Nicolas Babault
email: nicolas.babault@u-bourgogne.fr

Gaëlle Deley

Pascale Le Ruyet

François Morgan

François André Allaert

Effects of soluble milk protein or casein supplementation on muscle fatigue following resistance training program: a randomized, double-blind, and placebo-controlled study

Keywords: Muscle power, Endurance, Muscle thickness, Branched-chain amino acids

Background:

The effects of protein supplementation on muscle thickness, strength and fatigue seem largely dependent on its composition. The current study compared the effects of soluble milk protein, micellar casein, and a placebo on strength and fatigue during and after a resistance training program.

Methods: Sixty-eight physically active men participated in this randomized controlled trial and underwent 10 weeks of lower-body resistance training. Participants were randomly assigned to the Placebo (PLA), Soluble Milk Protein (SMP, with fast digestion rate) or Micellar Casein (MC, with slow digestion rate) group. During the 10-week training period, participants were instructed to take 30 g of the placebo or protein twice a day, or three times on training days. Tests were performed on quadriceps muscles at inclusion (PRE), after 4 weeks (MID) and after 10 weeks (POST) of training. They included muscle endurance (maximum number of repetitions during leg extensions using 70% of the individual maximal load), fatigue (decrease in muscle power after the endurance test), strength, power and muscle thickness.

Results: Muscle fatigue was significantly lower (P < 0.05) in the SMP group at MID and POST (-326.8 ± 114.1 W and -296.6 ± 130.1 W, respectively) as compared with PLA (-439.2 ± 153.9 W and -479.2 ± 138.1 W, respectively) and MC (-415.1 ± 165.1 W and -413.7 ± 139.4 W, respectively). Increases in maximal muscle power, strength, endurance and thickness were not statistically different between groups.

Conclusions:

The present study demonstrated that protein composition has a large influence on muscular performance after prolonged resistance training. More specifically, as compared with placebo or micellar casein, soluble milk protein (fast digestible) appeared to significantly reduce muscle fatigue induced by intense resistance exercise.

Background

Resistance training simultaneously stimulates catabolism and anabolism in active muscle fibers. The difference between these mechanisms is called net protein balance. When positive, the net protein balance favors increases in muscle mass, i.e., muscle hypertrophy. The effect of resistance training on net protein balance can persist up to 48 h [START_REF] Burd | Exercise training and protein metabolism: Influences of contraction, protein intake, and sex-based differences[END_REF]. In addition, any nutritional modification that could increase protein accretion in the muscle would maximize resistance training effects by enhancing muscle anabolism. In particular, it has now been well demonstrated that protein consumption after exercise shifts the balance in favor of muscle protein synthesis [START_REF] Rennie | Control of the size of the human muscle mass[END_REF].

Composition of supplements may play a key role in influencing net protein balance since previous studies have revealed that only essential amino acids could stimulate muscle protein synthesis [START_REF] Tipton | Nonessential amino acids are not necessary to stimulate net muscle protein synthesis in healthy volunteers[END_REF]. Furthermore, protein type, and not simply its amino acid composition, can differentially modulate protein synthesis depending on digestion kinetics. For instance, milk contains two protein fractions, soluble proteins and micellar casein, with rapid and slow digestion rates, respectively [START_REF] Boirie | Slow and fast dietary proteins differently modulate postprandial protein accretion[END_REF]. As a consequence, muscle protein synthesis has been shown to be greater with soluble proteins such as whey when compared with casein [START_REF] Tang | Ingestion of whey hydrolysate, casein, or soy protein isolate: Effects on mixed muscle protein synthesis at rest and following resistance exercise in young men[END_REF].

In addition to increases in muscle mass, functional adaptations, such as strength increases, are also obtained after essential amino acid (EAA) supplementation [START_REF] Cermak | Protein supplementation augments the adaptive response of skeletal muscle to resistance-type exercise training: A meta-analysis[END_REF]. For example, Vieillevoye et al. [START_REF] Vieillevoye | Effects of a combined essential amino acids/carbohydrate supplementation on muscle mass, architecture and maximal strength following heavy-load training[END_REF] found increases in lower body strength with EAA supplementation while no modification was obtained with placebo. Protein supplementation may also influence muscle fatigue. Indeed, previous authors [START_REF] Derave | Beta-alanine supplementation augments muscle carnosine content and attenuates fatigue during repeated isokinetic contraction bouts in trained sprinters[END_REF] have reported an attenuation of fatigue during repeated bouts of dynamic contractions after four weeks of protein supplementation as a result of an increased muscle buffering capacity during endurance exercise. Other amino acids, such as branched-chain amino acids (BCAA; leucine, isoleucine and valine), might also reduce fatigue by lowering perceived exertion and favoring mental performance during prolonged exercise [START_REF] Portier | Effects of branched-chain amino acids supplementation on physiological and psychological performance during an offshore sailing race[END_REF][START_REF] Blomstrand | A role for branched-chain amino acids in reducing central fatigue[END_REF]. Taken altogether, these results seem to suggest that the effects of protein supplementation are largely dependent on their composition, and it can be hypothesized that supplementation with a rapidly-digesting protein would be more efficient to improve both strength and resistance to fatigue. Given that different amino acid compositions could easily be obtained from protein milk extraction, the aim of the present study was to compare the effects of two different formulations of milk protein supplementation, one fast (a soluble milk protein) and one slow (micellar casein), on muscle performance (endurance, fatigue, strength and power) during and after a 10-week resistance training program.

Methods

Participants

A total of 68 male participants were recruited for the study. All were practicing two to six hours of physical activity per week (<3 sessions a week). None were engaged in any physical activity aimed at increasing the size and strength of knee extensor muscles. All were healthy and free of injury in the three months preceding the study. The study excluded subjects who had previously received treatment with corticoids. Participants who were currently taking any dietary supplement, sports drink, or functional food intended to enhance performance or muscle mass, or had taken any of these in the previous month, were also excluded. Moreover, subjects with known hypersensitivity to any of the constituents of the products under study (milk protein or lactose) were excluded. Throughout the study, subjects maintained their usual training routines and diets. All gave their written informed consent after being told about the experimental procedure. The study was conducted in accordance with the Helsinki Declaration and was approved by the local ethics committee (East I, number: 2011-38, 4 October 2011, AFSSAPS number: 2011-A00789-32).

After inclusion, participants were randomly divided into three experimental groups: 24 in the Placebo group (PLA), 22 in the Micellar protein group (MC), and 22 in the soluble milk protein group (SMP). Balanced randomization was made by blocks of four. The randomization code was not made available to anyone involved in conducting or evaluating the study and was released after the blind review and the freezing of the final database. The sample size was calculated ap r i o r iusing Nquery Advisor software (ver. 6.01, Statistical solutions Ltd, Cork, Ireland) based on the primary criterion (muscle endurance) and allowing for a power > 90%. This statistical analysis indicated a minimum of 22 participants per experimental group.

Experimental procedure

The primary objective of this randomized, double-blind study, conducted with parallel arms, was to evaluate the effects of different milk protein supplements on muscle endurance and fatigue following a resistance training program. A soluble milk protein beverage was compared with a micellar casein beverage and placebo. Body composition, knee extensor muscle thickness, maximal strength, power and perceived exertion were also determined.

The experiment involved four testing sessions: one at inclusion (PRE), one at the middle of the training program (after 28 days; MID) and two at the end of the 10 weeks training program (POST and POST + 5) (Figure 1). Testing sessions were conducted on non-training days and always at the same time of day for a given subject. PRE, MID and POST sessions included measurements of (i) right vastus lateralis muscle thickness using ultrasonography, (ii) lower limb muscle power during vertical jumps, (iii) maximal strength on a leg extension machine (one repetition maximum, 1-RM), (iv) muscle power on the same leg extension machine at 70% of the 1-RM, (v) muscle endurance during an all-out test (maximum number of repetitions performed with a load corresponding to 70% of the session's 1-RM) and (vi) recovery, as determined by muscle power, immediately, 30 min and 60 min after the endurance test. Tests were always performed in the aforementioned order after a standardized warm-up. Warm-up consisted of light pedaling followed by submaximal voluntary contractions and vertical jumps in order to familiarize subjects with testing procedures. The POST + 5 session only consisted of an all-out endurance test performed with the same load as the one used at PRE, followed by recovery power measurements.

All subjects followed the same 10-week resistance training routines, three times per week with a rest day between sessions. The program was based on three exercises involving knee extensors (knee extension machine and horizontal leg press) and knee flexors (hamstring curl machine) (Technogym, Gambettola, Italy). Throughout the training program, sets number, repetition number and recovery between sets varied: from the first to seventh week, sets increased from three to five and repetition maximum (RM) number also increased from eight to 15. During weeks eight and nine, training consisted of four sets of 20 RM. During the last week, training consisted of five sets of six RM. Recovery beteween sets was 1-2 minutes. The load used for each exercise was regularly adapted depending on the 1-RM evaluated every two weeks. All training sessions were supervised by specialized teachers.

Dietary supplementation

The three products under study were presented as 30-g sachets to be diluted in 200 mL water. Once the powders were diluted in cold water, drinks were of identical appearance, texture, taste, and all were isoenergetic (120 kcal per 30 g powder). Products were taken during the 10-weeks training period. On non-training days, protein intake was repeated twice, with the first in the morning after waking and the second during the afternoon. During training days, protein intake was repeated three times: in the morning and 30 min before and after the resistance training session. Protein supplementation was done either with Prolacta® or micellar casein.

Prolacta® (Lactalis, Retiers France) is a 90% soluble milk protein isolate. It is representative of native proteins in the non-casein phase of milk as it is a concentrate of native whey proteins extracted directly from skimmed cow milk by a soft membrane process (Lactalis Industry, Bourgbarré, France), and not from whey. Prolacta® is defined as a fast leucine-rich protein [START_REF] Boirie | Slow and fast dietary proteins differently modulate postprandial protein accretion[END_REF] differing from whey with a better amino acid composition. Moreover, Prolacta® has demonstrated larger postprandial protein retention than casein [START_REF] Gryson | Fast proteins" With a unique essential amino acid content as an optimal nutrition in the elderly: Growing evidence[END_REF]. Each 30 g Prolacta® sachet contained 10 g of protein from Prolacta®, 10.5 g sucrose, 8.2 g maltodextrine, 0.3 g lactose and 1 g soy lecithin. Each 30 g micellar protein sachet (produced by Lactalis, Retiers France) contained 10 g of Micellar Casein, 10.5 g sucrose, 7.5 g maltodextrine, 1gl a c t o s ea n d1gs o yl e c i t h i n .T h ea m i n oa c i dc o mposition of each product is detailed in Table 1. Placebo composition was 10.5 g sucrose and 19.5 g maltodextrine and did not contain any protein.

Measurements

Muscle thickness

The right vastus lateralis muscle thickness was measured in real time using an ultrasound machine (AU5, Esaote Biomedica, Florence, Italy) coupled to a 50 mm probe at a 7.5 MHz frequency. Subjects were in the supine position with the knee flexed at a 45°angle. The probe was placed perpendicular to the skin surface in the middle of vastus lateralis muscle, i.e., 39% of thigh length measured from the superior border of the patella to the anterior superior iliac spine [START_REF] Blazevich | Intra-and intermuscular variation in human quadriceps femoris architecture assessed in vivo[END_REF]. Thickness was calculated as the distance between superficial and deep aponeuroses measured at the ends and middle of each 3.8-cm-wide sonograph. Three images were independently obtained. The average value of these nine measures was calculated. Probe placement was carefully noted for reproduction during the other test sessions. Also, body composition (body weight, percent body fat) was quantified using a bio-impedance scale Fitness scale 7850 (Soehnle GmbH, Murrhardt, Germany).

Muscle strength

Subjects were seated on a leg extension machine (Multiform, La Roque d' Anthéron, France) with a 100°hip angle. The knee rotation axis was aligned with the machine rotation axis. The 1-RM was first determined after a standardized warm-up using five different loads and individually adjusted increments. Subjects were requested to lift each load only once. One minute of rest was permitted between trials. Care was taken to lift the load with a full range of motion (~100°). Range of motion was controlled using an electronic goniometer (Myotest, Sion, Switzerland).

Muscle endurance

Subjects were asked to lift a load corresponding to 70% of their 1-RM as many times as possible over a 100°r ange of motion. The test was stopped when subjects were unable to lift the load over a 80°range of motion during two consecutive repetitions. The number of repetitions was identified as subjects'" relative endurance" (PRE, MID and POST). Immediately after all endurance tests, the rating of perceived exertion was determined using a Borg scale [START_REF] Borg | Perceived exertion as an indicator of somatic stress[END_REF]. The endurance test procedure was repeated five days after the end of the experimental period (POST + 5), but using the same load as the one used at inclusion (here called "absolute endurance").

Muscle fatigue and recovery

Muscle power and vertical jump performance were measured just before, immediately after, 30 min after and 60 min after the endurance tests, to evaluate muscle fatigue and recovery. Muscle power was measured using a linear encoder (Globus, Codogne, Italy) on the leg extension machine with the same load as during the endurance test (70% of the 1-RM). Subjects were requested to lift the load as fast as possible (3 trials) throughout the 100°range of motion. The linear encoder measured the vertical velocity of the load being lifted and allowed measurements of muscle power during the entire range of motion. Peak power of the best repetition was considered for analyses.

Immediately after muscle power assessments, subjects performed two counter movement jumps on an Optojump system (Optojump, Microgate, Bolzano, Italy), starting from a standing position, then squatting down to a 90°k nee angle and extending the knees in one continuous movement. During these jumps, arms were kept close to the hips to minimize their contribution. The best jump height was retained for analyses.

Statistical analyses

Quantitative variables were presented as mean values and standard deviations (SD). Values were tested using a repeated measures analysis of variance. Groups (PLA, SMP and MC) were used as independent variables and time (PRE, MID, POST or POST + 5) was used as the dependent variable. A sensitivity analysis was also conducted and considered subjects with a muscle thickness at inclusion <22 mm (median value of study sample). Thirteen subjects were considered in both PLA and MC groups and 8 for SMP. In the case of significant main effects or interactions, Scheffé post-hoc tests were conducted. Qualitative variables (supplementation compliance or adverse effects) were presented as absolute and relative frequencies and were tested by using a Chi square test. Statistics were conducted using SAS software (Ver. 9.2, SAS Institute, Inc., Cary, NC, USA). P < 0.05 was taken as the level of statistical significance for all procedures.

Results

General observations

Initial values measured at PRE revealed comparable groups (Table 2). During the experimental protocol, compliance was evaluated by the percentage of products returned by subjects. The results show high average and comparable compliance between groups: 90.5%, 91.5% and 89.4% for PLA, MC and SMP groups, respectively (P = 0.769). In addition, tolerance to the three products under study was good and comparable in terms of frequency and nature. Of the 68 subjects who took products at least once, three subjects presented adverse events in each group. None of these events were due to the supplements ingested, but rather to personal convenience or injuries.

Muscle endurance

Absolute muscle endurance (Table 3), evaluated as the number of repetitions at 70% of the initial 1-RM, increased in all groups (+90.8 ± 58.8%, +81.2 ± 51.0% and +73.7 ± 32.2% for SMP, MC and PLA, respectively) with no significant difference between groups (P = 0.492). However, when subjects with small muscle thickness were considered separately (thickness < 22 mm, sensitivity study), increases reached the significant level between SMP and PLA (+103.0 ± 61.1% and +65.0 ± 33.9%, respectively, P < 0.05). Similar results were obtained for relative endurance measured POST with a significant time effect but no differences between groups (Table 3). For all subjects, the 1-RM load was significantly increased after training (Table 3). No significant interaction was obtained for the rating of perceived exertion.

Muscle fatigue and recovery

Muscle fatigue, i.e. power reduction during the endurance test, was similar between groups at PRE. This decrease in power was significantly lower for SMP as compared with MC and PLA after four weeks (-326. When considering PRE and POST + 5, slight betweengroups differences were registered for counter movement jump height. For SMP, counter movement jump height measured immediately after the endurance test slightly increased while it decreased for MC and PLA (+0.9 ± 4.6 cm, -1.6 ± 2.3 cm and -0.7 ± 2.8 cm, respectively; P=0.0686). A small difference was obtained for counter movement jump at POST + 5. As compared with baseline, 30 min after the endurance test, counter movement jump height significantly increased in SMP and PLA while it remained lower for MC (+1.9 ± 5.1 cm, +0.4 ± 2.1 cm and -0.6 ± 2.8 cm, respectively; P=0.0965).

Muscle thickness

Muscle thickness and body composition changes are indicated in Table 3. While a significant increase in muscle thickness was obtained with time, no difference was registered between groups. Also, no significant difference was noticed for body mass, percent body fat or percent body fat free mass (Table 3).

Discussion

The present study aimed to test the hypothesis that supplementation with milk proteins of varying compositions, used in combination with resistance training, would have different effects on physical performance. The main results revealed enhanced muscle endurance (i.e., number of repetitions), reduced fatigue (i.e., muscle power loss) and slightly enhanced recovery (i.e., vertical jump height) with a supplement composed of fast-digesting protein (soluble milk protein) as compared with a slow-digesting protein (micellar casein) or with a placebo. It should be noted that differences between groups were larger when subjects with lower initial muscle thickness were considered.

Increased muscle endurance and reduced muscle f a t i g u ew e r eo b s e r v e di nt h eg r o u pw i t hf a s tp r o t e i n supplementation. Similar results have previously been obtained with acute intakes [START_REF] Hoffman | Effect of a proprietary protein supplement on recovery indices following resistance exercise in strength/power athletes[END_REF][START_REF] Shimomura | Nutraceutical effects of branched-chain amino acids on skeletal muscle[END_REF][START_REF] Blomstrand | Administration of branched-chain amino acids during sustained exercise-effects on performance and on plasma concentration of some amino acids[END_REF] and with chronic diets [START_REF] Falavigna | Effects of diets supplemented with branched-chain amino acids on the performance and fatigue mechanisms of rats submitted to prolonged physical exercise[END_REF], while others failed to register any effects [START_REF] Kerksick | The effects of protein and amino acid supplementation on performance and training adaptations during ten weeks of resistance training[END_REF][START_REF] Davis | Effects of branched-chain amino acids and carbohydrate on fatigue during intermittent, high-intensity running[END_REF]. Three main mechanisms could be involved in the observed enhancement of muscle endurance and fatigue. The first may be related to glucose and glycogen availability. However, Falavigna et al. [START_REF] Falavigna | Effects of diets supplemented with branched-chain amino acids on the performance and fatigue mechanisms of rats submitted to prolonged physical exercise[END_REF] concluded that chronic BCAA supplementation has no effect on glucose metabolism and could therefore be excluded. The second may originate from reduced exercise-induced muscle protein degradation. Indeed, Hoffman et al. [START_REF] Hoffman | Effect of a proprietary protein supplement on recovery indices following resistance exercise in strength/power athletes[END_REF] reported lower creatine kinase activity following resistance exercise with acute protein supplementation. Moreover, they also registered improved recovery 24 and 48 h post exercise. Such results may be attributable to an enhanced repair process through reduced protein breakdown and increased protein synthesis [START_REF] Shimomura | Nutraceutical effects of branched-chain amino acids on skeletal muscle[END_REF]. However, due to the widely different experimental designs, such a mechanism is unlikely when considering our muscle endurance results. A third mechanism, related to a reduced central fatigue, could also be possible. Indeed, BCAA intake m a yr e d u c et h ep l as m ar a t i oo ff r e et r y p t o p h a n / BCA Aa n d therefore the transport of tryptophan into the brain. This would reduce the synthesis, concentration and release of 5-hydroxytryptamine that is directly related to the development of fatigue and to the consequent reduction of performance [START_REF] Blomstrand | A role for branched-chain amino acids in reducing central fatigue[END_REF]. In humans, such an effect has mostly been evidenced by a reduced feeling of fatigue during exercise and also during cognitive tasks such as shortterm memory [START_REF] Portier | Effects of branched-chain amino acids supplementation on physiological and psychological performance during an offshore sailing race[END_REF]. In the present study, no difference was registered in perceived exertion at the end of the muscle endurance test but the number of repetitions was increased with SMP. Therefore, our results are in general accordance with the literature. In addition, SMP induced a reduction in muscle fatigue. Although unclear, such an increase in muscle endurance could be attributed to the protein composition and digestibility. Indeed, with rapid digestibility, whey has been shown to induce a transient and more pronounced rise in whole body protein synthesis than casein [START_REF] Burd | Exercise training and protein metabolism: Influences of contraction, protein intake, and sex-based differences[END_REF][START_REF] Boirie | Slow and fast dietary proteins differently modulate postprandial protein accretion[END_REF]. Therefore, it could be speculated that soluble milk protein, tested here, may reduce central fatigue as a result of this enhanced whole body protein synthesis. Moreover, the amino acid composition of the two tested protein beverages is different, which could have a significant impact on the reported outcomes [START_REF] Tang | Ingestion of whey hydrolysate, casein, or soy protein isolate: Effects on mixed muscle protein synthesis at rest and following resistance exercise in young men[END_REF]. For example, cysteine content is seven times greater in SMP as compared to MC. It is well known that cystine enhances glutathione synthesis [START_REF] Mariotti | Acute ingestion of dietary proteins improves post-exercise liver glutathione in rats in a dose-dependent relationship with their cysteine content[END_REF], an endogenous muscle antioxidant. Using quite similar cysteine contents as in our study while comparing whey vs. casein, authors demonstrated the positive effects of daily cysteine supplementation on the augmentation of antioxidant defense and anaerobic cycling performance [START_REF] Lands | Effect of supplementation with a cysteine donor on muscular performance[END_REF]. Similar positive results were obtained for fatigue and performance during various exercises [START_REF] Medved | N-acetylcysteine enhances muscle cysteine and glutathione availability and attenuates fatigue during prolonged exercise in endurance-trained individuals[END_REF][START_REF] Pinheiro | Effect of n-acetylcysteine on markers of skeletal muscle injury after fatiguing contractile activity[END_REF] with increases in cystine, cysteine, and glutathione content as a result of N-acetylcysteine ingestion [START_REF] Medved | N-acetylcysteine enhances muscle cysteine and glutathione availability and attenuates fatigue during prolonged exercise in endurance-trained individuals[END_REF]. Therefore, beverages with various amino acid content and not only BCAA or leucine are beneficial during prolonged exercise.

Muscle strength, power and thickness, although improved after the experimental period, were not different between groups. Such results are quite surprising since both protein beverages should have enhanced muscle thickness and muscle strength increases as compared with the placebo. Indeed, protein and more particularly BCAA are well known to increase muscle protein synthesis. For example, leucine plays a major role in muscle protein synthesis [START_REF] Stock | The effects of adding leucine to pre and postexercise carbohydrate beverages on acute muscle recovery from resistance training[END_REF][START_REF] Ispoglou | Daily l-leucine supplementation in novice trainees during a 12-week weight training program[END_REF][START_REF] Stark | Protein timing and its effects on muscular hypertrophy and strength in individuals engaged in weight-training[END_REF] through the stimulation of the mammalian target of rapamycin signaling pathway [START_REF] Norton | Leucine regulates translation initiation of protein synthesis in skeletal muscle after exercise[END_REF]. The lack of differences in muscle thickness and strength between groups remains unclear but could be attributed to several factors such as the supplement characteristics, training type and training status.

Protein was supplemented twice on non-training days and three times on training days. On training days, protein was ingested before and after the exercise session. Although debated, such timing may appear as one of the most effective nutrient timing strategies for muscle protein synthesis [START_REF] Stark | Protein timing and its effects on muscular hypertrophy and strength in individuals engaged in weight-training[END_REF]. As compared to a morning/evening intake group, Cribb and Hayes [START_REF] Cribb | Effects of supplement timing and resistance exercise on skeletal muscle hypertrophy[END_REF] observed larger muscle mass increases with protein intake before and after training. The best stimulus for protein synthesis appeared to be with protein feeding in close proximity to training sessions [START_REF] Tipton | Stimulation of net muscle protein synthesis by whey protein ingestion before and after exercise[END_REF][START_REF] Tipton | Timing of amino acid-carbohydrate ingestion alters anabolic response of muscle to resistance exercise[END_REF], with feeding recommended within the first two hours postexercise [START_REF] Hartman | Consumption of fat-free fluid milk after resistance exercise promotes greater lean mass accretion than does consumption of soy or carbohydrate in young, novice, male weightlifters[END_REF][START_REF] Phillips | Mixed muscle protein synthesis and breakdown after resistance exercise in humans[END_REF][START_REF] Rasmussen | An oral essential amino acid-carbohydrate supplement enhances muscle protein anabolism after resistance exercise[END_REF].

Protein quantity was 20 or 30 g.day -1 (non-training and training days, respectively) with 10 g before and 10 g after resistance training sessions. Prima facie, these doses seem unlikely to be responsible for the lack of difference between groups for muscle thickness or muscle strength. However, associated with the timing, a dose-response relationship for protein synthesis is generally obtained. For example, although the contents of EAA and BCAA used in the current study would be sufficient to stimulate the mammalian target of rapamycin signaling pathway, a dose twice as large seems more efficient [START_REF] Kakigi | Whey protein intake after resistance exercise activates mtor http://www.jissn.com/content/11/1/36 signaling in a dose-dependent manner in human skeletal muscle[END_REF]. In a recent review [START_REF] Phillips | Dietary protein requirements and adaptive advantages in athletes[END_REF], Phillips recommended the ingestion of 20 g of high quality protein immediately after exercise to maximally stimulate protein synthesis; and several authors reported that consumption of 8 to 11.5 g of EAA containing 2 to 3 g of leucine after exercise may maximize the protein synthetic response [START_REF] Tang | Ingestion of whey hydrolysate, casein, or soy protein isolate: Effects on mixed muscle protein synthesis at rest and following resistance exercise in young men[END_REF][START_REF] Reidy | Protein blend ingestion following resistance exercise promotes human muscle protein synthesis[END_REF][START_REF] Burke | Effect of intake of different dietary protein sources on plasma amino acid profiles at rest and after exercise[END_REF]. Recently, authors have demonstrated that low doses of leucine (0.75 g) may stimulate muscle protein synthesis following resistance exercise in young healthy individuals [START_REF] Churchward-Venne | Supplementation of a suboptimal protein dose with leucine or essential amino acids: Effects on myofibrillar protein synthesis at rest and following resistance exercise in men[END_REF]. Higher doses (3 g leucine), however, maintained muscle protein synthesis longer and may be more effective for anabolism after resistance exercise [START_REF] Churchward-Venne | Supplementation of a suboptimal protein dose with leucine or essential amino acids: Effects on myofibrillar protein synthesis at rest and following resistance exercise in men[END_REF]. In our study, the suboptimal doses ingested after exercise might therefore have reduced the potential protein effects and the potential differences between groups for muscle strength and thickness. This dose-response relationship may also explain differences between groups for muscle endurance and fatigue. For example, Falavigna et al. [START_REF] Falavigna | Effects of diets supplemented with branched-chain amino acids on the performance and fatigue mechanisms of rats submitted to prolonged physical exercise[END_REF] registered increased or reduced time to exhaustion during prolonged swimming in rats with low or high BCAA diets. Intakes should therefore be adequately chosen to obtain optimal adaptations.

It should be remembered that training and supplementation effects are potentiated in subjects exhibiting lower muscle thickness at inclusion. Such a result is not surprising since training is well known to have larger effects in untrained subjects. For example, greater increases in muscle cross-sectional area have been reported in subjects who had not previously engaged in resistance training in comparison with more accustomed subjects [START_REF] Ahtiainen | Muscle hypertrophy, hormonal adaptations and strength development during strength training in strength-trained and untrained men[END_REF]. The effects of amino acids supply may also depend on training status, since greater disturbances in protein turnover (protein synthesis and degradation) are obtained following training in novice than in experienced athletes [START_REF] Phillips | Resistance training reduces the acute exercise-induced increase in muscle protein turnover[END_REF]. Moreover, the expected increase in protein synthesis following exercise appears to be smaller and shorter in trained athletes as compared with untrained subjects [START_REF] Phillips | Resistance-training-induced adaptations in skeletal muscle protein turnover in the fed state[END_REF][START_REF] Macdougall | The time course for elevated muscle protein synthesis following heavy resistance exercise[END_REF]. Thus, training status may influence muscle performance. Indeed, Vieillevoye et al. [START_REF] Vieillevoye | Effects of a combined essential amino acids/carbohydrate supplementation on muscle mass, architecture and maximal strength following heavy-load training[END_REF] found increases in lower body strength with an essential amino acid supplement while no modification was obtained with placebo. Surprisingly, in the same study, strength was similarly enhanced in both groups for the upper body. These authors concluded that supplementation and training adaptations seem to depend on the initial training status; the weaker the subjects, the larger the effect of protein supplementation on muscle strength. Moreover, the present study was conducted in physically active males. Hence, it is possible to speculate that, with untrained participants, differences between groups might have been revealed. Furthermore, a plateau, or 'ceiling effect', of the adaptive responses to training is generally observed for strength gains and the muscle protein synthetic response [START_REF] Phillips | Resistance training reduces the acute exercise-induced increase in muscle protein turnover[END_REF][START_REF] Alway | Effects of resistance training on elbow flexors of highly competitive bodybuilders[END_REF]. Hence, protein requirements and training stimulus are affected by training status and duration. For instance, greater protein intakes are required during the early stages of intensive bodybuilding training and more particularly in novices [START_REF] Lemon | Protein requirements and muscle mass/strength changes during intensive training in novice bodybuilders[END_REF]. Modification of the training program might also have exacerbated differences between groups for all studied parameters. Training volume [START_REF] Krieger | Single vs. Multiple sets of resistance exercise for muscle hypertrophy: A meta-analysis[END_REF] concomitant with t h el o a du s e di nt e r m so f1 -R M 's percentage [START_REF] Burd | Low-load high volume resistance exercise stimulates muscle protein synthesis more than high-load low volume resistance exercise in young men[END_REF] are possible parameters.

Conclusions

In conclusion, the present experiment demonstrated that protein supplementation may enhance possible adaptations induced by resistance training. Our results suggest that the effects of protein supplementation depend on the protein composition. More specifically, it appears that soluble milk protein is particularly efficient to improve resistance to fatigue. Therefore, supplementation with soluble milk protein may be recommended in combination with to resistance training.

Figure 1

 1 Figure 1 Illustration of the experimental procedure.

  8 ± 114.1 W, -415.1 ± 165.1 W and -439.2 ± 153.9 W, respectively; P =0.0483) and after 10 weeks of training (-296.6 ± 130.1 W, -413.7 ± 139.4 W and -479.2 ± 138.1 W, respectively; P =0.0004) (Figure2). Accordingly, when comparing PRE and POST, fatigue was significantly reduced for SMP while it increased for MC and PLA (-35.9 ± 133.5 W, +89.3 ± 97.7 W and +64.8 ± 154.6 W, respectively; P=0.0125).

Figure 2

 2 Figure 2 Muscle power decrease during the endurance test. Mean (±SD) decreases in muscle power during the endurance test before the training period (PRE), after 4 weeks training (MID) and at the end of the training (POST). *: significant differences between groups (P<0.05).

Table 1 Amino

 1

	SMP	MC

acids composition (g) for 100 g of soluble milk protein (SMP) or micellar casein (MC)

Table 2

 2 Main subjects' characteristics at inclusion (PRE)

		PLA	MC	SMP	ANOVA
	Age (years)	22.0 ± 3.9	22.2 ± 3.9	22.5 ± 4.1	P=0.912
	BMI (Kg/m 2 )	23.4 ± 3.7	23.7 ± 3.5	22.7 ± 2.4	P-0.546
	Physical activity (hours/week)	4.1 ± 2.2	5.1 ± 2.4	5.4 ± 3.0	P=0.193
	Vastus lateralis thickness (mm)	21.0 ± 3.2	21.5 ± 3.1	21.5 ± 3.5	P=0.845
	Counter movement jump (cm)	32.4 ± 6.0	32.2 ± 4.1	32.6 ± 4.5	P=0.971
	1-RM (kg)	87.7 ± 16.7	86.6 ± 17.7	83.6 ± 16.0	P=0.702
	Number of repetitions	16.5 ± 4.0	16.8 ± 3.4	15.5 ± 3.9	P=0.527
	Perceived exertion	13.9 ± 2.1	14.3 ± 2.2	14.0 ± 1.9	P=0.820
	Data are mean values ± SD. PLA: placebo; MC: micellar casein; SMP: soluble milk protein; BMI: body mass index; 1-RM: maximum load lifted by leg extension.

Table 3

 3 Changes of the main outcomes during and at the end of the experimental procedure

		PRE	MID	POST	POST + 5
		Muscle endurance (number of repetitions)*
	SMP	15.5 ± 3.9	17.3 ± 4.7	19.2 ± 7.1	25.2 ± 13.7
	MC	16.8 ± 3.4	19.3 ± 4.6	21.3 ± 6.0	25.8 ± 9.5
	PLA	16.5 ± 4.0	18.5 ± 6.0	20.5 ± 7.0	25.2 ± 10.4
			1-RM (kg)*
	SMP	83.5 ± 16.0	91.3 ± 13.7	96.1 ± 12.4	-
	MC	86.6 ± 17.7	98.9 ± 18.2	98.9 ± 17.4	-
	PLA	87.7 ± 16.7	100.3 ± 13.3	104.8 ± 11.3	-
			Muscle thickness (mm)*
	SMP	21.5 ± 3.5	-	22.2 ± 3.5	-
	MC	21.8 ± 3.1	-	22.1 ± 3.1	-
	PLA	21.0 ± 3.2	-	22.0 ± 3.6	-
			% body fat
	SMP	8.6 ± 2.2	8.5 ± 2.5	8.9 ± 2.6	-
	MC	9.4 ± 4.3	9.8 ± 5.1	10.0 ± 4.6	-
	PLA	9.9 ± 4.6	9.9 ± 4.5	10.3 ± 5.0	-
			% body fat free mass
	SMP	48.2 ± 2.0	47.9 ± 2.0	47.9 ± 2.3	-
	MC	47.1 ± 3.1	46.9 ± 3.5	46.7 ± 3.2	-
	PLA	46.9 ± 3.4	47.4 ± 4.0	46.7 ± 3.4	-
			Body mass (kg)

SMP 70.8 ± 8.7 72.5 ± 8.1 72.2 ± 8.9 -MC 77.7 ± 14.7 79.0 ± 15.1 79.2 ± 15.3 -PLA 76.6 ± 13.3 77.7 ± 12.7 78.3 ± 13.2 -Data are mean values ± SD. PLA: placebo; MC: micellar casein; SMP: soluble milk protein; BMI: body mass index; 1-RM: maximum load lifted by leg extension. *: significant effect (P<0.05).

http://www.jissn.com/content/11/1/36

Acknowledgements

The authors would like to thank Mr. Jeremy Denuziller for helping with data collection.

Abbreviations BCAA: Branched-chain amino acids; MC: Micellar casein; MID: Testing in the middle; PLA: Placebo; PRE: Testing at inclusion; POST: Testing at the end; RM: Repetition maximum; SMP: Soluble milk protein.

Competing interests

Lactalis Recherche et Développement provided financial support to conduct the study. The funders have no role in data collection and analysis or preparation of the manuscript. PLR and FM, two authors, have an affiliation (employment) to the commercial funders of this research.

Authors' contributions NB (corresponding author) was responsible for the study design, the execution of the measurements and the writing of the manuscript. GD participated in the study design and the writing of the manuscript. PLR and FM participated in the study design. FAA participated in the study design, the statistical analysis and the writing of the manuscript. All authors read and approved the final manuscript.


Virginie Girard
email: virginie.girard@irstea.fr

Dominique Monti
email: dominique.monti@univ-ag.fr

Pierre Valade
email: pierre.valade@ocea.re

Nicolas Lamouroux
email: nicolas.lamouroux@irstea.fr

Jean-Paul Mallet
email: jp.mallet@asconit.com

Henri Grondin
email: grondin.arda@wanadoo.fr

B P 16

Hydraulic preferences of shrimps and fishes in tropical insular rivers

Keywords: micro-habitat preferences, generalized additive models, transferability, Caribbean, Indian Ocean

Hydraulic habitat models based on the preferences of species for the hydraulic characteristics of their microhabitats are frequently used to evaluate the impact on the habitat of a change in river flow regime. Their application in a tropical insular environment is still limited as little is known about the hydraulic preferences of species. Hydraulic preferences models have been developed for 15 taxa (diadromous shrimps and fishes) sampled in 52 rivers in the Caribbean (the French West Indies) and the Indian Ocean (the Reunion island). Five datasets were used and group 8353 samples collected by electrofishing during 320 surveys (reach×date) performed between 1999 and 2011. Generalised additive models were used to link variations of taxa density within surveys to the hydraulic characteristics of the microhabitat (velocity, depth, substrate). Hydraulic preferences within each region (Caribbean and Indian Ocean) are significant for most of the taxa and vary little between rivers and surveys. The hydraulic variables explain up to 18.1% (univariate models) and 30.0% (multivariate models) of the deviance of densities within survey. Of the taxa selected, Atya scabra, Macrobrachium heterochirus, Xiphocaris elongata and the Sicydiinae are the most demanding.

INTRODUCTION

Tropical insular rivers are characterised by extreme and unpredictable hydrological events (typhoons, cyclones, [START_REF] Tew | Habitat and fish fauna structure in a subtropical mountain stream in Taiwan before and after a catastrophic typhoon[END_REF] that alternate with sometimes severe periods of low flow [START_REF] Covich | Dispersal -Limited biodiversity of tropical insular streams[END_REF] that are decisive for aquatic communities [START_REF] Poff | Implications of streamflow variability and predictability for lotic community structure -a regional-analysis of streamflow patterns[END_REF]. The communities of these rivers are characterised by low density and are mainly composed of three families of shrimp (Atyidae, Palaemonidae, Xiphocaridae) and four families of fish (Eleotridae, Gobiidae, Mugilidae and Anguillidae) [START_REF] Covich | The stream community[END_REF], all of which are diadromous [START_REF] Mcdowall | Ancestry and amphidromy in island freshwater fish faunas[END_REF]. The catadromous species (Mugilidae, Anguillidae) spend their lives in rivers and reach the sea and/or river outlets to reproduce. Amphidromous species (Atyidae, Palaemonidae, Xiphocaridae, Gobiidae) spawn and grow in freshwater except during larval stages which require a saline environment. The complex lifecycle of these species makes them especially vulnerable to increasing demographic pressure [START_REF] Mittermeier | Hotspots revisited: Earth's biologically richest and most endangered terrestrial ecoregions[END_REF][START_REF] Smith | A comparison of current and historical fish assemblages in a Caribbean island estuary: conservation value of historical data[END_REF], such as dams that modify flow regimes and river habitats, and impede their migrations [START_REF] March | Damming tropical island streams: Problems, solutions, and alternatives[END_REF][START_REF] Milton | Living in Two Worlds: Diadromous Fishes, and Factors Affecting Population Connectivity Between Tropical Rivers and Coasts[END_REF]. Many aspects of the flow regime may affect the habitats and the life cycle of tropical species (e.g. Welcomme and Halls, 2004). For example, the timing and the amplitude of floods may trigger fauna migration and reproduction (e.g. Way et al., 1998;Blanco and Scatena, 2005;Kikkert et al., 2009). However, little is known about the ecological impact of changes in low to moderate flows [START_REF] Pringle | Regional effects of hydrologic alterations on riverine macrobiota in the New World: tropical-temperate comparisons[END_REF][START_REF] Monti | Shifts between biotic and physical driving forces of species organization under natural disturbance regimes[END_REF]. Hydraulic habitat models have been used widely around the world to facilitate the management of low to medium discharges [START_REF] Tharme | A global perspective on environmental flow assessment: emerging trends in the development and application of environmental flow methodologies for rivers[END_REF][START_REF] Conallin | Instream physical habitat modelling types: An analysis as stream hydromorphological modelling tools for EU water resource managers[END_REF]. This method uses models that predict the amount of favourable habitat at the reach scale (~10 2 -10 3 m 2 ) as a function of discharge rate [START_REF] Bovee | A guide to stream habitat analysis using the instream flow incremental methodology[END_REF][START_REF] Ginot | EVHA, a Windows software for fish habitat assessment in streams[END_REF]. Habitat models combine the hydraulic model of a stream reach with models of hydraulic preferences. Classically, hydraulic preference models represent how the abundance or occurrence of a taxon (e.g. guild of species, species, life stage of a species) varies within the reach as a function of microhabitat parameters (e.g. velocity, depth, substrate). The applicability of habitat models is generally limited by their degree of transferability between rivers [START_REF] Jowett | Hydraulic constraints on habitat suitability for benthic invertebrates in gravel-bed rivers[END_REF][START_REF] Lamouroux | The generality of abundance-environment relationships in microhabitats: A comment on Lancaster and Downes[END_REF][START_REF] Lancaster | Linking the hydraulic world of individual organisms to Author-produced version of the article[END_REF]. Indeed, habitat preferences can vary as a function of a large number of biotic and abiotic factors such as competition, predation, and water chemistry [START_REF] Jackson | What controls who is where in freshwater fish communities -the roles of biotic, abiotic, and spatial factors[END_REF]. In order to better quantify the generality of hydraulic preference models, it is therefore interesting to study the hydraulic preferences of taxa using data collected from different rivers at different times (seasons, years) [START_REF] Leftwich | Factors influencing behavior and transferability of habitat models for a benthic stream fish[END_REF]Lamouroux et al., 1999a;[START_REF] Strakosh | Development and assessment of habitat suitability criteria for adult brown trout in southern New England rivers[END_REF][START_REF] Vilizzi | Assessing variation in suitability curves and electivity profiles in temporal studies of fish habitat use[END_REF][START_REF] Dolédec | Modelling the hydraulic preferences of benthic macroinvertebrates in small European streams[END_REF][START_REF] Mérigoux | Invertebrate hydraulic preferences and predicted impacts of changes in discharge in a large river[END_REF]. The models developed at several sites have led to contrasting conclusions, but have often highlighted the pertinence of models defined for a number of rivers sharing the same hydromorphological characteristics (Lamouroux et al., 1999b;Lamouroux et al. in press).

Not much is known about the hydraulic preferences of tropical river species [START_REF] Pringle | Regional effects of hydrologic alterations on riverine macrobiota in the New World: tropical-temperate comparisons[END_REF][START_REF] Scatena | A survey of methods for setting minimum instream flow standards in the Caribbean Basin[END_REF][START_REF] Boulton | Are tropical streams ecologically different from temperate streams? In Tropical Stream Ecology[END_REF]. Research into these species has mainly focused on (1) the altitudinal distribution of species and the importance of maintaining upstream/downstream connectivity [START_REF] Holmquist | High dams and marinefreshwater linkages: Effects on native and introduced fauna in the Caribbean[END_REF], (2) trophic links and the role of macro-consumers [START_REF] Crowl | Effects of different types of conditioning on rates of leaf-litter shredding by Xiphocaris elongata, a Neotropical freshwater shrimp[END_REF][START_REF] Coat | Trophic relationships in a tropical stream food web assessed by stable isotope analysis[END_REF], (3) the effects of extreme events on population structures (e.g. the harmful effect of low water levels on the abundance of Macrobrachium sp., [START_REF] Covich | Effects of drought and hurricane disturbances on headwater distributions of palaemonid river shrimp (Macrobrachium spp.) in the Luquillo Mountains, Puerto Rico[END_REF] and (4) the mechanisms and role of dispersion in population renewal [START_REF] Keith | Characterisation of post-larval to juvenile stages, metamorphosis and recruitment of an amphidromous goby, Sicyopterus lagocephalus (Pallas) (Teleostei: Gobiidae: Sicydiinae)[END_REF][START_REF] Crook | A method to assess longitudinal riverine connectivity in tropical streams dominated by migratory biota[END_REF]. However, few works have focused on the role of hydraulic constraints (e.g. shift of driving force under natural disturbance, [START_REF] Monti | Shifts between biotic and physical driving forces of species organization under natural disturbance regimes[END_REF] and the habitat selection mechanisms at the reach scale (e.g. habitat selection for Sicydiinae, Teichert et al. in press).

In this study, we examine how the density of 15 taxa of the families of Atyidae, Palaemonidae, Xiphocaridae, Gobiidae and Mugilidae vary as a function of the hydraulic parameters of the microhabitat (velocity, depth, substrate). The datasets used come from two regions: the French West Indies (Martinique, Guadeloupe) in the Caribbean and the island of Reunion in the Indian Ocean. These are recent volcanic islands with comparable hydromorphological characteristics (narrow valleys, abrupt reliefs, high waterfalls) [START_REF] Falkland | Small tropical islands: water resources of paradise lost[END_REF]. Our main objectives were to (1) build hydraulic preference models for different taxonomic groups in each of the two regions (Caribbean, Indian Ocean), (2) estimate the transferability of models among datasets and groups of surveys, and (3) compare hydraulic preferences among the species of the same family.

METHODS

Reaches and sampling dates

We used five datasets (Table 1), three collected from the Caribbean and two from the Indian Ocean (Figure 1). Abundance samples were taken in 121 reaches of 52 rivers during periods of 1 to 12 years. The reaches were distributed over the entire island, in river sections with persistent flows, and were sometimes located upstream or downstream of water intakes. The length of the reaches varied from 0.05 to 2.75 km so that each reach encompassed a variety of morphological units (e.g. riffles, rapids, pools, cascades). The reaches were located at an altitude between 5 and 670 m, at a distance from the sea from 0.1 to 22.8 km and at a distance of 1 to 34.5 km from the source. The surface area of the watersheds upstream of the reach was on average (± standard deviation) 15 (± 15 km 2 ) in the Caribbean and 70 (± 49 km 2 ) in the Indian Ocean. Most of the sampling was performed during the low flow period (when hydraulic selection occurs), i.e. from December to May in the Caribbean [START_REF] Chaperon | Les ressources en eaux de surface de la Guadeloupe[END_REF] and from November to April in the Indian Ocean [START_REF] Robert | Géographie de l'eau à l'île de La Réunion: Essai de distribution régionale de l'alimentation et de l'écoulement[END_REF]. 20% of the surveys (reach×date) in the Caribbean were carried out during the high flow period but when discharge rate was relatively low.

Sampling methods

Sampling of fishes and shrimps was done by electrofishing using three methods (Table 1) according to the objectives of previous studies (e.g. analysis of spatial patterns, [START_REF] Monti | Shifts between biotic and physical driving forces of species organization under natural disturbance regimes[END_REF]. The "points" method consisted in collecting 50 samples (~ 1 m 2 ) distributed randomly in the reach by positioning a portable electrode (e.g. DEKA 3000 Lord; DEKA-Gerätebau, Marsberg, Germany) without moving it [START_REF] Fiévet | Quantitative sampling of freshwater shrimps: Comparison of two electrofishing procedures in a Caribbean stream[END_REF]. The "habitat units" method consisted in fishing about 20 samples from wider surface areas distributed in the available morphological units. The surface area of the samples was 17 m 2 on average (± 8 m 2 ). The "quadrats" method consisted in fishing about 90 contiguous samples (squares of surface area = 4 m 2 ) in the reaches. These different methods have been used by different teams and were not intercalibrated in our reaches. However, point samples and habitat unit samples have already been combined in habitat modelling. For example, Lamouroux et al. (1999b) predicted fish density differences between reaches sampled by points from preference models made in other reaches sampled by habitat units.

Each sample was characterised by three microhabitat variables: average velocity (v), average water depth (h) and the size of the substrate (d). v, h and d were estimated on the basis of measurements along one vertical (44% of cases) or several verticals distributed in the sampling area (56% of cases). On each vertical, point velocities were measured using a current meter (a propeller for dataset Gq; an electromagnetic current meter for the other datasets) at 0.2h, 0.4h and 0.8h at the bottom of the bed when h > 0.20 m, otherwise at 0.4h. However, 5.4% of the velocities were estimated at the water surface as a function of the distance travelled by a float over a given time. The dominant substrate size d of the sample area was estimated visually using ordinal classes [START_REF] Cailleux | Limites dimensionnelles des noms des fractions granulométriques[END_REF][START_REF] Malavoi | Méthodologie de description et quantification des variables morphodynamiques d'un cours d'eau à fond caillouteux. Exemple d'une station sur la Filière (Haute Savoie)[END_REF]. The central value of the class of substrate was assigned to d.

Each individual fish was measured and identified at species level [START_REF] Keith | Atlas des poissons et crustacés d'eau douce de la Réunion[END_REF][START_REF] Lim | Atlas des poissons et crustacés d'eau douce de la Martinique[END_REF][START_REF] Monti | Atlas des poissons et des crustacés d'eau douce de la Guadeloupe[END_REF], except for three taxa in the Caribbean (Table 1):

Sicydium sp. groups two species of fish, Sicydium punctatum and Sicydium plumeri, of the Gobiidae family (subfamily Sicydiinae); Atya sp. groups the juvenile shrimps of Atya scabra et Atya innocous; and Macrobrachium sp. groups the juvenile shrimps of the family of Palaemonidae (subfamily Macrobrachium). However, the individuals of dataset Gq (Table 1) were not measured.

Taxa selected

Of the 21 to 28 species sampled in each dataset (Table 1) only the taxa with a total abundance > 200 individuals were chosen for the analysis. Therefore, we selected 11 taxa in the Caribbean belonging to three families of shrimp (Atyidae, Xiphocariidae, Palaemonidae) and two families of fish (Mugilidae, Gobiidae), and four taxa in the Indian Ocean belonging to two families of shrimp (Atyidae, Palaemonidae) and one family of fish (Gobiidae) (Table 2).

In order to get round the problem of different surface areas of the samples used in the analyses, the abundances of the samples were transformed to densities (Ď, number of each for an equivalent surface of 10 m 2 ). Taxa size classes were defined in order to infer the effect of size preferences on hydraulic. The limits of classes were chosen to obtain comparable numbers.

Statistical modelling

For each taxon of each region, we modelled the Ď as a function of microhabitat variables with a series of generalised additive models (GAMs). GAMs permit considering nonlinear relations, that are frequent when studying hydraulic preferences (e.g., Lamouroux et al., 1999a;[START_REF] Jowett | Habitat characteristics of crayfish (Paranephrops planifrons) in New Zealand streams using generalised additive models (GAMs)[END_REF]. They have a flexible structure that does not require prior determination of the form of the relation (Guisan andZimmerman, 2000, Guisan et al., 2002).

Six models (Eqs. 1-6) were defined with a log link function. The reference model M1 (Eq. 1) assumes that the density is constant by survey, so that there is no hydraulic preference. Model M2 (Eq. 2) defines the regional model where the abundance varies similarly in each survey as a function of a microhabitat variable. By comparing M2 in relation to M1, we quantify the strength of hydraulic preference independently of the other biotic and abiotic factors acting at the scale of the reach on a given date. In model M3 (Eq. 3), the hydraulic preferences can vary as a function of the dataset, i.e. the island or the sampling method. In model M4 (Eq. 4), the hydraulic preferences can vary as a function of the dataset and groups of surveys. The latter two models permit appreciating the transferability of the results between rivers. Different criteria were used to define two groups of surveys in M4 (with comparable numbers): the season of low and high flow in the Caribbean; the years < and ≥ 2009 in the Caribbean and the years < and ≥ 2005 in the Indian Ocean; the altitude (≤ or > 165 m in the Caribbean; < and ≥ 80 m in the Indian Ocean); the wind coast exposure (exposed or not); the size of the watershed (≤ or > 10 km 2 in the Caribbean; ≤ or > 50 km 2 in the Indian Ocean); the situation in relation to water intakes (upstream of an intake or not; only in the Caribbean). Lastly, models M5 and M6 (Eqs. 5-6) define multivariate models for a taxon with or without a term of interaction between microhabitat variables. Concerning the preference models of taxa size classes, we fitted M1 and M2 only. Our series of models is: p dataset is the term of interaction between the hydraulic variable and the dataset variable;

p:p survey is the term of interaction between the hydraulic variable and a group of surveys. The parameter a survey permits ignoring differences in density between surveys which do not reflect the microhabitat hydraulic preferences.

For all the models, we chose a negative binomial (NB) type error distribution, which can account for the over-dispersion of abundance data, as suggested by [START_REF] Gray | Selecting a distributional assumption for modelling relative densities of benthic macroinvertebrates[END_REF] freshwater macro-invertebrates) and Vaudor et al. (2009, freshwater fishes) for data of the same type. We set the dispersion parameter (θ) of the NB distribution for each taxon when fitting model M1. We also set the degree of freedom of the smoothing function of the GAMs at three to avoid any over-parameterisation [START_REF] Jowett | Habitat characteristics of crayfish (Paranephrops planifrons) in New Zealand streams using generalised additive models (GAMs)[END_REF]. Lastly, for each taxon we eliminated the surveys where the number of individuals was less than three. The models were fitted using the functions available in the mgcv package [START_REF] Wood | Generalized Additive Models: An Introduction with R[END_REF] of the R software (R Development Core Team, 2010).

We described the quality of our two series of nested models (M1-M4 and M1, M5-M6) using the additional deviance explained in comparison to the previous model. In addition, we used a likelihood ratio test to compare a given model with the previous one.

Author-produced version of the article published in River Research and Applications (2014), vol. 30, Issue 6, pages 766-779 The original publication is available at http://onlinelibrary.wiley.com, doi : 10.1002/rra.2675

RESULTS

A total of 8353 samples were collected from 320 surveys, making it possible to establish and study the preferences of 11 taxa and 16 size classes in the Caribbean, four taxa and eight size classes in the Indian Ocean (Table 2). The velocities of the samples varied from 0 to 2.80 m.s - 1 , the depths from 0.02 to 1.90 m and the substrate from 0 to 1.024 m.

The dispersion parameter θ varied from 0.06 (A. scabra) to 0.46 (M. poeyi) for the taxa of the Caribbean, and from 0.32 (A. serrata) and 1.10 (S. lagocephalus) for the taxa of the Indian Ocean (Table 2, standard error on θ ≤ 0.03 for all species). The low values of θ, which indicate over-dispersion of densities, were obtained for rare species (M. crenulatum, A.

monticola ) and those known to be gregarious (A. scabra; [START_REF] Lim | Atlas des poissons et crustacés d'eau douce de la Martinique[END_REF][START_REF] Monti | Atlas des poissons et des crustacés d'eau douce de la Guadeloupe[END_REF].

Univariate models M1 -M4 by taxon

The deviance explained by the survey in reference model M1 varied from 20.3 to 56.0% (on average 36%, Table 3). The addition of microhabitat variables in M2 explained from 0 to 18.1% (average 4.3%) of the residual deviance of M1. The model of velocity preferences was generally more efficient than that of preferences of depths and substrates (on average 6.9, 4.4 and 1.6% of explained deviance, respectively). Examples of fits of M2 models to the data observed are given in Figure 2.

Hydraulic preferences were significant for most of the species, except A. monticola (v and d), M. crenulatum, Atya sp. and X. elongata (d) ( 3 and4).

The preferences for velocities and depths, and to a lesser extent substrates (Table 3), differed between datasets. However, the additional deviance explained by M3 in comparison to M2 remained low: it was < 2.2% in the Caribbean and < 1.5% in the Indian Ocean (Table 3). The main differences observed in the Caribbean are the variations of preferences in the range of high velocities (> 0.5 m.s -1 ) (cf. A. scabra in Figure 3) and preferences for shallower or deeper water depths (cf. A. monticola in Figure 3). In the Indian Ocean, we observed preferences for lower velocities and an absence of preferences for depths in the dataset sampled by "points" in comparison to "habitat units" for C. acutipinnis (v, h) (Figure 4).

Regarding variations between groups of surveys, the comparison of M4 and M3 indicated that the localisation parameters of reaches and the sampling period generally had little influence on hydraulic preferences (explained relative deviance < 2.4%, Table 3). The highest values observed concerned A. scabra in the Caribbean, and A. serrata, M. australe and C. acutipinnis in the Indian Ocean.

Univariate models M1 -M2 by size classes

The hydraulic preferences according to size classes were mostly significant except for the preferences of X. elongata (h), A. monticola (h) and A. serrata (d) of small sizes and the preferences of adults of Sicydium sp. (d) and A. monticola (v) (Table 4, Figure 5). The deviance explained by the models by size classes was close to that of the model by taxon. The deviance explained was nonetheless higher for size classes concerning the preferences for

Multivariate models M5 -M6

The multivariate models without interactions (M5) explained from 2.8 to 24.4% of the residual deviance of M1 (Table 5). The addition of an interaction term (M6) improved the explained deviance between 3.7 and 9.4% for some taxa: Sicydium sp., X. elongata, C. acutipinnis and S. lagocephalus (Table 5).

DISCUSSION

The within-survey deviance explained by microhabitat variables varies between 0 and 18.1% (univariate regional models) and from 4.1 to 30.0% (multivariate regional models). The poor performances achieved by the models built are partly due to the small size of the scale of observation (~ 1 m 2 ) and likely result from the particular life cycle of the taxa (diadromous and opportunistic species). Similarly, the within-survey deviance of models of hydraulic preferences of the white shrimp Paranephrops planifrons, a diadromous species of the rivers of the north island of New Zealand, varies from 7 and 11% [START_REF] Jowett | Habitat characteristics of crayfish (Paranephrops planifrons) in New Zealand streams using generalised additive models (GAMs)[END_REF]. By way of

Author-produced version of the article published in River Research and Applications (2014), vol. 30, Issue 6, pages 766-779 The original publication is available at http://onlinelibrary.wiley.com, doi : 10.1002/rra.2675 comparison, [START_REF] Dolédec | Modelling the hydraulic preferences of benthic macroinvertebrates in small European streams[END_REF] showed that models of European regional preferences for macro-invertebrates explained on average 25% of within-survey variance.

Taxa showing the most marked hydraulic preferences are A. scabra, M. heterochirus, X.

elongata, Sicydium sp. in the Caribbean, and C. acutipinnis and S. lagocephalus in the Indian Ocean. For example, the density of A. scabra and X. elongata can be multiplied by a factor of ~ 5 for a change of velocity in the order of 0.5 m.s -1 .

The preferences of shrimps for high (A. scabra, M. heterochirus) or low (X. elongata)

velocities are probably linked to their capacity to resist currents or not and their foraging strategy. Indeed, A. scabra and M. heterochirus are larger in comparison to the other species of the same family (M. poeyi, M. faustinum) and have larger morphological attributes (pincers, legs, spines on pincers) [START_REF] Lim | Atlas des poissons et crustacés d'eau douce de la Martinique[END_REF] that allow them to move in rapids where densities of drifting benthos, particles and/or individuals are higher [START_REF] Orth | Microhabitat Preferences Of Benthic Fauna In A Woodland Stream[END_REF][START_REF] Brooks | Hydraulic microhabitats and the distribution of macroinvertebrate assemblages in riffles[END_REF]. Furthermore, the feeding mode of A. scabra is probably more specialised than for A. innocous, likewise with M. heterochirus in comparison to M. crenulatum. On the contrary, X. elongata has a strong preference for low velocities and deeper water since it lives essentially in the water column [START_REF] Lim | Atlas des poissons et crustacés d'eau douce de la Martinique[END_REF]. In addition, our results corroborate those of [START_REF] Monti | Shifts between biotic and physical driving forces of species organization under natural disturbance regimes[END_REF] who showed that M. heterochirus and X. elongata have notable preferences for velocities in environments with strong hydrological disturbances.

The fish of the subfamily of Sicydiinae in the two regions (Sicydium sp., C. acutipinnis and S. lagocephalus) prefer fast-flowing and shallow habitats. Their hydraulic preferences are probably linked to the presence of periphytic biofilm whose quality and development are conditioned by bed shear stress and light, respectively [START_REF] Julius | The survival of Sicyopterus stimpsoni, an endemic amphidromous Hawaiian gobiid fish, relies on the hydrological cycles of streams: evidence from changes in algal composition of diet through growth stages fish[END_REF]Lefrançois et al., 2011;[START_REF] Tabouret | Otolith microchemistry in Sicydium punctatum: indices of environmental condition changes after recruitment[END_REF]. The hydraulic preferences of S. lagocephalus and C. acutipinnis are comparable to those given by the logistic models built by Teichert et al.

(in press), except that the authors showed that C. acutipinnis has stronger preferences for depths than velocities. This difference in preference strength can be due to the longer sampling period covered by our dataset.

Taxa showing weaker hydraulic preferences are A. monticola, M. faustinum, M. crenulatum, M. australe, A. innocous, M. poeyi and the juvenile shrimps of the Caribbean.

These results are consistent with those of [START_REF] Monti | Shifts between biotic and physical driving forces of species organization under natural disturbance regimes[END_REF] who reported weak preferences for velocity for M. faustinum and M. poeyi. Concerning M. crenulatum, Monti and Legendre identified strong preferences for low velocities for four sites under strong and weak hydrological disturbance, suggesting that food predominates in influencing habitat selection. Some of these species are also described with preference variables according to diurnal phases (e.g. A. innocous) [START_REF] Keith | Atlas des poissons et crustacés d'eau douce de la Réunion[END_REF][START_REF] Lim | Atlas des poissons et crustacés d'eau douce de la Martinique[END_REF][START_REF] Monti | Atlas des poissons et des crustacés d'eau douce de la Guadeloupe[END_REF]. The weak preferences of the species observed here can also be partly due to variations in fishing efficiency or to our grouping of juveniles of some taxa (A. scabra and A. innocous; M. crenulatum, M. heterochirus and M. faustinum). Regarding fishing efficiency, it is as poor as the species are rare (M. crenulatum) and their mobility is high (A. monticola) [START_REF] Fiévet | Efficiency and sampling bias of electrofishing for freshwater shrimp and fish in two Caribbean streams, Guadeloupe Island[END_REF][START_REF] King | Evaluation of a sweep net electrofishing method for the collection of small fish and shrimp in lotic freshwater environments[END_REF]. It is therefore possible that fishing efficiency explains the absence of preference for velocity that we observed for the mobile A. monticola, whereas the taxon is expected to prefer fast and well oxygenated water [START_REF] Lim | Atlas des poissons et crustacés d'eau douce de la Martinique[END_REF]. Fishing efficiency can also explain that we observed weak preferences for A. innocous, which is an excellent swimmer and very reactive in the presence of predators [START_REF] Covich | Predator-prey interactions in river networks: comparing shrimp spatial refugia in two drainage basins[END_REF][START_REF] Hein | Running the predator gauntlet: do freshwater shrimp (Atya lanipes) migrate above waterfalls to avoid fish predation?[END_REF] or when disturbed by electrofishing [START_REF] Fiévet | Efficiency and sampling bias of electrofishing for freshwater shrimp and fish in two Caribbean streams, Guadeloupe Island[END_REF]. Regarding the shrimp M.

australe, its weak regional preferences can be explained by its sheltering behaviour during the day and variation due to its morphological plasticity (Zimmermann et al., 2012). The grouping of juveniles of different species of Macrobrachium is probably not very appropriate since we observed that the adults of M. faustinum and M. heterochirus have different preferences (forces and directions) regarding velocity and depth. This is less the case for the juveniles of the family of Atyidae since the adults (A. scabra, A. innocous and M. poeyi) share

Author-produced version of the article published in River Research and Applications (2014), vol. 30, Issue 6, pages 766-779 The original publication is available at http://onlinelibrary.wiley.com, doi : 10.1002/rra.2675 relatively similar preferences. Lastly, the more flexible mode of feeding (filtering and scraping) of M. poeyi and A. innocous in comparison to A. scabra (mainly filtering) can also explain less selective use of microhabitats.

The comparison of inter-species hydraulic preferences suggests that food strategies (filtering and/or scraping) and morphological adaptations (size, apical spine) effectively influence habitat selection. This result supports that aquatic taxa have developed strategies shaped by the flow regime [START_REF] Poff | Implications of streamflow variability and predictability for lotic community structure -a regional-analysis of streamflow patterns[END_REF]Poff, 1997). The use of life history traits is therefore encouraged for overcoming problems of identification and/or lack of data on a particular species [START_REF] Blanck | Relationships between life-history strategies of European freshwater fish species and their habitat preferences[END_REF].

The low additional deviance explained by datasets or groups of surveys suggests that regional models are pertinent for expressing taxa preferences. Likewise, the performance of models regarding size class does not suggest a notable improvement. These results should nonetheless be seen relatively. For example, it is possible that our definitions of groups are not very appropriate for expressing different competition conditions. These effects can exist for certain species (S. lagocephalus and C. acutipinnis, [START_REF] Lord | Femtosecond laser ablation ICP-MS measurement of otolith Sr:Ca and Ba:Ca composition reveal differential use of freshwater habitats for three amphidromous Sicyopterus (Teleostei: Gobioidei: Sicydiinae) species[END_REF] and might partially explain the variations observed between groups in our results (C. acutipinnis). Likewise, taking the hydrological regime [START_REF] Monti | Shifts between biotic and physical driving forces of species organization under natural disturbance regimes[END_REF] into account in more detail would undoubtedly lead to more thorough models. Lastly, a breakdown of taxa by life stage rather than by size class could be more pertinent to reflect the physiological needs of species (Bielsa et al., 2003, for S. lagocephalus) and their behaviours regarding different predators (e.g. longer rostrum in X. elongata in the presence of predatory fish, [START_REF] Covich | Predator-prey interactions in river networks: comparing shrimp spatial refugia in two drainage basins[END_REF].

To sum up, the hydraulic preferences of insular tropical taxa are generally weaker than in temperate regions. This observation, as well as the complexity of the life cycle of these taxa, tends to emphasise that the hydraulic preferences of taxa should only be taken into account within an approach that provides a more general description of their habitat during their life cycle. This approach would imply in particular taking into account the ecological effect of various flow regime attributes (e.g., timing and duration of floods, flashiness; [START_REF] Bunn | Basic principles and ecological consequences of altered flow regimes for aquatic biodiversity[END_REF], the longitudinal connectivity (e.g., Greathouse et al., 2006) and the potential threat of introduced species (Donlan and Wilcox, 2008). Nonetheless, our results

show that the hydraulic preferences of certain taxa are significant (e.g., A. scabra, M.

heterochirus) and consistent with the knowledge available on these taxa. Our regional models can contribute to describe the hydraulic preferences of these taxa and model how the management of low to average discharges modifies their available habitat. Martinique

! ! Guadeloupe Caribbean ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 1

C. acutipinnis

Ln-density

Author-produced version of the article published in River Research and Applications (2014), vol. 30, Issue 6, pages 766-779 The original publication is available at http://onlinelibrary.wiley.com, doi : 10.1002/rra.2675

((

  version of the article published in River Research and Applications (2014), vol. 30, Issue 6, pages 766-779 The original publication is available at http://onlinelibrary.wiley.com, doi : 10.1002/rra.2675 version of the article published in River Research and Applications (2014), vol. 30, Issue 6, pages 766-779 The original publication is available at http://onlinelibrary.wiley.com, doi : 10.1002/rra.2675

  Author-produced version of the article published in River Research and Applications (2014), vol. 30, Issue 6, pages 766-779The original publication is available at http://onlinelibrary.wiley.com, doi : 10.1002/rra.2675

	M1: log(Ď) ~ a survey	(1)
	M2: log(Ď) ~ a survey + s(p)	(2)
	M3: log(Ď) ~ a survey + s(p) + s(p:p dataset )	(3)
	M4: log(Ď) ~ a survey + s(p) + s(p: p dataset ) + s(p:p survey )	(4)
	M5: log(Ď) ~ a survey + s(v) + s(h) + s(d)	(5)
	M6: log(Ď) ~ a survey + s(v) + s(h) + s(d) + s(v, h, d)	(6)
	where p corresponds either to v, h or d; s() is a smoothing function (of cubic spline type); p:

Table 3

 3 Author-produced version of the article published in RiverResearch and Applications (2014), vol. 30, Issue 6, pages 766-779 The original publication is available at http://onlinelibrary.wiley.com, doi : 10.1002/rra.2675 intermediate (A. innocous, M. poeyi) or high (A. Scabra, M. heterochirus, A. serrata and Sicydiinae) velocities, whereas most taxa preferred shallow depths, except X. elongata, M. crenulatum and M. australe (Figures 3 and 4). A. scabra, M. heterochirus, A. serrata, C.

). The strongest preferences, relative to velocity and depth, concerned A. Scabra, M. heterochirus, X. elongata and Sicydium sp. in the Caribbean and S. lagocephalus and C. acutipinnis in the Indian Ocean.

The taxa had preferences for low (X. elongata, M. crenulatum, M. faustinum, M. australe), acutipinnis and S. lagocephalus preferred coarse substrates whereas M. australe preferred fine substrates (Figures

  Author-produced version of the article published in RiverResearch and Applications (2014), vol. 30, Issue 6, pages 766-779 The original publication is available at http://onlinelibrary.wiley.com, doi : 10.1002/rra.2675substrate. The preferences of larger individuals for velocities were weaker than those of smaller individuals for the species A. scabra and X. elongata; on the contrary, the preferences of larger individuals for velocities were stronger than those of smaller individuals for the species Sicydium sp.. Size classes generally preferred similar depth ranges except for the taxa

	X. elongata, where larger individuals tended to prefer deeper habitats (Figure 5). The
	preferences of larger individuals for substrate are weaker than those of smaller individuals for
	M. australe and M. heterochirus; conversely, larger individuals of A. scabra, A. serrata and S.
	lagocephalus have stronger preferences for substrate.

  Author-produced version of the article published in River Research and Applications (2014), vol. 30, Issue 6, pages 766-779The original publication is available at http://onlinelibrary.wiley.com, doi : 10.1002/rra.2675

Table 1 :

 1 Datasets characteristics

	176
	Dataset code	Island	Sampling method	Number of surveys	Number of samples (min-max per station)	Sampling Years	Number of taxa sampled
	Caribbean
	Gp	Guadeloupe	Points	32	1455	(10 -52) 2005, 2008, 2009	23
	Gq	Guadeloupe	Quadrats	27	1811	(79 -99)	2005	22
	Mp	Martinique	Points	21	946	(31 -50)	2008, 2010	21
	Indian Ocean
	Rp	Réunion	Points	17	1359	(13 -85)	1999, 2001	25
	Rh	Réunion	Habitat units	196	3284	( 3 -21)	2000-2011	28

Table 4 :

 4 Supplementary deviance M2/M1 (% of the residual deviance of M1 explained by M2) for size classes.Author-produced version of the article published in River Research and Applications (2014), vol. 30, Issue 6, pages 766-779The original publication is available at http://onlinelibrary.wiley.com, doi : 10.1002/rra.2675residual deviance of M i-1 explained by M i ). Additional deviance is shown for significant cases only (P-value <0.05).

	186
	187	Additional deviance is shown for significant cases only (P-value <0.05).
		Region	Taxon	Size class (mm)	v	M2/M1 h	d
		Caribbean	AIN	≤ 45 > 45	2.7 2.4	2.5 1.2	--
			AMO	≤ 115	1.8	-	1.1
				> 115	-	7.7	1.3
			ASC	≤ 50	20.9	10.1	3.1
				> 50	16.9	3.2	5.2
			MCR	≤ 55	1.3	2.5	-
				> 55	2.8	1.7	-
			MFA	≤ 40	2.1	0.5	0.5
				> 40	3.0	1.6	0.6
			MHE	≤ 45	7.4	3.3	2.0
				> 45	6.0	1.4	0.9
			MPO	≤ 15	2.1	4.3	0.5
				> 15	3.2	4.5	0.8
			SIC	≤ 45	2.6	5.4	-
				> 45	5.5	2.5	0.5
			XEL	≤ 40	11.0	-	-
				> 40	8.8	2.5	-
		Indian	ATY	≤ 20	3.1	6.0	-
		Ocean		> 20	3.7	5.2	4.3
			COA	≤ 35	8.2	9.4	0.5
				> 35	8.5	4.1	1.3
			MAA	≤ 40	7.4	2.1	3.6
				> 40	3.8	1.4	1.2
			SLA	≤ 55	11.6	13.6	1.0
				> 55	16.2	9.7	3.7
	188
	189

Author-produced version of the article published in River Research and Applications (2014), vol. 30, Issue 6, pages 766-779The original publication is available at http://onlinelibrary.wiley.com, doi : 10.1002/rra.2675

al.(1999),[START_REF] Lim | Atlas des poissons et crustacés d'eau douce de la Martinique[END_REF] and[START_REF] Monti | Atlas des poissons et des crustacés d'eau douce de la Guadeloupe[END_REF]. Abbreviations for feeding guilds are: F for filter feeding, H for Herbivore, D for Detritivore, O for Omnivore. θ (σ θ ) is

the dispersion coefficient (and its standard error) fitted for the taxa.

ACKNOWLEDGMENTS

We thank the staff from "Association Réunionnaise de Développement de l'Aquaculture" and from Asconit Consultants for their collaboration in this project and for collecting part of the data. We also thank Sylvie Mérigoux, Jean-Michel Olivier, Philippe Keith and Thierry Oberdorff for helpful discussions. The "Direction Régionale de l'Environnement, de l'Aménagement et du Logement" of Guadeloupe, the "Office de l'eau" of Martinique and "Office de l'eau" of Reunion provided financial support.

Table 3: Explained deviance for M1 (% of the initial deviance) and supplementary deviance M i /M i-1 (% of the residual deviance of M i-1 explained by M i ). Additional deviance is shown for significant cases only (P-value <0.05).

Taxon M1


Jacques Simonet

Electrochemical exfoliation in real time of natural graphite deposited onto glassy carbon. Doping and modifying carbons through ultra-thin graphite layers

Keywords: Graphite, Exfoliation, Graphene surfaces, Modified carbon electrodes

Natural graphite, deposited onto smooth glassy carbon, when reduced (around -2V vs.

Ag/AgCl) in the presence of tetraalkylammonium salts (TAAX) in aprotic polar solvents, lead to spectacular exfoliation processes followed in real time in the course of voltammetric scans.

The method permits to get extraordinary stable modified carbon surfaces, with quite simple voltammetric responses (1 to 4 reversible cathodic steps) that strongly depend on the nature of the cations used. Those cathodic steps exhibit currents quasi perfectly proportional to the scan rate. It is expected the formation of a graphene-like substrate. Surface modifications both by ferrocene and anthraquinone could underline the originality of those new materials

Introduction

Glassy carbon (GC) is considered as a convenient electrode material for achieving analysis and macro-electrolyses, especially within the cathodic domain [START_REF] Schäfer | Comprehensive Organic Synthesis[END_REF]. A large panel of electro-organic reactions were carried out within a quite extended cathodic domain at negatively polarized carbon and this material remains very useful to conduct electrochemical reactions at potentials < -2V in dry polar organic solvents. Morever, it has been reported that GC at E < -1.7 V, is not longer totally inert owing the presence of ribbons of graphite-like structures [START_REF] Jenkins | [END_REF][3]. Cathodic charging (by insertion of organic or inorganic cations) of these

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ACCEPTED MANUSCRIPT 2 different "impurities" has been used for inducing specific reactions with a large palette of electrophilic reagents [4]. Independently, it is worth recalling that, when polar organic solvents are used, cathodic charge of highly oriented pyrolytic graphite [5] specifically leads to well defined insertion stages [C n -,TAA + ] via concomitant insertion of electrons and tetraalkylammonium salts (TAAX). Fixed potential reduction and exfoliation of natural graphite by bulky ammonium salts has been reported [6]. The electrochemical synthesis of those "graphite salts", rather similar to {TAA-amalgams}, has to be considered both as a novel generation of reducing species and as poly-nucleophilic materials (then reactive with organic reagents for modification of graphites and carbons [7]).

Under these conditions, graphite appears to be a key substrate to create via several methods graphene plans. In electrochemistry, the basic method remains the reduction of graphene oxide [8]. The catholic reduction of commercial graphene has been achieved and 3D {graphene-GC} electrodes described. Recently, electrochemical functionalizations of graphene were reported [9].

The early method [6] for a facile exfoliation of graphite by means of electrochemistry (reduction at about -2.0 V) remains certainly a fascinating manner to generate in situ at electrified solid substrates thin graphene layers. We here describe a new process for achieving natural graphite exfoliation concomitantly with deposition onto glassy carbon.

Experimental section

Voltammetries were carried out in 0.1 M solutions of tetraalkylammonium salts (TAAX) such as tetramethylammonium (TMABF 4 ), tetraethylammonium (TEABF 4 ), tetra-nbutylammonium (TBABF 4 ), and tetra-n-octylammonium tetrafluoroborate (TOABF 4 ), and dissolved in upper grade dimethylformamide (DMF). Experiments described in this work did not need special treatment of electrolytic solutions.

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3 Potentials are referred to the aqueous Ag/AgCl/KCl (sat) . Voltammetric and coulometric measurements were performed using three-electrode cells separated with a fritted glass. The electrochemical instrumentation has been previously reported [7].

GC rods electrodes as substrates used for graphite deposition had geometric area of 1 and 7 mm 2 . All those carbon samples were purchased from Tokai Carbon C° (code: GC Rod) and Carbone Lorraine carbon vitreous VD 1500. Also, large GC plates (from Carbone Lorraine) were also employed for building flat electrodes of much larger area.

Prior to being modified by deposition of natural graphite, GC substrates were carefully polished first with silicon carbide paper (Struers 500) and then with a thinner polishing paper (Struers 1200). Then, natural graphite (from Ceylon) or highly oriented pyrolytic graphite (HOPG), both first partly exfoliated by the electrolytic method already quoted [6] were disposed on a carbon planar surface. Deposition of graphite was done using glassy cardboard and the surface was rubbed by firmly polishing electrodes until obtaining a shiny aspect of carbon. The final process for deposit final exfoliation is fully described below in §3.1.

Experimental results and procedures

Evidences for real time exfoliation procedures with natural graphite

First of all, an exfoliated graphite coating (electrolysis in DMF accordingly to reference [6] i.e. at mercury pool, potential -2.1 V, amount of electricity: 12 C/ 1 g of Ceylon graphite) was achieved at a GC disk and electrolyzed in DMF / TMAClO 4 . A typical voltammetry is displayed in Figure 1 A: while the GC disk polished according to the procedure given in the experimental does not display any strong signal, an intense signal assigned to the exfoliation process is obtained from the first scan. From -1.7 V, i.e. a potential reported to correspond to the cathodic charge threshold of graphite, the huge increase of current is immediately followed (≈ -2V) by a sudden decay. The anodic discharge, still rather intense is observed.

Furthers scans (scans 2 and 3) show a progressive crushing of charge/discharge processes until very reproducible signals (in potential and intensity) apparently specific of used TAA + salts. In general, with scans up to -2.3 V, peaks (their number ≤ 4 essentially depending on the

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M A N U S C R I P T employed TAA + electrolyte) are obtained. They are assigned to the electrochemical reduction (or charge) of a few remaining layers of graphite immobilized at the GC interface presumably by mean of π,π-interactions with the substrate. Exfoliation with TMA + is considered as a formatting process and allows testing behaviors of several ammonium salts that all exhibit specific responses. Some of them are displayed in Figure 1 B.

Reduction of graphite deposits onto GC (before exfoliation) revealed different points: in the course of the first sweep, some small reduction peaks are observed around -1V and are assigned to electroactive edge impurities provoked by the aging of graphite in contact with air (such as ketonyl and quinonyl groups, aromatic carboxylic acids). After three scans, reversible electrochemical steps are observed at E < -1.8 V and assigned to stable graphitic (or graphene) layer(s). Thus, in the specific case of tetraethylammonium of salts (triflate, tetrafluoroborate or perchlorate) in DMF, three well defined reversible steps are obtained (see curve C in Figure 1). Their peak currents are strictly proportional to the scan rate (until 1V s -1 )

and could be assigned to the reduction of graphene mono-layers although re-arrangement of those layers into "graphite-like" by means of π,π-interactions can reasonably envisaged. This kind of interaction could also contribute to fix graphene layers onto GC. Moreover, cathodic responses of those GC modified surfaces are particularly stable upon charge/discharge cyclings.

In TMAClO 4 / DMF, half-peak potentials of four reduction steps are with I (E I = -1.76 V),

II (E II = -1.97 V ), III (E III = -2.10 V ) and IV (E IV = -2.22 V). Those values are quite close and definitively do not correspond to charge potentials of graphite already reported with HOPG under very comparable conditions (chronopotentiometric curves with total filling of layers by TMA + [4]). Also, voltammetries of commercial graphene in the same electrolyte verified that similar responses were obtained, however without comparable results in terms of stability and intensity. Lastly, analytical determination of synthesized graphenes [START_REF] Ussano | Communication, 10th ECHEMS 2014[END_REF] at carbon electrodes led to comparable successive reversible cathodic peaks.

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Once the deposition of graphite in very thin layers (Scheme 1, A) was first formatted with TMA + , cycling's of those GC/graphite interfaces were undertaken with different TAA + salts in DMF. When radii of cations increase, global charge intensities (observed between -1.7 V and -2.3 V with a given scan rate) decay and noticed cathodic charge steps were progressively shifted to more negative potentials. From TMA + to TBA + , maximum charge (area of 7 mm 2 ) values decline from 25 x 10 -4 C to 3.9 x 10 -4 C. Very bulky salts like tetra-noctyl ammonium provoke a collapse of the charge value (1.8 x 10 -5 C). It is quite important to underline that a direct exfoliation is not obtained with tetra-n-octylammonium salts without a pre-formatting with cations of smaller radii (in particular, TMA + and TEA + ammonium salts).

The nature of the anion was checked to present a minor influence.

Nature of carbon-graphite layer interfaces

As mentioned above, charge currents of modified GC surfaces by means of deposited graphite exfoliation using a given cation led very reproducible electricity amounts (the total cathodic charge within a defined potential gap presents an average error no larger than 10 %).

These values are surprisingly small. The I/V responses appear to be quite similar to those of synthetically produced polyaromatic macromolecules by oxidative condensation of hexaphenylbenzene by means of organic chemistry under suitable conditions possibly leading to graphene nano-ribbons [START_REF] Ussano | Communication, 10th ECHEMS 2014[END_REF].

Deposition of graphene layers with the assumption that the external one in contact with solution is lonely reactive (Scheme 1, C) implying a quasi absence of diffusion of ions TAA + within the short times used in voltammetric sweeps) could be proposed as a plausible model.

Therefore, the reduction peaks found with each TAA + salt could be regarded as the formation of graphene poly-anions stabilized by a progressive coverage of surface as potential decreases. At each cathodic peak (or plateau) would correspond a coverage level. The statistic position of TAA + ions could be seen as a soft-grafting (or marking) of some carbon atoms of graphene. Assimilating reduction peaks with successive reduction levels could permit to estimate corresponding coverage by TAA + ions. For example, with tetraethylammonium tetrafluororate, the charge found was 4.8 x 10 -4 C at a surface of 7 mm 2 . The three steps are

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ACCEPTED MANUSCRIPT 6 supposed to be mono-electronic transitions. A value of 2.2 x 10 -8 ion /cm 2 seems high but should take into account the great surface roughness and that of the relative migration rate of those ions through the layers. Additionally, the weak electro-negativity of TAA + with the possibility to give multi-layers could be taken into account. With TOABF 4 , values based on mono-electronic transitions (2.5 x 10 -9 ion / cm 2 ) fit better with the proposal described above.

Functionalization of the interface GC-graphene

We aim to bring briefly additional supports to the proposed model by achieving different modifications of the external layer active towards TAA + cations (at E < -1.8 V, cathodic charge) and towards free radicals generated outside the charge zone (E > -1.8 V).

First of all, the grafting of 2-methylanthraquinone radical (from Br-CH 2 -AQ) was carried out both on bare GC and modified GC by graphitization. After two scans between 0V and -1.5 V (in DMF + TMABF 4 , scan rate: 50 mV s -1 ), the immobilization of AQ could be obtained: at bare GC Figure 2 A) , the half-peak width for the first step (radical anion of grafted AQ) is large (210 mV), total coverage: 5 ± 3 x 10 -9 mol cm -2 , peak potential: -0.88 V. Contrariwise, the same experiments at modified GC showed important discrepancies: first, an extremely sharp cathodic peak (half-peak width: 90 mV, first scan in TBABF 4 ), apparent AQ coverage: 12±3 x 10 -9 mol cm -2 , peak potential: 0.83 V (Figure 2,B). Definitively, it does exist a fundamental difference between the two kinds of surfaces possibly brought by the specificity graphene layer (two layers of grafting giving higher levels of immobilization). One may note (Figure 2,C) an evidence for the layer to be doped concomitantly by the presence of AQ layer and the charge of graphene.

Ferrocene (by means of Fc-(C 6 H 12 )-I) could be attached as well (curves D1 at graphene and D2 at GC) in both cases by using the cathodic charge of substrate (E = -2.0V). Superficial concentrations are Г Fc equal to 10 -8 and 5x10 -9 mol cm -2 respectively (Scheme 1,B). The ratio of coverages 2/1 should be noticed and Г Fc values within the same range than those given above for AQ. The method of direct exfoliation of graphite, described for the first time in this preliminary work, offers the possibility to create easily GC-graphene interfaces seen as new materials. These interfaces are specifically reactive, within the cathodic range, towards cations and organic electrophilic species. Moreover, the evidence for a doping at E <-1.8 V, suggests large possibilities for easy chemical reactions based on redox catalysis owing to deposited graphene. Produced materials are stable, of rather good conductivity and offer unusual surface modifications that quite certainly deserve further studies and clarifications.

Acknowledgements:

The author wishes to warmly thank Pr. V. Jouikov (University of Rennes, France) for his helpful support in the course of the present work.

Figure caption

Graphite charge followed by exfoliation

Highlights

Exfoliation of graphite was achieved at solid surfaces > Very thin and highly stable deposits of graphene layers were obtained at smooth glassy carbon > The specificity of several tetraalkylammonium salts (TAA+X-) was shown in the charge / cathodic reduction of those layers > Preliminary modifications by ferrocene and anthraquinone moieties were achieved at those graphene layers.

Figure 1 (Figure 2

 12 Figure 1

  Figure 2

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