domain: sde sdv chim

text:
Marine Uguen
email: marine.uguen@univ-lille.fr

Katy R Nicastro

Gerardo I Zardi

Sylvie M Gaudron

Nicolas Spilmont

Fleurine Akoueson

Guillaume Duflos

Laurent Seuront

Microplastic leachates disrupt the chemotactic and chemokinetic behaviours of an ecosystem engineer (Mytilus edulis)

Keywords: Chemical stimulus, Chemodetection, Predators, Blue mussel, Cues, Plastics, Intertidal species Graphical

Introduction

Chemical communication is a key process in ecology that has been extensively studied over a wide range of organisms, both terrestrial and marine [START_REF] Zimmer | Chemical signaling processes in the marine environment[END_REF]McClintock & Baker, 2001;[START_REF] Dicke | Chemical Ecology. From Gene to Ecosystems[END_REF][START_REF] Paul | Marine chemical ecology[END_REF][START_REF] Ng | Snails and their trails: the multiple functions of trail-following in gastropods[END_REF][START_REF] Tabata | Chemical Ecology of Insects[END_REF]. Specifically, chemical signals play a key role in intra-and inter-specific interactions (e.g. predation, reproduction, competition, aggregation) and others processes such as habitat selection, eventually affecting individual fitness, and community and ecosystems dynamics at larger scales [START_REF] Murlis | Odor plumes and how insects use them[END_REF]Bradbury & Vahrencamp, 1998;[START_REF] Dicke | Chemical Ecology. From Gene to Ecosystems[END_REF][START_REF] Bornancin | Chemical mediation as a structuring element in marine gastropod predator-prey interactions[END_REF]Zimmer & Butman, 2020). Many marine species rely on chemicals to get information about their environment [START_REF] Zimmer | Chemical signaling processes in the marine environment[END_REF]. For instance, the majority of molluscs possess sensory organs, such as the osphradium that contains chemoreceptors which allows the detection of e.g. food, conspecifics and predators [START_REF] Lucas | The distribution of the branchial nerve in Mytilus edulis and its relation to the problem of nervous control of ciliary activity[END_REF][START_REF] Morton | Habit and orientation in the small commensal bivalve mollusc, Montacuta ferruginosa[END_REF][START_REF] Lindberg | What is the molluscan osphradium? A reconsideration of homology[END_REF].

Intertidal mussels are considered as key ecosystem engineers on both soft and hard substrates [START_REF] Reise | Sediment mediated species interactions in coastal waters[END_REF][START_REF] Borthagaray | Mussels as ecosystem engineers: their contribution to species richness in a rocky littoral community[END_REF]. Noticeably, these organisms are motile; they use their foot both to move and attach themselves to their substrate through the production of byssal threads, extracellular collagenous structure [START_REF] Schneider | Implications of movement behavior on mussel dislodgement: exogenous selection in a Mytilus spp. hybrid zone[END_REF][START_REF] Waite | Mussel adhesion: finding the tricks worth mimicking[END_REF]. Both their motility and byssal production allow them to spatially self-organise into dense mono-and multi-layered beds, which decrease the vulnerability to predation, desiccation, heat, and wave dislodgement, while increasing fertilization success and survival [START_REF] Nicastro | Love thy neighbour: group properties of gaping behaviour in mussel aggregations[END_REF][START_REF] Iwasaki | Behavior and taxis of young and adult Limnoperna fortunei[END_REF][START_REF] Zardi | Density-dependent and species-specific effects on self-organization modulate the resistance of mussel bed ecosystems to hydrodynamic stress[END_REF]. Through the formation of beds, intertidal mussels locally dominate rocky shores and operate a biogenic transformation of the habitat [START_REF] Paine | Intertidal landscapes: disturbance and the dynamics of pattern[END_REF][START_REF] Menge | Community regulation: variation in disturbance, competition, and predation in relation to environmental stress and recruitment[END_REF][START_REF] Reise | Sediment mediated species interactions in coastal waters[END_REF]. These relatively stable and resilient structures enhance local biodiversity by facilitating the establishment and maintenance of a range of different species [START_REF] Reise | Sediment mediated species interactions in coastal waters[END_REF][START_REF] Palomo | Separating the effects of physical and biological aspects of mussel beds on their associated assemblages[END_REF][START_REF] Borthagaray | Mussels as ecosystem engineers: their contribution to species richness in a rocky littoral community[END_REF][START_REF] Arribas | Intertidal mussels as ecosystem engineers: their associated invertebrate biodiversity under contrasting wave exposures[END_REF][START_REF] Spilmont | First records of Ptilohyale littoralis (Amphipoda: Hyalidae) and Boccardia proboscidea (Polychaeta: Spionidae) from the coast of the English Channel: habitat use and coexistence with other species[END_REF]. Beyond their ecological role, mussels also play an important economic and heritage value through professional and recreational fishing, e.g. global aquaculture production for Mytilus edulis and M. galloprovincialis was 287,957 tons in 2016 (FAO Fishstat) with an estimated net worth ranging between US$2.5 10 6 and US$100 10 6 in the United States only [START_REF] Zippay | Effects of temperature change on mussel, Mytilus[END_REF].

Previous studies have shown that cues from damaged conspecifics and predators of intertidal mussels can significantly affect their movement and aggregation rate [START_REF] Côté | Predator-induced clumping behaviour in mussels (Mytilus edulis Linnaeus)[END_REF][START_REF] Nicastro | Behavioural response of invasive Mytilus galloprovincialis and indigenous Perna perna mussels exposed to risk of predation[END_REF][START_REF] Kong | Seawater acidification and temperature modulate anti-predator defenses in two coexisting Mytilus species[END_REF], though exceptions exist [START_REF] Commito | Separation anxiety: mussels self-organize into similar power-law clusters regardless of predation threat cues[END_REF][START_REF] Kong | Seawater acidification and temperature modulate anti-predator defenses in two coexisting Mytilus species[END_REF][START_REF] Manríquez | The combined effects of climate change stressors and predatory cues on a mussel species[END_REF]. An exposure to these cues also triggers morphological and physiological changes such as thicker shell, production and quality of byssal threads [START_REF] Leonard | Crab predation, waterborne cues, and inducible defenses in the blue mussel, Mytilus edulis[END_REF][START_REF] Kong | Seawater acidification and temperature modulate anti-predator defenses in two coexisting Mytilus species[END_REF]. Intertidal mussels can differentiate chemical signals from predators and behave accordingly to their specific prey handling techniques, e.g. the mussel Mytilus edulis increases byssus production when exposed to crab (Cancer irroratus) cue but not when exposed to starfish cue (Asterias rubens; [START_REF] Garner | Effects of injured conspecifics and predators on byssogenesis, attachment strength and movement in the blue mussel, Mytilus edulis[END_REF]. Though various mussel species share the sensory capacity to perceive, discriminate and adapt to different environmental chemical signals, the presence and intensity of behavioural responses to chemical stimuli, in particular in Mytilidae, is fundamentally cue-dependent [START_REF] Nicastro | Behavioural response of invasive Mytilus galloprovincialis and indigenous Perna perna mussels exposed to risk of predation[END_REF][START_REF] Garner | Effects of injured conspecifics and predators on byssogenesis, attachment strength and movement in the blue mussel, Mytilus edulis[END_REF][START_REF] Kong | Seawater acidification and temperature modulate anti-predator defenses in two coexisting Mytilus species[END_REF][START_REF] Manríquez | The combined effects of climate change stressors and predatory cues on a mussel species[END_REF]. Chemotaxis (i.e. a directional change in movement in response to a chemical stimulus) and chemokinesis (i.e. a non-directional change in movement properties such as speed, distance travelled or turning frequency in response to a chemical stimulus) are key behavioural processes involved in the response to chemical cues [START_REF] Bell | Chemo-orientation[END_REF][START_REF] Wilkinson | Random locomotion; chemotaxis and chemokinesis. A guide to terms defining cell locomotion[END_REF]. Only a few studies have addressed chemotaxis in bivalves and interactions with conspecifics, i.e., De [START_REF] De Vooys | Effect of a tripeptide on the aggregational behaviour of the blue mussel Mytilus edulis[END_REF] with M. edulis and [START_REF] Huang | Movement and aggregation in the fluted giant clam (Tridacna squamosa L.)[END_REF] with Tridacna squamosa, and between a bivalve (Montacuta ferruginosa) and its commensal species (Echinocardium cordatum; [START_REF] Morton | Habit and orientation in the small commensal bivalve mollusc, Montacuta ferruginosa[END_REF]. Specifically, the chemical communication on which an important part of behaviour is based, such as mate and food searching, as well as danger detection, can be altered by a range of anthropogenic chemicals [START_REF] Fleeger | Indirect effects of contaminants in aquatic ecosystems[END_REF][START_REF] Lürling | Info-disruption: pollution and the transfer of chemical information between organisms[END_REF][START_REF] Seuront | Zooplankton avoidance behaviour as a response to point sources of hydrocarbon-contaminated water[END_REF][START_REF] Seuront | Hydrocarbon contamination decreases mating success in a marine planktonic copepod[END_REF][START_REF] Seuront | Microplastic leachates impair behavioural vigilance and predator avoidance in a temperate intertidal gastropod[END_REF]. This disrupting effect of anthropogenic chemicals on intra and inter-species chemical communication has, to date, received considerably less attention. This issue is, however, particularly relevant given the growing awareness of the ubiquity and toxicity of plastic compounds and their leachates in marine systems [START_REF] Gall | The impact of debris on marine life[END_REF][START_REF] Jamieson | Bioaccumulation of persistent organic pollutants in the deepest ocean fauna[END_REF][START_REF] Gunaalan | The hidden threat of plastic leachates: a critical review on their impacts on aquatic organisms[END_REF]. Plastic pollution is one of the main challenges of the 21 st century through their deleterious physical and chemical effects on aquatic life [START_REF] Derraik | The pollution of the marine environment by plastic debris: a review[END_REF][START_REF] Gall | The impact of debris on marine life[END_REF][START_REF] Anderson | Microplastics in aquatic environments: implications for Canadian ecosystems[END_REF][START_REF] Rochman | The ecological impacts of marine debris: unraveling the demonstrated evidence from what is perceived[END_REF][START_REF] Sussarellu | Oyster reproduction is affected by exposure to polystyrene microplastics[END_REF][START_REF] Auta | Distribution and importance of microplastics in the marine environment: a review of the sources, fate, effects, and potential solutions[END_REF], leading to the death of millions of aquatic organisms annually (Ocean Conference United Nations, 2017). These effects can be due to either macroplastic (> 5 mm), microplastic (< 5 mm) and nanoplastic (< 100 nm) items either produced directly in the smaller form industrially or resulting from the breakdown of larger plastic items [START_REF] Cole | Microplastics as contaminants in the marine environment: a review[END_REF][START_REF] Paul-Pont | Constraints and priorities for conducting experimental exposures of marine organisms to microplastics[END_REF]. Plastic pollution has also a far more pernicious effect on marine life through the release of various molecules that are either absorbed (i.e. intrinsically bounded) to the polymer during the manufacturing process (e.g. light and heat stabilizers, antioxidants, nucleating and antistatic agents, flame retardants, plasticizers and colorants) or adsorbed at the surface of a polymer such as persistent organic pollutants, which include polycyclic aromatic hydrocarbons, polychlorinated biphenyls, polybrominated diphenyl ethers, pesticides or heavy metals (Delaeter et al., 2022). Recent studies have shown that direct exposure to either microplastics [START_REF] Crump | Microplastics disrupt hermit crab shell selection[END_REF] or microplastic leachates [START_REF] Seuront | Microplastic leachates impair behavioural vigilance and predator avoidance in a temperate intertidal gastropod[END_REF] can interfere with the cognitive system, sensory perception, and thus behaviour of marine invertebrates. For instance, the intertidal mussel M. edulis behaviourally responds to microplastic leachates through an increase in aggregation rate and frequency [START_REF] Seuront | Microplastic leachates induce species-specific trait strengthening in intertidal mussels[END_REF]. However, despite the ever-increasing concern about the ecological impact of plastic leachates [START_REF] Gunaalan | The hidden threat of plastic leachates: a critical review on their impacts on aquatic organisms[END_REF]Delaeter et al., 2022), it is still unknown whether plastic leachates interfere with conspecific or prey-predator chemical signals in bivalves in general, and in intertidal mussels in particular.

In this context, we aim to determine whether the chemotactic and chemokinetic responses in an ecologically and economically important intertidal mussel species are affected by microplastics leachates. To do so, we first assessed the presence of chemotactic and/or chemokinetic responses of M. edulis to cues from intact and injured conspecifics and the predatory cues from the invasive Asian shore crab Hemigrapsus sanguineus. This species is invasive along European coast and has been reported in other regions, it was first recorded in France in 1999 [START_REF] Breton | A new alien crab in Europe: Hemigrapsus sanguineus (Decapoda: Brachyura: Grapsidae)[END_REF] and became the dominant mussel predator of the rocky intertidal shores of the eastern English Channel [START_REF] Rolet | Etat de la colonisation des Décapodes invasifs du genre Hemigrapsus sur le littoral des Hauts-de-France en 2020[END_REF] less than two decades after its first report from our sampling area, i.e. 2006 [START_REF] Dauvin | Recent expansion of two invasive crabs species Hemigrapsus sanguineus (de Haan, 1835) and H. takanoi Asakura and Watanabe 2005 along the Opal Coast, France[END_REF]. Noticeably, given their short common evolutionary history of M. edulis and H. sanguineus, our behavioural approach provides a first step towards the assessment of M. edulis naivety status (i.e. the ability to recognize a new predator as a threat). We subsequently inferred how leachates from virgin polypropylene pellets may alter M. edulis chemotactic and chemokinetic responses. We further discuss how the observed behavioural changes are relevant for the ecology of the species in the context of increasing anthropogenic pressure on marine coastal ecosystems.

Material and Methods

Study organisms

Both individual Mytilus edulis (1.5-2.0 cm in shell length; Supplementary Materials, S1) and H. sanguineus (1.4-2.4 cm in length; Supplementary Materials, S1) were sampled in March 2021 at low tide on a rocky intertidal reef of the infra-littoral zone of the Fort de Croy (Wimereux, France; 50°45'52.3"N, 1°35'55.1"E) along the French coast of the English Channel.

Collected organisms were brought back to the laboratory within an hour. Prior to the experiments, mussels were acclimatised for 1 week under a natural 12:12 light:dark cycle in 85 L tanks filled with running aerated natural seawater (T = 10 °C, S = 33‰, pH = 8.08) directly pumped from the collection site, and no additional food was provided. H. sanguineus were sorted by sex, and kept in separate 85 L tanks in which crabs were acclimatised for 1 week and fed ad libitum daily with mussels previously crushed with a natural stone (to avoid the use of metal objects, hence to prevent metal ions from touching mussel tissue; [START_REF] Commito | Separation anxiety: mussels self-organize into similar power-law clusters regardless of predation threat cues[END_REF].

Crabs were kept in darkness during the acclimation to enhance food consumption [START_REF] Spilmont | Exogenous control of the feeding activity in the invasive Asian shore crab Hemigrapsus sanguineus (De Haan, 1835)[END_REF] and thus stimulate predator-induced alarm response in mussels as described here below.

Chemical cues

The effects of four natural chemical cues were assessed on the chemotactic and chemokinetic behaviour of M. edulis: (1) natural (i.e. control) seawater, (2) conspecific cues, (3) injured conspecific cues, and (4) fed crab cues. To assess the effect of microplastic leachates on the abovementioned natural cues, these treatments were subsequently mixed with microplastic (MP) leachate seawater. Note that the seawater used in all our experiments was consistently pumped directly from the collection site, hence could contain plastic leachates and cues naturally occurring at this site. As such, in the absence of additional experimentallygenerated cues, natural seawater was considered as cue less.

Conspecific cue seawater was prepared through the addition of 20 mussels which were placed for a 24 h period into 1 L of aerated seawater. Injured conspecific cue seawater was prepared through the addition of 20 injured mussels previously crushed with a stone, to 1 L of aerated seawater for a 24 h period. Crab cue seawater was prepared through the addition of 3 males and 3 females Hemigrapsus sanguineus (sex ratio of 1:1) into 1L of aerated seawater for a 24h period. For more information about exact sizes, see Supplementary Materials, S1.

For the mixed cues experiments, commercially available polypropylene pellets (typically 3.3-4.7 mm in diameter; Pemmiproducts, Aachen, Germany) were added to the previous treatment (i.e. natural seawater, conspecific, injured conspecific and crab cues). They were left 24 h in aerated tanks at a concentration of 12 g of pellets per litre (ca. 600 MPs per L, or equivalently 20mL of MPs per L; [START_REF] Seuront | Microplastic leachates impair behavioural vigilance and predator avoidance in a temperate intertidal gastropod[END_REF][START_REF] Seuront | Microplastic leachates induce species-specific trait strengthening in intertidal mussels[END_REF], in order to have four new solutions: seawater with leachates, conspecifics cues with leachates, injured conspecifics cues with leachates and crab cues with leachates. As a first approximation of the nature of the additive content of our polypropylene pellets, we ran a pyrolysis analysis coupled to a gas chromatography and a high-resolution spectrometer (see Supplementary Materials, S2), which led to the identification of 2 antioxidants (i.e. 4-tert-octylphenol, 4tOP, and Butyl hydroxytoluene, BHT) and 6 plasticizers (i.e. Diethyl phthalate, DEP, Diisobutyl phthalate, DIBP, Dibutyl phthalate, DBP, Bis(2-ethylhexyl)phthalate, DEHP, Di-n-octyl phthalate, DIOP, and Diisononyl phthalate, DINP).

Chemotactic and chemokinetic assay

Both acclimatation and experiments were consistently carried out under the same controlled conditions. Temperature was measured at the beginning and at the end of the experiment. Before the start of each experiment, byssal threads were carefully cut with scissors to separate the mussels from each other and from the substrate. Behavioural experiments were conducted in 22 cm diameter glass arenas with smooth, featureless surfaces (Fig. 1). To infer the presence of a chemotactic and/or chemokinetic response of the individual mussel to the different cues, their positions were recorded with respect to a point source. This point source was obtained by four equidistant holes (0.5 cm in diameter) drilled through the side of a clay pot (6.5 cm in diameter; Fig. 1) which had a top central hole. Arenas were filled with 600 mL unfiltered seawater saturated in oxygen. Five milliliters (5 mL) of either control or treatment seawater was injected in a clay pot through the top hole at a rate of 0.7 mL s -1 . Thus, the concentration of MP leachates, which is the same for all solutions containing plastic leachates, that flows out of the clay pot, was estimated at 1.66 mL L -1 (50 MPs per litre). Once fully mixed in the arena, this concentration reached 0.166 mL L -1 (5 MPs per litre), a concentration consistent with those found in coastal waters, i.e. 3.5 ± 2.0 MPs L -1 with concentrations between 1 and 6.4 MPs per wild M. edulis (United Kingdom; [START_REF] Li | Microplastics in mussels sampled from coastal waters and supermarkets in the United Kingdom[END_REF]. Immediately after the treatment or control injection, each specimen was placed individually at a distance of 4 cm from each side hole of the pot (n = 4 mussels; Fig. 1). Video recording started immediately after the injection of water cue in the clay pot, at a rate of 1 frame per minute for 1h30 using a GoPro camera (GoPro HERO7 Black, GoPro Inc., San Mateo, California, USA) placed 40 cm above the experimental container. The choice of using four mussels per arena was made to minimize aggregations between mussels, which decreases with the distance between the mussels [START_REF] Côté | Predator-induced clumping behaviour in mussels (Mytilus edulis Linnaeus)[END_REF][START_REF] Nicastro | Behavioural response of invasive Mytilus galloprovincialis and indigenous Perna perna mussels exposed to risk of predation[END_REF]. Each day, three treatments were run concurrently: seawater (control; n = 6 arenas) and two different treatments (n = 6 arenas for each treatment; Table 1; Fig. 2). Six additional arenas Point source Mussel Hole using seawater as control were also performed. Thus, the sample size for each cue was: seawater n = 30 arenas; seawater with leachates n = 12 arenas and n = 6 arenas all other cues tested with and without plastic leachates (see the experimental plan; Table 1; Fig. 2). An additional control was also performed. Each mussel was only used once and then discarded, no mussels showed injury or mortality signs during the experiment.

Behavioural analysis

Though we specifically chose to use of four mussels per arena to minimize the probability of occurrence of aggregation behaviour, overall four mussels ended-up as two aggregates of two mussels (i.e. physical contact between their shells) and two as almostaggregated (i.e. at a distance of less than 2 cm). These 6 mussels represent less than 2% (i.e.

6/312×100) of the total number of mussels used in the experiment, but could may have affected the behaviour of other mussels in the same arena [START_REF] Côté | Predator-induced clumping behaviour in mussels (Mytilus edulis Linnaeus)[END_REF][START_REF] Nicastro | Behavioural response of invasive Mytilus galloprovincialis and indigenous Perna perna mussels exposed to risk of predation[END_REF][START_REF] Commito | Recruitment facilitation and spatial pattern formation in soft-bottom mussel beds[END_REF]. Consequently, to avoid a potential bias related to the presence of aggregated conspecifics, the behavioural responses for the different endpoint were not studied at the level of the individual but at the level of the arena. Thus, for the different behavioural parameters studied, the data from the four mussels of each arena averaged and used as sample unit in the statistical analyses. Aggregated and almost-aggregated mussels have been included in the dataset.

First, motility was assessed by comparing the proportion of motile and non-motile mussels, to have an average percentage of mussel motility per arena. Chemokinesis was subsequently assessed through mean speed, gross distance (i.e. the total distance covered by the mussel between its initial and final position), net distance and confinement index (i.e. the ratio between net distance and gross distance) per arena. These parameters were measured only for mussels moving a net distance above 1 cm (i.e. approximately half mussel's shell length) using the TrackMate plugin of ImageJ [START_REF] Tinevez | TrackMate: An open and extensible platform for single-particle tracking[END_REF]. Note that the choice of a net distance greater than 1cm was specifically made because distances less than 1 cm were assigned by TrackMate to mussels oscillating around their center of mass without actually actively moving as their foot was never observed outside their valves (see Supplementary Material, S4).

Chemotaxis was estimated by recording the final motile mussel position with reference to the source point, in order to have an average percentage of mussels with a negative chemotaxis (repulsion from the source point) per arena.

Statistical analyses

To compare differences among all treatments and to take into account the potential confounding effect of running treatments on different days, the motility, speed, gross distance, net distance, confinement index and chemotaxis of mussels, observed in the control seawater, were compared between days with an ANOVA (for more details, see Supplementary Material, S5). As no significant differences (p > 0.05; see Supplementary Material, S5) were found among control replicates run in different days, behavioural data for each treatment from controls and treatments were pooled across days for further analyses.

Motility data were not normally distributed (Shapiro's test, p = 0.004), but showed a homogeneity of variance (Levene's test, p = 0.931). Parametric analysis was used based on the assumption that ANOVA is relatively robust to the effects of non-normality [START_REF] Zar | Biostatistical analysis[END_REF]; therefore, data were analysed using a 2-way ANOVA with treatment (control or microplastic leachates) and cues (seawater, conspecifics, injured conspecifics or crabs) as fixed factors.

Significant effects were examined using Tukey-HSD post-hoc test.

In order to measure the speed, gross distance, net distance, confinement index and chemotaxis, only mussels with a net distance greater than 1 cm (about half a body length) were considered, hence the sample size per treatment was: seawater n = 27, seawater with leachates n = 12 and n = 6 for the other treatments. The distribution of speed, gross distance, net distance and confinement index measurements were tested for normality (Shapiro's test, p = 0.00005, p = 0.00004, p = 0.070, p = 0.406, respectively) and homogeneity of variance (Levene's test, p = 0.141, p = 0.616, p = 0.582, p = 0.060, respectively). As all parameters showed a homogeneity of variance, but were not always normally distributed, parametric analyses were consistently used as described above. A 2-way ANOVA with treatment (control or microplastic leachates) and cues (seawater, conspecifics, injured conspecifics or crabs) as fixed factors was finally performed and significant effect were further examined using a Tukey-HSD post-hoc test.

To assess the chemotaxis, i.e. to compare the direction of motile mussels (with a net distance greater than 1cm), attraction (positive chemotaxis) or repulsion (negative chemotaxis)

to the source point, a 2-way ANOVA with treatment (control or microplastic leachates) and cues (seawater, conspecifics, injured conspecifics or crabs) as fixed factors was used, followed by a Tukey-HSD post-hoc test when significant differences were identified. Prior to this test, normality (p = 0.013) and homogeneity of variance (p = 0.109) were measured, as homogeneity of variance was confirmed, this test could be performed [START_REF] Zar | Biostatistical analysis[END_REF]. All statistical analyses were performed using the software R Core Team (2021).

Results

Chemokinesis

Motility

Polypropylene leachates inhibited significantly mussel motility for all cues tested (Treatment; p = 0.043; Fig. 3, Table 2). Specifically, a significantly higher percentage of mussels moved in the control treatment (no leachate) than when they were exposed to microplastic leachate (Tukey's test: p = 0.049).

Speed

There was a significant effect of Cue (Cue; p = 0.005; Fig. 4, Table 3). Specifically, regardless of the treatment, mussels always moved significantly slower when exposed to crab cues than when exposed to conspecifics cues or to control seawater (Tukey's test: p = 0.043 and p = 0.002 respectively). However, the speed of mussels exposed to conspecific cues, injured conspecific cues and control seawater did not significantly differ (Seawater; Tukey's test: p > 0.05). between treatments were identified by different letters and the number of arenas used was also indicated (n = ).

Gross Distance

The gross distance travelled by mussels varied significantly depending on the Cue (Cue; p = 0.0498; Fig. 5, Table 4). Specifically, regardless of the presence of leachates, the gross distance was always lower in mussels exposed to crab cues than when exposed to sea water (Tukey's test: p = 0.032 respectively). There were no significant differences for the mussel speed when exposed to conspecific cues, injured conspecific cues and the control (Seawater;

Tukey's test: p > 0.05).

Net Distance and Confinement index

There was a significant effect of the Treatment ´ Cue interaction (Treatment ´ Cue; p = 0.036; Fig. 6, Table 5). However, the Tukey's test revealed no significant differences (p > 0.05) for the net distance traveled by mussels between every cues and treatments, even if a marginal difference was found between conspecifics with and without microplastic leachates (p = 0.094).

The confinement index did not differ among factors nor treatments (Table 6).

Chemotaxis

There was a significant effect of the Treatment ´ Cue interaction on the mussel chemotaxis (Treatment ´ Cue; p = 0.038; Fig. 7, Table 7), as significantly more mussels showed a negative chemotaxis to crab cue without microplastic leachates than with microplastic leachates (Tukey's test: p = 0.047).

Discussion

Cue-specific chemotactic and chemokinetic responses in Mytilus edulis

Taken together, our results show that M. edulis exhibit both chemotactic and chemokinetic response to chemical cues, and that these responses are cue-dependent.

Lack of behavioural response towards conspecific cues

Mytilus edulis did not show any behavioural change in response to conspecific cues, in accordance with previous work on the effect of conspecific cues on M. edulis aggregation patterns [START_REF] Commito | Separation anxiety: mussels self-organize into similar power-law clusters regardless of predation threat cues[END_REF]. These observations are, however, in contrast with previous studies which showed that positive chemotaxis was the main driver of conspecific aggregation in bivalves [START_REF] Huang | Movement and aggregation in the fluted giant clam (Tridacna squamosa L.)[END_REF] and in particular in M. edulis [START_REF] De Vooys | Effect of a tripeptide on the aggregational behaviour of the blue mussel Mytilus edulis[END_REF]. These discrepancies may, however, be related to (i) potential behavioural divergence in M. edulis between those living on the bottom of a concrete ditch with running seawater or on beach groynes as seen in Netherlands [START_REF] De Vooys | Effect of a tripeptide on the aggregational behaviour of the blue mussel Mytilus edulis[END_REF] with those living on natural rocky shores habitats as seen in France (present work) and/or (ii) the presence of a seasonal component in the behavioural response of M. edulis to conspecific cues [START_REF] De Vooys | Effect of a tripeptide on the aggregational behaviour of the blue mussel Mytilus edulis[END_REF]. Though the identification of the mechanisms at the origin of the above-mentioned behavioural differences lies well beyond the scope of the present work, it warrants the need for further work.

Lack of behavioural response toward injured conspecifics cues

No behavioural changes were observed either in terms of chemotaxis or chemokinesis in response to injured conspecifics cues. These results are consistent with previous observations conducted on Mytilus edulis [START_REF] Commito | Separation anxiety: mussels self-organize into similar power-law clusters regardless of predation threat cues[END_REF]. They contrast, however, with the results of studies conducted on other mussel species, which may be indicative of a species-dependent behaviour. For instance, the motility of the zebra mussel (Dreissena polymorpha) decreases in response to crushed conspecifics cues [START_REF] Toomey | Factors affecting the movement of adult zebra mussels (Dreissena polymorpha)[END_REF][START_REF] Czarnołęski | Injured conspecifics alter mobility and byssus production in zebra mussels Dreissena polymorpha[END_REF].

Brachidontes variabilis has been shown to adapt its behaviour when exposed to injured conspecifics by seeking and selecting the most appropriate refuges [START_REF] Shin | Marine mussels Brachidontes variabilis selected smaller places of refuge and enhanced byssus production upon exposure to conspecific and heterospecific cues[END_REF]. Other studies showed an increase in aggregation rates, which is implicitly driven by a change in motility in both the Mediterrenean mussel Mytilus galloprovincialis and the brown mussel Perna perna in the presence of injured conspecifics [START_REF] Nicastro | Behavioural response of invasive Mytilus galloprovincialis and indigenous Perna perna mussels exposed to risk of predation[END_REF].

Crab cues induce a chemokinetic and potentially a chemotactic responses in Mytilus edulis

M. edulis behaviour was significantly altered by the presence of Hemigrapsus sanguineus cues through a chemokinetic response. Specifically, although motility is not affected, a decrease in both gross distance and speed compared to seawater control treatment was recorded. These results are consistent with previous experiments highlighting a decrease in movement of M. edulis and of another intertidal mussel (Hormomya mutabilis) in response to predator cues [START_REF] Reimer | Predator-induced changes in byssal attachment, aggregation and migration in the blue mussel, Mytilus edulis[END_REF][START_REF] Ishida | Reduced byssal thread production and movement by the intertidal mussel Hormomya mutabilis in response to effluent from predators[END_REF]. These observations are consistent with the activity reduction used by many aquatic animals as an antipredator strategy [START_REF] Clements | Behavioural responses to predators in Mediterranean mussels (Mytilus galloprovincialis) are unaffected by elevated pCO2[END_REF]. Indeed, low movement in mussels decreases water motion, visibility and odour dispersal and thus the probability of being spotted by predators relying on hydrodynamical, visual and chemical cues [START_REF] Ishida | Reduced byssal thread production and movement by the intertidal mussel Hormomya mutabilis in response to effluent from predators[END_REF][START_REF] Garner | Effects of injured conspecifics and predators on byssogenesis, attachment strength and movement in the blue mussel, Mytilus edulis[END_REF]Clement et al., 2020). In addition, our results suggest a negative chemotactic response of M. edulis to crab cues. This observation would be consistent with early evidence of chemodetection and chemotaxis in bivalves [START_REF] Morton | Habit and orientation in the small commensal bivalve mollusc, Montacuta ferruginosa[END_REF][START_REF] De Vooys | Effect of a tripeptide on the aggregational behaviour of the blue mussel Mytilus edulis[END_REF][START_REF] Huang | Movement and aggregation in the fluted giant clam (Tridacna squamosa L.)[END_REF]. However, to our knowledge, a chemotactic response of a bivalve to its predator has yet to be documented. Here, we move a step forward by showing that M. edulis was able to have an adapted behaviour response to predator cues by limiting its movements and by appearing to move in the opposite direction to the source. The combination of these two complementary adaptive strategies to predator cues may provide an important defence strategy against predators and may add a short-term component to the acknowledged long-term anti-predator traits of M.

edulis, such as its phenotypic plasticity (i.e. increase in its byssal attachment strength and the thickness of its shell following the presence of crab cues; [START_REF] Leonard | Crab predation, waterborne cues, and inducible defenses in the blue mussel, Mytilus edulis[END_REF][START_REF] Cheung | Induced anti-predator responses of the green mussel, Perna viridis (L.), on exposure to the predatory gastropod, Thais clavigera Küster, and the swimming crab, Thalamita danae Stimpson[END_REF].

We finally stress that the chemokinetic and possible chemotactic behavioural changes observed in the native M. edulis in response to the invasive crab predator H. sanguineus indicate that M. edulis is not naive to this predator. Prey naiveté is a lack of its ability to recognize a new predator as a threat and subsequently develop an effective anti-predator behaviour, due to the lack of coevolution between the predator and the prey [START_REF] Anton | Global determinants of prey naiveté to exotic predators[END_REF]. The invasion of H. sanguineus in the eastern English Channel is relatively recent, i.e. about 16 years at our study site [START_REF] Dauvin | Recent expansion of two invasive crabs species Hemigrapsus sanguineus (de Haan, 1835) and H. takanoi Asakura and Watanabe 2005 along the Opal Coast, France[END_REF]. The invader has now supplanted the native populations of the European green crab Carcinus maenas [START_REF] Rolet | Etat de la colonisation des Décapodes invasifs du genre Hemigrapsus sur le littoral des Hauts-de-France en 2020[END_REF]. The lack of naiveté of M. edulis reported in the present work noticeably contrasts with observations conducted in South Africa where, 28 years after its introduction, the invasive mussel (M. galloprovincialis) still had not developed any anti-predator responses to cues from a native predator (the spiny lobster Jasus lalandii), which instead had an effect on the native mussel (P. perna; [START_REF] Nicastro | Behavioural response of invasive Mytilus galloprovincialis and indigenous Perna perna mussels exposed to risk of predation[END_REF].

These observations suggest different predator-prey co-evolution history between M. edulis and M. galloprovincialis.

The overall M. edulis behavioural response to the different chemical cues inferred in the present work highlights a cue-specific response. Specifically, M. edulis responds to predatory crab cue previously fed on conspecifics (diet cue) but not to damaged released conspecific cues (alarm cue). This result is consistent with previous observation conducted on Macoma baltica, a marine bivalve which showed a strong response to fed crab effluents but a lack of response to injured conspecifics cues [START_REF] Griffiths | Chemically induced predator avoidance behaviour in the burrowing bivalve Macoma balthica[END_REF]. However, this behavioural response is likely to be species-dependent, as the same pattern was not observed for Mercenaria mercenaria [START_REF] Smee | Hard clams (Mercenaria mercenaria) evaluate predation risk using chemical signals from predators and injured conspecifics[END_REF]. This observation further suggests the existence of a hierarchy in the behavioural responses. Predator cues may be a more unambiguous threat compared to crushed conspecific cues which could instead be caused by a variety of risks, such as wave action, sand, storms, rockslides, and trampling [START_REF] Zardi | Hydrodynamic stress and habitat partitioning between indigenous (Perna perna) and invasive (Mytilus galloprovincialis) mussels: constraints of an evolutionary strategy[END_REF][START_REF] Nicastro | Between a rock and a hard place: combined effect of trampling and phototrophic shell-degrading endoliths in marine intertidal mussels[END_REF].

Alteration of Mytilus edulis chemotactic and chemokinetic responses by microplastic leachates

The addition of plastic leachates disrupted the chemotactic and/or chemokinetic behaviour of M. edulis from all treatments, indicating that polypropylene leachates can alter the neurosensory abilities of this species, hence its way of acquiring and processing information and therefore their decision-making.

Microplastic leachates alter M. edulis chemokinetic behaviour

The percentage of non-motile mussels was significantly greater in the presence of microplastic leachates, regardless of the cues. This may be the result of an inhibition of the neuromuscular performance of M. edulis by polypropylene leachates. Previous studies showed a deleterious effect of plastic additives on motility of mussel D-shape larvae (M. edulis; [START_REF] Capolupo | Chemical composition and ecotoxicity of plastic and car tire rubber leachates to aquatic organisms[END_REF], fish larvae (Danio rerio; [START_REF] Kim | Neurochemical and behavioral analysis by acute exposure to bisphenol A in zebrafish larvae model[END_REF], cladoceran (Daphnia magna; [START_REF] Lithner | Leachates from plastic consumer products-screening for toxicity with Daphnia magna[END_REF], drosophila (Drosophila melanogaster; [START_REF] Kaur | Effect of bisphenol A on Drosophila melanogaster behavior-A new model for the studies on neurodevelopmental disorders[END_REF], nematodes (Caenorhabditis elegans; [START_REF] Tseng | Phthalates induce neurotoxicity affecting locomotor and thermotactic behaviors and AFD neurons through oxidative stress in Caenorhabditis elegans[END_REF] and rats [START_REF] Vermeer | Exposure to bisphenol A exacerbates migraine-like behaviors in a multibehavior model of rat migraine[END_REF]. Thus, there is a significant effect of polypropylene leachates on mussel motility which could lead to larger scale disturbances. Indeed, any changes related to the chemosensory, referential, and behavioural abilities of M. edulis may have implications on their aggregation rate and consequently for the survival and reproduction of the species but also for intra-and interspecific competition for space with potentially cascading effects for coastal ecosystems [START_REF] Borthagaray | Mussels as ecosystem engineers: their contribution to species richness in a rocky littoral community[END_REF][START_REF] Van De Koppel | Experimental evidence for spatial self-organization and its emergent effects in mussel bed ecosystems[END_REF][START_REF] Nicastro | Love thy neighbour: group properties of gaping behaviour in mussel aggregations[END_REF][START_REF] Iwasaki | Behavior and taxis of young and adult Limnoperna fortunei[END_REF], Zardi et al., 2021).

Thus, in seawater contaminated with leachates, mussels were significantly less motile than in the control seawater treatment. This contrasts with the behaviour of the mollusc Littorina littorea, which was not affected by the presence of leachates from virgin polypropylene microbeads, although leachates concentration was higher (ca. 10-to 100-fold higher) than in our study [START_REF] Seuront | Microplastic leachates impair behavioural vigilance and predator avoidance in a temperate intertidal gastropod[END_REF]. This observation highlights distinct species chemosensitivity to plastic leachates.

Microplastic leachates alter M. edulis response to conspecific cues

In the present study, M. edulis behaviour towards conspecific cues was impacted by plastic leachates, via a decrease in their motility. There were less motile mussels, which in comparison to mussels exposed to conspecific cues without leachates, seems to cover a smaller net distance, however this last response was not significant. This observed decrease in motility and possible smaller net distance in response to conspecific cues with leachates may impact the aggregation rate of mussels which may have larger scale spatial and ecosystem implications.

However, M. edulis motility and aggregation rate have been shown to increase in seawater contaminated by plastic leachate [START_REF] Seuront | Microplastic leachates induce species-specific trait strengthening in intertidal mussels[END_REF]. Higher leachates concentration (ca.10to 100-fold more concentrated than in our study; [START_REF] Seuront | Microplastic leachates induce species-specific trait strengthening in intertidal mussels[END_REF] may, however, account for this discrepancy.

Microplastic leachates alter M. edulis response to injured conspecific cues

The combined signal of the injured mussels and leachates impact their chemokinetic response through a large percentage of non-motile mussels. Since no behavioural changes have been demonstrated in response to chemical cues from injured conspecifics, polypropylene leachates appear to affect the mussel itself and not its behaviour in response to injured conspecifics. Thus, the addition of leachates could lead to the reduction of motility found in the presence of injured conspecific cues.

Microplastic leachates alter M. edulis response to crab cues

When exposed to a mixture of crab cue and microplastic leachates, M. edulis showed a significantly lower proportion of motile mussels and a significant change in direction compared to the crab cue treatment without leachates, which may result in an inhibition of its negative chemotaxis. In addition, although the gross distance and speed did not significantly differ for this treatment than for the crab cues without leachates treatment, they were significantly reduced compared to the control and control with leachates. This change in speed and distance is related to the behavioural response of the mussel to the crab and does not appear to be affected by polypropylene leachates at the concentration used in this study. However, these leachates reduce mussel motility and affect chemotaxis, the latter suggesting a neurosensory impairment rather than a neuromuscular one. This could indicate that plastic leachates may be masking the signal and/or lead to sensory deficiencies. Plastics leachates have also been reported to have neurological effect and act on the cognition of organisms in their way of acquiring and processing information and thus their decision-making [START_REF] Seuront | Microplastic leachates impair behavioural vigilance and predator avoidance in a temperate intertidal gastropod[END_REF][START_REF] Crump | Microplastics disrupt hermit crab shell selection[END_REF]. For instance, the cognitive response of a prey to its predator is affected by plastic leachates in another mollusc, the gastropod Littorina littorea [START_REF] Seuront | Microplastic leachates impair behavioural vigilance and predator avoidance in a temperate intertidal gastropod[END_REF]. Indeed, the vigilance and anti-predation behaviours of gastropods (i.e. righting time, skioptic withdrawal, time to explore, avoidance response) have been shown to be altered and/or inhibited in the presence of plastic leachates [START_REF] Seuront | Microplastic leachates impair behavioural vigilance and predator avoidance in a temperate intertidal gastropod[END_REF].

The impairments described above are in line with previous work showing that phthalates (DEHP, DBP, DIBP) can alter the neurological behaviour in nematodes [START_REF] Tseng | Phthalates induce neurotoxicity affecting locomotor and thermotactic behaviors and AFD neurons through oxidative stress in Caenorhabditis elegans[END_REF].

Indeed, these are the dominant additives found in our polypropylene beads (Supplementary Materials, S2). Phthalates disrupt the nematode's antioxidant defence system, the morphology of some thermosensory neurons and the genes involved in this thermotaxis function [START_REF] Tseng | Phthalates induce neurotoxicity affecting locomotor and thermotactic behaviors and AFD neurons through oxidative stress in Caenorhabditis elegans[END_REF], altering their locomotion, i.e., body bends, head thrashes, and reversal frequencies [START_REF] Tseng | Phthalates induce neurotoxicity affecting locomotor and thermotactic behaviors and AFD neurons through oxidative stress in Caenorhabditis elegans[END_REF]. In similar fashion, these additives could induce a neurotoxicity in mussels, which would act on the neurosensory system and lead to an alteration of their movements and chemotactic capacities. A disturbance in M. edulis motility and neuro-sensory response could have consequences in aggregation rate and would impact the mussel growth, reproduction and anti-predator responses [START_REF] Harger | The role of behavioral traits in influencing the distribution of two species of sea mussels, Mytilus edulis and Mytilus californianus[END_REF][START_REF] Schneider | Implications of movement behavior on mussel dislodgement: exogenous selection in a Mytilus spp. hybrid zone[END_REF][START_REF] Van De Koppel | Experimental evidence for spatial self-organization and its emergent effects in mussel bed ecosystems[END_REF][START_REF] Zardi | Density-dependent and species-specific effects on self-organization modulate the resistance of mussel bed ecosystems to hydrodynamic stress[END_REF]. These impairments could lead to changes in mussel bed formations, to a decrease in mussel population and ultimately impact the ecosystem structure and functioning in M. edulis [START_REF] Borthagaray | Mussels as ecosystem engineers: their contribution to species richness in a rocky littoral community[END_REF].

Conclusion

Taken together, our results show that M. edulis behavioural responses are cue-specific.

Critically, exposure to plastic leachates altered mussel behavioural responses by altering their motility. The fitness of mussels is directly related to their motility through their ability to aggregate. The impact of microplastic leachates also altered the response of mussels to conspecific cues, which is a direct link to aggregation, and to their crab predator cues. This observation indicates that chemical communication among mussels may be disrupted by plastic leachates, suggesting that aggregation rate and bed formation and also prey-predator interaction may be impacted on the short-term, at least by polypropylene leachates and at the concentration tested here which is consistent to the contamination occurring in the marine environmental (i.e.

5 MPs L -1 ). Prey-predator interactions are essential traits of the ecology of marine systems and variations in the ability to detect predation risk can significantly influence population and community structures [START_REF] Ferrari | Chemical ecology of predator-prey interactions in aquatic ecosystems: a review and prospectus[END_REF]. The changes caused by plastic leachates may have important effects on the bioengineering role of M. edulis and have cascading effects on the associated communities. Given the predicted increase in plastic pollution and the relative limited information on plastic leachates compared to plastic ingestion [START_REF] Fauvelle | Organic additive release from plastic to seawater is lower under deep-sea conditions[END_REF], our work highlights effects of plastic leachates that may have fundamental knock-on effects on the functioning of intertidal ecosystems and the services provided to society. However, before extrapolating our results any further, it is crucial to look at the impact of plastic leachates on the predator behaviour, to see if its predatory performance is also affected or not in a plasticcontaminated environment.

Figure 1 :

 1 Figure 1: experimental design of one arena of the chemotaxis and chemokinesis experiments. Mussels

  edulis; (blue) stimuli from injured M. edulis; (orange) stimuli from fed Hemigrapsus sanguineus; stimuli with microplastic (MP) leachates (dark colour). For each treatment, 6 replicates were performed each day (two replicates of the same treatment 3 times a day at 2h interval) and an additional control (n = 6) was carried out on Day 5.

Figure 2 :

 2 Figure 2: Flow diagram of the treatments and their replications. Stimuli without plastic leachates (light

Figure 3 :

 3 Figure 3: percentage of motile Mytilus edulis by replicate in control seawater and natural cue treatments

Figure 4 :

 4 Figure 4: mean speed of Mytilus edulis by replicate in control seawater and natural cue treatments (light

Figure 5 :

 5 Figure 5: mean gross distance travelled by Mytilus edulis by replicate in control seawater and natural

Figure 6 :

 6 Figure 6: mean net distance travelled by Mytilus edulis by replicate in control seawater and natural cue

Figure 7 :

 7 Figure 7: percentage of M. edulis moving away from the point source by replicate in control seawater

Table 1 :

 1 Experimental timetable of the chemotaxis experiments. Stimuli without plastic leachates (light colour): (grey) Control, natural seawater, i.e. no stimulus; (green) stimuli from conspecifics Mytilus

Table 2 :

 2 results of 2-way ANOVA applied to the percentage of motile mussels with Treatment (control

	or microplastic leachates) and Cue (seawater, conspecifics, injured conspecifics or crabs) as fixed
	factors.

Table 3 :

 3 results of 2-way ANOVA applied to the mean speed by replicate with Treatment (control or microplastic leachates) and Cue (seawater, conspecifics, injured conspecifics or crabs) as fixed factors.

Table 4 :

 4

	Source of variation	df	MS	F	p
	Treatment	1	31.94	1.617	0.2080
	Cue	3	54.21	2.744	0.0498
	Treatment × Cue	3	30.38	1.538	0.228
	Residuals	67	19.76

results of 2-way ANOVA applied to the mean gross distance by replicate with Treatment (control or microplastic leachates) and Cue (seawater, conspecifics, injured conspecifics or crabs) as fixed factors.

Table 5 :

 5 results of 2-way ANOVA applied to the mean net distance by replicate Treatment (control or microplastic leachates) and Cue (seawater, conspecifics, injured conspecifics or crabs) as fixed factors.

Table 6 :

 6 Results of 2-way ANOVA applied to the mean confinement index by replicate with Treatment (control or microplastic leachates) and Cue (seawater, conspecifics, injured conspecifics or crabs) as fixed factors.

Table 7 :

 7 results of 2-way ANOVA applied to the percentage of mussels moving away from the source point with Treatment (control or microplastic leachates) and Cue (seawater, conspecifics, injured conspecifics or crabs) as fixed factors.

	Source of variation	df	MS	F	p
	Treatment	1	0.003253	0.257	0.614
	Cue	3	0.007719	0.609	0.611
	Treatment × Cue	3	0.024363	1.923	0.134
	Residuals	67	0.012667

cm

Acknowledgements

We thank J. Anquetin and A. Lutrand for their technical support. M. Uguen is funded by a PhD fellowship from the University of Lille and the Region-Hauts-de-France. This research was also financially supported by both the European Maritime and Fisheries Fund (FEAMP) and France Filière Pêche through the research project SOLACE (« Resilience of the mussel Mytilus edulis to anthropogenic and climatic stressors »), and by the Fundação para a Ciência e a Tecnologia (FCT -MEC, Portugal, grant number: UIDB/04326/2020) and the National Research Foundation of South Africa (grant number: 64801).

Credit author statement

Marine Uguen: Conceptualization, Formal Analysis, Methodology, Investigation, Writing -Original Draft, Editing. Katy R Nicastro: Investigation, Methodology, Resources, Writing -Review & Editing. Gerardo I Zardi: Investigation, Methodology, Resources, Writing -Review & Editing. Sylvie M Gaudron: Supervision, Resources, Writing -Review & Editing. Nicolas Spilmont: Investigation, Methodology, Resources, Writing -Review & Editing. Fleurine Akoueson: Methodology, Resources. Guillaume Duflos: Methodology, Resources. Laurent Seuront: Supervision, Conceptualization, Methodology, Resources, Writing -Review & Editing, Funding acquisition.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.