Ph.D Jessica Tallet
email: tallet@cict.f

Ph.D Viviane Kostrubiec

Pr Pier-Giorgio Zanone

Proactive transfer of learning depends on the evolution of prior learnt task

Keywords: Dynamic pattern theory, interference, facilitation, bimanual coordination, recall

The aim of the present study was to investigate the processes underlying proactive interference effect using bimanual coordination. Our rationale was that interference would develop only when the prior learnt A coordination pattern enters in competition with the subsequent required B pattern. We hypothesized that competition would arise only if the A pattern persist in memory before introducing the B pattern. In the experimental group, both A and B patterns were practiced and recalled whereas, in the control group, only the B pattern was practiced and recalled. In Experiment 1, involving initially bistable participants, the persistence of the A pattern led to interference, while, surprisingly, the A pattern forgetting entailed facilitation. In Experiment 2, carried out with tristable participants, no such transfer effects were evidenced. The apparently contradictory results can be interpreted coherently in the light of dynamical principles of learning.

In the beginning of the 19 th century, Hugo Münsterberg reported that, after having decided to change the usual place of his home key, he forgot the new place and kept searching for it at the previous place. This is the first anecdotic report of so-called proactive transfer of learning, namely, the impact of previously memorized experience on subsequent learning. In the field of motor learning, this effect has been commonly investigated through a proactive transfer paradigm: Typically, in an experimental group, participants practice two consecutive tasks, A and B, followed by a recall of the B task (b), hence a classic ABb design. In a control group, participants practice and recall the B task only (viz. _Bb). Proactive transfer effect occurs when experimental and control groups differ in learning and retention of the B task [START_REF] Hurley | The influence of augmented feedback and prior learning on the acquisition of a new bimanual coordintaion pattern[END_REF][START_REF] Schmidt | Motor control and learning: a behavioural emphasis[END_REF][START_REF] Magill | Motor learning: Concepts and applications[END_REF][START_REF] Adams | Historical review and appraisal of research on the learning, retention, and transfer of human motor skills[END_REF][START_REF] Schmidt | Transfer of movement control in motor skill learning[END_REF][START_REF] Wrisberg | Tests of the duomemory notion for movement information[END_REF][START_REF] Stelmach | Prior positionning responses as a factor in short-term retention of a simple motor task[END_REF][START_REF] Ascoli | Proactive interference in short-term motor retention[END_REF]. It captures the fact that practice of the prior A task can affect learning and retention of the subsequent B task. We focus here on so-called proactive negative transfer, or proactive interference effect, in which the experimental group under-performs the control group.

To date, several experimental factors are deemed to determine the degree of proactive interference effect: the delay between practice of the A and B tasks [START_REF] Shadmehr | Functional stages in the formation of human long-term motor memory[END_REF]; see also [START_REF] Brashers-Krug | Consolidation in human memory[END_REF][START_REF] Robertson | Current concepts in procedural consolidation[END_REF][START_REF] Ray | Proactive Inhibition. A function of time-interval[END_REF], the amount of practice for the B task [START_REF] Ascoli | Proactive interference in short-term motor retention[END_REF][START_REF] Stelmach | Prior positionning responses as a factor in short-term retention of a simple motor task[END_REF][START_REF] Wrisberg | Tests of the duomemory notion for movement information[END_REF] for a review) or the similarity between A and B tasks [START_REF] Schmidt | Transfer of movement control in motor skill learning[END_REF][START_REF] Blais | Components of proactive transfer in motor performance[END_REF]Lewis, McAllister, 1951;McAllister & Lewis, 1951;[START_REF] Shadmehr | Interference in Learning Internal Models of Inverse Dynamics in Humans[END_REF][START_REF] Brashers-Krug | Catastrophic interference in human motor learning[END_REF]. Theoretically, the degree of interference effect is often attributed to the process of competition developing between the memory traces for tasks A and B [START_REF] Stelmach | Retention of motor skills[END_REF]1982;[START_REF] Burwitz | Proactive interference and directed forgetting in short-term motor memory[END_REF]. In other words, competition is the process resulting in transfer effects: The stronger the A memory trace is, the larger competition develops between A memory trace and B learning task, and the larger the interference effect. This hypothesis implies that competition can only occur if the A trace is still present in memory when the B task is introduced. Yet, to our knowledge, the impact of the very persistence of the A memory trace on subsequent B task learning and retention has never been tested. The present study aims to fill this gap by tracking the evolution of the memory trace of the A task in order to predict the degree of proactive interference it may produce on learning and retention of the B task.

An original angle from which to consider the above issue comes from a dynamic approach to learning, which expounds the role of the pre-existing memory repertoire on subsequent learning (Zanone & Kelso, 1992;1994;1997;[START_REF] Walter | The formation and dissolution of "bad habits" during the acquisition of coordination[END_REF][START_REF] Kelso | Coordination dynamics of learning and transfer across different effector systems[END_REF][START_REF] Zanone | Searching for (dynamical) principles of learning[END_REF]. In 1989, Tuller and Kelso explored the spontaneous repertoire of bimanual coordination. To this end, they required naive participants to produce a broad range of coordination patterns according to a visual model, a paradigm called scanning (see also Zanone & Kelso, 1992;[START_REF] Hodges | Learning as a function of coordination bias: building upon pre-practice behaviours[END_REF][START_REF] Maslovat | Evaluation of scanning methodology in bimanual coordination[END_REF]; Atchy-Dalama, Peper, [START_REF] Atchy-Dalama | Movement-related sensory feedback mediates the learning of a new bimanual relative phase pattern[END_REF][START_REF] Temprado | Dynamics of learning and transfer and transfer of muscular and spatial relative phase in bimanual coordination: evidence for abstract directional codes[END_REF]. For each required coordination pattern, the relative timing between the two limbs was assessed by relative phase (RP) that captured the accuracy and the stability of the produced pattern and the performance bias. The findings showed that only two coordination patterns could be produced accurately and stably before any learning: the in-phase (0° of RP) and the anti-phase (180° of RP) patterns. The in-phase pattern results from the simultaneous activation of homologous muscles and the anti-phase pattern from the simultaneous activation of non-homologous muscles [START_REF] Kelso | Phase transitions and critical behaviour in human bimanual coordination[END_REF]. All other patterns manifested a bias toward one of these two stable patterns. This type of memory repertoire was coined as bistable. Similar findings were reported in a study by [START_REF] Yamanishi | Two coupled oscillators as a model for the coordinated finger tapping by both hands[END_REF] while coordination patterns were performed from memory, that is, after the model was withdrawn. Hence scanning proves to be an appropriate probe of the current state of the behavioral or memory repertoire [START_REF] Kostrubiec | How a new behavioral pattern is stabilized with learning determines its persistence and flexibility in memory[END_REF][START_REF] Tallet | Role of stability in the dynamics of learning, memorizing and forgetting coordination patterns[END_REF].

Regarding learning, a central contribution of a dynamical approach is that any new learning requirements must be considered on the background of pre-existing memory repertoire (Zanone & Kelso, 1992;[START_REF] Lee | Relative phase alterations during bimanual skill acquisition[END_REF][START_REF] Wenderoth | Learning a new bimanual coordination pattern is influenced by existing attractors[END_REF]Hurley & Lee, 2008). Before considering the competition between the two entirely novel A and B coordination patterns to be learned in accordance to the A and B tasks, a dynamical approach focuses on the initial competition that arises between the first to-belearned A pattern (required in the A task) and the spontaneously stable patterns preexisting in the memory repertoire (Schöner & Kelso, 1988a;[START_REF] Schöner | Learning as change of coordination dynamics: theory and experiment[END_REF][START_REF] Schöner | Learning and recall in a dynamic theory of coordination patterns[END_REF]Zanone & Kelso, 1992;[START_REF] Wenderoth | Learning a new bimanual coordination pattern is influenced by existing attractors[END_REF][START_REF] Fontaine | Learning a new bimanual coordination pattern: reciprocal influences of intrinsic and to-be-learned patterns[END_REF]. According to dynamical approach to learning [START_REF] Schöner | Learning and recall in a dynamic theory of coordination patterns[END_REF][START_REF] Kostrubiec | How a new behavioral pattern is stabilized with learning determines its persistence and flexibility in memory[END_REF], successful learning of any new coordination pattern requires overcoming the initial competition by stabilizing the to-be-learned pattern, hence altering the preexisting memory repertoire [START_REF] Lee | Relative phase alterations during bimanual skill acquisition[END_REF][START_REF] Smethurst | The acquisition of movement skills: practice enhances the dynamic stability of bimanual coordination[END_REF][START_REF] Walter | The formation and dissolution of "bad habits" during the acquisition of coordination[END_REF][START_REF] Wenderoth | Learning of a new bimanual coordination pattern is governed by three processes[END_REF]. From this perspective, the initial competition can be viewed as the process leading to proactive interference effect between the preexisting patterns and the to-be-learned A pattern, which is reflected by a bias toward a preexisting stable pattern at the beginning of learning (Zanone & Kelso, 1992;[START_REF] Walter | The formation and dissolution of "bad habits" during the acquisition of coordination[END_REF][START_REF] Lee | Relative phase alterations during bimanual skill acquisition[END_REF][START_REF] Fontaine | Learning a new bimanual coordination pattern: reciprocal influences of intrinsic and to-be-learned patterns[END_REF][START_REF] Wenderoth | Learning a new bimanual coordination pattern is influenced by existing attractors[END_REF]Hurley & Lee, 2008). Overcoming the initial competition corresponds to reducing such proactive interference effect, leading to a decrease of the initial bias toward a preexisting stable pattern as practice proceeds.

Recent findings suggest that the reduction of competition with learning can take two routes: by shift or by bifurcation [START_REF] Zanone | The coordination dynamics of learning: theoretical structure and experimental agenda[END_REF][START_REF] Zanone | Searching for (dynamical) principles of learning[END_REF]Zanone & Kelso, 1992;1997;[START_REF] Kostrubiec | Memory dynamics: distance between the new task and existing behavioural patterns affects learning and interference in bimanual coordination in humans[END_REF][START_REF] Kostrubiec | How a new behavioral pattern is stabilized with learning determines its persistence and flexibility in memory[END_REF][START_REF] Tallet | Role of stability in the dynamics of learning, memorizing and forgetting coordination patterns[END_REF]. The route by shift refers to the gradual displacement of an extant stable pattern toward the to-be-learned value whereas the route by bifurcation leads to the sudden creation of a new stable coordination pattern in the memory repertoire. Empirically, the two routes differ with regard to practice and long term retention. (1) During practice, the bifurcation route is characterized by a gain in stability, and possibly in accuracy, of the produced pattern, whereas the shift route leads to a gain in accuracy only. (2) After practice, the bifurcation route leads to a specific alteration in the memory repertoire, due to the creation of a new additional stable pattern, whereas the route by shift leads to a large alteration between the shifted and required patterns. (3) At recall, patterns learned by bifurcation durably persist in memory whereas patterns learned by shift are immediately forgotten by shifting back toward a preexisting stable but non-required pattern as soon as the model is withdrawn [START_REF] Kostrubiec | How a new behavioral pattern is stabilized with learning determines its persistence and flexibility in memory[END_REF]. It follows that only the bifurcation alters qualitatively and durably the memory repertoire, giving rise to a tristable repertoire, with three stable patterns now including the newly learnt pattern and the two old ones. Hence, only the bifurcation route to learning overcomes the initial competition definitely, thereby annihilating the initial proactive interference effect with the pre-existing stable patterns. Shift reduces the initial competition transiently, as revealed at recall by the reappearance of a proactive interference effect with the pre-existing stable patterns.

Turning back to the transfer issue, when two consecutive A and B contrived tasks are required, a major implication of the aforementioned findings is that proactive interference from the patterns A to B depends on how the A pattern is learnt on the background of preexisting stable patterns. The degree of competition between the A and B tasks should be modulated by the persistence of the A pattern in the memory repertoire. In our situation, two predictions may be drawn from the existence of different learning routes: (1) when the prior coordination pattern A is learned by bifurcation, it should persist in the memory repertoire and should thus enter in competition with the subsequent pattern B, leading to a large proactive interference effect between the A and B patterns. (2) In contrast, when the A pattern is learned by shift, it should be fairly rapidly forgotten, which would raise no competition. In this case, no proactive interference effect between the A and B patterns is predicted but proactive interference effect should arise between the required B pattern and the stable patterns preexisting before any practice in the memory repertoire, as if the A pattern had never been learnt.

In the present paper, we present two experiments aiming to test these hypotheses using the 45° and 135° patterns as A and B tasks in initially bistable (Experiment 1) and tristable (Experiment 2) memory repertoires. Previous results on 45° or 135° learning with bistable repertoire showed that after the same amount of practice, the 45° pattern persists in memory whereas the 135° pattern is forgotten [START_REF] Tallet | Role of stability in the dynamics of learning, memorizing and forgetting coordination patterns[END_REF]. Experiment 1 aims to replicate these results and to examine the transfer of learning from 45° to 135° patterns, and vice-versa.

Both A patterns are practiced until similar stability (and accuracy) levels are reached, in order (1) to guarantee that the patterns were learnt, (2) to test the learning speed, that is the number of trials necessary to reach the learning criterion and to (3) ensure that a possible transfer effects could not be attributed to the level of stability reached by the A pattern at the end of practice, given that lower stability may be associated with higher transferability [START_REF] Levin | Bimanual coordination involving homologous and heterologous joint combinations: when lower stability is associated with higher flexibility[END_REF] 1 . We expect that only the persistent 45° pattern should interfere with the subsequent 135° learning and retention. Experiment 2 follows the same rationale for learning these coordination patterns over an initial tristable repertoire.

1 [START_REF] Levin | Bimanual coordination involving homologous and heterologous joint combinations: when lower stability is associated with higher flexibility[END_REF] showed that the less stable the preexisting patterns are, the more a new required pattern is prone to be learnt by shift of the preexisting pattern, hence leading to a positive transfer of learning (or transferability).

Experiment 1

The transfer procedure used here is an adaptation of the classical proactive transfer paradigm (ABb). First, the originality of our procedure resides in introducing a recall test after each learning task in order to probe the retention of the required pattern just after its practice.

This adaptation led to an AaBb design where A and B refer to practice and a and b to recall of the A and B tasks, respectively. Second, all participants performed the two A and B patterns consecutively. The A pattern for one group was the same than the B pattern for the other group, and inversely. For both groups, the Aa pattern served as control for the Bb pattern. As explained below, this method allowed testing proactive interference effects using only two AaBb groups of participants (see Fig. 1).

Method

Participants. Twenty-eight participants (mean age: 24 years +/-2.8, 12 women and 16 men), students at the Faculty of Sport Sciences in Toulouse, volunteered to the experiment.

All were naïve to the purpose of the experiment and presented no impairment impeding the perception of the visual signal or the production of the required RP. They were not paid and approved an informed consent form, in agreement with the University guidelines and the ethical standards laid down in the declaration of Helsinki. Only sixteen (mean age: 23 years +/-2.8, 7 women and 9 men) were selected to continue the experiment (see the procedure below).

Materials. The apparatus consisted of a pair of joysticks fixed 10 cm apart on a table restricting two-dimensional movements to the left-right direction, with maximal amplitude of ±40°. Each joystick was connected to a linear potentiometer whose output was continuously sampled at 200 HZ and stored into a PC for further treatment. Participants were comfortably seated with their elbows on the table and grasped the joysticks at the distal end. A visual metronome, composed of two light-emitting diodes (LEDs) set 8 cm apart was displayed on a 33•× 24 cm black screen placed 80 cm in front of the participants. The onsets of the right and left LEDs were controlled via a microcomputer so that various RPs were displayed at a constant frequency of 1.25 Hz. A 15'' monitor provided a visual KR in the form of a plot displaying the cycle-by-cycle produced RP as a function of time, as well as its within-trial mean and SD.

Procedure. The task was to produce cyclical 1:1 frequency-locked oscillations of both hands in synchrony with the pacing signal from the visual metronome. The right and left LEDs paced the right and left hand motion, respectively. Each trial lasted 20 seconds.

As summarized in Fig. 1, two groups of participants exhibiting an initially bistable repertoire were exposed to two consecutive practice and recall sessions. The experimental design required participants to practice and recall the A task (Aa) first and the B task (Bb) second. All participants performed both 45° and 135° patterns: For the 45-135 Group, the prior A pattern was 45° and the subsequent B pattern was 135°. For the 135-45 Group, the prior A pattern was 135° and subsequent B pattern was 45°. In such a design, each group played the role of both a control and an experimental group. To test the impact of the 45° pattern on the 135° pattern learning and retention, the 135° pattern produced by the 45-135 Group was compared to the 135° pattern produced by the 135-45 Group. In this case, 135-45 Group was the control group. Second, we tested the impact of the 135° pattern on the 45° pattern learning and retention. To this aim, the 45° pattern produced by the 135-45 Group was compared to the 45° pattern produced by the 45-135 Group. In this case, 45-135 Group is the control group.

Insert Fig. 1 Familiarization The experiment lasted two days. The first day started with familiarization during which participants were required to perform the in-phase and the anti-phase patterns and an initially unstable pattern of 30°. KR was provided only after the 0° and 180° patterns trials to familiarize participants with the provided feedback. KR was presented on a screen in front of the participant. It displayed in red the RP produced during the 20 seconds trial and in blue the required RP. The mean RP and its standard deviation (SD) were also provided in order to inform the participant on the levels of accuracy and stability of the produced pattern. This task ensured that participants could perform 0° and 180° patterns accurately and stably, that is, with both absolute error (AE) and SD of RP showing values inferior to 20° (see [START_REF] Hodges | Learning as a function of coordination bias: building upon pre-practice behaviours[END_REF]. In addition, performing the 30° pattern helped participants to feel and understand what an inaccurate and unstable performance could mean (i.e., both AE and SD > 20°).

Pre-practice scan

Immediately after familiarization, a pre-practice scan (Initial scan in Fig. 1) was carried out to probe the initial memory repertoire. Participants were asked to perform 7 patterns with different RPs: 0°, 45°, 75°, 90°, 105°, 135° et 180°, specified by the LEDs and presented randomly. No KR was provided during or after the scan in order to limit uncontrolled learning. On the basis of the outcome of the initial scan, only sixteen so-called bistable participants were identified and selected to pursue the experiment. Three empirical features qualified for bistability: (1) to perform accurately and stably the 0° and 180° RPs, with a fixed criteria of both AE and SD of RP smaller than 20°; (2) to manifest both AE and SD of RP larger than 20° for the other phases, specially for the 90° requirement; and (3) to exhibit no sign of attraction 2 toward the 90°, neither toward 45° or 135° patterns (see [START_REF] Hodges | Learning as a function of coordination bias: building upon pre-practice behaviours[END_REF][START_REF] Tallet | Role of stability in the dynamics of learning, memorizing and forgetting coordination patterns[END_REF]. All the three conditions have to be fulfilled in order to be classified as bistable.

Practice of A task

Right after Initial scan, the prior pattern A was practiced (A in Fig. 1). For half of the participants (n = 8, selected randomly), a 45° pattern was required . For the other half, a 135° pattern was required (135-45 Group). Each practice trial was immediately followed by KR, presented in the same manner than during familiarization. For each participant, 20 mandatory practice trials were administrated. Then, they had to pursue practice until a double learning criterion demanding to produce the required pattern with AE ≤ 12° and SD ≤ 20° during three consecutive trials, in accordance with [START_REF] Kostrubiec | Memory dynamics: distance between the new task and existing behavioural patterns affects learning and interference in bimanual coordination in humans[END_REF]. Thus, participants performed at least 23 practice trials. The number of practice trials to reach the criterion (N) varied between participants, hence allowing a comparison of learning speed. Recall that at any rate, practice continued even if the criterion was reached before completion of 20 mandatory trials.

Recall of A task

Just after practice, one recall trial was administrated (a in Fig. 1). The required pattern had to be performed from memory during 20 seconds, that is, without visual model and without KR. The recall test examined the relative persistence of the practiced pattern.

Post-practice scan

2 Evidence for attraction is captured by a negative slope of the CE curve, reflecting systematic overestimation of the patterns below 90° and underestimation of the patterns above 90° (for details, see Yaminishi et al., 1980;Zanone & Kelso, 1992). Attraction is also captured by a peak of frequency at the neighborhood of the attractive RP in the RP distribution [START_REF] Kelso | Coordination dynamics of learning and transfer across different effector systems[END_REF]. More precisely, the explicit criterion for attraction toward 90° was to observe a 90° RP and its adjacent RPs (75° and 105°) produced with both EA and SD below to 25° and an overestimation (<30°) for the adjacent RPs before 90° (i.e. 75°) and/or an underestimation (<30°) for the adjacent RPs after 90° (i.e. 105°).

After recall, a post-practice scan (Post-practice scan after Aa task in Fig. 1) checked whether the practiced pattern altered the memory repertoire. This scan ended the first day of the experimental design.

Practice of B task

The following day, the second pattern was practiced (B in Fig. 1) following the very same procedure than in the first practice session. For participants having practiced the 45° pattern first, the 135° pattern was required (Group 45-135), and inversely (Group 135-45).

Recall of B task

An immediate recall test of the second practiced pattern (b in Fig. 1) was then administrated using the same procedure than for the recall test of task A.

Second post-practice scans

Just after the recall of the B task, a second post-practice scan (Post-practice scan after Bb task in Fig. 1) aimed to evaluate the alteration of the coordination repertoire after the two learning sessions.

Data reduction and analyses. The RP between the moving limbs was assessed by means of a cycle-by-cycle point-estimate method [START_REF] Scholz | A quantitative approach to understanding the formation and change of coordinated movement patterns[END_REF]Zanone & Kelso, 1992, 1997). RP represents the time difference between the consecutive maximal pronations of the right and left hands, reported to the period of the corresponding right-leading hand cycle, multiplied by 360°. For each trial, three dependant variables were studied: mean constant error (CE), mean AE of RP, and the associated SD of RP. CE characterized the possible bias of the produced pattern. Positive CE means that the produced pattern is overestimated as compared to the required pattern while negative CE means that the produced pattern is under-estimated. As mentioned above, AE assessed accuracy and SD stability. The number of trials, N, corresponded to the real number of trials necessary to reach the criterion, even if this number was inferior to 20 trials. It was also collected as a dependent variable assessing the learning speed.

Three series of statistical analyses have been carried out on practice, recall and scans.

(1) To replicate the findings by [START_REF] Tallet | Role of stability in the dynamics of learning, memorizing and forgetting coordination patterns[END_REF], the first series of analyses focused on the prior Aa tasks in both groups. We compared the performance of 45° and 135° patterns required as Aa tasks in 45-135 and 135-45 Groups. Next, the interference effect from A to B tasks was tested. (2a) In the second series of analysis, we compared the 135° pattern when produced as Aa task in the (control) 135-45 Group and when produced as Bb task in the (experimental) 45-135 Group. (2b) In the third series of analysis, we compared the 45° pattern when produced as Aa task in the (control) 45-135 Group and when produced as the Bb task in the (experimental) 135-45 Group.

For the first series of analyses (45° or 135 as Aa task), five independent variables were studied: Practiced Pattern (45° and 135°), Practice (21 practice trials: 20 first mandatory and the mean of the three last practice trials), Recall (the learning criterion and the recall test), Scan (pre-practice scan and post-practice scans) and Required Phase (0°, 45°, 75°, 90°, 105°, 135° and 180°). Except for Practiced Pattern, all the variables were treated as repeated measures.

For the analyses of practice, a 2 (Practiced Pattern) × 21 (Practice) ANOVA was carried out on CE, SD and AE in order to compare the evolution of the bias, stability and accuracy of the 45° and the 135° patterns during 21 practice trials. If necessary, deviation contrasts, comparing the value of each trial with the mean value of all trials, were performed in order to inspect more precisely the evolution of performance during practice (cf. [START_REF] Tallet | Role of stability in the dynamics of learning, memorizing and forgetting coordination patterns[END_REF]. A one-way 2 (Practiced Pattern) ANOVA was carried out on the AE and SD reached at the learning criterion to ensure that both 45° and 135° patterns reached equivalent levels of accuracy and stability at the end of practice. The same analysis was performed on N to compare the learning speed of the 45° and the 135° patterns.

For the analyses of recall, a 2 (Practiced Pattern) × 2 (Recall) ANOVA was carried out on CE, SD and AE in order to compare the evolution of the bias, stability and accuracy of the 45° and the 135° patterns between the end of learning and the recall test. If performance did not change significantly between the last practice trials and the recall test, the pattern was considered to be persistent. In the other case, the pattern is considered to be forgotten.

Forgetting, that is, the shift back to a non-required stable RP value was inferred from a decrease in accuracy (AE), with possible performance bias (CE), without change in stability (SD).

For the analyses of scans, a 2 (Practiced Pattern) × 7 (Required Phase) ANOVA was first performed on AE and SD of the required phases of the initial scan in order to ensure that the performance of the initial scans of both groups did not differ before practice. Student-Newman-Keuls contrasts were used as post-hocs in order to test whether scans reflected bistable repertoires (i.e., 0° and 180° more accurate and stable than other phases). Then, a 2 (Practiced Pattern) × 2 (Scan) × 7 (Required Phase) ANOVA performed on AE and SD of the required phases in scans 1 and 2 probed the impact of learning 45° or 135° pattern on such bistable repertoires.

For the two other series of analyses (135° as A and B task and 45° as A and B task), the same independent variables were used, except that Practice Pattern was replaced by Position. Hence, independent variables were: Position (A and B), Practice (21 practice trials), Recall (the learning criterion and the recall test), Scan (pre-practice scan and post-practice scan) and Required Phase (0°, 45°, 75°, 90°, 105°, 135° and 180°). Except for Position, all the variables were treated as repeated measures. Similar ANOVAs than above-mentioned were conducted to analyze practice, recall and scans, except that Practice Pattern was replaced by Position. This allowed comparing 135° (second series of analyses) or 45° (third series of analyses) when performed as A and B tasks.

Only significant results, with p <.05, are reported here. Main effects of Practice were evidenced on AE (F(20,280) = 3.345; p<.001) and SD (F(20,280) = 2.652; p<.001) and a main effect of Practiced Pattern was also found on AE (F(1,14) = 5.184; p<.04) (see Fig. 2). Hence, both patterns exhibited a gain in accuracy and stability, but the 45° pattern was globally more accurate that the 135° one (mean AE = 15° and 24°, respectively).

Results

No significant differences was evidenced on the levels of accuracy and stability reached at the end of practice of 45° and 135° patterns (F(1,14) = 1.928; ns for AE and F(1,14) = 0.279; ns for SD).

Finally, a main effect of Practiced Pattern was revealed on N (F(1,14) = 4.786; p<.05), indicating that the 45° was learnt more rapidly than the 135° (N=16 vs. N=26 trials to reach the learning criterion, respectively).

All the above results suggest that, although both patterns gained in accuracy and stability with practice, their evolution differed during practice. In particular, the 45° pattern was globally more accurate and the 135° pattern stabilized a range of under-estimated RP (about 110°) before reaching the required 135° RP.

Recall

Main effects of Recall and Practiced Pattern were revealed on AE only (F(1,14) = 9.15; p<.009 and F(1,14) = 4.6; p<.05, respectively) (see Fig. 2). This result indicates that (1) irrespective of the required pattern, accuracy decreased between the learning criterion and the recall (mean AE increased from 6° at the end of practice to 15° at recall), but (2) the 45° pattern was globally more accurate than 135° (AE = 8° and 13°, respectively). This suggests that 45° was more persistent in memory than 135°.

Scans

First, for the first scan, a significant effect of Required Phase was revealed on AE (F(6,84) = 9.004; p<.001) and SD (F(6,84) = 10.368; p<.001). This result indicates that, for the first scan, (1) accuracy and stability of the produced phases differed as a function of the required RP (Fig. 2, top Panel) and (2) accuracy and stability of the produced phases did not differ between the two groups. Student-Newman-Keuls contrasts revealed that the 0° and 180° phases exhibited the lowest AE and SD (p<.05; for 0°: mean AE = 4° and mean SD = 7°; for 180°: mean AE= 11° and mean SD = 15°). Moreover, SD of 0° was lower than SD of 180° (Diff = -7.904; p<.05). These results confirm that initial repertoires were bistable for both groups, since only 0° and 180° are accurate and stable before practice (see arrows in Fig. 2, top Panel).

Second, the analyses comparing the pre-and post-practice scans revealed a main effect of Practiced Pattern on AE (F(6,84) = 9.671; p<.001) and SD (F(6,84) = 14.479; p<.001), which indicated that the produced phases differed in terms of accuracy and stability (see Fig. 2). A main effect of Scan on AE (F(1,14) = 5.66; p<.04) and SD (F(1,14) = 8,246; p<.02) indicated that, whatever the practiced pattern (45° or 135°), accuracy and stability of all the required phases increased with practice (mean AE decreased from 30° to 24° and mean SD decreased from 20° to 17°). A main effect of Practiced Pattern of SD (F(1,14) = 8.132; p<.02) indicated that the stability of the required phases was globally larger after practice of 45° than after practice of 135° (mean SD = 19° and 14°, respectively). A significant interaction Scan × Required Phase on SD (F(6,84) = 2.272; p<.05) indicated that the stability of the required phases was affected by practice. An interaction Scan × Practiced Pattern × Required Phase on AE and SD (F(6,84) = 3.102; p<.01 and F(6,84) = 2.804; p<.02, respectively) indicated that the alterations in the repertoire due to practice differed as a function of the practiced pattern (45° or 135°). Student Newman-Keuls contrasts revealed that, after practice of 45°, this pattern became more accurate and stable after practice (see arrows in Fig. 2; mean AE from 43° to 14° and mean SD from 29° to 14°, respectively; giving that 45° became as accurate and stable as 0°: Diff = -4.201; ns and Diff = -6.637; ns respectively), whereas after the practice of 135°, all the phases between 75° and 135° became more accurate and more stable (see arrows in Fig. 2; mean AE from 38° to 32° and mean SD from 21° to 17°, giving that the 75° to 135° phases became as accurate as 0° and as stable as 180° after practice; Diff < 3.404; ns).

Insert Fig. 2 To sum up, the gain in stability underwent by 45° as well as 135° patterns suggest that they were learnt by bifurcation. However, 45° stabilized at the required value, whereas 135° led to the stabilization of an under-estimated value of RP that had a never been required. Thus, only 45° altered the memory repertoire specifically and persisted in memory at the required value. In contrast, practice of 135° led to the stabilization of a range of underestimated relative phases (from 75° to 135°) in the memory repertoire. At recall, the 135° pattern was less persistent than 45°.

Section 2. Position effects: comparison of 135° as A or B task Practice

A significant effect of Practice on AE only (F(20,280) = 2.287; p<.002) revealed that the accuracy of 135° increased whatever the practice position (see Fig. 3; from mean AE = 36° to mean AE = 8°). A significant effect on SD (F(1,14) = 4.585; p<.05) indicated that 135° was less stable at the end of B practice than of A practice (mean SD = 16° and 13°, respectively).

No significant effect was found on N (F(1,14) = 0.055; ns; N = 26 vs. 24 trials for A and B tasks respectively). All these results suggest that irrespective of the position, practice of the 135° pattern induced (1) a gain in accuracy, (2) no gain of stability (mean SD remained around 20°) and (3) the same learning speed. However, (4) 135° became less stable when practiced as a B task, namely after practice of 45°.

Recall

A significant effect of Recall on AE (F(1,14) = 4.902; p<.05) and a significant effect of Position on SD (F(1,14) = 7.912; p<.02) were found (see Fig. 3). These results suggest that, independent of the position, the accuracy of the produced pattern decreased between the last practice trials and the recall (mean AE increased from 7° at the end of practice to 15° at recall). However, 135° was less stable when recalled as a B task, namely after practice of 45° (mean SD = 17° and 12°, respectively).

Scans

First, a main effect of Required Phase on AE (F(6,84) = 5.625; p<.001) and SD (F(6,84) = 7.984; p<.001) indicated that all phases were produced with different degrees of accuracy and stability. Second, a significant effect of Position on SD (F(1,14) = 7.841; p<.05) indicated that all the phases of a scan were globally less stable when 135° was practiced as B task, that is after 45° (mean SD = 14° after 135° practiced as A task and mean SD = 19° after 135° practiced as B task). Third, a significant interaction Required Phase × Position was revealed on SD (F(6,84) = 2.619; p<.03). Student Newman-Keuls contrasts indicated that, when 135° was practiced as a A task, the phases of 75°, 90°, 105° et 135° became accurate and stable (as stable as 180°: Diff < -5.651; ns). When 135° was practiced as B task, namely after 45°, all phases became more accurate (as accurate as 180°: Diff < -14.237; ns) only the 45° pattern remained more stable (as stable as 0°: Diff = -9.142; ns), but not more accurate (see arrows in Fig. 3).

Insert Fig. 3 To sum up, learning 45° as A task induced an interference effect on the learning and retention of a subsequent 135° (Group 45-135 i.e., the experimental group). After the creation of the persistent 45° pattern in the memory repertoire, the 135° pattern induced (1) no gain in stability with practice; (2) a lower stability at the end of practice, (3) less specific alterations in the memory repertoire and (4) a larger decrease in accuracy and lesser stability at recall.

Section 3. Position effects: comparison of 45° as A or B task Practice

A main effect of Practice on CE (F(20,280) = 6.717; p<.001) indicated that, whatever the position, the 45° was first over-estimated and over-estimation decreased with practice (from mean CE = 38° to mean CE = 0°). Main effects of Practice was found on AE (F(20,280) = 7.177; p<.001) and SD (F(20,280) = 3.351; p<.001) and main effects of Position was found on AE (F(1,14) = 5.619; p<.04) and SD (F(1,14) = 9.827; p<.01) (see Fig. 4). These results suggest that, whatever the practice position, both accuracy and stability of the 45° pattern increased (mean AE decreased from 43° to 6° and mean SD from to 19° to 12°). However, 45° was globally more accurate and stable when practiced as B task, that is after 135° practice (mean AE = 19° and mean SD = 13° when practiced as A task and mean AE = 15° and mean SD = 9° when practiced as B task).

A significant effect of Position on SD (F(1,14) = 6.253; p<.03) indicated that 45° was more stable at the end of practice when it was practiced after 135° (mean SD = 14° at the end of practice as A task and mean SD = 10° at the end of practice as B task). A main effect of Position on N (F(1,14) = 7,636; p<.02) revealed that 45° was learnt twice faster when practiced as B task, that is after 135° (N = 8 as A task and 17 trials as B task).

Recall

A significant effect of Position was revealed on SD only (F(1,14) = 5.108 ; p<.05) (see Fig. 4). This suggests that, whatever the position, 45° was globally more stable as A task, i.e. after 135° practice (mean SD = 10° as B task and mean SD = 14° as A task).

Scans

A main effect of Required Phase was found on AE (F(6,84) = 6.862; p<.001) and SD (F(6,84) = 11.053; p<.001) and a main effect of Position was found on SD (F(1,14) = 4.744; p<.05) (see Fig. 4). This last result suggests that all the tested phases were more stable when 45° was practiced as B task, i.e. after 135° (mean SD = 19° after 45° as A task and SD = 14° after 45° as B task) (see arrows in Fig. 4).

Insert Fig. 4 To sum up, the prior practice of 135° induced a facilitation effect on the learning and retention of 45° (Group 135-45 i.e., the experimental group). After the 135° pattern practice, practice and recall of 45° manifested (1) a faster learning, (2) a larger accuracy and stability during practice, (3) a higher stability at the end of practice; (4) a specific alteration value in the memory repertoire at the required 45°, and (5) a higher stability during recall.

Discussion

This experiment aimed to test the impact of the persistence of a prior A task in memory on the learning and retention of a subsequent B task. Two groups of participants exhibiting an initial bistable memory repertoire practiced and recalled two coordination patterns consecutively, 45° and 135° or vice-versa. Our results are in line with our hypotheses. First, regarding learning and retention of the 45° and 135° patterns as Aa task, the two patterns exhibited different evolutions. In the case of the 45° pattern, the performed pattern (1) exhibited concomitant gain in accuracy and stability, (2) was more persistent in memory and (3) was stabilized in the memory repertoire. In the case of the 135° pattern, the performed pattern (1) stabilized an under-estimated pattern before reaching the required RP value, (2) was less persistent in memory at recall and (3) was accompanied by the stabilization of a large range of under-estimated RP values in the memory repertoire. These results are in accordance with those of Fontaine, [START_REF] Fontaine | Learning a new bimanual coordination pattern: reciprocal influences of intrinsic and to-be-learned patterns[END_REF], showing that during practice, the 45° was performed better than the 135° pattern, and replicate previous results by [START_REF] Tallet | Role of stability in the dynamics of learning, memorizing and forgetting coordination patterns[END_REF].

They suggest that the 45° pattern was learnt through the bifurcation route whereas the 135° pattern underwent a more complex evolution, that is, a bifurcation at an under-estimated RP value followed by a shift toward the required 135° value.

Second, regarding proactive transfer effects, in line with our hypotheses, the learning and recall of the B task differed as a function of persistence of the A task in memory. When the more persistent 45° pattern was learned as an Aa task, a proactive interference effect occurred on the subsequent 135° pattern learning and retention. In contrast, when the less persistent 135° pattern was learned as Aa task, an unexpected proactive facilitation effect was found on the subsequent learning and retention of 45°.

These effects can be interpreted in terms of the routes to learning. In the 45°-135° group, the bifurcation route to 45° learning created a specific and persistent alteration at a 45° RP value in the memory repertoire that could interfere with the subsequent learning / retention of the 135° pattern. By contrast, in the 135-45° group, the shift-based process caused by the practice of 135° pattern was followed by a general alteration of the memory repertoire. In this case, we would assume that the shift route developed a general capacity to learn a new pattern rapidly, resulting in a proactive facilitation effect. If so, the shift route can be viewed as a desirable (even if transient) form of general adaptation to the learning constraints, although it associated to forgetting of a preexisting pattern [START_REF] Kostrubiec | How a new behavioral pattern is stabilized with learning determines its persistence and flexibility in memory[END_REF]. This idea is in line with [START_REF] Altman | Forgetting to remember: the functional relationship of decay and interference[END_REF] who emphasized the functional role of forgetting, namely a benefic contribution to the reduction of a proactive interference effect. This position is also in line with previous work by [START_REF] Panzer | Learning of similar complex movement sequences: proactive and retroactive effects on learning[END_REF], suggesting that a proactive facilitation effect is the result of an adaptation (viz., a shift) of the prior A task in order to learn the subsequently required B task.

Experiment 2

Experiment 2 was devised to attest the generality of the above findings and tentative conclusions. It used identical experimental design and analyses with participants exhibiting an initial tristable repertoire, reportedly with a third stable coordination pattern mostly located at a 90° RP value [START_REF] Zanone | Coordination dynamics of learning and transfer: collective and component levels[END_REF][START_REF] Hodges | Learning as a function of coordination bias: building upon pre-practice behaviours[END_REF][START_REF] Kelso | Coordination dynamics of learning and transfer across different effector systems[END_REF][START_REF] Kostrubiec | How a new behavioral pattern is stabilized with learning determines its persistence and flexibility in memory[END_REF], and addressed the issue of whether the facilitation effect is consecutive to forgetting. Previous studies on such a tristable repertoire showed that, as practice of 45° or 135° patterns proceeds, learning is achieved through a shift of the preexisting 90° pattern toward the required pattern (for 45°, see [START_REF] Kelso | Coordination dynamics of learning and transfer across different effector systems[END_REF][START_REF] Amazeen | Is dynamics the content of a generalized motor program for rythmic interlimb coordination[END_REF] for 135°, see [START_REF] Kostrubiec | Memory dynamics: distance between the new task and existing behavioural patterns affects learning and interference in bimanual coordination in humans[END_REF][START_REF] Kostrubiec | How a new behavioral pattern is stabilized with learning determines its persistence and flexibility in memory[END_REF]. Moreover, recent results indicated that the 135° pattern is immediately forgotten after practice [START_REF] Kostrubiec | How a new behavioral pattern is stabilized with learning determines its persistence and flexibility in memory[END_REF]. To our knowledge, the evolution of the 45° pattern in memory has never been tested for a tristable repertoire. According to a theoretical model [START_REF] Zanone | Searching for (dynamical) principles of learning[END_REF], given that 45° is learnt by shift, this pattern should also be forgotten just after practice. Turning back to the transfer issue and on the basis of Experiment 1, we can predict that forgetting of 45° or 135° practiced as A tasks should lead to facilitation of the learning and retention of the B task.

Method

Participants. Twelve participants excluded from the Experiment 1 and exhibiting a tristable memory repertoire volunteered to participate in this second experiment (mean age: 25 +/-2,4 years; 5 women and 7 men).

Materials, protocol and data analyses. Materials, protocol and data analyses were identical than in Experiment 1 (see above Methods section), except that, for the initial scan, we ensured that the tristable participants were able 1) to perform accurately and stably the 0° and 180° RPs, 2) to manifest the 90° requirement with mean AE lesser than 20° and SD lesser 24 than 25° and 3) to produce spontaneously the 90° RP pattern or one of its two adjacent (75° or 105°) more accurately and stably than 45° and 135° RP. All these results indicate that (1) the 45° pattern was globally over-estimated but decreased toward the required value with practice (from mean CE = +30° to -5°), whereas the 135° pattern was globally underestimated but increased toward the required value (from mean CE = -22° to 0°), (2) both 45° and 135° became more accurate without changes in stability with practice (mean AE from 29° to 6°; mean SD = 17°), (3) the 45° pattern was globally more stable than the 135° pattern (mean SD = 14° and 20°, respectively), (4) 45° was learnt faster than the 135° (N=12 vs. N=31 trials to reach the learning criterion, respectively), and

Results

(5) both patterns reached a comparable level of accuracy and stability at the end of practice (mean AE = 7° and mean SD = 15° at the end of practice).

Recall

A main effect of Practiced Pattern on CE (F(1,10) = 5.639 ; p<.04) indicated that the 45° pattern was globally overestimated whereas the 135° pattern underestimated (mean CE = +8° and -3° respectively). The Practiced Pattern × Recall interaction on CE did not reach the level of significance (F(1,10) = 4 ; p=.07) but a finer look on the individual data indicated that CE of the 45° pattern increased for all participants, whereas CE of the 135° pattern globally decreased, except for one participant3 .

A main effect of Recall on AE (F(1,10) = 5.263 ; p<.05) indicated that the accuracy of both patterns decreased between the end of practice and the recall (from mean AE = 6° to 12°; see Fig. 5).

Scans

First, for the initial scans, a main effect of Required Phase on AE (F(6,60) = 5.321 ; p<.001) and SD of phases produced in the initial scan F(6,60) = 7.362 ; p<.001) was found. Student Newman-Keuls contrasts indicated that 0° was the most accurate and stable pattern (mean AE = 6° and mean SD = 7°), followed by 180° (mean AE = 11° and mean SD = 15°) and 90°

(mean AE = 13° and mean SD = 14°) (p<.05). Thus, the initial coordination repertoire was assumedly tristable (see arrows in Fig. 5, top Panel).

Second, a main effect of Required Phase was found on AE (F(6,60) = 7.92 ; p<.001) and SD (F(6,60) = 11.89 ; p<.001) and a Practiced Pattern × Required Phase interaction was significant on AE (F(6,60) = 3.741 ; p<.003) and SD (F(6,60) = 3.254 ; p<.01) (see Fig. 5). A significant Practiced Pattern × Scan × Required Phase was revealed on SD (F(6,60) = 2.742 ; p<.02). All these results suggest that the accuracy of the tested phases was not changed by practice of 45° or 135°. Student Newman-Keuls contrasts performed on stability showed that (1) after practicing 45° as A task, only this pattern became more stable (as stable as 0° and 180°; Diff = -5.275; ns and Diff = -6.527; ns respectively), whereas (2) after practicing 135° as A task, the 45°, 105° and 135° patterns became more stable (as stable as 0°; Diff < -8.966; ns).

Insert Fig. 5 Based on the experimental features of the shift route [START_REF] Kostrubiec | How a new behavioral pattern is stabilized with learning determines its persistence and flexibility in memory[END_REF], our results suggest that learning 45° or 135° patterns leads to a shift from the pre-existing 90° pattern toward the required RP value during practice and a shift back from the required RP value toward a preexisting stable pattern (probably the original 90° value) at recall. Moreover, as expected and shown in scans results, learning did not specifically alter the memory repertoire at the required RP values, given that gains in stability were not associated to gains in accuracy. All these results suggest that the process involved the shift route, from the preexisting 90° pattern toward the required RP and a shift back from the required RP toward the preexisting 90° pattern.

Section 2. Position effects: comparison of 135° as A or B task Practice

First, a main effect of Practice was revealed on AE only (F(20,200) = 2.335 ; p<.002) (see Fig. 6). Significant effect was found neither on N, nor on AE, nor on SD reached at the end of practice. These results suggest that, whatever the position, 135° exhibited (1) a gain in accuracy with practice (mean AE form 36° to 8°), (2) no gain in stability (mean SD around 20°), (3) similar learning speed (N=30 and N=26 after A and B practice respectively) and ( 4) the same level of accuracy and stability at the end of practice (mean AE = 7° and mean SD = 14°).

Recall

A main effect of Recall was revealed on CE (F(1,10) = 9.734 ; p<.01) and on AE (F(1,10) = 10.612 ; p<.01) (mean CE from -1° at the end of practice to -11° at recall and mean AE from 7° to 15°, see Fig. 6 middle Panel). Hence, whatever the practice position, RP decreased toward an under-estimated value between the end of practice and recall.

Scans

Main effects of Practiced Pattern was found on AE (F(6,60) = 6.136 ; p<.001) and SD (F(6,60) = 9.602 ; p<.001). Student Newman-Keuls contrasts showed that, whatever the practice position, the 105° and 135° patterns became more accurate (as accurate as 180°) and the 90° pattern less stable (no more as stable as 180°: Diff = 7.326; p<.05, see Fig. 6, bottom right Panel).

To sum up the ensemble of the above results, there was no transfer effect from 45° to 135° patterns.

Insert Fig. 6 Section 3. Position effects: comparison of 45° as A or B task Practice A significant effect of Practice was found on CE (F(20,280) = 6.715 ; p<.001) and on AE (F(20,200) = 5.335 ; p<.001) (see Fig. 7 for AE results). This suggests that, whatever the practice position, CE and AE decreased with practice (mean CE from +35° to +5° and mean AE from 42° to 7°). A main effect of Position was found on SD only (F(1,10) = 8.946; p<.02). These results suggest that, whatever the practice position, 45° exhibited (1) a gain in accuracy with practice, (2) no gain in stability (mean SD around 13°) and (3) similar learning speed (N = 12 and N = 8, respectively). After practice as B task (that is after 135°), 45° exhibited (4) similar level of accuracy but (5) higher stability than after practice as A task (mean AE=3° whatever the position; mean SD = 13° and 10° after practice as A and B task respectively).

Recall

A significant effect of Recall on CE (F(1,10) = 6.09 ; p<.04) and AE (F(1,10) = 5.283 ; p<.05) suggested that, whatever the practice position, accuracy decreased at recall (from mean AE = 4° at the end of practice to 8° at recall, see Fig. 7), tending toward an overestimated value (mean CE from 3° at the end of practice to 8° at recall).

Scans

A main effect of Practiced Pattern was significant on AE (F(6,60) = 8.464 ; p<.001) and SD (F(6,60) = 8.636 ; p<.001) and an interaction Required Phase × Position was significant on AE (F(6,60) = 2.488 ; p<.04) (see Fig. 7). Student Newman-Keuls contrasts pointed that, whatever the practice position, the 45° pattern became more stable (as stable as 0°; Diff = -5.253; ns). When 45° was practiced as A task, 75°, 105° and 135° became less accurate (Diff > --24.206; p<.05 as compared to 0°, 45° and 180°). When 45° was practiced as B task, 90° and 135° became less accurate (Diff > -20.655; p<.05 as compared to 0° and 45°).

To sum up, the higher end-practice stability of 135° pattern when practiced after 45° (in the 45-135 Group i.e., experimental group) is the only sign of possible facilitation effect found in Experiment 2.

Insert Fig. 7 Discussion This second experiment aimed to generalize the proactive transfer (viz facilitation) effect found in Experiment 1. Two groups of participants exhibiting an initial tristable memory repertoire practiced and recalled two coordination patterns, 45° and 135° consecutively, in this and the reverse order. Results showed that when practiced and recalled as A tasks, the 45° and 135° patterns exhibited similar evolutions. They gained in accuracy but not in stability 29 during learning, and rapidly deteriorated in accuracy at recall, with a possible bias toward another value, and without change in stability at recall. This suggests that both patterns were learnt by shifting the pre-existing 90° stable pattern and were forgotten by shifting it back toward the 90° value after practice. Hence, the 90° pattern persisted in the memory repertoire after practice. No qualitative alteration was created in the memory repertoire after practice of the 45° and 135° patterns as A tasks. Contrary to our hypotheses coming from Experiment 1, although the just-practiced patterns were forgotten, no facilitation effect was found on the subsequent B task (i.e. in the experimental group). The shift-based process did not systematically induce a general capacity to adapt to a new learning requirement.

It seems that transfer of learning effects can be understood by taking into account the impact of the A task in memory, that is, its persistence at recall and the alteration it created in the memory repertoire. In Experiment 2, patterns did neither persist at recall nor create a persistent alteration in the memory repertoire: (1) the 45° and the 135° patterns practiced as A task exhibited a deterioration of performance, with a possible shift back toward the initial 90° stable pattern at recall, and (2) they did not create any qualitative alteration in the memory repertoire. Eventually, it appears that the A tasks had never been learnt (or had been completely forgotten) so that no transfer effects occurred. In contrast, in Experiment 1, (1) the 135° pattern practiced as A task exhibited a deterioration of performance at recall, but (2) created a retained alteration in the memory repertoire. This persistent alteration may have induced a proactive facilitation effect on the subsequent 45° pattern learning and retention. This last finding suggests that even if patterns did not persist at recall, they can create a persistent alteration in the memory repertoire, leading eventually to proactive facilitation effect. This result comforts the claim that the evolution of the memory trace of the A task in the memory repertoire must be taken into account to predict transfer on the learning and retention of the B task. It challenges the traditional conceptions of memory, such as the theory of forgetting by trace decay (Ebbinghaus, 1885;[START_REF] Adams | Short-term memory of motor responses[END_REF][START_REF] Adams | Historical review and appraisal of research on the learning, retention, and transfer of human motor skills[END_REF] and the theory of forgetting by interference (Muller & Pilzecker, 1900;cited by Lechner, Squire & Byrne, 1999;[START_REF] Mcgeoch | Forgetting and the law of disuse[END_REF][START_REF] Lewis | Facilitation and interference in performance on the modified Mashburn apparatus: I. The effects of varying the amount of original learning[END_REF][START_REF] Mcallister | Retroactive facilitation and interference as a function of level of learning[END_REF]Lewis, 1951;[START_REF] Ascoli | Proactive interference in short-term motor retention[END_REF][START_REF] Stelmach | Prior positionning responses as a factor in short-term retention of a simple motor task[END_REF]1974), positing that forgetting leads to a complete loss of the memory trace. To sum up, what really matters is not only whether the A task is forgotten, but also, and mainly, to which RP value it shifts at recall and which patterns are really stabilized in the memory repertoire after the practice of the A task.

General discussion

The purpose of the present study was to address the issue of the proactive interference. We tested the effect of the persistence of a prior A task in memory on the learning and retention of a subsequent B task, using bimanual coordination as an experimental window. The classic proactive transfer paradigm was adapted to test the consecutive learning of a 45° and 135° RP patterns, or inversely, for bistable (Experiment 1) and tristable (Experiment 2) memory repertoires.

On the basis of a dynamic pattern learning model [START_REF] Schöner | Learning and recall in a dynamic theory of coordination patterns[END_REF][START_REF] Zanone | The coordination dynamics of learning: theoretical structure and experimental agenda[END_REF][START_REF] Zanone | Searching for (dynamical) principles of learning[END_REF]Schöner & Kelso, 1988a;[START_REF] Schöner | Dynamic pattern generation in behavioral and neural systems[END_REF][START_REF] Schöner | Learning and recall in a dynamic theory of coordination patterns[END_REF][START_REF] Blais | Components of proactive transfer in motor performance[END_REF][START_REF] Schöner | Learning as change of coordination dynamics: theory and experiment[END_REF], we posited that, when two A and B patterns are required consecutively, the degree of proactive interference effect should be modulated by the persistence of the A task in memory. Our results support this assumption. Results of Experiment 1 suggest that, in an initially bistable repertoire, the 45° pattern persisted in memory after practice, leading to a proactive interference effect with the subsequent 135° learning and retention. In line with previous results by [START_REF] Tallet | Role of stability in the dynamics of learning, memorizing and forgetting coordination patterns[END_REF], the 135° pattern was less persistent at recall time.

What is quite surprising is that the less persistent 135° pattern led to a facilitation in the subsequent learning and retention of the 45° pattern. This proactive facilitation effect may be understood by giving a closer look at the evolution of the 135° pattern during and after practice. Our findings suggest that practice gave rise to a bifurcation at an under-estimated (non-required) RP value, followed by a shift toward the required 135° pattern. This scenario is attested by the large alteration from 75° to 135° in the post-practice repertoire. It is thus possible that the stabilization of the 75° pattern have allowed a proactive facilitation effect on the subsequent 45° practice and retention.

We may now wonder on which ground the facilitation effect can be elicited.

According to a dynamic learning model, learning a new pattern depends on the distance between the required and the nearest pre-existing patterns in the memory repertoire [START_REF] Schöner | Learning and recall in a dynamic theory of coordination patterns[END_REF][START_REF] Zanone | The coordination dynamics of learning: theoretical structure and experimental agenda[END_REF][START_REF] Zanone | Searching for (dynamical) principles of learning[END_REF]. When the 45° pattern is required as a prior Aa task (control group), the distance to the nearest 0° stable pattern is 45°. When the 45° pattern is required as the Bb task (after 135° practice, in experimental group), the distance to the nearest 75° stable pattern is 30° only. Hence, the distance between the nearest preexisting stable pattern (75°) and the new required pattern (45°) is smaller when 45° is required as B task (in the experimental group) than when it is required as A task (in the control group). In this case, facilitation effect from 135° to 45° is thus found: when produced as Bb task, 45° is learnt twice faster, it is more accurate and stable during practice and it becomes more stable at the end of practice and at recall. This scenario can help interpreting the results concerning initially tristable repertoire in the second experiment. When practiced as A task, both 45° and 135° are learnt by shift of the initially stable 90° pattern and are then forgotten by shifting back toward 90° value as soon as the visual model was withdrawn (see also [START_REF] Amazeen | Is dynamics the content of a generalized motor program for rythmic interlimb coordination[END_REF][START_REF] Kostrubiec | Memory dynamics: distance between the new task and existing behavioural patterns affects learning and interference in bimanual coordination in humans[END_REF][START_REF] Kostrubiec | How a new behavioral pattern is stabilized with learning determines its persistence and flexibility in memory[END_REF]. Thus, when the B task was subsequently required, everything went as if the learning of the Aa task had not taken place at all in the experimental group. The current memory repertoire was similar to the initial tristable one: The distance between the required pattern and the pre-existing nearest 90° pattern was identical (45°) for both the control and the experimental groups, so that no (or little) facilitation was observed. Hence, these results are in accordance with the work by [START_REF] Miall | Adaptation to rotated visual feedback: a re-examination of motor interference[END_REF], suggesting that when two patterns A and B are consecutively practiced, whether the A task has really been learnt is crucial to predict a facilitation or an interference effect to the B task.

The present paper provides first evidence that the distance between the A and B tasks can lead to both proactive facilitation and interference effects on learning and retention of the B task. To date, proactive facilitation and interference effects have often been considered as two distinct phenomena, and have often been explained by different factors (see [START_REF] Adams | Historical review and appraisal of research on the learning, retention, and transfer of human motor skills[END_REF]. In the traditional approach, the two most convincing factors explaining both effects were the similarity of the A and B tasks (Thorndike and Woodworth, 1901) and the amount of practice [START_REF] Lewis | Facilitation and interference in performance on the modified Mashburn apparatus: I. The effects of varying the amount of original learning[END_REF][START_REF] Mcallister | Retroactive facilitation and interference as a function of level of learning[END_REF]Lewis, 1951). The similarity hypothesis predicts that the more two motor tasks are similar, the more they are prone to interact (see [START_REF] Schmidt | Transfer of movement control in motor skill learning[END_REF]. However, how similarity may be measured and when interference or facilitation will eventually happen remains obscure. The amount of practice hypothesis suggests that facilitation occurs when the amount of A task practice is low whereas interference effect occurs when the amount of the A task practice is large [START_REF] Lewis | Facilitation and interference in performance on the modified Mashburn apparatus: I. The effects of varying the amount of original learning[END_REF][START_REF] Mcallister | Retroactive facilitation and interference as a function of level of learning[END_REF]Lewis, 1951). However, these predictions are not supported by the present results: in bistable participants, the less practiced 45° pattern led to an interference effect whereas the more practiced 135° pattern led to facilitation (Experiment 1). Moreover, in tristable participants, no transfer effect was found although the 45° pattern was less practiced (Experiment 2). Thus, it is unlikely that the amount of practice account for transfer effects.

With regard to the dynamic approach to learning, one could suppose that proactive interference and facilitation could be explained in terms of the level of stability reached at a given instant. Recent work by [START_REF] Levin | Bimanual coordination involving homologous and heterologous joint combinations: when lower stability is associated with higher flexibility[END_REF] reports that the more stable the pattern is before the learning, the more it interferes with another pattern required subsequently, suggesting that the pre-learning level of stability of the A pattern may modulate the transfer effects. Accordingly, one could have supposed that the level of stability reached at the end of practice by the 45° and 135° patterns as A tasks could explain the proactive interference and facilitation effects. However, in the present experiments, 45° and 135° practiced as A tasks exhibited similar levels of stability (and accuracy) at the end of practice. Hence, transfer effects reported here cannot be attributed to the level of stability reached at the end of practice of the prior A task.

Our findings suggest, instead, that transfer effects can be attributed to the stabilization of the A pattern during practice, a process depending on the initial competition arising within the memory repertoire. In fact, the actually stabilized A pattern determines the distance with the subsequently required B pattern. In turn, distance determines the level of competition, thereby predicting whether facilitation or interference effect occurs. If the distance between A and B pattern in the experimental group is larger than the distance between B and the nearest pre-existing pattern in the control group, competition between A and B is larger than competition between B and the nearest pre-existing pattern, and interference occurs. In the converse case, facilitation occurs. If distance is equal, competition is equal, and no transfer effect appears. This scenario is in accordance with a previous study by [START_REF] Hurley | The influence of augmented feedback and prior learning on the acquisition of a new bimanual coordintaion pattern[END_REF]. These authors required a group of participants to first practice a 90° pattern and then a 135° pattern, while a second group of participants practiced 135° only. Their results showed that, for the first group, the 135° pattern competed with the 90° pattern just-stabilized by learning whereas, in the second group, the 135° pattern competed with the pre-existing 180° pattern. Altogether, these findings and ours indicate that, when two patterns are required consecutively, the first-stabilized pattern (A task) enters in competition with the subsequent pattern (B task), hence overshadowing the competition with spontaneously stable patterns (0° and 180° in a bistable repertoire, 0°, 90° and 180° in a tristable one).

To sum up, the present study suggests that the transfer effects depend on the alteration induced by the A task in the initial memory repertoire. What really matters is not only whether the A pattern is retained or forgotten, but also, and mainly, which pattern has been actually stabilized and recalled after practice. The stabilized pattern(s) depend(s) on competition between the initially stable pattern and the to-be-learned pattern, and the competition is modulated by the generic rule of distance among these patterns. Both proactive interference and facilitation effects can be interpreted as two distinct outcomes emerging from the background of the memory repertoire in virtue of the rule of distance. Thus, distance and competition between patterns in the memory repertoire may be critical, if tentative, concepts to provide an integrated view of proactive interference and facilitation effects. and as B task (right part of middle panel). In the top panel, Initial scan represents the mean performance of both groups given that no significant difference was found between the two

  Section 1. Practiced Pattern effects: comparison of 45° and 135° as Aa tasks Practice Main effects of Practice (F(2,280) = 1.693; p<.04) and Practiced Pattern (F(1,14) = 4.956; p<.05) and a significant interaction Practice × Practiced Pattern (F(2,280) = 2.409; p<.001) were revealed on CE of the 45° and 135° patterns. These results indicate that (1) whatever the pattern (45° or 135°), the RP evolved toward the required value with practice (mean CE = 0° at criterion); (2) 45° was globally overestimated, whereas 135° was globally underestimated (mean CE = +3° and -6°, respectively) and (3) the 45° and 135° patterns followed different evolutions with practice. Deviation contrasts revealed that, before evolving toward the required value, the 45° RP was overestimated in the first trials (F(1,7) = 14.035; p<.01; trial 1: mean CE = +52°), whereas the 135° RP was first overestimated (F(1,7) = 13.287; p<.01; 6 th trial: mean CE = +25°) and then under-estimated (F(1,7) = 5.691; p<.05; 11 th trial: mean CE = -22°).

Section 1 .

 1 Practiced Pattern effects: comparison of 45° and 135° as Aa tasks Practice A main effect of Practiced Pattern and a significant interaction Practiced Pattern × Practice were found on CE (F(1,10) = 11.33 ; p<.01 and F(2,200) = 1.784 ; p<.03, respectively). A main effect of Practice was revealed on AE (F(20,280) = 3.085 ; p<.001) and a main effect of Practiced Pattern on SD (F(1,10) = 13.581; p<.005) (see Fig. 5). A main effect of Practiced Pattern was found on N (F(1,10) = 8.846 ; p<.02). No significant effect of Practiced Pattern was revealed on AE and SD at the end of practice.

  Figures

  Figure 3

  Figure 4

Figure 1 .

 1 Figure 6

Figure 2 .

 2 Figure 2. Initial scan (left panel), Learning and Recall (middle panel) and Post-practice scan

Figure 3 .

 3 Figure 3. Initial scan (left panel), Learning and Recall (middle panel) and Post-practice scan

Figure 4 .

 4 Figure 4. Initial scan (left Panel), Learning and Recall (middle panel) and Post-practice scan

Without this participant, the main effect of Practiced Pattern as well as the Practiced Pattern × Recall interaction became significant on CE (F(1,9) = 17.447 ; p<.002 and F(1,9) = 8.358 ; p<.02 respectively). This result suggests that the performance of all but one participant was biased toward the 90° RP at recall. This interindividual variability comfort the previous experiment of[START_REF] Kostrubiec | How a new behavioral pattern is stabilized with learning determines its persistence and flexibility in memory[END_REF] which have already emphasized the importance to take into account individual data in order to understand memory bias.

 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0 2 1 2 2 2


S Viveiros

I Pournaras

L Couto

Amaral

S S Costa

E Ntokou

A Martins

M Viveiros

S Pournaras

I Couto

L Amaral

Identification of the plasmid-encoded qacA efflux pump gene in meticillin-resistant Staphylococcus aureus (MRSA) strain HPV107; a representative of the MRSA Iberian clone

Identification of the plasmid-encoded efflux pump gene

in meticillin-resistant (MRSA) strain HPV107; a representative of the MRSA Iberian clone

Introduction

Treatment, prevention and control of infectious diseases have undergone remarkable changes during the last century. Use of antibiotics to eliminate pathogenic bacteria, combined with the application of chemical compounds for disinfectant and/or antiseptic purposes (also known as biocides), has allowed the management of several pathogenic bacteria known to cause life-threatening conditions [START_REF] Gilbert | Potential impact of increased use of biocides in consumer products on prevalence of antibiotic resistance[END_REF]. However, the selective pressure derived from increasing usage of these antimicrobial compounds combined with the adaptability of bacteria has resulted in the emergence of bacterial strains resistant to representatives of one or more antibiotic classes as well as to biocides [START_REF] Gilbert | Potential impact of increased use of biocides in consumer products on prevalence of antibiotic resistance[END_REF]. Bacteria that possess a multidrug-resistant (MDR) phenotype are of foremost concern in healthcare settings inasmuch as they are problematic for therapy.

Amongst the known mechanisms of resistance to antibiotics, the activity of multidrug efflux pumps is a major contributor to the development of MDR phenotypes [START_REF] Poole | Efflux pumps as antimicrobial resistance mechanisms[END_REF]. In the pathogenic bacterium Staphylococcus aureus, several multidrug efflux pumps have been described and were found to be encoded by chromosomal genes (norA, norB, norC, mepA, mdeA, sepA and sdrM) as well as genes located on plasmids (e.g. qacA/B, smr, qacG, qacJ and qacH) [START_REF] Poole | Efflux pumps as antimicrobial resistance mechanisms[END_REF].

Usually, chromosomally encoded multidrug efflux pumps can confer reduced susceptibility to either antibiotics or biocides, whilst the plasmid-encoded pumps can only confer reduced susceptibility to biocides [START_REF] Poole | Efflux pumps as antimicrobial resistance mechanisms[END_REF]. Two such examples are the multidrug efflux pump QacA and the closely related QacB that extrude from S. aureus a wide variety of monovalent (both QacA and QacB) and divalent (only QacA) cationic compounds using the proton-motive force [START_REF] Brown | Staphylococcal multidrug efflux protein QacA[END_REF]. This study reports the finding of the multidrug efflux pump QacA in a clinical meticillin-resistant S. aureus (MRSA) strain HPV107, representative of the socalled MRSA Iberian clone that is currently disseminated throughout several European countries and the USA [START_REF] Deurenberg | The evolution of Staphylococcus aureus[END_REF].

Materials and methods

Strains

The strain HPV107, representative of the MRSA Iberian clone, was isolated at a Portuguese hospital in 1992 and is characterised by resistance to several classes of antibiotics, particularly -lactams, aminoglycosides, fluoroquinolones, macrolides, rifampicin and tetracycline [START_REF] Sanches | Evidence for the geographic spread of a methicillin-resistant Staphylococcus aureus clone between Portugal and Spain[END_REF][START_REF] Oliveira | The evolution of pandemic clones of methicillin-resistant Staphylococcus aureus: identification of two ancestral genetic backgrounds and the associated mec elements[END_REF][START_REF] Oliveira | Multiplex PCR strategy for rapid identification of structural types and variants of the mec element in methicillin-resistant Staphylococcus aureus[END_REF]. Strain HPV107 cured corresponds to MRSA strain HPV107 cured of plasmids. Staphylococcus aureus ATCC 25923, a clinical isolate collected in Seattle, WA, in 1945 and fully susceptible to antibiotics, was used as a reference strain. Strains were grown in tryptic soy broth or tryptic soy agar (TSA) (Oxoid Ltd., Basingstoke, UK) at 37 C. For minimum inhibitory concentration (MIC) determination, cultures were grown in Mueller-Hinton broth (Oxoid Ltd.) at 37 C.

Compounds

All antibiotics, biocides, dyes and efflux pump inhibitors (EPIs) were purchased from Sigma-Aldrich (St Louis, MO) or Fluka Chemie GmbH (Buchs, Switzerland).

Minimum inhibitory concentration determination

MICs of the study strains towards several antibiotics (oxacillin, ciprofloxacin, norfloxacin, levofloxacin, sparfloxacin, gentamicin, trimethoprim, kanamycin, chloramphenicol, tetracycline and erythromycin), biocides [tetraphenylphosphonium bromide, dequalinium chloride, benzalkonium chloride, hexadecyltrimethylammonium bromide, cetrimide, chlorhexidine (CHX) and pentamidine isethionate salt] and dyes [ethidium bromide (EtBr), berberine, acriflavine and crystal violet] were determined by the two-fold microdilution broth method according to Clinical and Laboratory Standards Institute (CLSI) guidelines [START_REF]Performance standards for antimicrobial susceptibility testing. Seventeenth informational supplement[END_REF], in the absence and presence of EPIs, namely thioridazine, chlorpromazine (CPZ), verapamil and reserpine.

Plasmid DNA isolation and plasmid curing

Plasmid DNA was isolated using a QIAprep Spin Miniprep Kit (QIAGEN, Hilden, Germany), with an additional incubation step with lysostaphin (35 mg/L) (Sigma-Aldrich) at 37 C for 90 min prior to cell lysis. HPV107 was cured of plasmids by successive passage in medium containing increasing subinhibitory concentrations of CPZ (from 2 mg/L to 22 mg/L) [START_REF] Molnár | Infectious plasmid resistance and efflux pump mediated resistance[END_REF] or novobiocin (NOV) (2 g/L to 125 g/L) [START_REF] Heir | The Staphylococcus qacH gene product: a new member of the SMR family encoding multidrug resistance[END_REF]. The efflux capacity of each subculture was evaluated by plating overnight cultures on TSA media containing the broad-range efflux pump substrate EtBr. Cells with lower efflux activity will not be able to extrude the fluorochrome EtBr substrate, which accumulates within the cell producing pink fluorescence under ultraviolet light. On the other hand, cells with higher efflux activity will be able to extrude EtBr, requiring higher EtBr concentrations to fluoresce. Thus, by inoculating each of the subcultures obtained on TSA plates containing 2.5 mg/L EtBr, it was possible to select non-fluorescent and fluorescent colonies (potentially lacking the plasmid) [START_REF] Heir | The Staphylococcus qacH gene product: a new member of the SMR family encoding multidrug resistance[END_REF].

Screening of efflux pump genes by polymerase chain reaction (PCR) and enzymatic restriction analysis

Plasmid-mediated efflux pump genes were screened by PCR using specific primers for qacA/B and smr [START_REF] Couto | Efflux-mediated response of Staphylococcus aureus exposed to ethidium bromide[END_REF]. The reaction mixture (50 L) contained 2.5 U of Taq polymerase (Fermentas Inc., Ontario, Canada), 1 Taq buffer (Fermentas Inc.), 25 pmol of each primer, 0.2 mM of dNTP and 1.75 mM of MgCl 2 . PCR reactions were conducted in a Mastercycler ® Personal 5332 thermocycler (Eppendorf AG, Hamburg, Germany) under the following conditions. DNA was denatured at 95 C for 1 min, followed by 30 cycles of denaturation at 95 C for 1 min, annealing at 40 C (qacA/B) or 48 C (smr) for 1 min and extension at 72 C for 1 min, followed by a final extension at 72 C for 5 min. Amplification products were visualised by 1% agarose gel electrophoresis. The resulting qacA/B fragment was purified and sequenced on both strands with the same set of primers. Sequence alignment and analysis were conducted with the aid of programs ClustalW and BioEdit v.7.0.8.0, respectively.

A c c e p t e d M a n u s c r i p t

3-8

The qacA/B internal fragment was digested with the restriction enzyme AluI (Fermentas Inc.) at 37 C for 2 h. Restriction products were analysed by 3.5% Metaphor agarose gel (Lonza Rockland Inc., Rockland, ME) electrophoresis.

Macrorestriction analysis

HPV107 and HPV107 cured were typed by pulsed-field gel electrophoresis (PFGE) using well established protocols. Briefly, agarose disks containing intact chromosomal DNA were prepared as previously described [START_REF] Couto | Efflux-mediated response of Staphylococcus aureus exposed to ethidium bromide[END_REF] and were restricted with SmaI according to the manufacturer's recommendations (New England Biolabs, Ipswich, MA). Restriction fragments were then resolved by PFGE, which was carried out in a contour-clamped homogeneous electric field (CHEF) apparatus (CHEF-DRIII; Bio-Rad, Hercules, CA) as previously described [START_REF]Performance standards for antimicrobial susceptibility testing. Seventeenth informational supplement[END_REF].

Semi-automated fluorometric method

Efflux activity of the study strains was evaluated using a previously described semi-automated fluorometric method [START_REF] Couto | Efflux-mediated response of Staphylococcus aureus exposed to ethidium bromide[END_REF][START_REF] Viveiros | Evaluation of efflux activity of bacteria by a semi-automated fluorometric system[END_REF]. This method, with the aid of the Rotor-Gene 3000 TM thermocycler and real-time analysis software (Corbett Research, Sydney, Australia), allows real-time detection of accumulation and efflux of the fluorescent efflux pump substrate EtBr.

Results and discussion

As expected, the susceptibility profiles of strain HPV107 and the reference strain S. aureus ATCC 25923 towards several antibiotics, biocides and dyes showed a fully resistant profile of HPV107 to most of the antibiotics tested together with reduced susceptibility to all biocides compared with the reference strain (Table 1). However, in the presence of EPIs, in particular thioridazine, a general decrease in the MICs for biocides and dyes was observed (Table 1). This observation suggests that HPV107 has an efflux activity that correlates with decreased susceptibility to biocides and dyes. Chromosomally encoded efflux pumps usually possess a range of substrates that include not only biocides and dyes, but also antibiotics such as fluoroquinolones. MICs for HPV107 of fluoroquinolones also decreased with some EPIs, although to a lesser extent than that observed for the biocides and dyes. Thus, the phenotype presented by HPV107 towards biocides and dyes probably results mainly from a multidrug efflux pump gene carried on a plasmid. To confirm this hypothesis, the presence of the plasmid-located efflux pump genes qacA/B and smr was screened by PCR in the S. aureus study strains. An internal fragment of the qacA/B gene was amplified by PCR (data not shown). To distinguish between the genes qacA and qacB, which only differ by seven nucleotides, the qacA/B internal fragment was digested with the restriction enzyme AluI, which recognises an additional restriction site within qacB [START_REF] Sekiguchi | Detection of the antiseptic-and disinfectant-resistance genes qacA, qacB, and qacC in methicillin-resistant Staphylococcus aureus isolated in a Tokyo hospital[END_REF]. Thus, when the qacA amplification product is restricted with AluI, several fragments are produced, the larger of which has 200 bp, whilst those obtained from the qacB gene are of a significantly smaller size. Since a 200-bp AluI fragment was detected (Fig. 1-A), we could infer that the plasmid present in HPV107 carries the qacA gene and not qacB. Sequencing of the qacA/B fragment corroborated the identification of the qacA gene. These conclusions were supported by the decrease in MIC 1).

To confirm the location of the qacA gene in HPV107, the strain was cured of plasmids by exposure to CPZ or NOV. The resulting cultures were then screened for the presence or absence of plasmids using EtBr as an indicator of efflux activity. The resulting fluorescent colonies presented morphology similar to the non-fluorescent colonies, albeit slightly smaller. Plasmid DNA extraction was carried out both from fluorescent and non-fluorescent colonies. No plasmids could be obtained from the fluorescent colonies in contrast to the nonfluorescent colonies that were found to carry a single 32-kb plasmid. These results indicate that the qacA gene was carried on a 32-kb plasmid in strain HPV107 and were supported by PCR amplification for the qacA/B internal fragment, which was positive for the non-fluorescent colonies and negative for the fluorescent colonies (Fig. 1-B). One of the fluorescent colonies obtained after successive passage with CPZ was selected for further testing and was designated HPV107 cured . PFGE patterns showed that no contamination occurred during the plasmid curing procedure (data not shown). A single difference was detected among the PFGE patterns of HPV107 and HPV107 cured , which corresponded to a fragment of ca. 30 kb, which was detectable for HPV107 only and may correspond to the qacA-carrying plasmid.

MICs of HPV107 cured for the antibiotics, biocides and dyes previously tested were determined and it was found that HPV107 cured maintained the antibioticresistant profile, but it became more susceptible to the biocides and dyes To confirm further that the activity of the QacA pump accounted for the reduced susceptibility to a diverse panel of biocides and dyes in HPV107, the efflux activities of HPV107, HPV107 cured and S. aureus ATCC 25923 were characterised by a semi-automated fluorometric method as previously described [START_REF] Couto | Efflux-mediated response of Staphylococcus aureus exposed to ethidium bromide[END_REF][START_REF] Viveiros | Evaluation of efflux activity of bacteria by a semi-automated fluorometric system[END_REF]. Data obtained showed that HPV107 had a higher efflux activity in comparison with that presented by S. aureus ATCC 25923 (Fig. 2-B) and, consequently, a lower retention of EtBr (Fig. 2-A). Moreover, strain HPV107 cured , without the qacA gene, showed a behaviour similar to the fully antibiotic-susceptible strain S. aureus ATCC 25923, with residual efflux activity and concomitantly a higher accumulation of EtBr (Fig. 2). The residual efflux activity detected in HPV107 cured could be the result of the basal activity of chromosomally encoded efflux pumps. These results confirmed that the activity of the efflux pump QacA was the main contributor to reduced susceptibility of the HPV107 to biocides and dyes.

Biocides are heavily used in healthcare settings for the management of nosocomial infections as well as in domestic and food industry settings [START_REF] Gilbert | Potential impact of increased use of biocides in consumer products on prevalence of antibiotic resistance[END_REF].

Consequently, nosocomial bacteria, and increasingly community bacteria, are constantly being exposed to these daily-use compounds, raising concerns about the role of biocides as a selective pressure for the appearance and maintenance of antibiotic-resistant bacteria. Inasmuch as bacteria with reduced susceptibility to biocides could be more prone to survive in such environments, A c c e p t e d M a n u s c r i p t 3-12 they can also create the opportunity for bacteria to develop an antibiotic resistant-phenotype, or even a MDR phenotype. Reduced susceptibility is mainly conferred by efflux pumps that have the capacity to expel from the cell interior a wide range of chemically dissimilar compounds, including one or several classes of antibiotics, and may render the cell with a MDR phenotype [START_REF] Poole | Efflux pumps as antimicrobial resistance mechanisms[END_REF].

In the particular case of S. aureus, prevention of nosocomial infections is mainly conducted by the use of antiseptics and disinfectants [START_REF] Smith | The association between biocide tolerance and the presence or absence of qac genes among hospitalacquired and community-acquired MRSA isolates[END_REF]. Hence, the emergence of biocide-resistant, and possibly antibiotic-resistant, bacteria can be of great importance. One such example of possible cross-resistance is the existence of plasmids that can harbour efflux pump genes that confer resistance to biocides as well as resistance determinants to antibiotics and heavy metals [START_REF] Poole | Efflux pumps as antimicrobial resistance mechanisms[END_REF]. Previous studies have shown that the plasmid-encoded qacA/B gene is the main efflux pump gene responsible for reduced susceptibility to biocides in S.

aureus isolates and that it is widespread in Europe [START_REF] Mayer | Distribution of the antiseptic resistance genes qacA, qacB, and qacC in 497 methicillinresistant and -susceptible European isolates of Staphylococcus aureus[END_REF]. Although some data do not support a link between increased biocide resistance and antibiotic resistance, other studies highlight the potential biocide/antibiotic crossresistance in S. aureus, particularly among hospital-acquired MRSA strains, and potentially community-acquired MRSA strains [START_REF] Smith | The association between biocide tolerance and the presence or absence of qac genes among hospitalacquired and community-acquired MRSA isolates[END_REF], owing to the common linkage of qacA/B and smr genes with -lactamase transposons as well as to resistance determinants to gentamicin and trimethoprim, among others [16]. This was not the case for MRSA HPV107, since both HPV107 and HPV107 cured showed identical levels of resistance to gentamicin, kanamycin and A c c e p t e d M a n u s c r i p t 3-13 trimethoprim (Table 1), thus excluding the presence of transposons on the plasmid carrying the qacA determinant.

However, conjugation of a highly successful genetic background, such as that of strain HPV107, a representative of the multiresistant international MRSA Iberian clone, with a plasmid carrying the structural gene for an efflux pump that confers resistance to antiseptic and disinfectant compounds usually employed in healthcare settings suggests that S. aureus strains carrying this determinant may be more prone to survive in such environments, thereby increasing their capacity to disseminate widely in a hospital setting. Fluorescence is an indicator of efflux capacity towards the broad-range efflux pump substrate ethidium bromide (EtBr). Cells with lower efflux activity will not be able to extrude the fluorochrome EtBr substrate, which accumulates within the cell producing pink fluorescence under ultraviolet light. On the other hand, cells with higher efflux activity will be able to extrude EtBr and will be nonfluorescent. Biocides and dyes

Monovalent

Edited Table 1

  author. Tel.: +351 21 365 2652; fax: +351 21 363 2105.E-mail address: lamaral@ihmt.unl.pt (L. Amaral).

  Staphylococcus aureus (MRSA) is a major nosocomial bacterium for which prevention and control measures consist mainly of the application of biocides with antiseptic and disinfectant activity. In this study, we demonstrated the presence of the plasmid-located efflux pump gene qacA in MRSA strain HPV107, a clinical isolate representative of the MRSA Iberian clone. The existence of efflux activity in strain HPV107 due to the QacA pump was also established and this QacA efflux activity was linked with a phenotype of reduced susceptibility towards several biocide compounds. No association could be made with antibiotic resistance. This work emphasises the potential of QacA pump activity in the maintenance and dissemination of important MRSA strains in the hospital setting and, increasingly, in the community.

  divalent cationic biocides in the presence of EPIs, which are substrates for QacA only (Table

  the exception of CHX (Table1). Analysis of other fluorescent and non-fluorescent colonies revealed similar phenotypes.

  Sidhu MS, Heir E, Leegaard T, Wiger K, Holck A. Frequency of disinfectant resistance genes and genetic linkage with -lactamase transposon Tn552 among clinical staphylococci. Antimicrob Agents Chemother 2002;46:2797-803.

Fig. 1 .

 1 Fig. 1. (A) Restriction products of the qacA/B internal fragment digested with

Fig. 2 .

 2 Fig. 2. Analysis of ethidium bromide (EtBr) accumulation and efflux by a semi-

A c c e p t e d M a n u s c r i p t 1 1

 11 TableCharacterisation of susceptibility profiles towards antibiotics, biocides and dyes, in the absence and presence of 0.5 MIC of efflux pump inhibitors (EPIs), for Staphylococcus aureus strain ATCC 25923, meticillin-resistant S. aureus (MRSA) strain HPV107 and its plasmid-cured derivative (HPV107 cured )

Acknowledgments

The authors would like to acknowledge Prof. Hermínia de Lencastre [Laboratório de Genética Molecular, Instituto de Technologia Química e Biológica, Universidade Nova de Lisboa (ITQB, UNL), Oeiras, Portugal] for providing strain HPV107 as well as Prof. Ilda Sanches (FCT, UNL) for access to PFGE facilities.

Funding SSC was supported by grant SFRH/BD/44214/2008 from Fundação para a Ciência e a Tecnologia (FCT, Portugal); EN was supported by an STSM grant from COST ACTION BM0701 (ATENS).

Competing interests

None declared.