.4 × 10

Carbohydrates (mg L ) 45.0 ± 3.5 198.0 ± 9.1 178.0 ± 6.9 690.0 ± 2.4

Carbohydrates (mg cell ) 7.7 × 10 3.4 × 10 2.9 × 10 1.1 × 10

Fig. 5

Effect of various treatments on the activity of the N. phyllepta D bound EPS. The inhibiting or stimulating effects of the N. phyllepta D bound EPS were determined using the microtiter plate assay. Biofilms were stained with crystal violet and quantified by measuring absorbance at 595 nm. The "100 %" values on the Y-axis are given to the biofilms grown without EPS fraction (aA). Samples + untreated EPS fraction (bB) correspond to reference values of the inhibiting or stimulating effects of the N. phyllepta EPS on the bacterial biofilm formation. The data represent mean values ± SD of three replicates. The effect of each treated EPS fraction (c C to hH) was compared with the effect of the untreated EPS fraction (bB). Highly significant differences were observed, only between not treated N. phyllepta D bound EPS and EPS treated with NaIO . These differences are indicated by *** (p < 0.001) on the upper part of the

Discussion

Successful strategy to remove the satellite bacteria from the N. phyllepta cultures Axenization of diatom cultures by the removal of bacteria was essential to ascertain that the studied active compounds were indeed produced by N. phyllepta and not by its associated bacteria. Depending on the diatom strain and its growth form, the axenization protocol can be very different [START_REF] Bruckner | Protocols for the removal of bacteria from freshwater benthic diatom cultures[END_REF]. In particular, benthic diatoms are often difficult to axenize because of the mucilage that surrounds the cells [START_REF] Bruckner | Protocols for the removal of bacteria from freshwater benthic diatom cultures[END_REF]. Moreover, many diatoms in co-culture with bacteria grow denser and faster than their axenic counterparts [START_REF] Bruckner | Bacteria associated with benthic diatoms from Lake Constance: phylogeny and influences on diatom growth and secretion of extracellular polymeric substances[END_REF]. We achieved to axenize the intertidal benthic diatom N. phyllepta, without changing its main biological properties (aspect, growth, and biofilm formation), thanks to a combination of antibiotics (kanamycin + streptomycin) used after a filtering step. This is in accordance with [START_REF] Bruckner | Protocols for the removal of bacteria from freshwater benthic diatom cultures[END_REF] who noticed that for most freshwater benthic diatoms, physical separations and antibiotic treatments have to be combined to fully remove associated bacteria. They succeeded in removing satellite bacteria from some diatom strains with a combination of penicillin G, streptomycin, and chloramphenicol without affecting their biological properties in cultures.

However, this combination could not be used in our case since chloramphenicol inhibits the growth of Navicula [START_REF] Khandeparker | Interactions of bacteria with diatoms: influence on natural marine biofilms[END_REF]. Recently, [START_REF] Windler | Technical note: purification of benthic diatoms from associated bacteria using the antibiotic imipenem[END_REF] described a simple protocol for freshwater benthic diatom axenization including treatment of the cultures with the antibiotic imipenem. Here, the imipenem treatment generated a disturbance of N. phyllepta biofilm formation. To optimize our axenization method, diatom cells were treated during the latency phase of the growth curve, during which the bacterial load is low. This approach allowed us to overcome problems due to the excretion of protective substances (such as EPS) by bacteria and diatoms during the growth phase [START_REF] Flemming | The biofilm matrix[END_REF][START_REF] Orvain | Tidal and seasonal effects on the short-term temporal patterns of bacteria, microphytobenthos and exopolymers in natural intertidal biofilms (Brouage, France)[END_REF]. Additionally, resuspending the cells by shaking at regular intervals enhanced the efficiency of the method by disturbing the diatom-bacterium biofilm formation and thus by decreasing the protection toward antibiotics.

EPS produced by N. phyllepta exhibit activity on bacterial biofilm formation

The main objective of our work was to investigate the effects of the EPS produced by axenic diatom N. phyllepta on the ability of three benthic bacterial models to form a biofilm. We worked intentionally with a concentration of EPS slightly higher than in the environment such as intertidal mudflats in order to better highlight biological activities. The examined bacterial strains reacted to one or several molecules secreted by N. phyllepta, suggesting that a direct diatom-bacterium contact was not required, as reported before [START_REF] Windler | Biofilm and capsule formation of the diatom Achnanthidium minutissimum are affected by a bacterium[END_REF]. Navicula phyllepta produced the active molecule(s) when cultivated separately from bacteria, which means that the synthesis and the secretion of these molecules were not induced by bacterial presence and/or activity. In our experimental conditions, the secretome part of N. phyllepta active on the early stages of bacterial biofilm formation was the bound EPS fraction harvested during the stationary phase of diatom growth. Its main begotten effects were (i) a strong inhibition (more than 40 %) of the biofilm formation of Flavobacterium sp. II2003 under both static and dynamic conditions and (ii) a stimulation of the biofilm formation, again under both conditions for Shewanella sp. IV3014 (up to 40 %) and only under static conditions for Roseobacter sp. IV3009. The results first depend on the bacterial strains and, to a lesser extent, on the experimental approach. In our case, it is likely that the difference in the surface properties between static (polystyrene) and dynamic (glass) conditions may influence early stages of surface colonization and subsequent biofilm formation of the bacterial strains [START_REF] Dang | Bacterial primary colonization and early succession on surfaces in marine waters as determined by amplified rRNA gene restriction analysis and sequence analysis of 16S rRNA genes[END_REF][START_REF] Jones | Bacterial community structure of biofilms on artificial surfaces in an estuary[END_REF][START_REF] Lee | Bacterial communities in the initial stage of marine biofilm formation on artificial surfaces[END_REF]. It was therefore essential to investigate different conditions of growth and/or biofilm formation to confirm (or not) the significance of the positive or the negative effect of the bound EPS fraction on the bacterial biofilm.

The active N. phyllepta compounds are bound EPS carbohydrates

The biochemical characterization of the active bound EPS fraction demonstrated that the inhibiting/stimulating activity can be assigned to saccharidic compounds, which were very abundant in the active fraction. In fact, the carbohydrate concentration in both bound and colloidal algal EPS fractions was approximately 4-fold higher during the stationary phase (D ) than during the exponential growth phase (D ). This is in accordance with [START_REF] Underwood | The importance of extracellular carbohydrate production by marine epipelic diatoms[END_REF] who described that the concentrations of extracellular carbohydrates in the medium significantly increased during the transition from exponential growth to stationary phase for benthic diatoms, including Navicula species, when grown in batch culture. Additionally, the amount of proteins was lower (up to 4.6-fold less for bound EPS) compared with the amount of carbohydrates, which is in agreement with previous reports on Navicula strains [START_REF] Staats | Isolation and characterization of extracellular polysaccharides from the epipelic diatoms Cylindrotheca closterium and Navicula salinarum[END_REF][START_REF] Underwood | The importance of extracellular carbohydrate production by marine epipelic diatoms[END_REF][START_REF] Scholz | Effects of varying pH on the growth and physiology of five marine microphytobenthic diatoms isolated from the Solthörn tidal flat (southern North Sea, Germany)[END_REF]. EPS (colloidal and bound fractions) extracted directly from mudflat microphytobenthic biofilms also predominantly consisted of polysaccharides, and only small quantities of proteins were present [START_REF] Underwood | The importance of extracellular carbohydrate production by marine epipelic diatoms[END_REF][START_REF] Pierre | Biochemical composition and changes of extracellular polysaccharides (ECPS) produced during microphytobenthic biofilm development (Marennes-Oléron, France)[END_REF].

Potential mode of action of the N. phyllepta EPS on the bacterial biofilm formation

In the phycosphere model of [START_REF] Bell | Chemotactic and growth responses of marine bacteria to algal extracellular products[END_REF], bacterial growth and metabolic activity were proposed to be stimulated by microalgal extracellular substances. Algal organic substances are generally considered as energy and carbon sources for bacteria [START_REF] Bellinger | Significance of diatom-derived polymers in carbon flowdynamics within estuarine biofilms determined through isotopic enrichment[END_REF], and EPS might favor bacterial metabolism by supplying nutrients (Middelburg et al. 2000 ;[START_REF] Goto | Biodegradation of photosynthetically produced extracellular organic carbon from intertidal benthic algae[END_REF][START_REF] Cook | Effect of nutrient availability on carbon and nitrogen incorporation and flows through benthic algae and bacteria in near-shore sandy sediment[END_REF][START_REF] Bellinger | Significance of diatom-derived polymers in carbon flowdynamics within estuarine biofilms determined through isotopic enrichment[END_REF]. Although no stimulation of bacterial growth has been observed in solid culture medium, the biofilm formation of Shewanella sp. IV3014 and Roseobacter sp. IV3009 was enhanced by N. phyllepta EPS. Indeed, failing to directly act on cell multiplication, microalgal polysaccharide could act as signaling molecules that modulate the expression of genes involved in biofilm lifestyle of recipient bacteria [START_REF] Rendueles | Antibiofilm polysaccharides[END_REF]. Diatom EPS are also assumed to facilitate cell attachment to a surface and to protect cells against desiccation and different environmental stress [START_REF] Lubarsky | The stabilisation potential of individual and mixed assemblages of natural bacteria and microalgae[END_REF][START_REF] Orvain | Tidal and seasonal effects on the short-term temporal patterns of bacteria, microphytobenthos and exopolymers in natural intertidal biofilms (Brouage, France)[END_REF], thus enhancing the development of associated bacteria biofilms.

In our work, N. phyllepta polysaccharides showed also a powerful biofilm inhibition activity on Flavobacterium sp. II2003, without any antibacterial activity, which leads to ascribe the N. phyllepta polysaccharide activity as specifically antibiofilm. Several studies identified antibiofilm molecules from bacteria, and exopolysaccharides were specifically shown (i) to interfere with bacterial adherence to surfaces and biofilm formation [START_REF] Valle | Broad spectrum biofilm inhibition by a secreted bacterial polysaccharide[END_REF][START_REF] Jiang | Antibiofilm activity of an exopolysaccharide from marine bacterium Vibrio sp[END_REF] ), (ii) to modulate expression of genes (such as curli genes) related to biofilm formation in bacteria [START_REF] Kim | Released exopolysaccharide (r-EPS) produced from probiotic bacteria reduce biofilm formation of 1 2 e[END_REF][START_REF] Rendueles | Antibiofilm polysaccharides[END_REF], or (iii) to disrupt established biofilms [START_REF] Qin | Pseudomonas aeruginosa extracellular products inhibit staphylococcal growth, and disrupt established biofilms produced by Staphylococcus epidermidis[END_REF][START_REF] Jiang | Antibiofilm activity of an exopolysaccharide from marine bacterium Vibrio sp[END_REF] ). However, very few polysaccharides from microalgae were described as negatively impacting bacteria or other microorganism activities, and none of them were studied in the marine context. They are (i) polysaccharides isolated from the Chlorella (green microalga) and Spirulina (cyanobacterium) inhibiting the binding of Helicobacter pylori to gastric mucin in vitro [START_REF] Loke | Antiadhesive property of microalgal polysaccharide extract on the binding of Helicobacter pylori to gastric mucin[END_REF] ) and (ii) a sulfated polysaccharide named naviculan and isolated from Navicula directa displaying antiviral activities against herpes simplex and influenza A viruses [START_REF] Lee | Antiviral sulfated polysaccharide from Navicula directa, a diatom collected from deep-sea water in Toyama Bay[END_REF] ). Recently, it has been shown that changes in the composition of carbohydrates produced by the diatom Asterionellopsis glacialis reduced bacterial abundance [START_REF] Da Silva | Carbohydrates produced in batch cultures of the surf zone diatom Asterionellopsis glacialis Sensu Lato: influence in vertical migration of the microalga and in bacterial abundance[END_REF].

As regards to the physico-chemical aspect of biofilm formation, the stimulating or inhibiting effect of microalgal EPS can also be due to modifications of the substratum (abiotic surface) and/or of the bacterial cell surface [START_REF] Grossart | Algae-bacteria interactions and their effects on aggregation and organic matterflux in the sea[END_REF][START_REF] Rendueles | Antibiofilm polysaccharides[END_REF]. Studies utilizing culture supernatants or purified polysaccharides as surface coating provide further evidence that microbial polysaccharides modify the wettability and the charge of abiotic surfaces and hence affect the interaction of bacteria with the substratum [START_REF] Valle | Broad spectrum biofilm inhibition by a secreted bacterial polysaccharide[END_REF][START_REF] Rendueles | Screening of Escherichia coli species biodiversity reveals new biofilm-associated anti-adhesion polysaccharides[END_REF]. Additionally, polysaccharides are suspected to change the physical properties of gram-negative and gram-positive bacteria cell surfaces such as the cell surface hydrophobicity [START_REF] Sayem | Antibiofilm activity of an exopolysaccharide from a sponge-associated strain of Bacillus licheniformis[END_REF] ). These properties are essential during the adhesion phase of biofilm formation and the further development of microcolonies [START_REF] O'toole | Biofilm formation as microbial development[END_REF].

Interest for the diatoms to modulate the formation of bacterial biofilms and potential biotechnological applications Our experiments with axenic diatom secretomes and different benthic bacterial biofilm models clearly demonstrate that specific interactions between algae and bacteria are instrumental for bacterial biofilm formation. Specific algae-bacteria interactions have largely been neglected so far but are presumably as important as, for instance, nutrient supply and grazing in controlling the development of microphytobenthic biofilms [START_REF] Grossart | Algae-bacteria interactions and their effects on aggregation and organic matterflux in the sea[END_REF] ). In our work, it is striking that the active diatom bound EPS fraction showed two opposite effects on bacterial biofilm formation, depending on the strains. One can hypothesize that N. phyllepta would favor some satellite bacteria for its own (or reciprocal) benefit and, conversely, would inhibit attachment of others, useless or detrimental to diatom cells [START_REF] Amin | Interactions between diatoms and bacteria[END_REF]. Bound EPS might be closely involved into the formation of the extracellular matrix of the microphytobenthic biofilm, facilitate attachment of the cell to a surface, and also improve cell-cell interactions between bacteria and diatoms [START_REF] Lubarsky | The stabilisation potential of individual and mixed assemblages of natural bacteria and microalgae[END_REF][START_REF] Pierre | Biochemical composition and changes of extracellular polysaccharides (ECPS) produced during microphytobenthic biofilm development (Marennes-Oléron, France)[END_REF]. It strongly suggests the existence of complex physico-chemical interactions between microorganisms in natural biofilms, especially in our model, i.e., the transitory biofilm forming at the surface of sediment of intertidal mudflats during daily emersion. These interactions could allow a process of selection/adaptation to changing environmental conditions, and populations able to co-exist or to exploit algal/bacterial species would be promoted, as it has been shown for estuaries [START_REF] Haynes | Diatom-derived carbohydrates as factors affecting bacterial community composition in estuarine sediments[END_REF] ).

The study of marine biofilms raises a growing interest, especially for human activities, and marine diatoms are considered as attractive sources of new active compounds for biotechnological applications [START_REF] Fu | Developing diatoms for value-added products: challenges and opportunities[END_REF].

Flavobacterium species are important pathogens in aquaculture setting [START_REF] Duchaud | Complete genome sequence of the fish pathogen Flavobacterium psychrophilum[END_REF] ) and have been detected in industrial, domestic, and medical environment biofilms [START_REF] Basson | Evaluation of adherence, hydrophobicity, aggregation, and biofilm development of Flavobacterium johnsoniae-like isolates[END_REF]. In that framework, it is of particular interest to identify a diatom secretome that strongly inhibits the biofilm formation by a Flavobacterium strain. On the contrary, Shewanella sp. IV3014 and Roseobacter sp. IV3009, whose biofilm formation is positively impacted by N. phyllepta secretome, belong to genera known to be dominant among the pioneers in marine environments [START_REF] Salta | Marine biofilms on artificial surfaces: structure and dynamics[END_REF]. It is crucial to understand the first steps of marine biofilm formation in order to efficiently prevent biofouling or conversely to stimulate biofilm formation, which improves for instance larval settlement and metamorphosis of farmed oysters [START_REF] Yu | Larval settlement and metamorphosis of the pearl oyster Pinctada fucata in response to biofilms[END_REF]. Therefore, the molecules of the N. phyllepta EPS bound fraction responsible for Shewanella sp. IV3014 or Roseobacter sp. IV3009 and Flavobacterium sp. II2003 biofilm stimulation and inhibition, respectively, need to be identified.

  Cip ciprofloxacin, Sm streptomycin, Km kanamycin Antibiotics not toxic for N. phyllepta;

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  Fig. 4 Effect of EPS bound fraction D from axenic N. phyllepta cultures on bacterial biofilm formation in dynamic conditions. Biofilms of Flavobacterium sp. II2003 (a), Roseobacter sp. IV3009, (b) and Shewanella sp. IV3014 (c). 3D images of the biofilms (top and side views) are shown on the right (bars = 67.3 µm) and the corresponding COMSTAT analysis output on the left. Untreated control biofilm: bacterial biofilm without diatom EPS fraction. The data represent mean values ± SD of three replicates

  http://eproofing.springer.com/journals/printpage.php?token=nuGKxB4... 16 sur 31 30/08/2016 11:43 the most concentrated in sugars. Navicula phyllepta D bound EPS fraction was further analyzed. It was submitted to different treatments to gain information on the chemical nature of the putative active compounds (Fig. 5 ). Neither proteases nor lipase nor nuclease treatments significantly affected the activity of the D bound EPS fraction. Only the NaIO treatment significantly reduced the antibiofilm activity of the D bound EPS fraction, by 90 % (t = 18.53, p < 0.001), against Flavobacterium sp. II2003, and totally prevented the stimulating activity in Roseobacter sp. IV3009 (t = -13.8, p < 0.001) and Shewanella sp. IV3014 (t = -39.5, p < 0.001). NaIO very efficiently hydrolyzes saccharides by oxidizing the carbons bearing vicinal hydroxyl groups and cleaving the C-C bonds

  figure. T values are -18.53, 13.8, and 39.5 for Flavobacterium sp. II2003 (a), Roseobacter sp. IV3009 (b), and Shewanella sp. IV3014 (c), respectively

Table 1 Antibiotics tested alone or in combination for the axenization of the N. phyllepta cultures

	Cip	Water	5
	Ipm	Water	98
	Ap	Ethanol/water (50/50)	100
	Tc	Ethanol/water (50/50)	50
	Sm	Water	100
	Km	Water	50
	Km + Sm	Water	100/100

Ap ampicillin, Tc tetracycline, Sm streptomycin, Km kanamycin, Cip ciprofloxacin, Ipm imipenem -1 e.Proofing http://eproofing.springer.com/journals/printpage.php?token=nuGKxB4... 7 sur 31 30/08/2016 11:43 Ap ampicillin, Tc tetracycline, Sm streptomycin, Km kanamycin, Cip ciprofloxacin, Ipm imipenem Ap + Sm Ethanol/water (25/75) 100/100 Ap + Tc + Km Ethanol/water (25/75) 100/50/30 Ap + Tc + Sm + Km Ethanol/water (25/75) 100/50/100/30

  . To elucidate the biochemical nature of the active component(s) of diatom EPS 20× fractions,

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	8	-1	600
		-1
			600
			-1
			-1
	3	-2

Table 2

 2

	-1
	-1
	-1
	-1
	4
	4
	4

) without affecting viability and culture aspect of diatoms. This pretreatment was therefore chosen. After validation of this step, different antibiotics (ampicillin [Ap], ciprofloxacin [Cip], imipenem [Ipm], kanamycin [Km], streptomycin [Sm],

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Acknowledgments

The authors thank the Conseil Général de la Charente Maritime (France) for the Ph.D. grant of Ibtissem Doghri, the CPER littoral 2007-14 (France), the CNRS EC2CO program MicroBiEn 2013-14 (France), the Région Poitou-30/08/2016 11:43 Charente (France) and the Région Bretagne (France), and the European FEDER funds, for their financial support.