Noémie Deldicq
email: noemie.deldicq@outlook.fr

Laurent Seuront

Dewi Langlet

Vincent M P Bouchet

Assessing behavioural traits of benthic foraminifera: implications for sediment mixing

Keywords:

The assessment of behavioural traits of marine organisms is increasingly recognized as a key issue to understand their role in ecosystem processes, such as bioturbation and nutrient cycling.

The movement ability of intertidal foraminifera suggest that they may have a role, yet to be quantified, in benthic-pelagic coupling through their movement on the sediment, at the sediment-water interface and within the sediment. In this context, we investigated the behavioural traits of 5 benthic foraminiferal species typical of European temperate mudflats under standardized trophic light and temperature conditions. Behavioural traits related to motion of Ammonia tepida, Haynesina germanica, Cribroelphidium williamsoni, Miliammina fusca and Quinqueloculina seminula species were assessed through their travelled distance, velocity, tortuosity of the path, position in the sediment and activity index. By analogy with macrofauna bioturbation functional groups, we describe the studied foraminifera as biodiffusors-species with 3 sub-groups defined according to their vertical position in the sediment. C. williamsoni belongs to the epifaunal-biodiffusors group while A. tepida and H. germanica belongs to surficial-biodiffusors. Finally, the species Q. seminula and M. fusca are considered as gallery-biodiffusors species. Our results further suggest that features such as velocity, activity and tortuosity may mediate sediment-mixing intensity. Therefore, Q. seminula, H. germanica and C. williamsoni which are the most active species, would have larger effect than the less active A. tepida and M. fusca on particles reworking rates. Taken together, our results suggest that benthic foraminifera may play an underestimated role in bioturbation processes.

Introduction

Trait-based studies have largely been implemented to describe ecosystem functioning, especially over the last decade [START_REF] Braeckman | Role of macrofauna functional traits and density in biogeochemical fluxes and bioturbation[END_REF][START_REF] Cardinale | The functional role of producer diversity in ecosystems[END_REF][START_REF] Gothland | Biological traits explain the distribution and colonisation ability of the invasive shore crab Hemigrapsus takanoi[END_REF]). Specifically, traits -defined as "the morphological, physiological or phenological features measurable at the individual level, from the cell to the whole-organism level" [START_REF] Violle | Let the concept of trait be functional![END_REF]) -can either be demographical (e.g. birth, mortality), biological (e.g. size, growth), ecophysiological (e.g. nutrient assimilation, resource uptake) or behavioural (e.g. locomotion, species interactions). These features determine the role of a species in the ecosystem; hence allow defining functional traits [START_REF] Violle | Let the concept of trait be functional![END_REF][START_REF] Gagic | Functional identity and diversity of animals predict ecosystem functioning better than species-based indices[END_REF]. For instance, a comprehensive assessment of species-specific behavioural traits associated to locomotion is one way to understand the role of species in the structure and functioning of coastal ecosystems.

Specifically in soft sediment environment, the behavioural traits related to faunal motion are intrinsically considered as functional traits involved in bioturbation processes through the displacement of sediments particles and the related enhancement of fluxes of both dissolved and particulate materials [START_REF] Mermillod-Blondin | Influence of bioturbation by three benthic infaunal species on microbial communities and biogeochemical processes in marine sediment[END_REF][START_REF] Mermillod-Blondin | Ecosystem engineering: the impact of bioturbation on biogeochemical processes in marine and freshwater benthic habitats[END_REF][START_REF] Kristensen | What is bioturbation? the need for a precise definition for fauna in aquatic sciences[END_REF][START_REF] Aller | Sedimentary diagenesis, depositional environments and benthic fluxes[END_REF]. For instance, surface sediment displacement intensity is directly linked to the type of activity (feeding, tube building) in the terebellid polychaete Eupolymnia nebulosa (Maire et al. 2007c). Recent methodological improvements such as highfrequency image analysis and automated acquisition help to quantify behavioural traits associated to bioturbation, see [START_REF] Maire | Quantification of sediment reworking rates in bioturbation research: a review[END_REF] for a review. Continuous observation of Abra ovata showed that this bivalve's behavioural activity is linked to sediment-mixing intensity (Maire et al. 2007a). Depending on their behavioural traits, bioturbating species can induce various changes on the benthic compartment such as the microbial community structure [START_REF] Banta | Effects of two polychaete worms, Nereis diversicolor and Arenicola marina, on decomposition in an organic-poor and an organic-enriched marine sediment[END_REF][START_REF] Marinelli | Experimental investigation of the control of bacterial community composition in macrofaunal burrows[END_REF][START_REF] Papaspyrou | Microbial reaction rates and bacterial communities in sediment surrounding burrows of two nereidid polychaetes (Nereis diversicolor and N. virens)[END_REF]) and in the biogeochemical reactions occurring in the sedimentary column [START_REF] Gutierrez | Physical ecosystem engineers as agent of biogeochemical heterogeneity[END_REF]. Trait-based approaches hence allow the description of different functional groups of macrofauna species such as gallerydiffusors, biodiffusors, regenerator, upward-and downward-conveyors [START_REF] François | A new approach for the modelling of sediment reworking induced by a macrobenthic community[END_REF].

There is still, however, a limited knowledge on the role of the meiobenthic compartment in bioturbation processes (see review in [START_REF] Schratzberger | Meiofauna matters: the roles of meiofauna in benthic ecosystems[END_REF].

Meiofaunal taxa activity (nematodes and copepods) increase bacterial growth, oxygen fluxes, denitrification and organic matter mineralisation [START_REF] Aller | Meiofauna and solute transport in marine muds[END_REF][START_REF] Bradshaw | The use of tracers to evaluate the importance of bioturbation in remobilising contaminants in Baltic sediments[END_REF][START_REF] Middelburg | Burial at sea[END_REF][START_REF] Nascimento | Meiofauna enhances organic matter mineralization in soft sediment ecosystems[END_REF][START_REF] Piot | Meiofauna affect the macrobenthic biodiversityecosystem functioning relationship[END_REF][START_REF] Bonaglia | Meiofauna increases bacterial denitrification in marine sediments[END_REF]. Benthic foraminifera, despite their high abundance and ecological importance in the marine meiobenthos [START_REF] Murray | Ecology and applications of benthic foraminifera[END_REF][START_REF] Schönfeld | The FOBIMO (FOraminiferal BIo-MOnitoring) initiative-towards a standardised protocol for soft-bottom benthic foraminiferal monitoring studies[END_REF], have essentially been ignored in most studies dealing with meiofaunal bioturbation. Furthermore, little is known on foraminiferal traits (e.g. habitats, metabolism, feeding modes, displacements), and only a few studies quantified the motion behaviour of foraminifera (e.g. [START_REF] Kitazato | Observation of behaviour and mode of life of benthic foraminifers in laboratory[END_REF], 1988[START_REF] Seuront | The devil lies in details: new insights into the behavioural ecology of intertidal foraminifera[END_REF], Jauffrais et al. 2016b). Hence, all benthic foraminiferal functions may not yet be identified, particularly those involved in bioturbation processes. Benthic foraminifera can move over relatively long distances [START_REF] Seuront | The devil lies in details: new insights into the behavioural ecology of intertidal foraminifera[END_REF] and their movements types (rotating, spinning, crawling) are driven by both their number of pseudopods [START_REF] Kitazato | Foraminiferal microhabitats in four marine environments around Japan[END_REF] and the morphology of their tests (i.e. spiral or quinqueloculine, [START_REF] Wetmore | Burrowing and sediment movement by benthic foraminifera, as shown by time-lapse cinematography[END_REF]. Foraminiferal motion varies in intensity [START_REF] Severin | Laboratory observations of the rate of subsurface movement of a small miliolid foraminifer[END_REF][START_REF] Gross | Sediment interactions of foraminifera: implications for food degradation and bioturbation processes[END_REF] and induces sediment displacements [START_REF] Kitazato | Locomotion of some benthic foraminifera in and on sediments[END_REF][START_REF] Hemleben | Deep-sea foraminifera under long time observation in the laboratory[END_REF] that have so far only been quantified for deep-sea communities [START_REF] Gross | Sediment interactions of foraminifera: implications for food degradation and bioturbation processes[END_REF]. These displacements create trails at the sediment surface (e.g.

Quinqueloculina impressa, Cribroelphidium excavatum) and galleries (e.g. Ammotium cassis, Cribroelphidium excavatum subsp. clavatum) in the sediment [START_REF] Richter | Zur ökologie der Foraminiferida II: lebensraum und lebenweise von Nonion depressulum, Elphidium excavatum und Elphidium selseyense[END_REF][START_REF] Severin | Burrows and trails produced by Quinqueloculina impressa Reuss, a benthic foraminifer, in fine-grained sediment[END_REF][START_REF] Linke | Microhabitat preferences of benthic foraminifera-a static concept or a dynamic adaptation to optimize food acquisition?[END_REF][START_REF] Hemleben | Deep-sea foraminifera under long time observation in the laboratory[END_REF][START_REF] Bornmalm | Laboratory observations of rates and patterns of movement of continental margin benthic foraminifera[END_REF][START_REF] Gross | Sediment interactions of foraminifera: implications for food degradation and bioturbation processes[END_REF].

Recently, [START_REF] Seuront | The devil lies in details: new insights into the behavioural ecology of intertidal foraminifera[END_REF] showed a negative geotaxis in Ammonia tepida and a positive geotaxis in both C. excavatum and Haynesina germanica suggesting that these species move on and in the sedimentary column to colonize different microhabitats depending on their ecological requirements [START_REF] Wetmore | Burrowing and sediment movement by benthic foraminifera, as shown by time-lapse cinematography[END_REF][START_REF] Linke | Microhabitat preferences of benthic foraminifera-a static concept or a dynamic adaptation to optimize food acquisition?[END_REF][START_REF] Murray | Ecology and applications of benthic foraminifera[END_REF]. Hence, benthic foraminiferal functional traits associated to motion behaviour may exist and be involved in sediment mixing. This further suggests that the assessment of behavioural traits is an absolute prerequisite to a comprehensive description of foraminiferal role in particulate fluxes at the sediment-water interface. Yet, behavioural traits such as activity, motion intensity and vertical position are poorly described in benthic foraminifera. In this context, the objectives of this study are (i) to assess the horizontal and vertical dynamic of 5 intertidal foraminiferal species at the sediment-water interface, (ii) to quantify the motion behaviour of these species and (iii) to use the emergent vertical position and behavioural traits as a stepping stone to objectively classify these species into functional groups. In intertidal mudflats from temperate environment, oxygen penetration depth rarely reached 1 cm and foraminifera are mainly distributed in the 0-1 cm layer [START_REF] Geslin | Oxygen respiration rates of benthic foraminifera as measured with oxygen microsensors[END_REF][START_REF] Cesbron | Vertical distribution and respiration rates of benthic foraminifera: contribution to aerobic remineralization in intertidal mudflats covered by Zostera noltei meadows[END_REF]). This study hence specifically focused on foraminifera living in the top 1 st cm of sediment.

Material and Methods

Sediment sampling and experimental set-up

Surface sediment (0-1 cm) was collected from February to June 2018 at low tide in the Authie Bay (50°22'20"N, 1°35'45"E), an intertidal mudflat located on the French coasts of the English Channel for living benthic foraminifera. Sampled sediment was stored in plastic containers (100 ml) and transported to the laboratory, where it was washed through a 125 μm mesh-size sieve. Living benthic foraminifera were subsequently individually sorted with a brush, identified and their pseudopodial activities checked with an inverted phase-contrast microscope. Five intertidal species were considered in this study due to their high density at the study site: the planispiral species Haynesina germanica and Cribroelphidium williamsoni, the trochospiral species Ammonia tepida, the agglutinated species Miliammina fusca and the porcelaneous species Quinqueloculina seminula. Recent molecular investigations showed that the phylotypes H. germanica S16, C. williamsoni S1 and A. tepida T6 occur in the Authie Bay (Schweizer, unpublished data). Individual sizes ranged from 300 to 800 µm in diameter.

Depending on the abundance at the time of sampling, 8 to 33 individuals per species were used for the experimentation (Table 1). Before behavioural observations, individuals were kept overnight 12h-long for acclimation under the experimental condition in a controlledtemperature room at 18°C. Behavioural observations were performed in 300 ml aquaria filled with 25-30ml defrost Authie-Bay sediment and 250 ml unfiltered eastern English-Channel seawater (30 PSU). We used defrost sediment to make sure sediment was free of moving macroand meio-organisms (since nematodes, copepods and macrofaunal organisms are killed during sediment freezing) and that only foraminiferal tracks could be observed at the sediment surface.

After the 12-h acclimation period, foraminifera were placed randomly at the surface of sediment (Fig. 1). Note that the overlaying water was fully saturated in oxygen though a 10-min vigorous air bubbling immediately before spreading living foraminifera on the sediment surface. At the end of the 24h-experiment, dissolved-oxygen saturation was about 56% in the overlaying seawater right above the sediment-water interface (HI9829 Multiparameter Meter, Hannah instruments).

Quantification of behavioural traits

Individual displacements in and on the sediment were recorded by time-lapse photography (1 image every 10 min for 24 hours) using a digital camera (Nikon V1 with a Nikkor 10-30 mm lens; Fig. 1). For each foraminifera, 144 images were combined in image-analysis software Fiji to extract (x,y) coordinates using the Manual tracking plugin [START_REF] Schindelin | Fiji: an open-source platform for biological-image analysis[END_REF]. The behavioural traits of the above-mentioned species were investigated adapting the method described in [START_REF] Seuront | The devil lies in details: new insights into the behavioural ecology of intertidal foraminifera[END_REF]. Five parameters were used to characterise the traits of each study species.

First, the level of activity i.e. time allocated to motion by each individual, was estimated with the activity index Ai. This index is based on the ratio between the total time taken by one individual to move from its initial to its final position (tmove) and the time that the individual spent moving between these positions (tactive):

Ai =100´(tactive/ tmove)
The distance travelled by each individual between two images (i.e. 10 min) was calculated as:

Dt = √ ((xt -xt+1) 2 + (yt -yt+1) 2 )
where (xt,yt) and (xt+10, yt+10) are the coordinates between two successive images taken at times t and t+10 min and the velocity (mm h -1 ) of each individual was subsequently calculated considering the total distance travelled in 24 h.

The complexity (or tortuosity) of movement paths was assessed using fractal analysis. The fractal dimension measures the degree to which the trajectory fills the available space and is bounded between D = 1 for a line (i.e. the simplest instance of a trajectory) and D = 2 for a movement so complex that it actually fills the whole available space. The fractal dimensions of foraminifera trajectories were estimated using the box dimension method (see Seuront, 2010[START_REF] Seuront | On uses, misuses and potential abuses of fractal analysis in zooplankton behavioral studies: A review, a critique and a few recommendations[END_REF] for reviews), which relies on the "l cover" of the object, i.e. the number of boxes of length l required to cover the object. A more practical alternative is to superimpose a regular grid of boxes of length l on the object and count the number of boxes occupied by a subset of the object. This procedure is repeated using different values of l. The surface occupied by the trajectory of a foraminifera is then estimated using a series of counting boxes spanning a range of surfaces down to some small fraction of the entire surface. The number of occupied boxes increases with decreasing box size, leading to the following power-law relationship:

N (l) ∝ l -D b
where l is the box size, N(l) is the number of boxes occupied by the trajectory, and Db is the box fractal dimension. The fractal dimension Db is estimated from the slope of the linear trend of the log-log plot of N(l) versus l.

Finally, to assess the preferential position of the 5 studied species, the vertical position in the sediment of each individual was determined for every picture based on a 3 depths categories classification. When the test remained visible at the surface and the width of the path was indistinguishable, the individual was considered to be crawling on the sediment surface (Fig. 2A,D). In turn, when the individual was burrowing into the sediment, its position was divided in two categories: it was considered as moving at the sediment-water interface when half of the test was visible (Fig. 2B,E) and to be burrowed in the sediment when a swelling at the sediment surface was the only indication of the presence of the test in the sediment (Fig. 2C,F).

Statistical analyses

Because activity index, distance travelled, and velocity were non-normally distributed (Kolmogorov-Smirnov test, p < 0.05), multiple comparisons between species were conducted using the Kruskal-Wallis test, and a subsequent multiple comparison procedure based on the Mann-Whitney test was eventually used to identify distinct group of measurements. The presence of significant differences in fractal dimensions between species was assessed using an analysis of covariance [START_REF] Zar | Biostatistical analysis[END_REF]. All statistical analyses were performed using © R.3.5.2. software (R Core Team 2019).

Results

Among the 230 individuals picked for the experiment, 103 individuals were analysed as we kept only those possible to track from the start to the end of the experiment (Table 1).

Activity index

Cribroelphidium williamsoni was the most active species with a mean activity index at 89% corresponding to 21/24h of displacement in the sediment (Fig. 3A, Table 1). This species activity index is significantly larger (p < 0.05) than Ammonia tepida, Haynesina germanica and Quinqueloculina seminula, which are not significantly different from each other (p < 0.05).

These 4 species have a significantly higher activity index than Miliammina fusca (p < 0.05) which was the less active species with a mean activity index at 50% (Fig. 3A).

Distance and velocity

Quinqueloculina seminula moved significantly faster (p < 0.05, Fig. 3C) and over longer distances (Fig. 3B) than the other. The four other species were discriminated in two other homogeneous groups with species moving slowly over short distances (Miliammina fusca and Ammonia tepida; Fig. 3B,C, Table 1) and species that moved at intermediate speed over intermediate distances (Cribroelphidium williamsoni and Haynesina germanica; Fig. 3B,C, Table 1).

Tortuosity

Fractal dimensions D were in the range 1-1.3, indicating relatively linear trajectories. The analysis of covariance identified a group of 4 species (Miliammina fusca, Ammonia tepida, Haynesina germanica and Quinqueloculina seminula) moving significantly more tortuously than Cribroelphidium williamsoni (Fig. 3D).

Vertical position

Miliammina fusca and Quinqueloculina seminula individuals were essentially observed (i.e.

100% and 70 to 90%, respectively) burrowed in the sediment throughout the experiment (Fig. 4A,B). Conversely, the vast majority (90-100%) of Cribroelphidium williamsoni individuals remained on the sediment surface during the first three hours, before progressively moved to the sediment-water interface (10 to 100%) and were barely observed down into the sediment (Fig. 4C). Haynesina germanica gradually burrowed from the sediment surface down into the sediment with time to be essentially (75 to 80%) located into the sediment after 15 hours of observations (Fig. 4D). Finally, Ammonia tepida exhibited a slightly different temporal pattern.

First, a vast majority of them (95%) dug from the sediment surface down into the sediment in less than an hour, before progressively spreading out back up towards to sediment-water interface (Fig. 4E).

Discussion

This study is based on 24-h long observations assessing the behavioural traits of five species of benthic foraminifera to further understand their implication in sediment mixing. For instance, macrofaunal species behavioural traits affect the way they displace sediment particles [START_REF] François | A new approach for the modelling of sediment reworking induced by a macrobenthic community[END_REF][START_REF] Maire | Effects of food availability on sediment reworking in Abra ovata and A. nitida[END_REF], 2007a). It allowed for their classification in functional groups i.e. biodiffusors, regenerators or conveyors [START_REF] François | A new approach for the modelling of sediment reworking induced by a macrobenthic community[END_REF][START_REF] Kristensen | What is bioturbation? the need for a precise definition for fauna in aquatic sciences[END_REF]. The intensity of species activity is further directly linked to their life-mode which drives sediment mixing and bio-irrigation [START_REF] Gérino | Macro-invertebrate functional groups in freshwater and marine sediments: a common mechanistic classification[END_REF][START_REF] Gilbert | Sediment reworking by marine benthic species from the Gullmar Fjord (Western Sweden): importance of faunal biovolume[END_REF][START_REF] Maire | Quantification of sediment reworking rates in bioturbation research: a review[END_REF], 2016[START_REF] Kristensen | What is bioturbation? the need for a precise definition for fauna in aquatic sciences[END_REF]. Considering that foraminifera fundamentally displace sediment over short distances due to their small size (Gross 2002, present work), we hereafter consider foraminifera as biodiffusors since they are "organisms with activities that usually results in a constant and random local sediment bio-mixing over short distance" [START_REF] Kristensen | What is bioturbation? the need for a precise definition for fauna in aquatic sciences[END_REF]).

More specifically, biodiffusors are typically divided in 3 subgroups (epifaunal-, gallery-and surficial-biodiffusors) depending on their life-mode in the sediment [START_REF] Kristensen | What is bioturbation? the need for a precise definition for fauna in aquatic sciences[END_REF]. In this context, our high-frequency image analysis of foraminiferal behaviour showed speciesspecific preferential depths of activity, that we used hereafter to classify the studied intertidal foraminiferal species in different biodiffusors functional subgroups (Fig. 5). Furthermore, motion-behaviour features (activity index, travelled distance, velocity and tortuosity) drive intra-functional group variability.

Foraminifera as biodiffusors

Epifaunal-biodiffusors

In our experiments, Cribroelphidium williamsoni only moved on the sediment surface and at the sediment-water interface. This observation is consistent with previous studies describing this species as epifaunal [START_REF] Allison | Culture studies of the benthic foraminifera Elphidium williamsoni: evaluating pH, Δ[CO ! "# ] and inter-individual effects on test Mg/Ca[END_REF] and reporting the highest density of the species in the uppermost oxygenated sediment layers [START_REF] Alve | Temporal variability in vertical distributions of live (stained) intertidal foraminifera, Southern England[END_REF][START_REF] Bouchet | Influence of the mode of macrofauna-mediated bioturbation on the vertical distribution of living benthic foraminifera: first insight from axial tomodensitometry[END_REF]). This kleptoplastic species can host 10 times more active chloroplasts in its cytoplasm than other temperate-water species [START_REF] Lopez | Algal chloroplasts in the protoplasm of three species of benthic foraminifera: taxonomic affinity, viability and persistence[END_REF]. Kleptoplasty suggesting a preference for well-lighted surface sediment, this species most likely have a surface-limited effect on the sediment mixing and bio-irrigation. Consequently, C. williamsoni may be considered as an epifaunal-biodiffusor (Fig. 5) which include "organisms that occur predominantly above the sediment-water interface. Their activities are limited to near-surface sediments and generally redistribute fine particles randomly over very short distances along the surface" [START_REF] Kristensen | What is bioturbation? the need for a precise definition for fauna in aquatic sciences[END_REF].

Surficial-biodiffusors

Haynesina germanica and Ammonia tepida tended to avoid the surface sediment and both preferred the sediment-water interface and burrowed position. Specifically, at the end of the experiment, A. tepida individuals were evenly distributed between these two positions while H. germanica ones preferred to be completely burrowed. These results are consistent with in situ observations where A. tepida is found on and in the sediment [START_REF] Goldstein | Microhabitats of salt marsh foraminifera: St. Catherines Island, Georgia, USA[END_REF][START_REF] Bouchet | Influence of the mode of macrofauna-mediated bioturbation on the vertical distribution of living benthic foraminifera: first insight from axial tomodensitometry[END_REF]) while H. germanica mainly occur at the sediment surface [START_REF] Alve | Temporal variability in vertical distributions of live (stained) intertidal foraminifera, Southern England[END_REF][START_REF] Bouchet | Influence of the mode of macrofauna-mediated bioturbation on the vertical distribution of living benthic foraminifera: first insight from axial tomodensitometry[END_REF]). The later can sequester photosynthetically active chloroplasts, which might be used as food sources under low-light condition (Jauffrais et al. 2016a). Our results suggest that both species could alternate between epifaunal and infaunal micro-habitats (Fig. 5). As a consequence, we classify these species in the surficial-biodiffusors group which are "organisms with activities mostly restricted to the uppermost few centimetres of the sediment, and these species rarely venture above the sediment-water interface" [START_REF] Kristensen | What is bioturbation? the need for a precise definition for fauna in aquatic sciences[END_REF].

To be consistent with this definition, further assessments are needed to understand how deep H. germanica and A. tepida can mix the sediment.

Gallery-biodiffusors

Quinqueloculine-shaped species (i.e. Quinqueloculina seminula and Miliammina fusca) moved immediately from the surface down into the sediment layer and stayed buried during the remaining time of experiment. These two species are hence clearly not restricted to the sediment-water interface. Quinqueloculina seminula has, however, previously been described as an epifaunal species (Di [START_REF] Bella | Benthic foraminiferal assemblages in active volcanic area of the Azores Islands (North Atlantic Ocean)[END_REF][START_REF] Martins | Environmental quality assessment of Bizerte lagoon (Tunisia) using living foraminifera assemblages and a multiproxy approach[END_REF] preferring oxic-zone [START_REF] Moodley | Subsurface activity of benthic foraminifera in relation to porewater oxygen content: laboratory experiments[END_REF], but is also known to be tolerant to low-oxygen events [START_REF] Bernhard | Benthic foraminiferal proxy to estimate dysoxic bottom-water oxygen concentrations: Santa Barbara basin, US Pacific continental margin[END_REF][START_REF] Martins | The response of benthic foraminifera to pollution and environmental stress in Ria de Aveiro (N Portugal)[END_REF], 2013[START_REF] Langlet | Foraminiferal species responses to in situ, experimentally induced anoxia in the Adriatic Sea[END_REF]. Since in our 24-hours long observations specimens never went back to the surface, these species may rather be considered as infaunal; with the ability to create galleries in the sediment [START_REF] Severin | Laboratory experiments on the vertical movement of Quinqueloculina impressa Reuss through sand[END_REF][START_REF] Severin | Burrows and trails produced by Quinqueloculina impressa Reuss, a benthic foraminifer, in fine-grained sediment[END_REF]. As a consequence, Q. seminula and M. fusca should be considered as gallery-biodiffusors (Fig. 5) which are organisms "inducing diffusive local bio-mixing of particles and vertical transport of particles from the upper regions of the sediment to the lower limit of burrow penetration" [START_REF] Kristensen | What is bioturbation? the need for a precise definition for fauna in aquatic sciences[END_REF].

Features explaining intra-functional group variability

Life traits such as individual size and foraging strategy affects role in species-specific functional role in bioturbation processes [START_REF] François | A new approach for the modelling of sediment reworking induced by a macrobenthic community[END_REF][START_REF] Gérino | Macro-invertebrate functional groups in freshwater and marine sediments: a common mechanistic classification[END_REF][START_REF] Solan | In situ quantification of bioturbation using time-lapse fluorescent sediment profile imaging (f-SPI), luminophore tracers and model simulation[END_REF][START_REF] Mermillod-Blondin | The functional significance of bioturbation and biodeposition on biogeochemical processes at the water-sediment interface in freshwater and marine ecosystems[END_REF]. For instance, macrofauna species displace a quantity of sediment which is a direct proportion to their volumetric size [START_REF] Dorgan | Burrow extension by crack propagation[END_REF]. In our study, quinqueloculine-shaped species (i.e. Quinqueloculina seminula and Miliammina fusca) are species with the larger test volume suggesting that they would displace more sediment than the other species investigated in the present work. However, effects of benthic fauna on fluxes at the sediment-water interface depend on sediment reworking and bio-irrigation modes rather than on the biogenic structure volume produced [START_REF] Bouchet | Influence of the mode of macrofauna-mediated bioturbation on the vertical distribution of living benthic foraminifera: first insight from axial tomodensitometry[END_REF]. Intensity of bioturbation is hence a complex interplay between numerous life traits.

More specifically, the rate by which particles are physically moved also depends on feeding strategies [START_REF] Gérino | Quantification of bioturbation processes and method to identify related functional groups[END_REF]). Tortuosity gives key information on that aspect [START_REF] Pyke | Optimal foraging theory: a critical review[END_REF][START_REF] Bell | Searching behaviour: the behavioural ecology of finding resources[END_REF]. In our study, the five species exhibited a relatively low tortuosity value. It implies that they explored their environment extensively with close-to-linear trajectory. Such extensive search strategy (or transecting e.g. [START_REF] Bell | Searching behaviour: the behavioural ecology of finding resources[END_REF] in the case of the 5 studied species, is optimal under spatially-located food patches [START_REF] Pyke | Optimal foraging theory: a critical review[END_REF][START_REF] Seuront | Anomalous diffusion and multifractality optimises mating encounters in the ocean[END_REF][START_REF] Seuront | The devil lies in details: new insights into the behavioural ecology of intertidal foraminifera[END_REF]. This foraging strategy is consistent with what is known of the trophic ecology of the five species used in this study e.g. microphytobenthos, bacteria, metazoans [START_REF] Nomaki | Benthic foraminifera as trophic links between phytodetritus and benthic metazoans: carbon and nitrogen isotopic evidence[END_REF][START_REF] Dupuy | Predation of mudflat meio-macrofaunal metazoans by a calcareous foraminifer, Ammonia tepida (Cushman, 1926)[END_REF], Jauffrais et al. 2016b[START_REF] Chronopoulou | Metabarcoding insights into the tropic behaviour and identity of intertidal benthic foraminifera[END_REF]. For instance, Haynesina germanica and Ammonia tepida are both herbivorous, feeding on benthic diatoms [START_REF] Ward | Feeding of benthic foraminifera on diatoms and sewage-derived organic matter: an experimental application of lipid biomarker techniques[END_REF][START_REF] Pascal | Bacterivory in the common foraminifer Ammonia tepida: isotope tracer experiment and the controlling factors[END_REF]. Recently, an in situ study showed that the feeding behaviour of intertidal benthic foraminifera is more complex that what is observed experimentally [START_REF] Chronopoulou | Metabarcoding insights into the tropic behaviour and identity of intertidal benthic foraminifera[END_REF]. Intertidal foraminifera exhibited clear varied and species-specific trophic behaviour and were actually able to feed on different food sources. Specifically, A.

tepida may prefer to feed on algae but it is actually also able to feed on bacteria [START_REF] Pascal | Bacterivory in the common foraminifer Ammonia tepida: isotope tracer experiment and the controlling factors[END_REF]. Since microphytobenthos, bacteria and metazoans microscale (i.e. centimetre-scale) distributions are extremely patchy [START_REF] Pinckney | Spatial autocorrelation analysis of meiofaunal and microalgal populations on an intertidal sandflat: scale linkage between consumers and resources[END_REF][START_REF] Danovaro | Small-scale distribution of bacteria, enzymatic activities, and organic matter in coastal sediments[END_REF][START_REF] Spilmont | There's more to the picture than meets the eye: sampling microphytobenthos in a heterogeneous environment[END_REF]), all the studied species would benefit from the extensive search strategy to optimize their likelihood to locate food patches. Such extensive displacement tends to maximize sediment mixing (Seuront 2010[START_REF] Viswanathan | The physics of foraging: an introduction to random searching and biological encounters[END_REF]. However, species feeding strategy has not been investigated throughout our experiment since we used homogenised defrost surface-sediment therefore containing labile organic-matter, living bacteria, dead macro-and meio-faunal organisms and the non-filtered overlaying contains microalgae. To further assess a supprimé: is 322 a supprimé: This behavioural strategy is consistent with an 323 optimal foraging strategy.

whether foraging-strategy would affect sediment-mixing rate, motion-behaviour of foraminiferal species in a patchy and homogeneous controlled-food-condition needs to be assess.

The five studied species showed significant differences between their respective activity index, travelled distance and velocity. Within the functional groups above mentioned, species may not have the same sediment mixing intensity and this intensity may most likely depend on the detailed properties of species-specific motion behaviour. Within the gallery-biodiffusors, Quinqueloculina seminula was the second most active species and travelled the longest distances, while Miliammina fusca was the less active species and over lowest distances.

Similarly, the surficial-biodiffusor Haynesina germanica was more active and moved further than Ammonia tepida. As a consequence, Q. seminula and H. germanica may rework a larger volume of sediment. Furthermore, irrespective of their functional group, the most active species (i.e. Cribroelphidium williamsoni, Q. seminula and H. germanica) may contribute more to sediment mixing than the less active species such as A. tepida and M. fusca (Fig. 5).

Conclusion

Based on the monitoring of species-specific behavioural traits this study assigned for the first time intertidal benthic foraminiferal species into the biodiffusors-functional group. Hence, the preferential depth of activity being fundamentally the prime feature allowing differentiating between species. Secondary features like (i) size, (ii) feeding-modes, (iii) activity index, (iv) travelled distance (v) velocity and (vi) tortuosity would most likely mediate the intensity of bioturbation and explain the intra-functional group differences. To definitively validate our classification, further work is needed on the quantification of sediment reworking rate of the five studied species. Furthermore, our observations showed that foraminifera create physical disturbances at the sediment surface, previously described as sediment pellets [START_REF] Chandler | Foraminifera may structure meiobenthic communities[END_REF]. Such a pelletised surface layer created by benthic organisms activity can ease the resuspension of sediments by tidal currents [START_REF] Davis | The role of bioturbation in sediment resuspension interaction with physical shearing[END_REF][START_REF] Willows | Influence of an infaunal bivalve on the erosion of an intertidal cohesive sediment: a flume and modeling study[END_REF][START_REF] Orvain | A model of fluff layer erosion and subsequent bed erosion in the presence of the bioturbator, Hydrobia ulvae[END_REF], 2004) and affect sediment bio-irrigation. These physical and chemical changes will affect microbial communities [START_REF] Bertics | Biodiversity of benthic microbial communities in bioturbated coastal sediments in controlled by geochemical microniches[END_REF][START_REF] Piot | Meiofauna affect the macrobenthic biodiversityecosystem functioning relationship[END_REF], organic matter mineralization and nutrient cycle [START_REF] Gilbertson | Differential effects of microorganism-invertebrate interactions in benthic nitrogen cycling[END_REF][START_REF] Aller | Sedimentary diagenesis, depositional environments and benthic fluxes[END_REF]. Meiofaunal species activity e.g. copepods, nematodes and foraminifera will consequently increase organic matter and NOx fluxes which strongly affect benthic-pelagic coupling and therefore ecosystem functions [START_REF] Danovaro | Exponential decline of deep-sea ecosystem functioning linked to benthic biodiversity loss[END_REF][START_REF] De Goeij | Surviving in a marine desert: the sponge loop retains resources within coral reeds[END_REF]. This suggest that foraminifera might play a underestimated role on sediment cohesiveness and dissolved-elements benthic fluxes [START_REF] Schratzberger | Meiofauna matters: the roles of meiofauna in benthic ecosystems[END_REF].

This study supports the fact that, in a context of biodiversity change, assessing life traits of benthic foraminifera is critically needed to understand their role in ecosystem functioning.

Figure 2 .

 2 Figure 2. Schematic representation (A, B and C) and images (D, E and F) of the three vertical position categories, which can be taken by a foraminifer. (A and D) Surface -(B and E) Sediment-water interface -(C and F) Burrowed. Examples from Haynesina germanica (D and E) and Quinqueloculina seminula (F). Scale bars = 0.2 mm.

Figure 3 .

 3 Figure 3. Box and whiskers plots of the four calculated parameters (A) Activity index (%) -(B) Distance (mm) -(C) Velocity (mm.h -1 ) -(D) Tortuosity for all species. The letters 'a','b','c' indicate significant distinct groups of measurements (Mann-Whitney test, p < 0.05). The box represents the first, second and third quartiles and the whiskers extend to 1.5 times the interquartile range values outside this range are represented by open circles.

Figure 4 .

 4 Figure 4. Temporal evolution of the vertical position of each studied species

Table captions 619

 captions Table1. Number N of individuals of each species used for experimentations and the subsequent 620 number n of individuals for which (x,y) coordinates were extracted with mean (X ̅ ) and standard Figure 1. Experimental set-up for time-lapse assessment of foraminiferal life trait

	621
	622	deviation (σ ) for each parameters.
	Species			Q. seminula	H. germanica	C. williamsoni	A. tepida	M. fusca
	Total individual	N	30	90	30	60	20
	Analysed individual	n	16	30	16	33	8
	Distance (mm)	X ̅ σ	88.25 32.95	51.51 19.63	44.18 13.50	17.29 5.94	14.06 4.67
			X ̅	3.67	2.15	1.84	0.72	0.59
			σ	1.37	0.82	0.56	0.25	0.19
	Activity index (%)	X ̅ σ	76.33 17.41	83.31 12.21	89.80 7.08	78.49 13.17	50.77 15.52
	Tortuosity	X ̅ σ	1.16 0.05	1.15 0.03	1.10 0.05	1.14 0.03	1.12 0.03
	623
	624	Figures captions
	625
	626
	627
	628

Velocity (mm.h -1 )

Acknowledgements

The authors thank the Région Hauts-de-France, the Ministère de l'Enseignement Supérieur et de la Recherche and the European fund for regional economic development for their financial support through the attribution of the CPER research project CLIMIBIO. Noémie Deldicq PhD fellowship is funded by the Ministère de l'Enseignement Supérieur et de la Recherche. Dewi Langlet Post-doctoral fellowship is supported by the STARS research project COFFEE of the Région Hauts-de-France and the CPER research project CLIMIBIO. Molecular investigations of spiral-shaped foraminifera phylotype were done within the research project AMTEP, funded by the CNRS-INSU program EC2CO.


Dewi Langlet
email: dewi.langlet@univ-lille.fr

Vincent M P Bouchet

Camille Delaeter

Laurent Seuront

Motion behavior and metabolic response to microplastic leachates in the benthic foraminifera Haynesina germanica

Keywords: Benthic foraminifera, Plastic leachates, Polypropylene, Survival, Behavior, Respiration

Plastic is one of the major sources of pollution in modern oceans. When in seawater, toxic plasticizers (the additives incorporated in plastic polymers during manufacturing processes) typically diffuse and accumulate in sediments and in benthic and pelagic organisms' tissues. These plastic leachates affect survival, behavior and metabolism of various marine metazoans, but little effort was placed in studying their effect on protists. In this contribution we monitored the short-term effect of polypropylene (PP) leachates at both environmentally realistic and chronic concentrations on Haynesina germanica locomotion and metabolism. We found that PP leachates has no lethal nor effects on this species activity.

Taken together, these results suggest that benthic foraminifera may be more resistant than marine metazoans to plasticizers pollutants.

Introduction

Plastics are acknowledged as one of the most ubiquitous and conspicuous sources of pollution of the Anthropocene, especially in the marine environment (W. C. Li et al., 2016). Microplastics (MP) can either be small plastic particles (smaller than 5mm) released in the environment or result from the breakage and aging of macroplastics. They are now considered the most numerically abundant form of solid waste on the planet [START_REF] Eriksen | Plastic Pollution in the World's Oceans: More than 5 Trillion Plastic Pieces Weighing over 250,000 Tons Afloat at Sea[END_REF] and a potential threat to marine ecosystems globally [START_REF] Galloway | Interactions of microplastic debris throughout the marine ecosystem[END_REF]. Hence, they are widely observed from coastal waters to the deep-ocean floor and from tropical to polar regions [START_REF] Barnes | Remote Islands Reveal Rapid Rise of Southern Hemisphere Sea Debris[END_REF][START_REF] Chiba | Human footprint in the abyss: 30 year records of deep-sea plastic debris[END_REF].

Microplastics are also responsible for a range of sub-lethal effects related to their pernicious role as a vector of chemical pollutants. These pollutants leaching from MP to the marine environments originate from the additives compounds (e.g. plasticizers, flame retardant, UV stabilizers, antioxidant, and antistatic molecule) incorporated in plastics during the manufacturing process to modify the plastic polymers physical properties and durability, but also from the chemical compounds already present in the water (i.e. coming from another source of pollution) which are adsorbed at the MP's surface when aging in the environments. Plastic additives such as phthalates, bisphenol A, nonyphenols and brominated flame retardants can reach high concentrations in coastal waters [START_REF] Hermabessiere | Occurrence and effects of plastic additives on marine environments and organisms: A review[END_REF][START_REF] Sánchez-Avila | Organic micropollutants in coastal waters from NW Mediterranean Sea: Sources distribution and potential risk[END_REF] and accumulate in marine organisms tissues [START_REF] Vered | Using solitary ascidians to assess microplastic and phthalate plasticizers pollution among marine biota: A case study of the Eastern Mediterranean and Red Sea[END_REF]. This work specifically focuses on the toxicity of virgin MP leachates since they have recently been identified as one of the most critical threat related to the presence of plastics in the ocean [START_REF] Hahladakis | An overview of chemical additives present in plastics: Migration, release, fate and environmental impact during their use, disposal and recycling[END_REF][START_REF] Paluselli | Phthalate Release from Plastic Fragments and Degradation in Seawater[END_REF]. The toxic effects of virgin microplastic leachates have been reported in various marine faunal taxa, such as barnacles (H.-X. Li et al., 2016), crustacean larvae [START_REF] Lithner | Leachates from plastic consumer products -Screening for toxicity with Daphnia magna[END_REF], gastropods [START_REF] Seuront | Microplastic leachates impair behavioural vigilance and predator avoidance in a temperate intertidal gastropod[END_REF], bivalves [START_REF] Ke | Impacts of leachates from single-use polyethylene plastic bags on the early development of clam Meretrix meretrix (Bivalvia: Veneridae)[END_REF] and sea urchins [START_REF] Oliviero | Leachates of micronized plastic toys provoke embryotoxic effects upon sea urchin Paracentrotus lividus[END_REF]. Desorption of these chemicals in the surrounding environment causes a range of harmful effects on embryo development, reproduction, behavior or induce genetic aberrations (see [START_REF] Oehlmann | A critical analysis of the biological impacts of plasticizers on wildlife[END_REF] for a review).

To date and to the best of our knowledge, there is still a critical lack of information available on effect of microplastic on protists, despite a recent urge to fill this knowledge gap [START_REF] Rillig | Microplastic and soil protists: A call for research[END_REF].

However, MP ingestion is likely to be common in protists [START_REF] Setälä | Ingestion and transfer of microplastics in the planktonic food web[END_REF], including foraminifera [START_REF] Ciacci | Nanoparticle-Biological Interactions in a Marine Benthic Foraminifer[END_REF], and subsequently negatively impact their metabolic activity [START_REF] Ciacci | Nanoparticle-Biological Interactions in a Marine Benthic Foraminifer[END_REF][START_REF] Su | Microplastic exposure represses the growth of endosymbiotic dinoflagellate Cladocopium goreaui in culture through affecting its apoptosis and metabolism[END_REF]. Benthic foraminifera were targeted in this work due to their importance in the structure and function of benthic ecosystems [START_REF] Geslin | Oxygen respiration rates of benthic foraminifera as measured with oxygen microsensors[END_REF][START_REF] Gooday | The Role of Benthic Foraminifera in Deep-Sea Food Webs and Carbon Cycling[END_REF], their ability to respond to various types of pollutant both under laboratory conditions [START_REF] Denoyelle | Innovative use of foraminifera in ecotoxicology: A marine chronic bioassay for testing potential toxicity of drilling muds[END_REF][START_REF] Ernst | Benthic foraminiferal response to experimentally induced Erika oil pollution[END_REF][START_REF] Nigam | Effects of sudden stress due to heavy metal mercury on benthic foraminifer Rosalina leei: Laboratory culture experiment[END_REF] and in situ (see [START_REF] Alve | Benthic foraminiferal responses to estuarine pollution: a review[END_REF] for a review). Like any benthic organisms, they are directly exposed to the range of pollutants, including microplastics [START_REF] Schwarz | Sources, transport, and accumulation of different types of plastic litter in aquatic environments: A review study[END_REF] which cannot be degraded by bacteria [START_REF] Nauendorf | Microbial colonization and degradation of polyethylene and biodegradable plastic bags in temperate fine-grained organic-rich marine sediments[END_REF] and therefore accumulate in coastal sediments [START_REF] Galgani | Accumulation of debris on the deep sea floor off the French Mediterranean coast[END_REF]. In this context, the present study assessed the potential short-term effects of the leachates from virgin polypropylene pellets considered at both environmentally realistic and chronic concentrations on the stress level of the benthic foraminifera Haynesina germanica. Specifically, movement behavior [START_REF] Seuront | Microplastic leachates impair behavioural vigilance and predator avoidance in a temperate intertidal gastropod[END_REF] and respiration rate [START_REF] Su | Microplastic exposure represses the growth of endosymbiotic dinoflagellate Cladocopium goreaui in culture through affecting its apoptosis and metabolism[END_REF] were considered as proxies of the stress level of H. germanica following an exposure to polypropylene leachates. This foraminiferal species and this plastic polymer were specifically chosen for their high abundances along the French coast of the eastern English Channel (Armynot du [START_REF] Armynot Du Châtelet | Benthic foraminifera in transitional environments in the English Channel and the southern North Sea: A proxy for regional-scale environmental and paleo-environmental characterisations[END_REF][START_REF] Francescangeli | Does elevation matter? Living foraminiferal distribution in a hyper tidal salt marsh (Canche Estuary, Northern France)[END_REF][START_REF] Hermabessiere | Microplastic contamination and pollutant levels in mussels and cockles collected along the channel coasts[END_REF].

Material and methods

Haynesina germanica collection

Surface sediment (0-1cm) from Boulogne-sur-Mer harbor mudflat (eastern English Channel, 50°43'06.4"N 1°34'22.0"E) was sampled in June 2019 and stored in 100 ml polypropylene containers.

Sediment was kept at ambient temperature during transportation and placed within one hour in English Channel seawater aquarium (12°C and 35 PSU) under a natural day-light cycle conditions until the experiment took place. Sediment was sieved over a 125 µm stainless-steel mesh and colored-cytoplasm Haynesina germanica were subsequently sorted. Only the active specimens (i.e. leaving a displacement track on a thin layer of sediment) were considered as living and selected for the experiment. Living individuals were transferred in artificial seawater (ASW) prepared with 35 grams of sea salt (RedSea Fish Farm, Israel) per liter of Milli-Q water (Merck Millipore, Germany) and gently cleaned with a brush to remove any surrounding particles.

Experimental conditions

Both behavioral experiments and metabolic measurements were conducted exposing H. germanica to artificial seawater as control and to microplastic leachates seawater. Microplastic leachates seawater was prepared from commercially available virgin polypropylene pellets (typically 3.3 to 4.7 mm in diameter; Pemmiproducts, Germany) mixed with artificial seawater at a concentration of 20 ml and 200 ml of pellets per liter (hereafter respectively referred to as PP20 and PP200) and aerated for 24 h before the beginning of the experiments following the protocol developed in [START_REF] Seuront | Microplastic leachates impair behavioural vigilance and predator avoidance in a temperate intertidal gastropod[END_REF] to monitor the effect of plastic leachates on a marine gastropod. Although not quantified in this experiment, polypropylene leachates typically contain bisphenol A, octylphenol and nonylphenol [START_REF] Hermabessiere | Occurrence and effects of plastic additives on marine environments and organisms: A review[END_REF].

Behavioral experiment

For each experimental condition (i.e. control seawater and the two leachate treatments PP20 and PP200), 15 living Haynesina germanica (maximum diameter range: 300-440 µm) were spread randomly on the bottom of 15-cm wide glass-Petri dishes filled with ASW, PP20 or PP200 (Figure 1). Petri dishes were placed in a light and temperature-controlled incubator (MIR-154, Panasonic, Japan) set at 12°C. The movements of H. germanica were recorded every 10 minutes using a digital camera (V1 with a 10-30 mm lens, Nikon, Japan) under homogenous dim light conditions (photosynthetically active radiation <100 µmol photon m -² s -1 ; SA-190 quantum sensor, LI-COR, USA) provided by a horizontal array of LEDs (YN-160 III, Yongnuo, China). Each experiment lasted 10 hours.

Images were compiled in the open-source image analysis software Fiji [START_REF] Schindelin | Fiji: an open-source platform for biological-image analysis[END_REF] and (x,y) coordinates were measured for each individual H. germanica using the Manual Tracking plugin (Figure 1). The distance travelled (Dt) by each individual between two images was calculated as:

Dt = √ [(xt - xt+1) 2 + (yt -yt+10) 2 ]
where (xt, yt) and (xt+10, yt+10) are the coordinates between two successive images taken at 10-minute intervals. The total distance travelled in 10 hours was calculated from the sum of all Dt and subsequently converted to locomotion speed (mm h -1 ). These behavioral parameters were measured using trajr package (McLean and Skowron Volponi, 2018) in R v.3.5.3 (R Core Team, 2019).

Trajectories complexity was assessed using fractal analysis. The fractal dimensions of foraminifera trajectories were estimated following the box dimension method [START_REF] Seuront | On uses, misuses and potential abuses of fractal analysis in zooplankton behavioral studies: A review, a critique and a few recommendations[END_REF][START_REF] Seuront | Fractals and Multifractals in Ecology and Aquatic Science[END_REF].

Respiration measurements

Five H. germanica specimens were randomly selected from the individuals used in behavioral experiments and transferred from the Petri dish to a 1-mm wide glass microtube containing the three tested seawater (ASW for control and PP20 and PP200 to test the effect of polypropylene leachates).

Steady-state oxygen consumption gradient (dC/dz, in pmol cm -4 ) in the millimeter above the organisms were measured using a 50-µm Clark-type oxygen microelectrode (Unisense, Denmark). Oxygen fluxes (J, pmol cm -1 s -1 ) in the microtube were calculated using Fick's first law of free diffusion as J = D´dC/dz [START_REF] Li | Diffusion of ions in sea water and in deep-sea sediments[END_REF] with D being the free diffusion coefficient for oxygen (D = 1.6 10 -5 cm 2 s -1 at 12°C and 35PSU). Individual respiration rate (R, pmol ind -1 day -1 ) was then calculated as R = J´S/n (considering the microtube inner section S = 7.9 10 -3 cm 2 and the number of individuals n = 5). Note that our measurements were conducted on groups of 5 individuals both to take into account the low individual respiration rate of benthic foraminifera and to overpass the sensor detection limit [START_REF] Geslin | Oxygen respiration rates of benthic foraminifera as measured with oxygen microsensors[END_REF]. Respiration rate measurements were replicated 6 times in control seawater and triplicated in both P20 and PP200 leachate treatments. Since respiration is influenced by individual size, specimens were measured to normalize the respiration rates by the foraminiferal biovolume (8.10 6 µm 3 in average; estimated following [START_REF] Geslin | Oxygen respiration rates of benthic foraminifera as measured with oxygen microsensors[END_REF]. All respiration measurements were carried out in the dark in a 12°C temperature-controlled water bath (Huber CC-K12, Germany).

Data analysis

Due to our small size samples, the effect of the 3 experimental conditions on movement speed, fractal dimension and foraminiferal respiration rate was tested using Kruskal-Wallis test [START_REF] Hollander | Nonparametric Statistical Methods[END_REF]) in R v.3.5.3 (R Core Team, 2019).

Results

Image analysis show that 100% of the individuals tested were moving throughout the experiments and were still alive after being exposed to PP20 and PP200 for 10 hours. Haynesina germanica moved over distances ranging from 7 to 32 mm, at locomotion speed ranging from 0.7 to 3.2 mm h -1 , 1.6 to 2.8 mm h -1 and 1.1 to 3.1 mm h -1 for ASW, PP20 and PP200 respectively (Figure 1A). All the trajectories considered in this work were significantly described in terms of fractal dimensions that ranged between 1.02 and 1.13 with average values of 1.07, 1.06 and 1.06 in ASW, PP20 and PP200 respectively (Figure 1B). Finally, respiration rate ranged from 41 to 114 10 -6 pmol µm -3 day -1 in the ASW control, from 66 to 164 10 -6 pmol µm -3 day -1 in PP20 and from 84 to 98 10 -6 pmol µm -3 day -1 in PP200 (Figure 1C).

Neither locomotion speed, fractal dimensions nor respiration rates exhibited any significant differences between the three experimental conditions (Kruskall-Wallis-test: p>0.05; Table 1).

Discussion

The additives leaching from polypropylene (i.e. essentially antioxidant additives such as bisphenol A, octylphenol and nonylphenol; [START_REF] Hermabessiere | Occurrence and effects of plastic additives on marine environments and organisms: A review[END_REF] have lethal effects on mollusks [START_REF] Oehlmann | Effects of Endocrine Disruptors on Prosobranch Snails (Mollusca: Gastropoda) in the Laboratory. Part I: Bisphenol A and Octylphenol as Xeno-Estrogens[END_REF], barnacle larvae (H.-X. Li et al., 2016), amphibians [START_REF] Hogan | Exposures to Estradiol, Ethinylestradiol and Octylphenol Affect Survival and Growth of Rana pipiens and Rana sylvatica Tadpoles[END_REF], annelids and crustaceans [START_REF] Staples | Characterizing the effects of bisphenol A on sediment-dwelling benthic organisms[END_REF]. In contrast, the present work showed a lack of any lethal effect on Haynesina germanica of PP leachates.

Similarly, no sublethal effect were perceptible through H. germanica locomotion and metabolism. Specifically, locomotion speed was nearly 2-fold lower than those reported previously on the same species (here ~2 mm h -1 vs. ~4 mm h -1 in [START_REF] Seuront | The devil lies in details: new insights into the behavioural ecology of intertidal foraminifera[END_REF] probably due to the lower experimental temperature (12°C here vs. 22°C in [START_REF] Seuront | The devil lies in details: new insights into the behavioural ecology of intertidal foraminifera[END_REF] since decreasing temperature is known to reduce foraminiferal activity [START_REF] Bradshaw | Laboratory experiment on the ecology of foraminifera[END_REF]. Our results nevertheless clearly indicated that PP leachates did not affect foraminiferal behavior (Figure 1A,B). This is consistent with the observed lack of behavioral impairment in the intertidal gastropod Littorina littorea; as PP20 leachates impaired their chemosensory ability without impacting their neuromuscular abilities [START_REF] Seuront | Microplastic leachates impair behavioural vigilance and predator avoidance in a temperate intertidal gastropod[END_REF]. In turn, our results contrast with previous evidence that PP-plasticizers reduce fish larvae velocity in the first days after hatching [START_REF] Inagaki | Low dose exposure to Bisphenol A alters development of gonadotropin-releasing hormone 3 neurons and larval locomotor behavior in Japanese Medaka[END_REF][START_REF] Wang | Bisphenol A affects axonal growth, musculature and motor behavior in developing zebrafish[END_REF] and negatively impact adult-fish locomotion and reproductive behavior after at least 2 months of exposure [START_REF] Gray | Reproductive success and behavior of Japanese medaka (Oryzias latipes) exposed to 4-tert-octylphenol[END_REF][START_REF] Xia | Effects of chronic exposure to nonylphenol on locomotor activity and social behavior in zebrafish (Danio rerio)[END_REF]. Note that the apparent discrepancy observed between the aforementioned studies and our experiment might be due to differences in exposure duration as reported in Table 2.

Plasticizers have previously been reported to lead to an immediate increase followed by a decrease in respiration rates with rising phenols concentration in mollusks [START_REF] Levine | Metabolic responses to acute toxicity of alkylphenols and alkylphenol polyethoxylates in Elliptio complanata measured by calorespirometry[END_REF]. They can also induce energetical impairments in crustaceans anaerobic metabolism in less than 2 days [START_REF] Nagato | Metabolomics reveals energetic impairments in Daphnia magna exposed to diazinon, malathion and bisphenol-A[END_REF]. In contrast, we did not find any significant effects of PP leachates on foraminiferal respiration, even under very high leachates concentrations, i.e. PP200 (Figure 1C). To the best of our knowledge, the only other study that investigated the effect of PP leachates on a unicellular organism found a decrease in dinoflagellate photosynthesis [START_REF] M'rabet | Impact of two plastic-derived chemicals, the Bisphenol A and the di-2-ethylhexyl phthalate, exposure on the marine toxic dinoflagellate Alexandrium pacificum[END_REF] after 1 day of exposure, in accordance with the reduced growth and oxygen production observed in the marine cyanobacteria Prochlorococcus following a 24h-long exposure to leachates of common plastic items (i.e. HDPE shopping bags and PVC matting; [START_REF] Tetu | Plastic leachates impair growth and oxygen production in Prochlorococcus, the ocean's most abundant photosynthetic bacteria[END_REF]. Note that, conversely to M'Rabet et al. ( 2018) who specifically worked with bisphenol A (Table 2), we did not have any control on the composition of the PP leachates. This is a clear limitation of our study that will need to be improved in future works.

Overall, both the behavioral and metabolic activity data gathered in this preliminary study indicate that the benthic foraminifera Haynesina germanica do not respond to MP unlike other unicellular and metazoan organisms. Though this is highly speculative, this observation may suggest that their resistance to leachates from virgin PP might induce a competitive advantage for benthic foraminifera. Such a competitive advantage for foraminifera has previously been observed in relation to some anthropo-natural phenomena such as organic-matter enrichment and anoxia [START_REF] Langlet | Foraminiferal survival after long-term in situ experimentally induced anoxia[END_REF][START_REF] Stachowitsch | Coastal hypoxia and anoxia: a multi-tiered holistic approach[END_REF]. Note, however, that the observed lack of effect of MP on foraminiferal activity may also be due to the relatively short-term exposure used in our experiments. More fundamentally, the diversity of methods reported in the literature related to the type of polymer considered, the use of unidentified leachates or a specific plasticizer, the pollutant concentrations, the duration of exposure as well as the biology of the organisms considered (Table 2) dramatically prevent to reach a general consensus when comparing the effect of MP on foraminifera with other organisms in our study. In this context, future experiments aiming to assess the effects of MP leachates on benthic foraminifera should benefit from (i) being more specific about the acute or chronic nature of their exposure, and (ii) identifying and quantifying the plasticizers used or present in the leachates. Finally, further work is also needed to assess the potential effects of leachates from (i) weathered PP in particular as they have shown to have significantly stronger effects that virgin plastics [START_REF] Bejgarn | Toxicity of leachate from weathering plastics: An exploratory screening study with Nitocra spinipes[END_REF][START_REF] Gandara E Silva | Leachate from microplastics impairs larval development in brown mussels[END_REF][START_REF] Kedzierski | Threat of plastic ageing in marine environment. Adsorption/desorption of micropollutants[END_REF][START_REF] Nobre | Assessment of microplastic toxicity to embryonic development of the sea urchin Lytechinus variegatus (Echinodermata: Echinoidea)[END_REF][START_REF] Seuront | Microplastic leachates impair behavioural vigilance and predator avoidance in a temperate intertidal gastropod[END_REF], (ii) different plastic polymers (H.-X. Li et al., 2016;[START_REF] Lithner | Comparative acute toxicity of leachates from plastic products made of polypropylene, polyethylene, PVC, acrylonitrile-butadiene-styrene, and epoxy to Daphnia magna[END_REF][START_REF] Lithner | Leachates from plastic consumer products -Screening for toxicity with Daphnia magna[END_REF][START_REF] Tetu | Plastic leachates impair growth and oxygen production in Prochlorococcus, the ocean's most abundant photosynthetic bacteria[END_REF] and (iii) ingested plastic particles [START_REF] Ciacci | Nanoparticle-Biological Interactions in a Marine Benthic Foraminifer[END_REF]. Table 1 caption: results of the Kruskal-Wallis statistical analyses testing the effect of the experimental conditions (ASW as a control, PP20 and PP200) on the three measured response variables.

Response variable Kruskal-Wallis Χ² degrees of freedom p-value Locomotion speed 1.6 2 0.44 Fractal dimension 3.9 2 0.14 Respiration rate 1.3 2 0.52

Figure 1

 1 Figure 1 caption: A and B: schematic representation of the experimental setup with lateral view (A) and top view (B) of the position of the foraminifera (black ovoid shape) placed on the petridish. C: photograph of the initial position of the 15 individuals used in ASW control conditions. D: example of 3 extracted trajectories for ASW (full black line), PP20 (full grey line) and PP200 (dotted grey line).

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Figure 2 :

 2 Figure 2: Locomotion speed (A), fractal dimension (B) and respiration rate (C) of Haynesina germanica under the three experimental conditions (ASW: artificial seawater, i.e. control conditions; PP20 and PP200: seawater prepared with 20 and 200 ml l -1 polypropylene pellets, respectively). The box represents the first, second and third quartiles and the whiskers extend to 1.5 times the interquartile range. Values outside of this range are represented by open circles.

Acknowledgments

The authors thank the Région Hauts-de-France, and the Ministère de l'Enseignement Supérieur et de la Recherche (CPER Climibio), and the European Fund for Regional Economic Development for their financial support. Dewi Langlet Post-doctoral fellowship is supported by the STARS research project COFFEE of the Région Hauts-de-France and the CPER research project CLIMIBIO. We also thank Lionel Denis, Dominique Menu and Laurent Brutier for their logistical support. We thank the two anonymous reviewers and the editor Pablo Munguia for their constructive comments who helped improving significantly this manuscript.