M.-N Avettand-Fènoël
email: marie-noelle.avettand-fenoel@univ-lille.fr

M Marinova

R Taillard

Atomic scale characterization of a pure Al -galvanized steel spot magnetic pulse joint interface

Keywords: Aluminum -galvanized steel spot magnetic pulse welding, Interface, Microstructure, HAADF, HR(S)TEM, STEM-EDX

The bonding interface of pure Al -galvanized steel spot magnetic pulse welds was characterized at the atomic scale. The Al-Zn interface microstructure is worthy of attention as it contains a few micrometers thick biphased layer. This singular layer consists of an aggregate of grains with a 200 to 400 nm size containing islands of either Al or Zn solid solutions oversaturated in Zn or Al, respectively. Inside a grain, some mottled contrasts with a size in-between 5 and 10 nm correspond to fluctuant solute contents. Besides, some native oxides have been dissolved at the interface suggesting the incorporation of oxygen atoms in the biphased layer. Large and elongated (Al,Fe,Si) particles primitively contained in the base Al were also dissolved which gives rise to some round shaped Si particles in the biphased layer. All these microstructural features and changes are consistent with a ballistic origin. Due to both the absence of solidification defects and the rather planar morphology of the bonding interface, the process is expected to occur at the solid state.

Introduction

Dissimilar assemblies with partial replacement of steel by light materials are of crucial importance in automotive industry for both energy cost-savings and reduction of exhaust fumes required for environment protection. Galvanized steel is used in automotive industry for corrosion resistance. However joining aluminum with galvanized steel remains a challenge since these materials are characterized by different melting points, thermal conductivities, coefficients of thermal expansion, mechanical properties and flow laws [1-3]. In addition, the high chemical affinity between Al and Fe [START_REF] Brandes | Smithells Metal Reference Book[END_REF] may lead to the precipitation of brittle intermetallic compounds (IMCs) [START_REF] Möller | Handbook of laser welding technologies, Chapter 9: Laser welding and brazing of dissimilar materials[END_REF][START_REF] Shah | Review of research progress on aluminium-steel dissimilar welding[END_REF] at the joint interface. Diffusion is all the more favored that joining proceeds at the liquid state in the case of welding or brazing. The presence of Zn at the steel surface, with a melting point far lower than those of Al and steel, may further promote Al and Fe diffusion [START_REF] Kutsuna | Laser roll welding of dissimilar metal joint of zinc coated steel and aluminum alloy[END_REF]. The formation of thick continuous IMCs at a joint interface is well known to be harmful for the ductility and then the soundness of the joint [START_REF] Tanaka | Comprehensive analysis of joint strength for dissimilar friction stir welds of mild steel to aluminum alloys[END_REF]. The use of solid state thermomechanical processes is thus particularly recommended since they should limit the formation of IMCs. Nevertheless, the latter processes are characterized by high strains and strain rates which generate very high densities of vacancies favoring again atomic diffusion [6-97-10].

High velocity impact welding techniques, among which Magnetic Pulse Spot Welding (MPSW), are smart processes able to solve the problem of dissimilar joining. Derived from high speed magnetic pulse forming, MPSW is an environmental friendly and energy saving solid state process which only lasts a few microseconds [START_REF] Kapil | Magnetic pulse welding: an efficient and environmentally friendly multi-material joining technique[END_REF]. This time is far shorter than the duration of usual solid state thermomechanical processes, like friction or friction stir welding processes, which are characterized by welding cycles of a few seconds. MPW is a shock welding process where the current pulse passing through an electromagnetic coil produces high magnetic forces. Such forces propel a flyer sheet towards a static sheet, which leads to the bonding of both sheets [START_REF] Kapil | Magnetic pulse welding: an efficient and environmentally friendly multi-material joining technique[END_REF]. MPSW brings about a low heat input to materials, which should limit the formation of IMCs and reduce the heat affected zone extent [10-1211-13].

With regards to the Al-steel system, various MPW joints were obtained in a tubular [13-2314-24] or planar [12,24-2913,25-30] configuration. No work reports MPSW between galvanized steel and aluminum except a paper of the present authors who have considered magnetic pulse spot joining of hot dip galvanized steel with AA1050 pure aluminum [START_REF] Avettand-Fènoël | Effect of steel galvanization on the microstructure and mechanical performances of planar magnetic pulse welds of aluminum and steel[END_REF]. The architecture of the interface was investigated at a mesoscopic scale. Going from pure Al to steel, the interface is constituted of four successive layers: (i) a 10 at.% Zn oversaturated Al solid solution, (ii) an (Al+Zn) aggregate of Al and Zn grains, (iii) a Zn layer and (iv) the inhibition layer. The total thickness of the (Al+Zn) mixture and of the remaining Zn layer corresponds to the initial thickness of the Zn coating on steel.

The present paper characterizes the (Al+Zn) mixed layer at the atomic scale. This in-depth investigation will provide clues about the mechanism of formation of the bi-phased layer which can be deleterious for the joint mechanical properties. This joint indeed fractured in this bi-phased layer during shear-tensile tests [START_REF] Avettand-Fènoël | Effect of steel galvanization on the microstructure and mechanical performances of planar magnetic pulse welds of aluminum and steel[END_REF], very likely because of the low plasticity of Zn.

Experimental procedure

The base materials are 40 mm wide coupons of AA1050 aluminum (90 mm long and 0.5 mm thick, hardness of 40 HV 0.05 ) and of hot-dip galvanized cold rolled DP450 steel (110 mm long and 1.17 mm thick, hardness of 160 HV 0.05 ). The galvanized coating on DP450 steel is 11 µm thick. The chemical compositions of the base materials supplied by the provider are indicated in Table 1. Aluminum which is the better electrical conductor constitutes the flyer sheet whereas steel with its higher yield strength is the target workpiece. A 8 mm wide and 12 mm long hump was deep drawn on the Al flyer sheet. The sheets were cleaned with acetone and stacked over a lap-length of 50 mm. They were finally joined by magnetic pulse spot welding with a discharge energy of 10 kJ. The setting is displayed in figure 1. The generator is characterized by a capacitance of 408 µF, an inductance of 100 nH and a resistance of 14 mΩ. The Cu inductor presents a 20 mm long and 2.7 mm thick active zone, a 44 nH inductance and a 0.1 mΩ resistance. An insulating sheet with a thickness of 0.1 mm is inserted at the Al -inductor interface. Then the stand-off distance corresponds to the hump depth that is 1.5 mm. Further details are given in the previous paper [START_REF] Avettand-Fènoël | Effect of steel galvanization on the microstructure and mechanical performances of planar magnetic pulse welds of aluminum and steel[END_REF]. The transverse cross section of the joints was prepared using standard procedure and finally polished with 1 µm diamond paste.

In order to complete its previous mesoscopic analyses [3031], the joint interface was studied by high resolution scanning transmission electron microscopy (STEM) coupled to Xray energy dispersive spectroscopy (EDX). The analysis was achieved by means of a TITAN Themis 300 S/TEM with a probe aberration corrector and monochromator, allowing spatial resolution of 70 pm and energy resolution of 150 meV. The microscope is equipped with a super-X windowless 4 quadrant SDD (silicon drift detector) detection system for the STEM-EDX mapping and with several annual dark field detectors. The experiment has been performed with a 0.5 nm probe size, a convergence angle of 21 mrad and a probe current of approximately 100 pA. For the high angle annular dark field (HAADF) images collection angles have been between 50 and 200 mrad. Orientation maps were acquired on a Tecnai G2 20 Twin microscope operating at 200 kV and equipped with a LaB 6 filament and the ASTAR™ tool. The TEM was set in micro-beam mode (5 nm probe size) and smallest condenser aperture (50µm) with precessed illumination (precession angle of 1 deg), a scanning step size of 10 nm and an exposure time of 20 ms. The thin foils (figure 2) have been prepared by the Focused Ionic dual Beam (FIB) technique. During cutting, the thin foil edge facing the ion beam was protected by a 3.5 µm thick deposit of platinum. A 300 pA beam intensity was applied for the final thinning.

Results

As aforesaid, from steel to pure aluminum, the interfacial zone is successively composed of (i) a 150 nm thick inhibition layer, (ii) a 3 µm thick Zn layer, (iii) a 8 µm thick (Al+Zn) mixed layer (figure 2) and (iv) an Al solid solution containing 10 at.% of Zn [START_REF] Avettand-Fènoël | Effect of steel galvanization on the microstructure and mechanical performances of planar magnetic pulse welds of aluminum and steel[END_REF].

Given the too small thickness of the inhibition layer compared to the size of the X-rays irradiated zone, the determination of its chemical composition was not possible by EDX. It is however well known that the inhibition layer is generally constituted of Fe 2 Al 5 Zn x and FeAl 3 Zn x intermetallic compounds [START_REF] Marder | Metallurgy of zinc coated[END_REF].

Figures 4a, 4b and 4c depict the ragged morphology of the microstructure in the Al + Zn mixed zone. This layer consists in a random distribution of aggregates of either Al-rich or Zn rich grains. The 50 to 450 nm size of the latter grains is markedly smaller than the 1 µm mean grain size of both the Al and Zn base material. It is thus concluded that recrystallization has occurred during either welding or the subsequent thin foil preparation by ion polishing.

Besides, it is worth noting the presence of oxygen as well as of silicon bearing spheroidal areas in the (Al+Zn) mixed zone (figure 3). In addition, silicon is not linked to iron in this intermediate layer (figure 3). The mean chemical composition of these O and Si rich particles (marked by a white arrow in figure 4) is 47.6% Al-28.3% Si-24.1% Zn (at.%) according to the EDX analyses. Due to the larger volume of the analyzed zone compared to the particles' size, their Al and Zn contents are however overestimated. The presence of Al and Zn may also be an artifact due to the particle embedment within the mixed (Al+Zn) layer. With regard again to the intermediate (Al + Zn) mixed zone, the micrographs at a microscopic (figures 3 and 4) and at an atomic scale (figures 5 and 6) display some mottled contrasts in both Al and Zn rich zones. These changes of contrast are randomly dispersed. The contrast in HAADF images is proportional to the sample thickness and to the atomic number Z to the power of 1.7 [3233] of the elements present within the material. Thus areas with higher Zn (Z=30) concentration will appear brighter than Al (Z=13) rich zones. In order to understand further the variations of contrast in the HAADF images we have looked through the change in chemical composition by EDX coupled to local changes in crystal lattice. The variation of the HAADF contrast does not correspond to change in crystal orientation as confirmed by FFT (Fast Fourier Transform) patterns in the respective areas. No subgrains boundaries are detected either. Some examples are depicted in figures 5 and 6. Figure 5 shows the atomic column arrangement in an Al grain oriented along a [-112] zone axis. According to EDX analyses, the grain is locally enriched with Zn. Its atomic content is in between 3.5 at% and 7.8 at%. It then suggests the existence of (Al) Zn solid solutions. Figures 6 and7 display the atoms arrangement along the (0001) basal plane of Zn. The EDX analysis reveals the existence of a (Zn) Al solid solution with Al content in-between 9.4 at% and 22.4 at.%.

The analysis of the atomic resolution HAADF images (figures 5 and 6) gives the distances and angles between the atom planes which identifies their Miller indexes and their zone axis. The precision on interplanar distances and lattice parameters amounts to 0.04 nm. Then the Zn and Al apparent crystal lattice parameters are deduced from the data. The experimental atomic volumes have finally been calculated by taking into account the crystallographic data and the difference of compactness of the two crystal lattices. Although Al and Zn present distinct crystal lattice structures, respectively face cubic centered and hexagonal, the Vegard's law has been applied to the Al-Zn system. It is worth noting that this reasoning has already been used in literature for elements with different crystal structures [START_REF] Cahn | Physical Metallurgy, Part I[END_REF]. From the experimental atomic volumes and the Vegard's law, it has been possible to estimate the amount of Zn (or Al) in solid solution in Al (or in Zn), respectively (red and green squares in figure 8). The latter contents compared with those determined locally in the same areas by EDX are summarized in figure 8. The analysis of the inter-planar distances confirms the existence of Al and Zn solid solutions but the solutes contents determined by application of the Vegard's law are generally slightly higher, in particular for the Al solid solutions, than those measured by EDX (figure 8). Figure 8 In addition, for a given crystal orientation, various atom spacings have been observed in particular in the Al rich zones (see for instance the L profile in figure 9). The distances of 0.218 and 0.260 nm between the atoms rows are close to the 0.234 (0.2025) nm spacing between {111} ({002}) planes in pure Al.

The accurate observation of high resolution HAADF micrographs also puts into evidence some distortion (see for instance inside the yellow rectangle in figure 9). The distortion may result either from the presence of solute atoms in Al which leads to crystal lattice accommodation or from the proximity of strain fields due to lattice defects (dislocations, disclinations …), internal stresses or from an undulating thin foil.

Finally, figure 10 displays the grains orientation map at the interface between the AA1050 parent metal and the (Al+Zn) mixed zone. By comparison with the Al (figures 10b and c) and Zn (figures 11b and c) base materials, the (Al + Zn) mixed zone is characterized by important changes of grain orientation in both kinds of grains. In agreement with the various Al grains orientations in the mixed zone (figure 10b), figure 10c depicts additional and less intense poles.

Discussion

The analysis of the AA1050 -galvanized DP450 steel magnetic pulsed joint interface puts into evidence the successive presence of an inhibition layer, of a 3 µm thick Zn layer and of a 8 µm thick (Al + Zn) mixed layer from the steel to pure aluminum (figure 2).

In spite of its IMC nature, and thus brittle behavior, the inhibition layer has not been seriously fragmented during welding (see Figure 3). This impact resistance has very likely to do with both its reduced thickness and the hydrostatic state of stresses generated by its Al and Zn continuous surroundings [3536].

Incomplete destruction of the primitive Zn layer

The noticeable remains of the Zn layer at the joint interface can first be questioned. It is however consistent with literature results given the 11 µm initial thickness of the Zn layer. This width is expected to exceed the usual close to 10 µm thickness of the material layer ejected to form the MPW jet which is constituted of materials from both sides of the interface [START_REF] Stern | Jet composition in magnetic pulse welding: Al-Al and al-Mg couples[END_REF]. The existence of a pure Zn layer without any defects of shrinkage and of a rather planar interface after welding proves that Zn has not melted during MPW. This result agrees with simulation data according to which a 200°C temperature is reached during MPW of Al alloy [START_REF] Miranda | Magnetic pulse welding on the cutting edge of industrial applications[END_REF]. It however contradicts some explanations [3738] and multiphysics numerical simulation data according to which temperatures, as high as 1200°C, are reached during MPW of Al alloy [START_REF] Sapanathan | In situ metallic porous structure formation due to ultra high heating and cooling rates during an electromagnetic pulse welding[END_REF]. The absence of Al-Zn eutectic further pleads for a solid state process.

(Al+Zn) mixed layer

Origin of the (Al+Zn) interfacial layer

As aforesaid, the origin of the 8 µm thick (Al+Zn) mixed layer must be considered. Two mechanisms may be proposed a priori, namely a solid state diffusion mechanism and/or a ballistic mechanism. a) Diffusion mechanism -The diffusion coefficient of Zn in Al amounts to 2.17×10 -17 cm²/s and 3.63×10 -10 cm²/s at 110 and 415°C, respectively [4] while the diffusion coefficient of Al in Zn is equal to 3.16×10 -9 cm²/s at 300°C [START_REF] Cui | Assessment of atomic mobilities in fcc Al-Zn and Ni-Zn alloys[END_REF]. According to these data, the diffusion distances of Al and Zn would be lower than 1.78×10 -3 and 1.47×10 -7 µm in Zn and Al for the 10 µs duration of MPSW. These distances are far smaller than the 8 µm thickness of the (Al+Zn) mixed layer. Solid state diffusion alone, even if promoted by the high strain and high strain rates inherent to MPSW, cannot explain the distance crossed by the Al and Zn atoms during MPW. High strains and strain rates actually generate very high densities of vacancies [START_REF] Militzer | Modelling the effect of deformation-induced vacancies on segregation and precipitation[END_REF]. Thus the (Al+Zn) layer thickness cannot result from single atom diffusion during MPSW. The actual microstructure may however result from the metallographic sample preparation. The ion milling, used to prepare the thin foil, is expected to proceed at a temperature close to 400°C for 3 hours [4041] which should entail Al and Zn diffusion over distances of about 58.0 and 19.8 µm, respectively. These rough estimations exceed the 8 µm thickness of the (Al+Zn) mixed (Al+Zn) layer. Complementary experiments are therefore planed in order to conclude on this eventual effect of post-welding. b) Ballistic mechanism -In the present case, Al (low density 2.7 g/cm 3 , high melting temperature 660°C) and Zn (high density 7.1g/cm 3 , low melting temperature 419°C) very likely present a similar sensitivity to ejection during MPSW like for the couple Al -Mg for which the jet is essentially constituted of the lighter and lower melting point metal that is Al. A jet comprising the Al and Zn fragments generated by the disintegration of the surfaces of the AA 1050 and Zn coating under the high pressure impact at high velocity is therefore formed. These fragments are of various sizes. The largest ones present a 200 to 400 nm size, while others are either nanometric clusters of atoms or even isolated atoms. Some oxide particles are also extracted from the surfaces and dragged in this chaotic material flow. In close contact with the surface of material not affected by the jet, the flow of a part of the resulting ballistic mixture is then very likely stopped by the surfaces asperities created by the former extracted fragments. The various fragments are propelled towards each other with a huge pressure up to 10 5 MPa [26,37,4327,38,44]. Under such a high pressure, the largest fragments are deformed and their atoms are forced to share and exchange electrons, leading to a compact agglomerate of fragments. Thus a part of the ballistic mixture remains trapped at the joint interface and constitutes the mixed (Al+Zn) layer. This interpretation is consistent with (i) the absence of crystal orientation relationships between the Al and Zn rich grains constituting the mixed (Al+Zn) layer and (ii) the numerous miscellaneous crystal orientations of these grains (figure 10). The ballistic explanation is further consistent with literature results which showed some Fe fragments and discrete Al x Fe y intermetallic particles [2627] or amorphous fragments [4445] at Al/steel and Al/metallic glass MSW joint interfaces, respectively.

In addition, the (Al+Zn) mixed area contains some 300 nm sized oxygen and silicon bearing particles with a spheroidal morphology (figure 4) which differs from the elongated shape of the 1 µm long and 100 nm wide Fe and Si rich precipitates present in the primitive AA 1050 alloy (see yellow arrows in figure 2). Silicon as well as iron is an aluminum impurity. In the primitive aluminum alloy, and as it is the rule for raw Al 1050 alloys [4546], some Al X Fe Y Si Z precipitates are detected [3031] while silicon is not linked to iron in the mixed zone (figures 3 and 4). The presence of Si rich particles suggests that the Fe and Si bearing precipitates rich were dissolved during MPW in spite of their large dimensions. Precipitate dissolution during MPW cannot have a pure thermal origin because the lowest temperature at which Al x Fe y Si z particles become liquid is 577°C via a eutectic reaction, at least at equilibrium [4647]. This temperature is significantly higher than the 200°C expected MPSW peak temperature [START_REF] Miranda | Magnetic pulse welding on the cutting edge of industrial applications[END_REF]. In addition, in pure thermal conditions, the respective distances covered by Fe and Si atoms in Al at room temperature for 10 µs are shorter than 2.1×10 -4 and 1.5×10 -3 µm, these values being calculated at 520°C according to available data [START_REF] Brandes | Smithells Metal Reference Book[END_REF]. Therefore, we must conclude that the high density of vacancies generated by the intensive plastic deformation together with the disintegration during MPSW has significantly promoted the dissolution of Al X Fe Y Si Z precipitates, Si and Fe atomic diffusion and formation of big Si rich particles. The formation of Si particles has also to do with the lack of Si solubility in Al and Zn, at least in equilibrium conditions [START_REF] Brandes | Smithells Metal Reference Book[END_REF]. At last, the presence of oxygen and silicon rich particles in the mixed (Al+Zn) zone is likely to result from the oxidation of the silicon particles trapped at the joint interface.

Besides, some oxygen may have penetrated into the mixed zone during the impact. According to literature, the solubility of oxygen into Al is 3×10 -8 at.% in equilibrium conditions [START_REF] Wriedt | The Al-O (aluminum-oxygen) system[END_REF]. Literature also indicates that oxygen in oversaturation has been put into evidence by atom probe tomography at the interface of Al and Fe in Al-steel welds elaborated at the solid state by ultrasonic additive manufacturing which proceeds at a 10 5 s -1 shear strain rate [START_REF] Sridharan | Colossal super saturation of oxygen at the iron-aluminum interfaces fabricated using solid state welding[END_REF]. In such a weld, the decomposition of oxides into oxygen atoms during welding was suggested to result from the extensive plastic deformation which led to oxygen segregation to dislocations and vacancies. This interpretation may be validated in the present case of MPSW and would be consistent with the theory of ballistic mixing in metals [START_REF] Lund | Driven alloys in the athermal limit[END_REF]. This approach requires huge increase in the non equilibrium defects density. As already said, numerous vacancies may have been created during MPSW since local strain rate can reach values up to 10 7 s -1 for a few µs [29,3830,39]. Instead of being completely ejected by the jet as usually established for MPW [START_REF] Psyk | Electromagnetic forminga review[END_REF], some oxides may have dissolved into the (Al+Zn) layer under the impact effect.

Another assumption would suggest that the adsorption of oxygen within the mixed zone may have occurred during the specimen preparation and transport. It is however less probable since, in the latter case, the preferential localization of oxygen in the mixed zone (figures 3 and 4) is not consistent with the differences of chemical affinity between O and Al, Fe, Zn and Si [START_REF] Brandes | Smithells Metal Reference Book[END_REF].

Thus the ballistic mechanism with the trapping of fragments of both the Zn and Al layers at the interface seems to be the most probable and predominant mechanism explaining the formation of the mixed (Al+Zn) area at the joint interface during MPSW. However the presence of well crystallized grains as evidenced by the FFT spot patterns (figures 5, 6 and 7) differs from other MPW results showing existence of narrow amorphous layers at Al/Fe interfaces [14,1515,16]. It must nevertheless be noticed that amorphization as well as crystallization may arise from usual conditions of ion milling [START_REF] Viguier | Heating of TEM specimens during ion milling[END_REF]. This assumption could be checked by performing cryogenic ion milling or using oscillating (pulsed) current to avoid excessive heating [4041].

Mottled areas in Al and Zn grains

The dimensional features of the mottled contrasts in the Al and Zn rich zones (figures 5 and 6) are close to those due to spinodal decomposition in the Al-Zn system. This conclusion arises from the similarity of the present microstructural features with those displayed in a TEM bright field image [5051] or found by numerical simulations of spinodal decomposition [START_REF] Lopez-Hirata | Analysis of spinodal decomposition in Al-Zn and Al-Zn-Cu alloys using the non-linear Cahn-Hilliard equation[END_REF]. Nevertheless the present microstructure cannot arise from a spinodal separation because the 9.4 to 14.3 at% Al, and the 5.9 to 9.7 at% Zn mean solute content in the Zn and Al-rich zones, respectively, are largely outside the 41 to 83.5 at% Al limits of the incoherent miscibility gap at 277°C in the equilibrium Al-Zn phase diagram [START_REF] Brandes | Smithells Metal Reference Book[END_REF].

The mottled contrasts are due to the presence of islands of correspond to oversaturated Al and Zn solid solutions (figures 5 and 6). Actually, according to the Al-Zn equilibrium phase diagram, the solubility of Al into Zn is inexistent while that of Zn into Al is 2 at.% at room temperature [START_REF] Brandes | Smithells Metal Reference Book[END_REF]. These solubility limits are clearly exceeded in the present study where the Al rich zones within the Zn based grains solid solution contains up to 22.4 at.% Al and the solute rich areas in the Al one based grains can reach up to 7.8 at.% Zn.

Like the architecture of the mixed (Al+Zn) layer, the mottled areas are very likely formed by the ballistic mixture. The presence of these solute enrichments in one grain is assumed to result from the inclusionincorporation of tiny nanometric fragments or clusters of atoms of the other material followed by their dissolutiondispersion during the impact. The incorporation of the foreign material pieces originates either from the shock between pieces or from the viscous like material flow generated by the impact. Actually, at the interface, Al and Zn very likely behave as high velocity fluids because of the high strain rates and the great pressures up to 10 5 MPa [26,37,4327,38,44]. The viscous state would be induced by the great atomic disorder due to a high density of vacancies generated by the high strain rate reaching values up to 10 7 s -1 for a few µs [29,3830,39]. It is worth noting that the very short duration of welding and the temperature and duration of ion milling were not sufficient to reach equilibrium, which should imply repulsion between Al and Zn rather than homogenization of solid solutions.for the homogenization of the solute content in the grains. The anomalous Si contents of the grains in the (Al+Zn) layer probably have a similar ballistic origin. According to the EDX results in figures 5 and 6, and in contradiction with the equilibrium solubility limits the Zn rich grains are more oversaturated in Si than the Al rich grains. Besides it is worth noting that the Si content of the Zn rich grains exceeds its nominal content in the AA1050 alloy (figure 6).

Conclusions

The characterization at the atomic scale of the singular biphased layer contained in the Al -galvanized steel MPW joint supplies clues about its ballistic origin. This conclusion originates from the following observations:

(i) The layer displays a random distribution of Al and Zn rich grains with various crystal orientations and heterogeneous chemical compositions. (ii) The grains are Some oversaturated Al and Zn based solid solutions with local solute enrichments are locally observed in the grains.

Besides, the important layer thickness and the dissolution of very large particles must be noted since they cannot result from single atomic diffusion even aided by the very high strain rates inherent to MPW. The dissolution of the large particles rather arises from their desintegration.

The absence of both solidification defects and Al-Zn eutectic as well as the planar interface morphology prove that the bonding interface has formed without melting.

Figure 1 :

 1 Figure 1: Setting for Magnetic Pulse Spot Welding.

Figure 2 :

 2 Figure 2: General aspect of a thin foil cut along the joint transverse section (HAADF image).

Figure 3 :

 3 Figure 3: Details of the rectangular zone 1 in figure 2. On-line HAADF image (a) and STEM-EDX maps of Al K α (b), Zn K α (c), Fe K α (d) , Si K α (e) and O K α (f).The brightest zone in the Fe K α X-ray map is due to local thin foil thickening.

Figure 4 :

 4 Figure 4: Details of zone 2 in figure 2. On-line HAADF image (a) and STEM-EDX maps of Al K α (b), Zn K α (c), Fe K α (d) , Si K α (e) and O K α (f). It is worth noting that the Fe distribution is rather homogeneous low at thea mean 0.25 at.% content. whatever the Al or Zn based grainsBesides Fe is more concentrated in Zn areas.

  displays a negative deviation to Vegard's law. It can be explained by the competition between electronic interactions between both elements and the volume change entailed by the size difference of the substitutional element [3435]. The presence of a random distribution of brighter atoms (corresponding to Zn atoms) in the zone bordered by a blue circle in figure 9 is a clear indication of the presence of random solid solutions.

Figure 5 :

 5 Figure 5: Al rich zone in the intermediate area: on-line HAADF image (a) with associated Al K α (b), Zn K α (c) X-ray maps. HAADF image (a), FFT pattern (d) and EDX results of the areas marked in the HAADF image (a). Micrograph (a) is presented at two magnifications.

Figure 6 :

 6 Figure 6: Zn rich zone in the intermediate area: i) HAADF image (a) and FFT pattern (d) of the zones marked in the HAAADF image, ii) on-line HAADF image (a) with associated Al K α (b), Zn K α (c) X-ray maps. Micrograph (a) is presented at two magnifications.

Figure 7 :

 7 Figure 7: Atomic scale analysis of a Zn rich zone inside the intermediate area: HAADF image (a) and FFT pattern (b) of the zone marked in the HAADF image. The EDX spectrum (c) of this encircled zone indicates the following composition: 76.8% Zn -22.4% Al -0.8% Si (at%).

Figure 8 :

 8 Figure 8: Al and Zn amounts in solid solution in Zn and Al, respectively according to i) the Vegard's law applied to the apparent lattice parameters (red and green squares), and to ii) the EDX analyses (in blue). The coordinates of the crosses are the experimental results corresponding to the Al content estimated by the EDX analyses and the atomic volume deduced from the FFT patterns. Their position below the straight line corresponding to the Vegard's law suggests a negative deviation to the Vegard's law for the Al-Zn system.

Figure 9 :

 9 Figure 9: Al rich areas in the intermediate layer: the area surrounded with a yellow frame shows atom planes distortion along one direction.

Figure 10 :

 10 Figure 10: Orientation map of zone 3 in figure 2. Map (a) shows the distribution of Al and Zn rich phases; the Al and Zn phases are colored in red and blue, respectively. Map (b) depicts the crystal orientations of the Al and Zn grains along the normal direction, the meaning of the

Figure 11 :

 11 Figure 11: Orientation map of zone 4 in figure 2. The EDX map (a) shows the distribution of the Fe and Zn phases colored in red, green and blue, respectively. Map (b) depicts the crystal orientations of the Fe and Zn grains along the normal direction, the meaning of the colors are indicated in the caption. (c) is the Zn (0003) pole figure. Given the uncertainty about the nature of the phases contained in the inhibition layer, this layer has not been considered for the analyses.

[ 3637 ]

 3637 , Al -Al [4142] and contrary to the couples of materials Al -Cu or Al -Ni [4142] or Al -Fe [12,26,4213,27,43],

Table 1 : Chemical composition of the base materials (wt.%)

 1

		Al	Fe	Si	Cu	Mn	Mg	Zn	Ti	C	P	Cr	Mo
	AA1050 balance	0.40	0.25	0.05	0.05	0.05	0.07	0.05	-	-	-	-
	DP450 steel	0.015 -0.08	balance	0.400 max	-	1.600 max	-	-	Ti+ Nb 0.050	0.100 max	0.040 max	0.800 max	0.300 max
									max

Data availability

All the data in this manuscript are available upon request to the corresponding author.

Aknowledgments

The authors are very grateful to Pr. G. Racineux, Ecole Centrale de Nantes -France, to have provided the joints and to D. Troadec, IEMN, France for having performed thin foil cutting by FIB. The TEM facility in Lille (France) is supported by the Conseil Régional du Nord-Pas de Calais, and the European Regional Development Fund (ERDF).


Anne-Sophie Pavaux
email: annesophie.pavaux@gmail.com

Rostan Julie

Laurence Guidi-Guilvard

Marro Sophie

Ternon Eva

Olivier P Thomas

Lemée Rodolphe

Gasparini Stéphane

Pavaux Anne-Sophie

Guidi-Guilvard Laurence

Ternon Eva²

Effects of the toxic dinoflagellate Ostreopsis cf. ovata on survival, feeding and reproduction of a phytal harpacticoid copepod

Keywords: Benthic HABs, Ostreopsis cf. ovata, chemical ecology, meiobenthic copepods, reprotoxicity

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Over the past decades, the occurrence of Harmful Algal Blooms (HABs) has been increasing in frequency, intensity and geographic distribution [START_REF] Berdalet | Harmful Algal Blooms in Benthic Systems: Recent Progress and Future Research[END_REF][START_REF] Glibert | The Global, Complex Phenomena of Harmful Algal Blooms[END_REF]. These blooms negatively impact human health and wellbeing, by affecting coastal ecosystem services [START_REF] Berdalet | Marine harmful algal blooms, human health and wellbeing: challenges and opportunities in the 21st century[END_REF] as well as marine organisms and ecosystems [START_REF] Dolah | Health and Ecological Impacts of Harmful Algal Blooms: Risk Assessment Needs[END_REF]. In this regard, the strong geographical expansion of the toxic dinoflagellate Ostreopsis cf. ovata in the Mediterranean Sea constitutes an emerging problem [START_REF] Parsons | Gambierdiscus and Ostreopsis: Reassessment of the state of knowledge of their taxonomy, geography, ecophysiology, and toxicology[END_REF]. This dinoflagellate has long been regarded as a potential vector involved in ciguatera fish poisoning [START_REF] Glaziou | The epidemiology of ciguatera fish poisoning[END_REF][START_REF] Hallegraeff | A Review of Harmful Algal Blooms and Their Apparent Global Increase[END_REF], and it has more recently been shown to produce palytoxin (PLTX) and palytoxin-like compounds [START_REF] Ciminiello | The Genoa 2005 Outbreak. Determination of putative palytoxin in mediterranean Ostreopsis ovata by a new liquid chromatography tandem mass spectrometry method[END_REF] named ovatoxins (OVTXs) a potential cause of toxic bio-aerosols causing human respiratory illnesses [START_REF] Ciminiello | First Finding of Ostreopsis cf. ovata Toxins in Marine Aerosols[END_REF].

Ostreopsis cf. ovata is a benthic dinoflagellate usually described as epiphytic on macroalgae and seagrasses growing on rocky shallow seabeds [START_REF] Mangialajo | The toxic benthic dinoflagellate Ostreopsis ovata: Quantification of proliferation along the coastline of Genoa, Italy[END_REF][START_REF] Totti | Ostreopsis ovata bloom along the Conero Riviera (northern Adriatic Sea): Relationships with environmental conditions and substrata[END_REF] but has also been reported to have a planktonic phase [START_REF] Vila | Establishing the link between Ostreopsis cf. ovata blooms and human health impacts using ecology and epidemiology[END_REF].

Several studies on invertebrate mass mortalities during Ostreopsis cf. ovata blooms [START_REF] Graneli | Sea urchins poisoning by the benthic dinoflagellate Ostreopsis ovata on the Brazilian Coast[END_REF][START_REF] Shears | Blooms of benthic dinoflagellates of the genus Ostreopsis; an increasing and ecologically important phenomenon on temperate reefs in New Zealand and worldwide[END_REF] suggest that this dinoflagellate may be harmful to benthic organisms living nearby even if its effects may be confounded with other stress factors such as pollution or hypo-/anoxic conditions (Shears andRoss, 2010, 2009). The toxic effect of O. cf. ovata has previously been investigated on several organisms using ecotoxicological bioassays. For instance, the jellyfish Aurelia sp., has been shown to be particularly sensitive at the ephyra (or larval) stage [START_REF] Giussani | Effects of the harmful dinoflagellate Ostreopsis cf. ovata on different life cycle stages of the common moon jellyfish Aurelia sp[END_REF] and the mussel Mytilus galloprovincialis reacted to O. cf. ovata exposures by modifying immunological, histological and oxidative levels [START_REF] Gorbi | Effects of harmful dinoflagellate Ostreopsis cf. ovata exposure on immunological, histological and oxidative responses of mussels Mytilus galloprovincialis[END_REF]. Also, fertilization and early development of the sea urchin Lytechinus variegatus were affected by O. cf. ovata [START_REF] Neves | Effects of the toxic benthic dinoflagellate Ostreopsis cf. ovata on fertilization and early development of the sea urchin Lytechinus variegatus[END_REF]. Moreover, a decrease of more than 98.5% of gill cell viability was observed in response to O. cf. ovata exposure [START_REF] Verma | Molecular and phylogenetic characterization of Ostreopsis (Dinophyceae) and the description of a new species, Ostreopsis rhodesae sp. nov., from a subtropical Australian lagoon[END_REF]. Even though meiofauna living on macrophytes play important roles in benthic biochemical and ecological processes, little is known on the effects of this toxic microalga on the surrounding community. Only one in situ study has previously investigated the impact of O. cf. ovata on phytal meiofauna, and results showed a severe decrease in the number of nauplii during the toxic blooms, suggesting a reprotoxic effect of the dinoflagellate on harpacticoid copepods [START_REF] Guidi-Guilvard | The negative impact of Ostreopsis cf. ovata on phytal meiofauna from the coastal NW Mediterranean[END_REF].

The aim of the present study was to provide new insights on the potential effect of the toxic benthic dinoflagellate Ostreopsis cf. ovata on meiofauna, and more specifically on benthic copepods. A harpacticoid copepod belonging to the genus Sarsamphiascus (formerly Amphiascus) was isolated from the natural environment where O. cf. ovata summer blooms occur with densities reaching 4. 10 6 cells.gFW -1 [START_REF] Cohu | Proliferation of the toxic dinoflagellate Ostreopsis cf. ovata in relation to depth, biotic substrate and environmental factors in the North West Mediterranean Sea[END_REF] and was here used as a model organism. Another copepod species belonging to the same genus has already been widely used as a model in toxicity tests [START_REF] Bejarano | Reproductive and developmental effects of atrazine on the estuarine meiobenthic copepod Amphiascus tenuiremis[END_REF][START_REF] Cary | Phenylpyrazole Insecticide Fipronil Induces Male Infertility in the Estuarine Meiobenthic Crustacean Amphiascus tenuiremis[END_REF][START_REF] Chandler | Fipronil effects on estuarine copepod (Amphiascus tenuiremis) development, fertility, and reproduction: a rapid life-cycle assay in 96-well microplate format[END_REF]. This organism was shown to be relevant for ecotoxicological assays due to their small size, short life cycle, simplicity to maintain and manipulate in laboratory conditions and sensitivity to a wide array of toxic substances. In the present laboratory study, the selected copepod was exposed to ecologically relevant concentrations of the dinoflagellate O. cf. ovata [START_REF] Accoroni | Ecology of Ostreopsis cf. ovata Blooms in the Northwestern Adriatic Sea[END_REF][START_REF] Cohu | Proliferation of the toxic dinoflagellate Ostreopsis cf. ovata in relation to depth, biotic substrate and environmental factors in the North West Mediterranean Sea[END_REF] as well as Licmophora paradoxa, a diatom frequently found associated with O. cf. ovata in benthic assemblages (Lemée, personal communication), which will here be used as a control. The effects of O. cf. ovata were investigated by recording three biological parameters of the copepod: survival, food uptake (assessed through the number of fecal pellets produced) and reproduction (fertility and fecundity ratios). The toxicity of the strain used in these experiments was evaluated using Artemia franciscana toxicity bioassay and confirmed by UHPLC-HRMS quantification of ovatoxins.

Materials and Methods

Algal cultures

The dinoflagellate Ostreopsis cf. ovata (MCCV 054) and the diatom Licmophora paradoxa (MCCV 033) used as a control microalga were isolated from macroalgal samples collected at the Rochambeau site (Bay of Villefranche-sur-mer, N-W Mediterranean, 43°41'35.64''N-7°18'31.54''E). The MCCV is the Mediterranean Culture Collection of Villefranche, where strains are maintained and available for the scientific community.

Both strains were cultured in 150 Ml flasks containing L1 medium [START_REF] Guillard | Stichochrysis immobilis is a diatom, not a chrysophyte[END_REF] prepared with autoclaved aged and 0.2µm filtered seawater, adjusted to a salinity of 38. All the cultures were maintained at 24°C, under a 14:10 light/dark cycle (light intensity 250 µmol.m -2 .s -1 ). Cells of O. cf. ovata used for the experiments were collected from the cultures during the exponential phase. They were counted in 12 mL aliquots (triplicates) fixed with acidic lugol (4% v/v) using a liquid particle counter (HIAC/Royco 9703, Pacific Scientific Instruments) following a size range of 2-80 µm [START_REF] Stramski | Effects of temperature, nitrogen, and light limitation on the optical properties of the marine diatom Thalassiosira pseudonana[END_REF].

Cell diameter range of O. cf. ovata and L. paradoxa in culture were 20-40 µm and 15-20 µm, respectively. Biovolumes were calculated assuming O. cf. ovata (1) had the shape of a cone with a half sphere, and L. paradoxa (2) was a truncated cone, using the following equations [START_REF] Olenina | Biovolumes and size-classes of phytoplankton in the Baltic Sea Helsinki Commission Baltic Marine Environment Protection Commission[END_REF]: (1) (2)

where V = volume, h = height, D = diameter, d1 = large diameter, d2 = small diameter. The biovolume ratio between the two microalgae was 1:6, i.e. O. cf. ovata (average biovolume = 17 360 µm 3 ) was 6 times larger than L. paradoxa (average biovolume = 2 968 µm 3 ).

These biovolumes were calculated using 15 cells for each strain.

Toxicity of the strain

Artemia franciscana toxicity bioassay

The level of toxicity of the O. cf. ovata strain used for this study was checked by performing a standard Artemia franciscana test [START_REF] Faimali | Toxic effects of harmful benthic dinoflagellate Ostreopsis ovata on invertebrate and vertebrate marine organisms[END_REF][START_REF] Neves | Toxicity of benthic dinoflagellates on grazing, behavior and survival of the brine shrimp Artemia salina[END_REF].

The high sensitivity of Artemia to toxic substances makes it suitable for ecotoxicological assessment [START_REF] Kalčíková | Artemia salina acute immobilization test: a possible tool for aquatic ecotoxicity assessment[END_REF][START_REF] Nunes | Use of the genus Artemia in ecotoxicity testing[END_REF]. Such bio-assays, described as simple, inexpensive and convenient, can be used to test the toxicity of harmful algal strains [START_REF] Neves | Toxicity of benthic dinoflagellates on grazing, behavior and survival of the brine shrimp Artemia salina[END_REF]. Cysts of A. franciscana (Ocean Nutrition Sep-art) maintained at 4°C in the dark, were incubated in 2 L of filtered seawater (salinity 38), at 20°C with vigorous and continuous aeration until reaching the most sensitive development stages (Stages 2-3, [START_REF] Kerster | Brine shrimp (Artemia salina) nauplii as a teratogen test system[END_REF]. The larvae were collected using a glass pipette and phototactism and further transferred to 6-well plates. Five larvae were placed in each wells, using one 6-well plate per experimental condition. A total of 4 well-plates were used to investigate the effects of the following cell concentrations of O. cf. ovata: 4, 40, 400 and 4000 cells.mL -1 .

The ratio number of algal cells: number of target animals was then between 20:5 to 20 000:5. The number of dead larvae was estimated after 2, 4, 24 and 48 h exposure times. Larvae were considered dead when there was no reaction to strong light or to glass pipette aspiration or when a shift to white coloration of the body was observed. LC50 and LT50 values were calculated, corresponding to the concentration which induced the mortality of 50% of the organisms and the lethal time (in hours) to reach 50% mortality, respectively.

UHPLC-HRMS analyses

The toxicity of the strain was confirmed using chemical analyses. Ostreopsis cf. ovata cells were grown in 300 mL flasks at previously described conditions, and harvested at day 10. The culture was further centrifuged at 600 g during 10 min at ambient temperature. The cell pellet was flash-freezed using liquid nitrogen and stored at -80 °C until extraction. The metabolites were extracted using 4 mL of MeOH/H2O (80:20;v:v) and sonicating during 5 min in a cooled ultra-sonic bath. The extracts were centrifuged at 2500 g during 10 min at ambient temperature and the supernatants were transferred into 20 mL vials. These steps (MeOH, ultra-sonic bath and centrifugation) were repeated 3 times. The resulting organomethanolic extracts were evaporated using a stream of nitrogen avoiding dryness; a volume of 500 µl of DMSO was added in each extract and the remaining H2O was fully evaporated. All samples were therefore stored in 100% DMSO (500 µl) at -20°C until UHPLC-HRMS analysis.

Ovatoxins quantification by UHPLC-UV-HRMS were performed using an Agilent 1290 system (Agilent Technologies, USA) equipped with a diode array detector and coupled to an Agilent 6540 Qtof mass spectrometer (Agilent Technologies, USA) by the injection of 10µL on T3 column (Acquity UPLC HSS T3 1.8µm, 2.1mm x 100mm, Waters). Separation was achieved using a linear elution gradient of H2O:MeOH (80:20, v:v)/MeOH with 0.1mM of ammonium formate and 0.1% formic acid from 90:10 (v:v, isocratic from 0 to 2mins) to 0:100 (v:v, isocratic from 12 to 13 mins) with a 0.4 mL.min -1 flow rate. UV detection was set at 210, 233 and 263 nm. Ions detections was recorded in positive mode (ESI +) in the range 60 -3000 Da. Collision energies (CE) of 30, 70 and 110 eV were applied to confirm the presence of ovatoxins moieties [START_REF] Brissard | Complex toxin profile of French Mediterranean Ostreopsis cf. ovata strains, seafood accumulation and ovatoxins prepurification[END_REF][START_REF] Ciminiello | Isolation and structure elucidation of ovatoxin-a, the major toxin produced by Ostreopsis ovata[END_REF]. The spectrometer analyzer parameters were set as follows: nebulizer sheath gas, N2 (35 psig); drying gas, N2 (11 L.min -1 ); Gas Temperature, 300°C; capillary, 4.129 µA; Vaporizer/Sheat Gas Temp, 350°C.

Ovatoxins quantification was performed by extracting and integrating all tri-charged ions (from 858 to 910 m/z) from the base peak chromatogram. The concentration of ovatoxins is given in palytoxin equivalent as commercially available standard of palytoxin (Wako Chemicals GmbH, Neuss, Germany) was used to perform a calibration curve, assuming their ionization pattern is similar.

The model copepod

The harpacticoid copepod used in the experiments thrives in the shallow rocky shore of the Marinières site (Bay of Villefranche-sur-Mer, N-W Mediterranean, 43°42'21.51"N -7°19'07.44"E) where it was collected in 2012 using a WP2 net towed over the macroalgal cover. In the laboratory, samples were maintained in 10 L tanks together with a planktonic copepod (Acartia clausi) in 0.2-µm filtered aged seawater (salinity 38) at 22°C, in the dark to avoid phototactism, and fed three times a week with a mixture of the microalgae Dunaliella salina (MCCV 20) and Tisochrysis lutea (CCAP 927/14). The copepod belongs to the family Miraciidae Dana, 1946 (formerly Diosaccidae), and is part of the varians-Group. Within this group, the genus Amphiascus, renamed Sarsamphiascus by [START_REF] Huys | Unresolved cases of type fixation, synonymy and homonymy in harpacticoid copepod nomenclature (Crustacea: Copepoda)[END_REF], is well known for taxonomic discrepancies and extreme difficulty in species identification [START_REF] Hicks | Some littoral harpacticoid copepods, including five new species, from Wellington, New Zealand[END_REF][START_REF] Wells | An annotated checklist and keys to the species of Copepoda Harpacticoida 4[END_REF]. In the present study, the species name remains uncertain due to inter-individual variation in the parapodial setation. However, among the possible varians-Group species, only Amphiascus propinquus had been previously reported in areas close to the Bay of Villefranche-sur-mer (i.e. the Italian Ligurian shore, Ceccherelli, personal communication). For all these reasons, this species was named Sarsamphiascus cf. propinquus [START_REF] Sars | Copepoda (Harpacticoida): Georg Ossian Sars | NHBS Shop[END_REF] in our study.

Experimental setup

All experiments were run in 6-well plates, each well containing 4 mL of autoclaved aged and 0.2-µm filtered seawater (salinity 38), at 24°C, in the dark. One milliliter of microalgae at the appropriate cell concentration was added to the wells as food, except in the no-food controls which received instead 1mL of clean culture medium. The content of each well was renewed every 48 hours and wells were checked daily under a binocular microscope (ZEISS, SteREO Discovery V12). Experiments involving the copepod S. cf.

propinquus started with adults and late copepodites. Prior to the experiments, individuals were collected from the culture tanks, sorted with an elongated Pasteur pipette, flushed twice in seawater and transferred to the 6-well plates. They were left for 2 days without food to allow gut clearance, except in the reproduction experiments where the food was added from the start. At the end of some of the experiments, copepods were fixed with formalin (4% v/v) to determine sex and overall size (from the tip of the cephalosome, excluding the rostrum, to the end of the last body somite, excluding the caudal rami) using a NIKON AZ100 microscope coupled to a Digital Imaging System equipped with the NIS-Elements software (Nikon Instruments Inc., New-York, U.S.A.).

Acute toxicity of O. cf. ovata on Copepods

To estimate and compare the level of toxicity of O. cf. ovata, the copepods were exposed to five increasing cell concentrations (500, 1 000, 2 000, 4 000 and 20 000 cells.mL -1 ) and one no-food control in six-well plates, as previously described for Artemia bioassay. The test involved 72 individuals (12 per concentration and no-food control, 1 per well) and lasted 9 days. The ratio of algal cells/ number of target animals was between 2500:1 to 100 000:1. Mortality (as defined above for Artemia bio-assay) was daily recorded in order to calculate LC50 and LT50.

2.6. Impact of Ostreopsis cf. ovata on copepod survival and food uptake

To investigate potential toxic effects of O. cf. ovata cells, mono-and mixed diets were fed to the copepod. Five different treatments were applied: (i) O. cf. ovata at a concentration of 100 cells.mL -1 reflecting the realistic bloom alert threshold measured in the natural environment [START_REF] Lemée | Interactions between Scientists, Managers and Policy Makers in the Framework of the French MediOs Project on Ostreopsis (2008-2010)[END_REF][START_REF] Tichadou | Health impact of unicellular algae of the Ostreopsis genus blooms in the Mediterranean Sea: experience of the French Mediterranean coast surveillance network from 2006 to 2009[END_REF]

Statistical analyses

Mean fecal pellet production (number of pellets.cop -1 ) at a given time was calculated only for the copepods that had survived at that given time. Mean egestion rate (number of pellets.cop -1 .day -1 ) for a given treatment and experiment was the slope of the regression line between the corresponding mean fecal pellet production values and time.

The R package ecotoxicology was used to calculate median lethal time (LT50) and concentration (LC50). Note that median lethal times were only calculated for the second experiment since a mortality of 100% of the copepods is required to estimate these values.

Kruskal-Wallis tests were used to assess the influence of O. cf. ovata on egestion; a Dunn's post hoc tests was used a posteriori to identify which experimental treatment differed from the others when the Kruskal-Wallis test showed significant differences (p<0.05). Kaplan Meier curves and a log-rank test were applied to evaluate the influence of O. cf. ovata on mortality of S. cf. propinquus. The Fisher Exact Test was used to assess the potential toxicity of O. cf. ovata on the reproduction performances of the copepod.

All tests were performed using the PAST software [START_REF] Hammer | PAST: Paleontological Statistics Software Package for Education and Data Analysis[END_REF].

Results

Quantification of the toxins

The Ostreospis cf. ovata strain used in the present study (MCCV 054) was shown to produce 44 ± 17 pg PLTX eq/cell at the end of its exponential phase (day 10).

Acute toxicity of the Ostreopsis cf. ovata on Artemia franciscana

Mortality of Artemia franciscana larvae in the control conditions (without Ostreopsis cf.

ovata) remained below 10% even after 48 hours of experiment (Figure 1), following standard Artemia toxicity tests which is less than 10% of mortality in the control.

Results showed that O. cf. ovata induced mortalities of A. franciscana larvae and values increased rapidly with exposure time and concentration of the dinoflagellate. Two hours exposure at the lowest concentration (4 cells.mL -1 ) were sufficient to impact 13% of the larvae and more than 70% were dead at the highest concentration (4 000 cells.mL -1 ). After 4 hours of exposure, larval mortality almost reached 50% at the lowest concentration of microalgae, and more than 86% at the highest concentration. After 48 hours of exposure, irrespective of the cell concentration tested, all the larvae died while more than 80% survived in the no-food control. Standard measures of toxicity showed a median lethal concentration (LC50) below 4 cells.mL -1 after a 48-hour exposure, and a median lethal time (LT50) of 1.3 hours when exposed to the highest cell concentration (4 000 cells.mL -1 ).

Acute toxicity of Ostreopsis cf. ovata on Copepods

Ostreopsis cf. ovata exposures induced mortality of Sarsamphiascus cf. propinquus and the impact increased with exposure time and cell concentration (Figure 2). However, results were highly variable and remained relatively low irrespective of the microalgal concentration tested. Even after 9 days of exposure at the highest cell concentration, mortality did not exceed 50% (on average), while it reached more than 58% in the nofood control. However mortality was higher at low concentration (i.e. below 1 000 cells.mL -1 ) than at medium concentration (i.e. between 1 000 cells.mL -1 to 4 000 cells.mL - 1 ). Indeed after 9 days of experiment, 40% of the copepods died at 500 cells.mL -1 although only 20% died at 4 000 cells.mL -1 . Median lethal concentration (LC50) calculated after an exposure time of 48 hours was more than 20 000 cells.mL -1 , and half lethal time (LT50) for the highest cell concentration (20 000 cells.mL -1 ) was 120 hours. This latter value was lower compared to that found in the no-food control, i.e. LT50 (no-food) = 192 h.

Impact of Ostreopsis cf. ovata on food intake of Copepods

Fecal pellet production (or egestion) was used as a proxy to estimate food intake. In both the 10-day and 14-day experiments, the number of fecal pellet increased with time in the four feeding treatments, except in the no food treatment (Figure 3). The Kruskal-Wallis analysis of variance showed that mean values differed significantly between treatments (p < 0.001, for the two experiments). In the absence of food, egestion was extremely low, and ceased shortly after the start of the experiments. Only 3 and 6 fecal pellets were collected in total from the 10-day and 14-day controls, respectively. When the copepods were fed with O. cf. ovata alone, fecal pellet production first increased sharply, and slowed down after a few days. A total of 189 and 45 fecal pellets were collected from the 10-day and 14-day experiments with mean egestion rates reaching 0.62 and 0.19 pellets.copepod -1 .day -1 , respectively. The Dunn's post hoc test showed that the mean pellet production measured in the O. cf. ovata 14-day experiment was not significantly different from the corresponding no-food control, but was significantly different from measurements in the 10-day experiment. Mean fecal pellet production was significantly different between all treatments (including controls), except for the above stated treatment. It was also significantly different between the O. cf. ovata fed as mono-diet and the half concentration of both microalgae mixed-diet as well as between both mixed diets. The highest fecal pellet production values were measured with the diatom monodiet. Egestion increased linearly with time (α < 0.005) with a mean production rate of 5.1 and 4.0 pellets.copepod -1 .day -1 in the 10-day and 14-day experiments, respectively. When the diatom was supplemented together with O. cf. ovata in the food (mixed diets), copepod egestion followed the same trend, but mean egestion rates were lower. With the full concentration of both microalgae in the mixed diet, copepods produced on average 2.5 and 3.8 pellets.copepod -1 .day -1 , while with the half-mixed diets the mean pellet production decreased to 1.6 and 1.4 pellets.copepod -1 .day -1 in the 10-day and 14day experiments, respectively.

Copepods fed with O. cf. ovata alone (mono-diet) were observed under an epifluorescence microscope and images revealed an intense fluorescence of their digestive tracks due to the autofluorescence of chlorophyll indicating the presence of dinoflagellate chloroplast (Figure 4).

Impact of Ostreopsis cf. ovata on survival of Copepods

Results obtained in the 10-day and 14-day experiments showed similar trends (Figure 5).

Mortality of S. cf. propinquus increased with the duration of the experiment, but the magnitude of this increase differed significantly between treatments (log rank test, p < 0.001 for the two experiments). The highest mortalities occurred in the absence of food, while when O. cf. ovata alone was fed to the copepods (mono-diet), mortalities were less pronounced. Feeding a mixed diet by introducing the diatom L. paradoxa with O. cf. ovata in the food, reduced mortalities. Finally, when the diatom alone was used as food, mortality along time was almost null and only slightly increased towards the end of the experiments. The log-rank test showed that mortality associated to the diatom mono-diet did not significantly differ from mortality measured when copepods were fed with the mixed diet (iii) (i.e. O. cf. ovata at 100 cells.mL -1 + L. paradoxa at 600 cells.mL -1 ). On the other hand, mortality values with O. cf. ovata differed significantly from mortality values observed in all other treatments. Moreover, mortality due to the absence of food differed significantly from mortality observed in all the other treatments. In other words, O. cf.

ovata alone offered to the copepods and the absence of food, both, strongly affected their survival.

However, mortality associated to O. cf. ovata in the food always remained lower compared to starvation, excepted during the first 7 days. This difference is reflected in the median lethal time values, i.e. LT50 under starvation was 9 days, while LT50 with O. cf.

ovata (at 100 cells.mL -1 ) was 11 days. When the copepods were fed with O. cf. ovata alone at 100 cells.mL -1 , mortality was significantly different from results obtained with mixed diets. For example, after 14 days, ca. 67% of the copepods died with the O. cf.

ovata mono-diet, while they were only ca. 13% when fed with the same concentration of O. cf. ovata supplemented with the diatom at 600 cells.mL -1 . However, when reducing the cell concentrations of both micro-algae by two fold in the mixed diet (i.e. O. cf. ovata at 50 cells.mL -1 + L. paradoxa at 300 cells .mL -1 ), copepod mortality increased to ca. 37%.

Finally, when the diatom was fed alone to the copepods, mortality values remained below 10%.

Impact of Ostreopsis cf. ovata on reproductive performances of Copepods

Depending on the experiment, when the copepods were fed with the diatom L.

paradoxa, 55 to 88% of the females present were gravid (Table 1). On the other hand, when O. cf. ovata was used as food, only 13 to 18% of the females were gravid, and in the absence of food (starvation) gravid females did not exceed 10%. The Fisher test showed that fecundity ratios obtained with the O. cf. ovata mono-diet were actually not significantly different from values in the no-food controls, but significantly lower than with the diatom mono-diet (Table 2).

Depending on the experiment, between 88 and 210 nauplii hatched in the presence of the diatom L. paradoxa (Table 1). Only 2 to 11 nauplii hatched in the presence of the dinoflagellate O. cf. ovata, and between 0 and 5 nauplii hatched in the absence of food. More specifically, each gravid female produced on average 9.3 nauplii with the diatom mono-diet, 2.7 nauplii with the O. cf. ovata mono-diet, and 1.7 nauplii when not fed. Here again, the Fisher test showed no significant difference in the fertility ratios derived from the two latter treatments (Table 2). Moreover, the number of nauplii per ovigerous female was significantly lower when the copepods were exposed to O. cf.

ovata compared to copepods fed L. paradoxa. Irrespective of the experimental treatment, all the nauplii that hatched were viable, at least until the end of the experimental period.

Discussion

The physiological state of the copepod Sarsamphiascus cf. propinquus was altered when exposed to the dinoflagellate Ostreopsis cf. ovata. Although this copepod appeared as very resistant to the toxic dinoflagellate during acute toxicity evaluation tests, changes in fecal pellet production as well as reduced fecundity and fertility indexes were observed.

Acute toxicity of Ostreopsis cf. ovata

The toxicity of the dinoflagellate strain used in this study was estimated by a simple ecotoxicological assay using Artemia franciscana larvae and the content in ovatoxins was further assessed by UHPLC-UV-HRMS. Using a palytoxin standard for calibration, we estimated that the ovatoxin content of the dinoflagellate strain used in this study was comparable to other Mediterranean strains [START_REF] Brissard | Complex toxin profile of French Mediterranean Ostreopsis cf. ovata strains, seafood accumulation and ovatoxins prepurification[END_REF], although more enriched than previously assessed for this same strain (18 pg PLTX eq/cell, [START_REF] Ternon | Allelopathic interactions between the benthic toxic dinoflagellate Ostreopsis cf. ovata and a co-occurring diatom[END_REF]. This enrichment can be easily explained by the protocol for cell extraction and data analysis that differed from the previous study.

The Artemia larvae were highly sensitive to O. cf. ovata with a half lethal concentration (LC50) below 4 cells.mL -1 and a median lethal time of 2 hours at a 4 000 cells.mL -1 cell concentration. These crustacean larvae have previously shown high sensitivity towards other Mediterranean strains of O. cf. ovata [START_REF] Faimali | Toxic effects of harmful benthic dinoflagellate Ostreopsis ovata on invertebrate and vertebrate marine organisms[END_REF][START_REF] Pezzolesi | Influence of temperature and salinity on Ostreopsis cf. ovata growth and evaluation of toxin content through HR LC-MS and biological assays[END_REF] with median lethal concentrations varying from 4 to 8 cells.mL -1 . The results obtained in the present study were consistent with previous studies undertaken on other toxic dinoflagellates such as Gambierdiscus excentricus, Prorocentrum lima [START_REF] Neves | Toxicity of benthic dinoflagellates on grazing, behavior and survival of the brine shrimp Artemia salina[END_REF] and Ostreopsis siamensis [START_REF] Rhodes | Uptake of palytoxin-like compounds by shellfish fed Ostreopsis siamensis (Dinophyceae)[END_REF]. The extreme sensitivity exhibited by the Artemia larvae in the present study confirms that Artemia is a suitable model for assessing the toxicity of O. cf. ovata.

The toxicity of O. cf. ovata has previously been investigated on benthic and pelagic model organisms. Regarding crustaceans, previous studies have shown clear differences between species. For instance, the isopod Sphaeroma serratum exhibited lower acute toxicity compared to the amphipod Corophium insidiosum [START_REF] Prato | Preliminary assessment of Ostreopsis cfr. ovata acute toxicity by using a battery bioassay[END_REF]. Our study highlighted (1) a higher mortality of the copepod S. cf. propinquus at low concentration due to a starvation. At medium concentration, the mortality was the lowest suggesting an optimal balance between food needs and toxicity effects due to O. cf. ovata;

(2) a strong resistance of S. cf. propinquus, with a median lethal concentration (LC50), reaching more than 20 000 cells.mL -1 after 48 hours of exposure. Indeed, S. cf. propinquus was at least 80 times more tolerant compared to Tigriopus fulvus, for which the LC50 after a 48 hour exposure was 250 cells.mL -1 [START_REF] Faimali | Toxic effects of harmful benthic dinoflagellate Ostreopsis ovata on invertebrate and vertebrate marine organisms[END_REF]. Such differences could be explained by variable environmental conditions experienced by each species [START_REF] Prato | Preliminary assessment of Ostreopsis cfr. ovata acute toxicity by using a battery bioassay[END_REF] or strains used for toxicity assays. We suggest that S. cf. propinquus might have adapted to the toxicity of O. cf. ovata due to the natural co-occurrence of both organisms. Resistance of grazers facing toxic dinoflagellates has already been reported for calanoid copepod populations that have frequently experienced blooms of the highly toxic Alexandrium spp.

These copepods were reported to be more resistant to toxic blooms compared to naive populations and had a relatively better fitness in the presence of the toxic dinoflagellates [START_REF] Colin | Testing for resistance of pelagic marine copepods to a toxic dinoflagellate[END_REF]. The difference of sensitivity to O. cf. ovata exposure observed between the two benthic copepods could also be explained by the strains used for toxicity assays. Indeed, very few studies have tested the variability among strains (see [START_REF] Faimali | Toxic effects of harmful benthic dinoflagellate Ostreopsis ovata on invertebrate and vertebrate marine organisms[END_REF][START_REF] Giussani | Effects of the harmful dinoflagellate Ostreopsis cf. ovata on different life cycle stages of the common moon jellyfish Aurelia sp[END_REF] and it was impossible to realise this test for technical constraints.

In some studies, the toxicity of O. cf. ovata was also evaluated by using lysed cells or cellular extracts. For exemple, [START_REF] Faimali | Toxic effects of harmful benthic dinoflagellate Ostreopsis ovata on invertebrate and vertebrate marine organisms[END_REF] compared the effects of different states of O. ovata cultures (such as filtered and resuspended O. ovata cells, growth medium devoid of algal cells and sonicated cells) on crustacean and fish larvae and found a significant toxic effect only when the whole algal cells were physically present.

However, it is difficult to compare such treatments as little is known about released PLTX and OVTXs toxins which are released, their solubility as well as their stability in seawater.

Moreover, the lack of harmonization between extraction procedures applied within studies also make comparisons difficult. It is extremely difficult to identify which chemical cues induced these toxic effects.

4.2. Was Ostreopsis cf. ovata ingested by Sarsamphiascus cf. propinquus?

Although fecal pellet production is rarely used to evaluate the food uptake in copepods [START_REF] Souza-Santos | Feeding rate cycle of the epibenthic harpacticoid copepod Harpacticus flexus: Laboratory experiments using fecal pellets counts[END_REF], the method is simple, fast and quite satisfying when only estimates are needed. In our study, mean egestion rates was between 0.19 and 0.62 pellets.copepod -1 .day -1 when copepods were fed with O. cf. ovata. This suggests a true ingestion of the toxic dinoflagellate by S. cf. propinquus even if the egestion rates were significantly lower than those obtained with all other food treatments.

This ingestion was confirmed by epifluorescence microscopy observations showing the presence of algal chlorophyll in the gut content. The copepods even seemed to have obtained an energetic advantage since mortality rates were lower when they were fed with O. cf. ovata compared to the no-food control. Nevertheless, O. cf. ovata alone was not enough to sustain the energy requirements of the copepods as mortality rates were higher in the mono-diet experiment compared to the mixed diet.

4.3. Negative impacts of Ostreopsis cf. ovata on the copepod physiology

The food uptake results show a clear decrease of fecal pellet production when the copepods fed on O. cf. ovata. Significant differences of fecal pellet production observed when the diatom was offered in mono-diet and in mixed-diet clearly, show that the presence of O. cf. ovata is deleterious. This difference could be due to the higher biovolume of O. cf. ovata (compared to the control diatom L. paradoxa) or to its toxicity.

Copepods are known to actively select the size of their prey [START_REF] Mullin | Some factors affecting the feeding of marine copepods of the genus Calanus[END_REF]. However, some studies suggest that prey size might not be the main factor controlling ingestion.

For example, De [START_REF] De Troch | Is diatom size selection by harpacticoid copepods related to grazer body size?[END_REF] showed that the harpacticoid copepod Harpacticus obscurus had no clear preference for small diatoms. In the present study, the difference in fecal pellet production by S. cf. propinquus between the feeding treatments could then rather be explained by the presence of toxic secondary metabolites produced by O. cf.

ovata since mortality was also higher in the presence of O. cf. ovata. The addition of the toxic dinoflagellate to the diet clearly contributed to increase copepods mortality. It might hence be suggested that the copepod could avoid toxic cells by chemosensory detection of toxic cues. Some studies have previously shown the ability of pelagic copepods to select a non-toxic prey in a mixed algal culture including the toxic dinoflagellate Alexandrium excavatum [START_REF] Turriff | Toxin accumulation and feeding behaviour of the planktonic copepod Calanus finmarchicus exposed to the red-tide dinoflagellate Alexandrium excavatum[END_REF]. Such a selection capacity, as suggested for S.

cf. propinquus, is highlighted by a difference in fecal pellet production between mixedand mono-diets of the diatom L. paradoxa. Even if the concentration of the diatom was the same in both diets, the decrease of egestion might be due to an effort to select non-toxic preys, leading to a lower ingestion rate of the algal particles. In addition to the decrease in fecal pellet production of copepods fed O. cf. ovata, hatching success was also significantly lower. In fact, compared to the no-food treatment, the presence of the toxic dinoflagellate reduced the fecundity and fertility ratios by 4.7-fold and 3.5-fold, respectively. These results agree with in situ observations of [START_REF] Guidi-Guilvard | The negative impact of Ostreopsis cf. ovata on phytal meiofauna from the coastal NW Mediterranean[END_REF] who showed changes in the composition of the meiobenthic community when O. cf.

ovata blooms occurred in summer 2008, with a 72% decrease in the number of nauplii, hence suggesting that the dinoflagellate affected benthic harpacticoid copepods reproduction. These impacts on reproduction are comparable to results obtained in previous studies for planktonic copepod species, such as Temora stylifera fed

Prorocentrum micans, Gymnodinium sanguinium, and Gonyaulax polyedra, [START_REF] Ianora | First evidence of some dinoflagellates reducing male copepod fertilization capacity[END_REF][START_REF] Laabir | Effect of specific dinoflagellate and diatom diets on gamete ultrastructure and fatty acid profiles of the copepod Temora stylifera[END_REF], as well as Calanus finmarchicus [START_REF] Roncalli | The effect of the toxic dinoflagellate Alexandrium fundyense on the fitness of the calanoid copepod Calanus finmarchicus[END_REF] and

Calanus sinicus [START_REF] Liu | Feeding and reproductive responses of marine copepods in South China Sea to toxic and nontoxic phytoplankton[END_REF] fed Alexandrium fundyense and Alexandrium tamarense respectively. The reprotoxicity of microalgae on copepods could have various origins. For Temora stylifera fed on Prorocentrum micans, the reduced fertilization was due to an effect on the sperm. Maternal effects or male age are excluded since hatching rates returned to normal when new males were introduced in the experiment [START_REF] Ianora | First evidence of some dinoflagellates reducing male copepod fertilization capacity[END_REF].

It is worth noting that, due to technical constraints, our results were obtained for only a single O. cf. ovata strain. These effects on survival and reproduction should be ascertained using other strains from different sites even if these effects of O. cf. ovata on reproduction have already been demonstrated on sea urchin [START_REF] Migliaccio | Subtle reproductive impairment through nitric oxide-mediated mechanisms in sea urchins from an area affected by harmful algal blooms[END_REF].

4.4. Were these negative effects due to toxins produced by O. cf. ovata?

After ingestion of O. cf. ovata cells, the release of toxins could have caused a gradual physical incapacitation of the copepod leading to a reduced feeding efficiency. Indeed, during the first 7 days mortality rates were higher with O. cf. ovata than in the no-food control. Moreover, during the first 4 days of the "food uptake" experiments, there was no significant difference between ingestion when the diatom was offered alone or when it was mixed with O. cf. ovata, suggesting again that the ingestion decrease observed after the first 5 days could be due to an alteration of the copepod's physiology due to chemical cues produced by the dinoflagellate. This is supported by a previous study by [START_REF] Sykes | Acute physiological reactions of Calanus pacificus to selected dinoflagellates: direct observations[END_REF], reporting that ingestion of Gonyaulax grindleyi and Karenia brevis (ex.

Ptychodiscus brevis) cells can cause physiological reactions such as regurgitation or elevated heart rate. Other studies have moreover demonstrated an induction of domoic acid production by Pseudo-nitzschia sp. exposed to herbivorous copepods [START_REF] Lundholm | Induction of domoic acid production in diatoms-Types of grazers and diatoms are important[END_REF] which could act as zooplankton grazing deterrents. The question arises about the role of toxins produced by O. cf. ovata in the bloom formation and maintenance by deterring copepods. The ability of Alexandrium minutum to increase its toxin production in response to the exposition to copepods has already been demonstrated as a way to facilitate its bloom formation by being more resistant to grazers [START_REF] Selander | Copepods induce paralytic shellfish toxin production in marine dinoflagellates[END_REF].

However, the question concerning the digestibility of O. cf. ovata arises. Acartia clausi fed with the dinoflagellate Prorocentrum micans, showed a longer gut transit time suggesting that the digestibility of these cells was difficult, probably because of the presence of cellulose-rich compounds in the theca hence requiring additional enzymes to break down these compounds [START_REF] Tirelli | Relationship between functional response and gut transit time in the calanoid copepod Acartia clausi: role of food quantity and quality[END_REF].

Very little information is available regarding the effects of PLTX and OVTXs on sperm viability or as antimitotic compounds. Nonetheless, this impairment on reproductive ability caused by O. cf. ovata has previously been described in two species of sea urchins.

In Paracentrotus lividus, fertilization success and progeny development processes were both compromised [START_REF] Migliaccio | Subtle reproductive impairment through nitric oxide-mediated mechanisms in sea urchins from an area affected by harmful algal blooms[END_REF] and in Lytechinus variegatus, fertilization and early stage development were also affected [START_REF] Neves | Effects of the toxic benthic dinoflagellate Ostreopsis cf. ovata on fertilization and early development of the sea urchin Lytechinus variegatus[END_REF]. The mode of action of toxins (i.e. OVTXs) produced by O. cf. ovata is poorly documented. PLTX, for instance, is known as a potent inhibitor of sperm motility in several species including the sea urchin

Tripneustes gratilla [START_REF] Morton | Potent inhibition of sperm motility by palytoxin[END_REF]. However, PLTX represents only 8 % of the known toxins contained in Villefranche O. cf. ovata [START_REF] Brissard | Complex toxin profile of French Mediterranean Ostreopsis cf. ovata strains, seafood accumulation and ovatoxins prepurification[END_REF] and OVTXs toxicity still needs to be evaluated.

Reprotoxicity could also be related to the inadequate nutritive values of the dinoflagellate. A previous study has shown that the number of copepod nauplii depends on the quality of the ingested food, suggesting that the biochemical composition of the toxic dinoflagellate Cochlodinium polykrikoides may significantly affect Acartia omorii reproduction [START_REF] Kyoungsoon | Influence of food quality on egg production and viability of the marine planktonic copepod Acartia omorii[END_REF]. Such a hypothesis needs to be explored in detail by performing an assessment of the reproductive impacts of O. cf. ovata on other copepods or meiobenthic organisms, to confirm if the reproductive effect was due to an inadequate or poor nutritive value or to the toxicity of O. cf. ovata secondary metabolites.

Interaction between O. cf. ovata and its environment could also be mediated by the presence of an extracellular mucilage [START_REF] Giussani | Active role of the mucilage in the toxicity mechanism of the harmful benthic dinoflagellate Ostreopsis cf. ovata[END_REF]. Indeed, this dinoflagellate produces an abundant mucilaginous matrix, which helps the cells to adhere to substrates.

This mucus, which plays diverse roles such as to improve competition with other microorganisms (for space and nutrients) or as a physical barrier against predators, is actually increasingly reported for dinoflagellates [START_REF] Barone | Behavioural trait of Ostreopsis ovata (Dinophyceae) in Mediterranean rock pools: the spider's strategy[END_REF][START_REF] Giussani | Active role of the mucilage in the toxicity mechanism of the harmful benthic dinoflagellate Ostreopsis cf. ovata[END_REF]. During the present study, we often observed an intense production of mucus when O. cf. ovata concentrations were high. This mucilage helped the algal cells to adhere to the body of the copepods, mostly on their furca. This could have compromised the copepod's movements, and hence their ability to find food, which furthermore could explain the clear decrease in fecal pellet production when the toxic dinoflagellate was used as food. It could also have altered fertilization [START_REF] Gasparini | Mesozooplankton grazing during the Phaeocystis globosa bloom in the southern bight of the North Sea[END_REF].

Conclusion

This study highlights the high resistance of S.

  cf. propinquus to acute toxicity from O. cf. ovata, compared to other tested animals. The observed resistance can result from an acclimatization process developed by this copepod species after frequent exposures to the toxic algae. The present study, together with the previous in situ analyses undertaken by Guidi-Guilvard et al (2012), clearly shows the impact of O. cf. ovata on the reproduction of benthic copepods, and more precisely on the fecundity and fertility ratios. These deleterious effects on reproduction and consequently on population growth, will modify the composition of benthic communities and can also suggest the existence of chemical defence mechanisms exposed by O. cf. ovata as a survival strategy against grazers. ACKNOWLEDGMENT This work was supported by the French ANR project (OCEAN 15: Ostreopsis Chemical Ecology and Allelopathy Network; project ANR-15-CE35-0002) and the project COCLIME (which is part of ERA4CS, an ERA-NET initiated by JPI Climate, and funded by EPA (IE), ANR (FR), BMBF (DE), UEFISCDI (RO), RCN (NO) and FORMAS (SE), with co-funding by the

  The potential effects of O. cf. ovata on the reproduction capacity of S. cf. propinquus were assessed through 3 identical experiments, which ran for 7 days with only mono-diets. The treatments were the same as (i), (ii) and (v) in the previous experiments, i.e. O. cf. ovata at 100 cells/mL -1 , L. paradoxa at 600 cells/mL -1 and a no-food control. For each experiment, 120 males and females were first equally distributed in 3 glass crystallizing dishes and acclimatized to the corresponding treatment for 72h. Then, copepods were randomly transferred to 6-well plates (10 individuals per well to facilitate fecundation).

; (ii) L. paradoxa at a concentration of 600 cells.mL -1 corresponding to the microalgae supplied in treatment

(i)

in terms of bio-volumes ; (iii) a mixed diet of O. cf. ovata and L. paradoxa (i.e. treatments (i) and (ii) combined) to investigate if the presence of the two microalgae could change the response of the copepods; (iv) the mixed diet (iii) at half concentrations (i.e. O. cf. ovata at 50 cells.mL -1 + L. paradoxa at 300 cells.mL -1 ) ; (v) a no-food treatment. To study both mortality and ingestion of S. cf. propinquus, two experiments were performed during 10 days and 14 days, each involving 150 individuals (30 per treatment, 1 per well).

Mortality (as previously defined) was assessed daily, while feeding (assessed through the number of fecal pellets produced by alive copepods ;

[START_REF] Souza-Santos | Feeding rate cycle of the epibenthic harpacticoid copepod Harpacticus flexus: Laboratory experiments using fecal pellets counts[END_REF]

every 48 h, at the time of medium renewal. Copepods that had been in contact with O. cf. ovata alone were observed under an epifluorescence inversed microscope (Axio Scope.A1, UV excitation, red fluorescence) to check for the presence of O. cf. ovata cells in the digestive track.

The formation of egg sacs was checked every day to estimate fecundity ratios, i.e. number of ovigerous females to total number of females. Ovigerous females were isolated immediately after egg sacs were observed, but were still exposed to algal treatments described above. Hatching was monitored daily by counting the number of nauplii.

Fertility ratios, i.e. number of nauplii to number of ovigerous females, were subsequently calculated, assuming that egg mortality and cannibalism were negligible. Indeed, we assumed that no egg mortality occurred only in control condition because the effect of the toxins on egg mortality was not tested. It is noteworthy that no treatment was used to induce reproduction in this experiment. At the end of the experiments, sex and size of all adult individuals were determined. Body length (Mean ± Standard Deviation) of adult S. cf. propinquus reared in the laboratory and used in the experiments was 479 ± 65 µm (n=68) for females and 407 ± 43 µm (n = 79) for males. No mortality was observed in this experiment since it only lasted 7 days and with very low concentrations of O. cf. ovata.

European, Union; Grant 6904462). The research leading to these results received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No 730984, ASSEMBLE Plus project. We gratefully thank Professor V.-U.

Checcerelli (retired from the University of Bologna, Italy) for his kind taxonomic assistance and advice concerning the copepod. Authors are members of the French National GDR PhycoTox.