Pablo Griffiths

Gildas Coativy

Florent Dalmas

Guillaume Falco

Liuyin Jiang

Ziyin Xiang

Minh-Quyen Le

Benjamin Ducharne

Damien Le Roy

Françoise Méchin

Julien Bernard

Sylvain Meille

Guilhem P Baeza

Ultrafast Remote Healing of Magneto-Responsive Thermoplastic Elastomer Based Nanocomposites

Keywords: induction heating, stimulus healing, nanocomposites, additive manufacturing, thermoplastic elastomers

We describe herein, a general, efficient and scalable process to design magneto-responsive thermoplastic elastomer-based (nano)composites that can be repeatedly healed in a few tens of seconds by triggering polymer melting upon exposure to a high frequency magnetic field. Three series of composites loaded with 1-15 vol.% of Fe3O4 nanoparticles, Fe nanoparticles or Fe microparticles were produced and characterized in depth with the aim to identify the physical properties required for two applications: 1. Materials healing, that we evaluate through rewelding of pre-cut samples and subsequent tensile tests, and 2. Surface smoothening of 3D-printed objects, serving to remove superficial defects and improve their appearance. The optimal formulation consisting of a composite reinforced with 5 vol.% of Fe nanoparticles ensures a high ability to heat while keeping a low viscosity in the molten state being ideal for polymer processing.

INTRODUCTION

Recent societal trends oriented towards the reuse of technological goods have emerged as the antithesis of the built-in obsolescence philosophy. The development of such sustainable approaches is however not an easy task, most often burdened by a limited number of effective repairing and recycling steps of the materials. A typical example is that of filled elastomers, solicited in a cyclic manner over long periods until their inevitable disintegration. This is the case for tires, shoe soles, seals and wire sheaths that generate a massive pollution on land [START_REF] Boucher | Primary microplastics in the oceans: a global evaluation of sources[END_REF][START_REF] Sommer | Tire abrasion as a major source of microplastics in the environment[END_REF] and in the oceans. [START_REF] Boucher | Primary microplastics in the oceans: a global evaluation of sources[END_REF] Yet, a technological gap exists to design repairable and recyclable elastomers. Indeed, although the vulcanization process provides rubber-based materials with unrivalled mechanical properties, the presence of chemical crosslinks prevents their healing and any possible second life. The challenge resides therefore in finding an alternative satisfying highly demanding requirements in terms of modulus, toughness, wear resistance, chemical stability, and cost. Self-healing rubbers based on supramolecular polymer networks gave reason for hope which unfortunately failed to materialize due to their limits in terms of mechanics, chemical endurance (e.g., sensitivity to humidity [START_REF] Louhichi | Humidity affects the viscoelastic properties of supramolecular living polymers[END_REF] ) and scalability at the industrial level.

A viable solution resides in thermoplastic elastomers (TPE, or TPU standing for thermoplastic polyurethanes) which represent a credible alternative for the production of both rubber-based mass goods and specialty polymers. TPEs are often made of crystallizable multiblock copolymers which form biphasic materials at room temperature. While assemblies of hard segments, playing the role of physical cross-links, confer the rubber its elasticity, the soft phase ensures its flexibility. [START_REF] Gaymans | Segmented copolymers with monodisperse crystallizable hard segments: Novel semi-crystalline materials[END_REF][START_REF] Drobny | Handbook of Thermoplastic Elastomers[END_REF][START_REF] Baeza | Multiscale Filler Structure in Simplified Industrial Nanocomposite Silica/SBR Systems Studied by SAXS and TEM[END_REF] Above their melting temperature, hard-domains dissociate, resulting in chains diffusion and macroscopic solid-to-liquid transition. On the other hand, when cooled down at service temperature, hard-segments gather back from the liquid state providing solid-like properties to the material. This dual behavior considerably eases the processing of TPEs, enabling reuse, repair and recycling, conversely to vulcanized rubbers.

The pioneering work of Corten and Urban [START_REF] Corten | Repairing polymers using oscillating magnetic field[END_REF] showed the possibility to repair a polymer-based responsive composite through the use of an oscillating magnetic field. However, this study was restricted to a low-(costly) fluorinated terpolymer. Alternatively, Ramanujan et al. developed promising healable rubbers based on copolymers (either polyethylene-vinyl acetate or acrylonitrile butadiene) [START_REF] Ahmed | Magnetic field triggered multicycle damage sensing and self healing[END_REF][START_REF] Panigrahi | Bio-Inspired Multiple Cycle Healing and Damage Sensing in Elastomer-Magnet Nanocomposites[END_REF] but the lack of systematic physical characterization inhibits the adaptation of the concept to more conventional, i.e., industry-oriented, chemistries. Alternatively, several research groups attempted to control the solid-to-liquid transition in model supramolecular networks through induction heating. [START_REF] Hohlbein | Remote-controlled activation of self-healing behavior in magneto-responsive ionomeric composites[END_REF][START_REF] Falco | Supramolecular Superparamagnetic Nanocomposites Based on a Magnetite-Filled Unentangled Terpyridine-Functionalized Polymer[END_REF][START_REF] Ogliani | A thermo-reversible silicone elastomer with remotely controlled self-healing[END_REF] In these examples, ionic aggregates, terpyridine (TPy) and ureidopyrimidinone (UPy) moieties were respectively used, without providing strong mechanical properties to the materials, which displayed a shear modulus below 1 MPa or a flow regime at room temperature. Importantly, all the above-mentioned materials (excluding ref. 11) were prepared through solvent casting, precluding large scale production. In summary, although magneto-responsive thermoplastic rubbers are henceforth quite popular to serve as actuators, [START_REF] Chen | Light-and magneticresponsive synergy controlled reconfiguration of polymer nanocomposites with shape memory assisted self-healing performance for soft robotics[END_REF][START_REF] Erb | Actuating soft matter with magnetic torque[END_REF][START_REF] Deng | Laser reprogramming magnetic anisotropy in soft composites for reconfigurable 3D shaping[END_REF] it appears that triggering their phase transition in view of a possible healing or post-processing treatment remains a largely uncharted territory, limited to the field of adhesives [START_REF] Bae | Study on the heating behavior of Ni-embedded thermoplastic polyurethane adhesive film via induction heating[END_REF][START_REF] Salimi | Composite polyurethane adhesives that debond-on-demand by hysteresis heating in an oscillating magnetic field[END_REF] and bioengineering-oriented hydrogels. [START_REF] Nardecchia | Synthesis and rheological properties of 3D structured self-healing magnetic hydrogels[END_REF] Besides, one can also mention the remarkable work of Yang et al. on model magnetic nanocomposites based on (more conventional) polymethyl methacrylate [START_REF] Yang | Defect-targeted self-healing of multiscale damage in polymers[END_REF] and polypropylene [START_REF] Yang | Selfhealing of electrical damage in polymers using superparamagnetic nanoparticles[END_REF] matrixes where microcracks are healed through an optimized induction heating process.

In the present work, we propose to upgrade commercial TPEs towards (nano)composites filled with magnetic particles making them responsive (stimulus-healable), while enhancing their stiffness. We wish to step away from the "self-healing" character that is still of limited interest for structural applications, and to focus on a convenient "healing on-demand" faculty based on induction-heating. The latter feature, based on magnetic hysteresis losses in the case of micro-and nanoparticles, [START_REF] Deatsch | Heating efficiency in magnetic nanoparticle hyperthermia[END_REF] presents several advantages with respect to usual convective heating:

(i) Remote control enables to generate heat through insulating materials (see e.g., the Teflon film we use during healing), being of particular interest for TPE used as adhesives. [START_REF] Bae | Study on the heating behavior of Ni-embedded thermoplastic polyurethane adhesive film via induction heating[END_REF] (ii) Spatio-selective heating is possible through particles' localization in macro-architectured materials enabling localized phase transitions.

(iii) High tunability of the heating process is achievable because the heat generation does not depend only on the external stimulus but also on the material's properties (e.g., content and nature of particles as well as matrix viscosity). [START_REF] Deatsch | Heating efficiency in magnetic nanoparticle hyperthermia[END_REF] We first describe in details the structure of three series of magnetic nanocomposites (filled with either Fe or Fe3O4 nanoparticles, or Fe microparticles) and their physico-chemical properties, i.e., thermal, rheological and magnetic characteristics, with regard to their composition. Next, we study in a systematic way the heating efficiency displayed by these materials upon exposure to oscillatory magnetic fields and show how the nature and the volume fraction of magnetic particles enable precise tuning of the temperature increase in the composites. With possible applications in mind, we then investigate the solid-state mechanics of pristine and magnetically healed composites to demonstrate that recovery can be achieved within a minute through induction heating. Finally, based on our optimal formulation containing 5 vol.% of Fe nanoparticles, we produce samples through additive manufacturing and use our technology to smoothen their surface.

MATERIALS AND METHODS

2.1.Materials

Polymer Matrix: The polymer matrix is a commercial TPU, Desmopan 85085A (Covestro, Germany) with Mw = 120 kg mol -1 and molar-mass dispersity = 2. The content in hard-segments (HDI-HDO-HDI) is 28 wt.%. The soft-segments are made of two different motifs (SS1: butanediol polyadipate) and SS2 (polypropylene oxide) respectively representing 50 wt. % and 22 wt.% of the TPU (see SI section 1 for the full NMR characterization of the TPU).

Magnetic Fillers: The three magnetic nanoparticles (powders) were purchased from Nanografi (Turkey). For the sake of simplicity and possible upscaling no surface functionalization was performed. We refer to these particles as Fe3O4 (magnetite nanoparticles), Fe-n (iron nanoparticles) and Fe-µ (iron microparticles). Particles sizes are displayed in Table 1. for thermogravimetric analysis).

Methods

Atomic Force Microscopy (AFM): AFM phase micrographs were performed on a Dimension 3100 equipped with a Nanoscope V scanning probe controller (Bruker, USA) using tapping mode.

All experiments were performed in air and at room temperature using noncontact tips with frequencies around 330 kHz, a radius of curvature smaller than 7 nm, and a nominal spring constant of 42 N m -1 (PPP-NCH from Nanosensor, Switzerland). The neat TPU displayed in Figure 1B was extruded similarly as the composites and subsequently hot-pressed at 160 °C for 3 minutes.

Small Angle X-ray Scattering (SAXS): The SAXS experiment presented in Figure 1C was carried out on a Xeuss 2.0 apparatus (Xenocs, FR) at the Laboratoire Léon Brillouin (LLB, CEA Saclay, France). The instrument uses a microfocused Cu-Kα1 source with a wavelength of 1.54 Å and a Pilatus3 detector (Dectris, Switzerland). The TPU was shaped into 1 mm thick films and the sample-detector distance was set to 2.5 m to achieve a q-range from 0.03 -2 nm -1 . Data were corrected from the background, transmission and thickness sample to obtain the scattering intensity in absolute units (cm -1 ). SAXS results measured on Fe3O4 and Fe-n based nanocomposites are presented in SI Section 3.

X-ray Diffraction (XRD): XRD measurements were performed at room temperature on a D8 advance apparatus (Bruker, USA) using Cu-Kα1 radiation. Diffraction patterns of our powders were recorded in the 2θ range of 20-90°.

Transmission Electron Microscopy (TEM): For conventional TEM observations, thin sections were prepared by cryo-ultramicrotomy using a Leica Ultracut UCT microtome (Germany) with a diamond knife from DiAtome, United Kingdom. The temperature of the sample and the cutting speed were set to -80 °C and 0.3 mm s -1 respectively. Sections of about 90 nm thick were drycollected and placed on a 400-mesh copper grid. TEM images were acquired using a CM120

Philips transmission electron microscope operating at an accelerating voltage of 120 kV.

Scanning Electron Microscopy (SEM): Powders and composites micrographs were obtained from a VEGA3 apparatus (TESCAN, USA). The acceleration voltage of the electrons was set to 10-20 kV, and the beam intensity "BI" was varied from 3 to 13. Either secondary or backscattered electrons were used (see micrographs). Focused Ion Beam -Scanning Electron Microscope (FIB-SEM) required a two-step milling procedure on the bulk nanocomposite surface, which had previously been metalized with gold. This procedure used high and fine current beams (4 nA and 80 pA respectively) to analyze a polished surface using an in-lens secondary electron (SE) detector.

Differential Scanning Calorimetry (DSC): A Perkin Elmer DSC 8000 was systematically used.

Experiments were run under a nitrogen flow of 20 mL min -1 at 10 °C min -1 . Samples were first cooled down to -90 °C and heated up to 220 °C (-80 to 200 °C displayed). This cycle was then repeated once, resulting in the measurements displayed in Figure 3A-C (2 nd heating segment).

Rheology: Frequency sweep measurements at 180 °C (100 > % > 0.1 rad s -1 and & = 10-50 % according to the materials) were all performed in a strain-controlled rheometer (ARES 2KFRTN1 from Rheometric Scientific, currently TA, USA) using stainless steel parallel plates of 8 mm diameter (ca. 0.8 mm in sample thickness). A nitrogen convection oven allowed maintaining inert atmosphere with a temperature control better than ±1 °C.

Tensile Tests: Uniaxial tensile tests were performed at room temperature on a 1/ME device (MTS, USA) equipped with a 1 kN load cell at a fixed of rate 10 mm min -1 . The samples were shaped into dumbbell pieces having working dimensions of 10.0 × 4.0 × 1.0 mm 3 . A minimum of three tests was performed for each configuration.

Induction Heating and Thermal Imaging: Induction heating was performed with a master controller v3+ apparatus (CEIA, Italy) connected to a pancake-shaped (flat spiral) inductor made of two concentric turns. A 0.1 mm-thick Teflon composite tape covered the inductor to protect it from material flowing. The frequency of the magnetic field was set to 855 kHz and its maximum amplitude to ca. 10 mT corresponding to an intensity of 19 A in the inductor. The alternative current is delivered through electric pulses. The effective irradiation of the samples is referred to as ' ()*+, /' (,-.01 and ranges from 17% to 100%; ' ()*+, and ' (,-.01 are respectively the duration of the electromagnetic pulse and the time in-between two pulses such as illustrated in Figure 4C.

Because of the square decrease of the magnetic field amplitude with the distance, the healing procedure was performed at a coil-sample distance not exceeding 5 mm. Disk-shaped samples (6 mm in diameter and 0.48-0.52 mm thick) were heated and their temperature variations were recorded with a PI 450 infrared camera (Optris, Germany) equipped with a 13° lens characterized with an optical resolution of 382 × 288 pixels and a measurement rate of 27 Hz. Temperatures reported in this work refer systematically to the maximal temperature recorded by the device. A schematic representation and photographs of the whole dispositive are displayed in Figure 4A-B.

Data were processed with the software Optix PIX Connect in the 20-250 °C temperature range.

The healing of dumbbell specimens used for tensile test experiments consisted of cutting samples in two identical pieces and placing them in a silicone mold of corresponding dimensions equipped with an anti-adhesive bottom part. After a minute of induction heating such as illustrated in Figure 8C, the healed specimen was extracted from the mold and measured again (see mechanical tests in Figure 8A).

X-ray microtomography: Tomography imaging was performed on a Phoenix v|tome|x (GE Sensing & Inspection Technologies, USA) with a PaxScan digital X-ray image detector (Varian, USA) at room temperature (ca. 25 °C). We used 80 kV and 180 µA as X-ray parameters, placing the sample vertically at an optimal distance for 3-4 µm for voxel size. 3D reconstructed structures revealing the presence of pores in non-dried healed samples loaded with 5 vol.% in Fe-n particles are available as Supporting Materials. Analogous videos highlighting the good distribution of particles are also provided.

Additive manufacturing: Fused filament fabrication of dumbbell-shaped samples and pyramids was performed on a T333 device from Tobeca (France). The machine was equipped with a pellet's extruder MPE (Tobeca, France) avoiding the additional production of filament. The tests were limited to the case of our optimal formulation consisting of a Desmopan matrix reinforced with 5

vol.% of Fe-n particles. The temperature of the nozzle and the printing stage were set to 210 °C and 50 °C respectively. The printing rate was set to 50 mm s -1 .

RESULTS AND DISCUSSION

Formulation and Structure of the Nanocomposites

Nanocomposites were routinely prepared through extrusion from a commercial TPU and magnetic particles (magnetite, nano-iron and micro-iron) respectively referred to as Fe3O4, Fe-n and Fe-µ hereafter. The chemical structure of the TPU is illustrated in Figure 1A emphasizing the alternance of soft-and hard segments (denoted respectively SS and HS). The HS are based on 1,6hexanediol and 1,6-hexamethylene diisocyanate, well-known for providing highly symmetrical crystalline structure, [START_REF] Matějka | Structure evolution during order-disorder transitions in aliphatic polycarbonate based polyurethanes. Self-healing polymer[END_REF] yet with a reasonable melting temperature (ca. 150 °C). As expected for this class of polymers, [START_REF] Versteegen | Properties and morphology of segmented copoly (ether urea) s with uniform hard segments[END_REF][START_REF] Nébouy | Process-oriented structure tuning of PBT/PTHF thermoplastic elastomers[END_REF][START_REF] Sijbrandi | Synthesis, morphology, and properties of segmented poly (ether amide) s with uniform oxalamide-based hard segments[END_REF] AFM experiments reveal that HS aggregate into long (ca. 100-200 nm) and rather straight ribbon-like crystallites evenly spaced every 11-14 nm (Figure 1B), as confirmed statistically through SAXS ( ! = 2"/# ! $ 13 nm -Figure 1C). XRD diffractograms emphasizing the purity of the three magnetic powders, and SEM micrographs taken from these "as received"

products, are respectively presented in Figure 1D and Figures 1E-G.

After extrusion and subsequent hot pressing, the distribution of fillers within the nanocomposite films is systematically homogeneous at a large lengthscale -see TEM micrographs in Figure 2A, 2E and 2I, representative of the histograms presented in Figures 2D, 2H and 2L. However, a closer look reveals important features. Most Fe3O4 elementary particles ( %& ' ( ) = 14-29 nm) are merged into fractal aggregates of ca. 100 nm of diameter (Figure 2B-C), possibly gathering into branches, reminiscent of the morphology of widely used fumed silica. [START_REF] Benane | Multiscale structure of super insulation nano-fumed silicas studied by SAXS, tomography and porosimetry[END_REF] Then, larger spherical Fe-n particles ( %&*+ = 90-100 nm) aggregate into a relatively wide range of objects containing several tens of nanoparticles (Figure 2F-G). For both types of nanoparticles, we refer the reader to SAXS patterns provided in SI section 3 where the high polydispersity limits the quantitative analysis. Finally, Fe-µ particles appear to be even more polydisperse in size and shape. They contain a great number of micro-particles and a few much larger ones. In addition, needle-like particles, with a typical length of 300 nm and a diameter of 50 nm are observed in Figure 2J-K.

Thermal and Rheological Properties of the Nanocomposites

The choice of a TPU as composite matrix resides in its flexibility at room temperature and its melting point situated below 200 °C. The latter, which depends mainly on the fraction and nature of the HS, ensures a good thermal stability in service while enabling a quick solid-to-liquid transition well before the polymer degradation temperature. However, once filled with nanoobjects, TPUs are likely to behave differently, owing to hydrodynamical effects and to interactions between their polar groups (ethers, esters, urethanes) and the surface of the fillers. [START_REF] Bailey | Dynamics of Polymer Segments, Polymer Chains, and Nanoparticles in Polymer Nanocomposite Melts: A Review[END_REF] As a result, physical properties such as glass transition (, -), melting temperature (, . ) and rheological behavior may be modified.

DSC thermograms of our neat TPU and the three series of composites are presented in Figure 3A-C showing that soft domains exhibit a glass transition at -55 °C regardless of the nature and content in magnetic fillers. A first endothermic peak is observed from 0 to 50 °C corresponding to the melting of SS crystallites made of butanediol polyadipate segments (PABD). The corresponding fraction of crystalline SS is 0 1 33 $ 10 wt.% in the neat TPU (see details in SI section 1). While Fe3O4 and Fe-µ fillers do not show any particular effect on the SS melting, indicating limited interactions between the polymer and the nanoparticles, the situation is different with Fen objects whose incorporation progressively results in the disappearance of the SS melting signature. This effect must be understood based on the favorable interactions existing between Fe 2+ ions at the oxidized Fe-n particles surface and the most polar segments of the TPU (PABD SS and HS) whereas OH group detected on Fe3O4 particles seem not to interact favorably with them (see FTIR spectra in SI section 4). More expectedly, the lack of visible interaction between the polymer and the Fe-µ particles undoubtedly stems from their much lower specific surface area.

Beyond the impact on the SS crystallites, increasing the content in Fe-n nanoparticles is further seen to reduce both the melting point and the fraction of crystalline HS, synonymous of a significant alteration of the whole polymer network. All these observations are summarized in Figure 3G, where we represent both the SS and HS melting enthalpy as a function of the filler fraction. Importantly, all the crystalline domains of TPU have completed their melting above 150 °C. The HS crystalline content, which systematically decreases with increasing the filler content, varies in-between 0 1 43 $ 11 % and 15 % in the Fe3O4 and Fe-µ series, while it is slightly lower in the Fe-n one, varying in-between 0 1 43 $ 56 % and 12.6 %.

While necessary, TPU melting is not a sufficient condition to make the composites flow and promote macroscopic healing or surface smoothening. Indeed, it is well known that the addition of micro-and/or nanoparticles to a polymer melt changes its rheological properties. The latter are mostly dependent on the particles fraction and dispersion as well as their interactions with the polymer chains. Series of frequency sweep measurements, (storage (7') and loss (7'') shear moduli as a function of the pulsation 8), performed on the three types of nanocomposites far above the TPU melting point (, = 180 °C) are presented in Figure 3D-F. While in the case of homogeneous liquid-like materials such as molten TPUs, one expects 79 < 799 with 79~8 : and 799~8 ; (Maxwell fluid [START_REF] Rubinstein | Polymer Physics[END_REF] ), adding progressively particles is expected to make them more elastic until reaching a possible viscoelastic solid or "gel" state satisfying 79 > 799 with both 79 and 799 being independent of 8.

This usual trend is visible in both Fe3O4 and Fe-n series where the nanocomposite melts see their elasticity growing (or ?@AB CDE = B799/79 decreasing) with the particles content until reaching solidlike properties at ca. 15 vol.% (?@AB CDE < 5). Nevertheless, we believe that the physical origin of such gelation is different in those two systems. Indeed, Fe3O4 particles are much smaller and organized in a different way with respect to the Fe-n ones. In particular, their higher specific surface and their pre-aggregated morphology enhances their ability to form branches likely to percolate across the TPU. In spite of a lower specific surface area, DSC thermograms clearly underlined the preferential interactions of larger and well-spherical Fe-n particles with the TPU, likely to explain the creation of an elastic network in the composite loaded with 15 vol.% in nanoparticles, being as strong as in the case of its Fe3O4 counterpart. In great contrast with Fe3O4

and Fe-n series, the addition of Fe-µ particles into the TPU are seen to have a quite limited impact on its rheology that we assign to a much lower specific surface area and no significant interaction with the polymer chains, in good agreement with DSC results reported in Figure 3C. For the sake of clarity, we report in Figure 3H the global evolution of the storage modulus at 10 rad s -1 (7'C8 ;F E)

for the three series of composites. From the sole rheological point of view (Figure 3D-F), i.e., because they flow on a reasonably short timescale (terminal relaxation time well below 0.01 s resulting in 7'' > 7'), the nanocomposites filled with 5 vol.% in particles appear to be promising candidates for the design of healable materials.

Induction Heating and Thermal Imaging

We next look at the ability to heat of our composites in the presence of an oscillatory magnetic field, targeting 150-200 °C to trigger the TPU melting without degradation. In view of establishing suitable conditions of healing and mechanical recovery, it is mandatory to precisely monitor the temperature evolution during the sample's treatment in order to optimize its flow, i.e., to tune its viscosity so that cracks and micro-defects can be erased without any visible polymer degradation.

The induction-heating device used in this work is described in Figure 4A. It includes a highfrequency generator (855 kHz) delivering 19 A in a 2-turns pan-cake coil (Figure 4B). The current is generated in the form of pulses (see Figure 4C). A 3D model of the pancake coil is presented in The evolution of the temperature as a function of oscillatory magnetic field exposition time is given in Figure 5A-C for the three series of nanocomposites. The Fe3O4 series appears to be the simplest to rationalize since it seems to follow the case in which particles dissipate a constant amount of heat in their surrounding medium regardless of the experimental time and temperature.

This well-established situation results in an increase of the temperature of the material with a continuously decreasing rate ,/ ? until reaching a permanent regime. This profile, fitted with Equation 1, being the 0-dimension solution of the heat equation for a constant energy flux, indicates that a single heating mechanism, assigned to the hysteresis losses occurring in magnetic particles is involved. We refer the reader to SI section 5 for electric and magnetic characterizations of the composites and SI section 6 for more information on the heating mechanisms. In Equation 1, , F , G, and H stand respectively for the room temperature (≈ 22 °C), the amplitude of the heating process (°C) and its characteristic time (s).

,C?E = , F I G,C5 J e *K L M E (1)
While a "single heating mechanism" behavior is also observed for Fe-n and Fe-µ composites at 1 vol.%, the temperature profiles recorded for at higher filler fractions are qualitatively different.

In fact, it is clear that a second mechanism is triggered in both series when the materials temperature approaches the TPU melting point at ca. 150 °C. This additional heat dissipation, stems from the dramatic fall of viscosity at the TPU melting point favoring the rotation of nanoparticles (see section 3.4). This motion is not spontaneous as for Brownian relaxation, [START_REF] Torres | The relevance of Brownian relaxation as power absorption mechanism in Magnetic Hyperthermia[END_REF][START_REF] Abenojar | Structural effects on the magnetic hyperthermia properties of iron oxide nanoparticles[END_REF] (H N O 5/2"P -see SI section 6) but results from the applied magnetic field generating a torque in the particles that leads to high-frequency friction with the surrounding polymer and extra-heat dissipation. [START_REF] Hiergeist | Application of magnetite ferrofluids for hyperthermia[END_REF][START_REF] Bayerl | Induction heating of thermoplastic materials by particulate heating promoters[END_REF][START_REF] Yassine | Highly efficient thermoresponsive nanocomposite for controlled release applications[END_REF] In analogy with Fe3O4 data, Fe-n and Fe-µ results were fitted with the Equation 2A-B below

,C? < ? . E = , F I G, ; C5 J e *K L Q M E (2A) ,C? > ? . E = , F I G, ; R5 J e *K L Q M S I G, : C5 J e *CK*T U E L V M E (2B)
where ? . is the time at which melting happens.

Coming back to the case of Fe3O4, no extra heating contribution is seen in the 10 and 15 vol.% composites in spite of , > , . . This result is understood based on both rheological and structural information. First, Fe3O4 nanobeads are much aggregated so that, free rotation is hindered when the magnetic field is flipped. Second, heating temperatures above , . can only be reached above 10 vol.% in particles, corresponding to the apparition of a 7' elastic plateau at low frequency (Figure 3D), indicating limited motion of beads. However, it seems that increasing the pulse duration of the magnetic field allows the melting of the TPU from 5 vol.% in Fe3O4, where gelation is not reached yet, allowing motion of least aggregated nanoparticles to generate a slight extraheating (see Figure 5D).

For the sake of comparison, we collected in Figure 5E all the specific absorption rates (WXY) in W g -1 for ? Z[\]& /? Z&^_`a = 17%, calculated from:

WXY = b Z c Z , ? d TfF (3)
where b Z and c Z are the specific heat capacity and mass fraction of fillers. While the WXY order of magnitude is similar for the three series of composites (30-80 W g -1 ), Fe-n particles appear to be more efficient than Fe3O4 and Fe-µ ones in terms of heat generation, which is explained through a higher value of magnetic saturation resulting in a broader gChE hysteresis loop at high frequency. [START_REF] Coey | Magnetic materials[END_REF] It is further noteworthy that WXY values remain almost constant with increasing regardless of the particles nature, indicating a limited impact of dipolar interactions. [START_REF] Deatsch | Heating efficiency in magnetic nanoparticle hyperthermia[END_REF][START_REF] Linh | Magnetic fluid based on Fe3O4 nanoparticles: Preparation and hyperthermia application[END_REF][START_REF] Piñeiro-Redondo | The influence of colloidal parameters on the specific power absorption of PAA-coated magnetite nanoparticles[END_REF][START_REF] Urtizberea | Specific absorption rates and magnetic properties of ferrofluids with interaction effects at low concentrations[END_REF] This is corroborated in Fe3O4 Fe-n series by SAXS diffractograms showing no further aggregation of the particles with increasing i (SI section 3).

Solid-State Tensile Properties of Nanocomposites

The last requirement in terms of physical properties regards the mechanical performances in service. While adding micro-or nanoparticles to the TPU matrix is expected to enhance its stiffness at low deformation (reinforcement [START_REF] Song | Concepts and conflicts in nanoparticles reinforcement to polymers beyond hydrodynamics[END_REF] ), the behavior at large amplitude may on the other hand, be strongly impacted by the creation of defects favoring crack initiation and propagation. In Figure 7A-C, we present quasi-static tensile tests performed on the three series of nanocomposites revealing these effects. As expected, the drop of toughness is visible in Figure 7D, which gathers the whole data set in terms of energy at failure ( !"#$% ) versus filler fraction, showing a similar trend regardless of the particle's nature. Besides, the Young's modulus enhancement (Figure 7E)

indicates that Fe3O4 particles reinforce significantly more the TPU than Fe-n and Fe-µ ones as one may have expected from their higher specific surface and pre-aggregated nature favoring mechanical percolation. Here again, the composites loaded with 5 vol.% appear as a good compromise in terms of solid-state mechanics in spite of their limited reinforcement (ca. 30% stronger than the neat TPU for Fe3O4 and Fe-n series). Strikingly, the stress-strain response of the healed materials systematically overlaps with the non-damaged samples up to above 1.2 true strain unit, far above the standard use of common TPU based goods. This is further emphasized in Figure 8B where Original, Reshaped and Healed I samples exhibit identical tensile behaviors, regardless of the particles' nature. Distinctive behaviors are exclusively observed at very high strain where Healed I samples systematically show a lower strain at fracture than their Reshaped counterparts. This is assigned to a different microstructure around the healed zone caused by the persisting phase separation within the liquid TPU, as observed at low frequency in Figures 3D-F. In fact, this lack of dissociation during healing inhibits the whole memory erasure, limiting the recovery of the TPU structure once cooled down.

In addition, structural reorganization of the particles in Fe-n and Fe-µ composites (Figure 6) are also expected to impact the behavior at large strain.

To track the potential formation of defects upon mending, we further performed X-ray tomography on a 5 vol.% Fe-n composite before and after the induction healing process (see Figure 8E-G and corresponding videos in Supplementary Materials). Although samples looked similar macroscopically, X-ray tomography revealed the presence of large holes in Healed I materials that were formed during the healing process owing to evaporation of residual water in the TPU (see Figure 8F). Consistent with our thoughts, no hole was detected in mended composites when the samples were vacuum-dried before healing; Figure 8G and its corresponding video shows complete structural recovery. Note though that such sample treatment is generally not mandatory as similar mechanical recovery was observed in Figure 8A for hole-containing and hole-free composites. This is explained by the large size of the critical defect admissible in relatively soft rubber-based materials.

Beyond this high mechanical recovery, another asset of our procedure is that the composites healing is extremely fast -1 minute or less-making any comparison with self-healing mechanisms irrelevant (see the Graphical Abstract adapted from ref. 39, which further illustrates the high mechanical properties of the so-healed materials). For example, the pioneer work of Cordier et al.

demonstrated 85% recovery of the tensile properties after 180 minutes. [START_REF] Cordier | Self-healing and thermoreversible rubber from supramolecular assembly[END_REF] To illustrate the general character of our approach, we have further proven that the same procedure can be effectively applied for the reparation of any TPE (see SI section 8). We prepared a 5 vol.% Fe-n composite using another TPE made of polybutylene-terephthalate and polytetrahydrofuran with a higher HS melting point (i.e., 180 °C). As expected, an additional heating contribution coming from particles reorientation was observed above this temperature when induction heating was used. Tensile tests on healed samples confirmed that this material could also be successfully repaired albeit suffering from the same limitations at high strain.

Our methodology is therefore effective regardless of the nature of the magnetic filler and of the TPU and can be optimized based on the properties we identified in this work. For example, it is worth pointing out that Fe3O4 and Fe-µ based materials are slightly more fragile (Original and Reshaped) and less healable (Healed I) than their Fe-n-based counterparts (see Figures 7A-C and8A). As suggested from DSC thermograms, we believe that lower elongations at break are due to weaker interactions between the polymer chains and the particles.

Induction heating as a post-treatment of 3D printed objects

In this last part, we present some opportunities that offers our technology regarding the posttreatment of objects produced through fused filament deposition, unavoidably presenting topographic defects that are sometimes smoothened by using solvent vapor treatments. Here, the solid-to-liquid phase transition triggered by the magnetic field enables to melt the surface of the so-prepared object, letting the surface tension of the resulting liquid driving the surface quality.

The contactless nature, the rapidity, the limited penetration of the magnetic field and the possibility to use co-deposition to tailor the location of the nanoparticles are strong assets in view of a possible upscaling at the industrial level.

To illustrate the potential of this approach, we first manually applied the magnetic field on a face of a truncated pyramid (Figure 9A) made through fused filament deposition of our TPU based composites loaded with 5 vol.% in Fe-n particles. While the surface imperfections related to the manufacturing process are well visible on the left-hand side, a much smoother surface is obtained after only a few seconds of irradiation (right hand-side). Besides, we repeated this procedure in Figure 9B, where we treated a porous dumbbell-shaped sample. Here again, after a few seconds the matter is densified resulting in a much shinier surface, demonstrating the possibility to reach an "injected look" within a short time.

CONCLUSION

We developed a simple and straightforward approach to produce ultrafast healable and strong rubber-like materials scalable at the industrial level. These materials are based on common components and can be processed using extrusion, injection as well as fused filament fabrication.

This strategy can be successfully applied to a range of magnetic fillers with different dimensions and chemistry so that induction heating parameters can be precisely tuned to fit with the material's solid-to-liquid phase transition. The healed materials exhibit a satisfying recovery of the mechanical properties. Also, the only requirement for the polymer matrix lies in a good compromise of viscosity enabling the material to flow through the cut while preventing the deformation of the sample. This offers a vast field of opportunities for TPU and more conventional thermoplastic homopolymers.

Figure 1 .

 1 Figure 1. A) Schematic representation of the TPU architecture and corresponding topological formulae of each type of segment. B) AFM micrograph of a hot-pressed sample of Desmopan 85085A. C) SAXS pattern of the Desmopan emphasizing the inter-ribbon distance observed in B. D) XRD patterns of the three magnetic powders. E-G) SEM micrographs of Fe3O4, Fe-n and Fe-µ powders.

Figure 2 .

 2 Figure 2. TEM and SEM micrographs of composites containing ca. 5 vol.% in Fe3O4 (A-C), Fe-n (E-G) and Fe-µ magnetic particles (I-K). Corresponding number and volume distributions are provided in the histograms (D, H, L); see section 2 for average values. Note that (K) is split into two images representing respectively an isotropic and an aligned arrangement of Fe-µ needles.

Figure 3 .

 3 Figure 3. A-C) DSC thermograms measured from the TPU matrix and composites loaded with ca. 1, 5, 10 and 15 vol.% in Fe3O4, Fe-n and Fe-µ respectively. The heat flux is normalized by the TPU mass in the sample. Nanocomposites data are shifted for clarity. G) HS and SS melting enthalpy extracted from A-C, dotted lines are guides for the eye. D-F) Rheology frequency sweep measurements performed at 180 °C (1 vol.% data is not shown) for the three composites. Full and open patterns correspond to the shear storage (7') and loss (7'') moduli respectively. H) Shear storage modulus value at 10 rad s -1 and 180 °C as a function of the filler volume fraction for the three series of composites -extracted from D-F. Dotted lines are guides for the eye.

Figure 4D .

 4D Figure 4D. The resulting magnetic field profile is established through Comsol simulations (see

Figure 4 .

 4 Figure 4. A) Schematic representation of the induction heating setup. B) Photograph of the heating stage made of the pancake-shaped coil, a silicone mold and the sample. The shiny red dot comes from a pyrometer. C) Oscillogram of the voltage applied to the coil during the healing procedure. (? Z[\]& /? Z&^_`a = 17%) D) 3D model of the pancake coil. E) Comsol simulation of the magnetic field generated in the surroundings of the pancake coil during the induction heating process evidencing that its amplitude within the sample does not exceed ca. 6 mT. (top view). F) Analogous figure in side view.

Figure 5 .

 5 Figure 5. A-C) Temperature profiles as a function of time during induction heating for the three series of composites loaded with ca. 1, 5, 10 and 15 vol.% in Fe3O4, Fe-n and Fe-µ respectively (color), the grey line stands for the neat TPU. ? Z[\]& /? Z&^_`a was fixed to 17%. Dashed lines are fitted to the Fe3O4 and Fe-n (short time) data with Equation 1, and to Fe-µ data with Equation 2. D) Temperature profile of a composite loaded with ca. 5 vol.% in Fe3O4 for growing magnetic pulse times. The slight extra-heating indicates possible motion of nanoparticles. E) WXY as a function of the filler volume fraction for the whole set of composites. Dotted lines are guides for the eye representing the evolution of the first heating process only.

Figure 6 .

 6 Figure 6. A) SEM micrographs obtained from a composite containing ca. 5 vol.% in Fe-µ particles. Needles are clearly seen to align under a permanent magnetic field. B) FIB-SEM micrographs obtained from a composite containing ca. 5 vol.% in Fe3O4 after being healed 60 s, no alignment is observed. C-D) FIB-SEM micrographs from a composite containing ca. 5 vol.% in Fe-n, C) before and D) after healing, where dipolar chains are observed.

Figure 7 . 3 . 6

 736 Figure 7. A-C) Tensile tests (true stress vs. true strain) carried out on the TPU matrix and on composites loaded with ca. 1, 5, 10 and 15 vol.% in Fe3O4, Fe-n and Fe-µ respectively. D) Energy at failure as a function of the volume fraction in filler for the whole set of composites. The dotted line is a linear guide for the eye. E) Normalized Young's modulus as a function of the filler fraction showing the reinforcement effect. Dashed lines stand for the hydrodynamic models of Einstein-Smallwood & ' ' ( = 1 + 2.5)* and Guth-Gold & ' ' ( = 1 + 2.5) + 14.1,) -* giving an idea of the

Figure 8 .

 8 Figure 8. A) Tensile tests performed on nanocomposites loaded with ca. 5 vol.% in Fe3O4, Fe-n and Fe-µ. See text for Original, Reshaped, Healed I and Healed II mentions. Reported heating duration and / 0367# 8/ 0#"9:; were adapted according to the particle's <>?. B) Zoom-in tensile curves at low and moderate strain amplitudes. C) Photograph of the setup during the healing process. Thermal imaging indicates the zone of the sample affected by induction heating. D) Photograph of a sample at different stages of the healing procedure. E-F-G) Tomography micrographs of Reshaped, Healed I and Healed II composites loaded with 5 vol.% in Fe-n particles, respectively. Corresponding videos and 3D reconstructions are provided as Supplementary Material.

Figure 9 .

 9 Figure 9. A) Impact of the smoothening procedure on the topography of a truncated pyramid. Optical micrographs of untreated (left) and treated (right) faces. B) Analogous figure for a dumbbell-shaped sample.

Table 1 :

 1 Particles size as provided by the supplier and determined through image analysis from

	nanocomposites micrographs. " # $$$ and " % $$$$ refer to the average diameters in number and in weight
	respectively. Corresponding histograms are provided in Figure 2D, 2H and 2L.
		Fe3O4	Fe-n	Fe-µ
	Supplier	14-29 nm	90-100 nm	"325 mesh" (< 44 microns)
	Image analysis ( " #### , $ #### )	14 nm, 16 nm	74 nm, 101 nm	3 µm, 39 µm

ACKNOWLEDGMENT All the authors thank Vivien Truchot and Laura Courty (IMP, INSA-Lyon) for their help with the samples' preparation and 3D-printing respectively. Eric Maire and Jérôme Adrien (MATEIS, INSA-Lyon) are gratefully acknowledged for their technical support with the tomography investigations. Jacques Jestin (LLB, CEA Saclay) performed the SAXS measurement on the neat TPU. The NMR Polymer Center of the "Institut de Chimie de Lyon" (FR3023) and especially Dr Fernande Da Cruz-Boisson are acknowledged for assistance and access to the NMR facilities. P.G., G.C., F.M., J.B., S. M. and G.P.B. acknowledge the financial support of the Institut Carnot I@L for the funding assigned to the project POMMADE. G.P.B. acknowledges the financial support of IDEX-Lyon and INSA-Lyon through the program ELAN-ERC. All the authors are indebted to the ''Microstructure Technological Center'' (CTµ) of University of Lyon for the access to the transmission electron microscope and the ultramicrotomy device.

Evidence for particles reorientation under oscillatory magnetic field

The extra-heat generation detected in Fe-n and Fe-µ composites at the TPU melting (see Figure 5B-C) strongly suggests the possibility for the nanoparticles to move under the application of the magnetic field, acting as an external force. On the contrary, the absence of extra-heating in Fe3O4 based composites (Figure 5A), including at , > , . , indicates that the motion of such aggregated particles is unlikely. To verify these hypotheses, we present several micrographs in Figure 6. First, we show that Fe-µ needles can be oriented easily in a composite hot-pressed at 180 °C in presence of a permanent magnet (Figure 6A). Then, we used a dual column FIB-SEM to characterize the cross-section of nanocomposites containing ca. 5 vol.% in either Fe3O4 or Fe-n. While, Fe3O4 particles do not reveal any alignment after 60 s of induction heating (Figure 6B), the same experiment generates a clear alignment of particles in the case of Fe-n fillers (Figure 6C-D, respectively obtained before and after induction heating). We can thus confirm that the highfrequency magnetic field enables both rotational and translational Fe-n particles diffusion, resulting into the creation of dipolar chains likely to impact the heating efficiency and the mechanics of materials healed several times. This important "durability-oriented" question will be examined in a future work involving X-ray photon correlation spectroscopy and thermal imaging performed in-situ during induction heating. ASSOCIATED CONTENT Supporting Information.

1. Full chemical characterization of the Desmopan 85085A. 2. Thermogravimetric analysis of all the composites. 3.Small angle X-ray scattering performed on the Fe3O4 and Fe-n series of composites. 4. Fourier transform infrared spectroscopy of all the powders. 5. Electric and magnetic characterization of the composites. 6. Quantitative rationalization of the heat generation. 7.

Complementary tensile tests. 8. Transposition of the healing procedure onto an alternative polymer.

The following files are available free of charge.

X-ray Tomography: Figure8E(Reshaped).mp4, Figure8F(HealedI).mp4, Figure8G(HealedII).mp4 3D-reconstructed animations : 8F_Healed-non-dried, 8G_Healed-dried

AUTHOR INFORMATION

Corresponding Author *G.P.B.: guilhem.baeza@insa-lyon.fr

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Notes

The authors declare no competing financial interest.

GRAPHICAL ABSTRACT


Eva Gfrerer

Danae Laina

Rüdiger Wagner

Marc Gibernau

Anja C Hörger

Hans Peter Comes

Stefan Dötterl
email: stefan.doetterl@plus.ac.atorcid

Arum Maculatum

L Eva Gfrerer

COMES Hans Peter

Antennae of psychodid and sphaerocerid flies respond to a high variety of floral scent compounds of deceptive Arum maculatum L

Keywords: Arum maculatum, brood-site mimicry, Coproica ferruginata, cow dung, GC-EAD, GC-MS, Psychoda spp., physiologically active volatiles

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

Introduction

Floral scents are important mediators of plant-animal interactions [START_REF] Raguso | Wake up and smell the roses: the ecology and evolution of floral scent[END_REF] . Typically, a floral scent bouquet is composed of 20-60 volatiles per plant species [START_REF] Knudsen | Diversity and distribution of floral scent[END_REF] , but some species emit highly complex bouquets consisting of more than 100 volatiles [START_REF] Hadacek | Club-shaped organs as additional osmophores within the Sauromatum inflorescence: odour analysis, ultrastructural changes and pollination aspects[END_REF][START_REF] Schlumpberger | Geographic variation in floral scent of Echinopsis ancistrophora (Cactaceae); evidence for constraints on hawkmoth attraction[END_REF][START_REF] Gfrerer | Floral scents of a deceptive plant are hyperdiverse and under populationspecific phenotypic selection[END_REF] . In generalist pollination systems, widespread compounds (e.g., 2-phenylethanol, phenylacetaldehyde, benzaldehyde [START_REF] Primante | A syrphid fly uses olfactory cues to find a non-yellow flower[END_REF][START_REF] Knauer | Bees use honest floral signals as indicators of reward when visiting flowers[END_REF][START_REF] Theis | Fragrance of Canada thistle (Cirsium arvense) attracts both floral herbivores and pollinators[END_REF] ) are typically involved in pollinator attractions, whereas in specialised systems reviewed by [START_REF] Bouwmeester | The role of volatiles in plant communication[END_REF] , the specificity in pollinator attraction is often reached by the emission of either specific blends composed of common compounds e.g. [START_REF] Schiestl | Orchid pollination by sexual swindle[END_REF] or highly specific compounds. Examples of such particular compounds are diacetin, which attracts highly specialised oil-collecting bees [START_REF] Schäffler | Diacetin, a reliable cue and private communication channel in a specialized pollination system[END_REF][START_REF] Castañeda-Zárate | Food reward chemistry explains a novel pollinator shift and vestigialization of long floral spurs in an orchid[END_REF] , 4methyl-5-vinylthiazole, which attracts cyclocephaline beetles [START_REF] Dötterl | Evidence for behavioral attractiveness of methoxylated aromatics in a dynastid scarab beetle-pollinated Araceae[END_REF][START_REF] Maia | The key role of 4-methyl-5-vinylthiazole in the attraction of scarab beetle pollinators: a unique olfactory floral signal shared by Annonaceae and Araceae[END_REF][START_REF] Stamm | absolute configurations, and biological activities of floral scent compounds from night-blooming Araceae[END_REF] , or p-cresol as well as dimethyl trisulphide, which attract flies seeking oviposition sites e.g., [START_REF] Jürgens | Chemical mimicry of insect oviposition sites: a global analysis of convergence in angiosperms[END_REF][START_REF] Zito | Dimethyl disulfide and dimethyl trisulfide: so similar yet so different in evoking biological responses in saprophilous flies[END_REF] . Although the number of compounds known to be involved in pollinator attraction is increasing, the biological role of the more than 2,000 floral volatile organic compounds (VOCs) that have already been described [START_REF] Knudsen | Diversity and distribution of floral scent[END_REF][START_REF] El-Sayed | The Pherobase: database of pheromones and semiochemicals[END_REF] is largely unknown.

In plant species with highly complex bouquets, it is often not possible to obtain all floral compounds as pure substances and to disentangle their individual behavioural effects.

Additionally, many scent compounds are often not identifiable, because characterised references are missing, and are then listed as unknowns, which complicates investigations of their biological role [START_REF] Schlumpberger | Geographic variation in floral scent of Echinopsis ancistrophora (Cactaceae); evidence for constraints on hawkmoth attraction[END_REF][START_REF] Gfrerer | Floral scents of a deceptive plant are hyperdiverse and under populationspecific phenotypic selection[END_REF] . In consequence, there are large gaps in our understanding of the chemical communication, especially between plants with complex scents and their pollinators.

The fly-pollinated and brood-site deceptive Arum maculatum L. (Araceae, Fig. 1), a wide-spread European perennial herb, emits a remarkable diverse scent. A single individual releases up to 150 different scent compounds, and overall more than 300 compounds have been recorded for this plant species so far [START_REF] Gfrerer | Floral scents of a deceptive plant are hyperdiverse and under populationspecific phenotypic selection[END_REF][START_REF] Kite | The floral odour of Arum maculatum[END_REF][START_REF] Chartier | Do floral odor profiles geographically vary with the degree of specificity for pollinators? Investigation in two sapromyophilous Arum species (Araceae)[END_REF][START_REF] Chartier | Geographical variations of odour and pollinators, and test for local adaptation by reciprocal transplant of two European Arum species[END_REF][START_REF] Marotz-Clausen | Incomplete synchrony of inflorescence scent and temperature patterns in Arum maculatum L. (Araceae)[END_REF][START_REF] Szenteczki | Spatial and temporal heterogeneity in pollinator communities maintains within-species floral odour variation[END_REF] . However, most of these compounds have not yet been identified [START_REF] Gfrerer | Floral scents of a deceptive plant are hyperdiverse and under populationspecific phenotypic selection[END_REF] . Among the identified compounds, some are widespread floral scents (e.g., methyl salicylate, germacrene D) and/or well-known from brood-site deceptive plants (e.g., indole, p-cresol, 1-octen-3-ol [START_REF] Jürgens | Chemical mimicry of insect oviposition sites: a global analysis of convergence in angiosperms[END_REF] ), while others are rarely found as floral scents (e.g., βlutidine, p-cresyl butyrate [START_REF] Knudsen | Diversity and distribution of floral scent[END_REF][START_REF] El-Sayed | The Pherobase: database of pheromones and semiochemicals[END_REF] ).

In Central and much of Western Europe, A. maculatum is predominantly visited and pollinated by female Psychoda phalaenoides L. (Psychodidae, Diptera) [START_REF] Espíndola | Variation in the proportion of flower visitors of Arum maculatum along its distributional range in relation with community-based climatic niche analyses[END_REF][START_REF] Laina | Local insect availability partly explains geographical differences in floral visitor assemblages of Arum maculatum L. (Araceae)[END_REF] . Yet, in Western France and the Mediterranean region, the most abundant visitors are often other psychodid species (e.g., P. grisescens TONN., P. trinodulosa TONN., P. zetterstedti JEŽEK) as well as nonpsychodid Diptera, such as Sphaeroceridae [START_REF] Espíndola | Variation in the proportion of flower visitors of Arum maculatum along its distributional range in relation with community-based climatic niche analyses[END_REF][START_REF] Tonnoir | A synopsis of the British Psychodidae (Dipt.) with descriptions of new species[END_REF][START_REF] Roháček | Sphaeroceridae associated with flowering Arum maculatum (Araceae) in the vicinity of Tübingen, SW-Germany (Insecta: Diptera)[END_REF] . Psychodidae and Sphaeroceridae are rare pollinators of angiosperms, but apart from A. maculatum also pollinate other species of Arum (e.g., A. concinnatum, A. cylindraceum, A. cyrenaicum, A. italicum,) and other species/genera of Araceae (e.g., Typhonium eliosurum, Arisaema heterocephalum) [START_REF] Sayers | Dung mimicry in Typhonium (Araceae): explaining floral trait and pollinator divergence in a widespread species complex and a rare sister species[END_REF][START_REF] Gibernau | Pollination in the genus Aruma review[END_REF][START_REF] Kakishima | Pollinator assemblages of Arisaema heterocephalum subsp. majus (Araceae), a critically endangered species endemic to Tokunoshima Island, Central Ryukyus[END_REF][START_REF] Urru | Pollination strategies in Cretan Arum lilies[END_REF][START_REF] Diaz | A comparison of the pollination ecology of Arum maculatum and Arum italicum in England[END_REF] . In A. maculatum, insects are attracted on the first day of anthesis (female stage) to the pitfall-trap inflorescence (Fig. 1). They slip and fall into the floral chamber (i.e., the lower part of the inflorescence where the fertile flowers are situated), where they are trapped overnight and are only released on the second and final day of flowering, after being dusted with fresh pollen [START_REF] Kite | The floral odour of Arum maculatum[END_REF][START_REF] Diaz | A comparison of the pollination ecology of Arum maculatum and Arum italicum in England[END_REF][START_REF] Lack | The pollination of Arum maculatum L. -a historical review and new observations[END_REF] .

The strong, dung-like inflorescence scent of A. maculatum attracts its flypollinators [START_REF] Chartier | Geographical variations of odour and pollinators, and test for local adaptation by reciprocal transplant of two European Arum species[END_REF][START_REF] Kite | Inflorescence odours and pollinators of Arum and Amorphophallus (Araceae)[END_REF] which breed in (and mate on) a range of different decaying organic matter, such as moist leaf litter, mushrooms, cow or horse dung [START_REF] Laurence | The larval inhabitants of cow pats[END_REF][START_REF] Wagner | Zur Kenntnis der Psychodidenfauna des Allgäus[END_REF][START_REF] Satchell | The ecology of the British species of Psychoda (Diptera: Psychodidae)[END_REF][START_REF] Withers | A preliminary account of the Irish species of moth fly (Diptera: Psychodidae)[END_REF] . Although the pollination biology of A. maculatum is well-studied [START_REF] Urru | Pollination strategies in Cretan Arum lilies[END_REF][START_REF] Diaz | A comparison of the pollination ecology of Arum maculatum and Arum italicum in England[END_REF][START_REF] Lack | The pollination of Arum maculatum L. -a historical review and new observations[END_REF] , the role of the individual floral volatiles for pollinator attraction remains largely unknown. To date, only five compounds of A. maculatum (i.e., indole, p-cresol, 2-heptanone, α-humulene, and skatole), all also released by cow dung [START_REF] Dormont | Innate olfactory preferences in dung beetles[END_REF][START_REF] Sládeček | Succession of dunginhabiting beetles and flies reflects the succession of dung-emitted volatile compounds[END_REF] , have been tested for attractiveness to potential pollinators, and their attractive function in (female) Psychodidae has been proven [START_REF] Sayers | Dung mimicry in Typhonium (Araceae): explaining floral trait and pollinator divergence in a widespread species complex and a rare sister species[END_REF][START_REF] Kite | Inflorescence odours and pollinators of Arum and Amorphophallus (Araceae)[END_REF][START_REF] Scheven | MS Untersuchungen des Appendixduftes blühender Pflanzen von Arum maculatum L. und Arum italicum MILLER; Nachweis der attraktiven Wirkung der Duftbestandteile Indol, Humulen und p-Kresol auf Psychoda phalaenoides L[END_REF] . However, preliminary bioassays at two natural sites of A. maculatum (Salzburg/Austria; Daone/North Italy) could not verify that these compounds, alone or as a blend, are attractive to psychodid (and sphaerocerid) flies, suggesting that other compounds are more likely to be responsible for the main attractiveness of the inflorescence Gfrerer, E. et al., unpubl .

Compounds potentially behaviourally active in plants with complex scents, such as A. maculatum, can be pre-selected by measuring the insects' peripheral olfactory detection of floral scents, using gas chromatography coupled to electroantennographic detection (GC-EAD [START_REF] Schiestl | Detection of physiologically active flower volatiles using gas chromatography coupled with electroantennography[END_REF][START_REF] Jhumur | Electrophysiological and behavioural responses of mosquitoes to volatiles of Silene otites (Caryophyllaceae)[END_REF] ). In previous studies testing scent of various plant species (e.g., Ceropegia sandersonii [START_REF] Heiduk | Ceropegia sandersonii mimics attacked honeybees to attract kleptoparasitic flies for pollination[END_REF] , Encephalartos villosus [START_REF] Suinyuy | Geographical matching of volatile signals and pollinator olfactory responses in a cycad brood-site mutualism[END_REF] ), such measurements frequently pointed to candidate attractants and subsequently allowed the identification of behaviourally active compounds of (complex) scent blends [START_REF] Heiduk | Ceropegia sandersonii mimics attacked honeybees to attract kleptoparasitic flies for pollination[END_REF][START_REF] Suinyuy | Geographical matching of volatile signals and pollinator olfactory responses in a cycad brood-site mutualism[END_REF][START_REF] Dötterl | Nursery pollination by a moth in Silene latifolia: The role of odours in eliciting antennal and behavioural responses[END_REF] . A comparison of the physiologically active floral compounds with the physiologically active compounds of the imitated substrate (or element) also helped to identify the key compounds of the interaction [START_REF] Schäffler | Diacetin, a reliable cue and private communication channel in a specialized pollination system[END_REF][START_REF] Schiestl | The chemistry of sexual deception in an orchid-wasp pollination system[END_REF][START_REF] Stensmyr | Rotting smell of dead-horse arum florets[END_REF] . Here, we investigated the antennal responses of four psychodid and one sphaerocerid species to the inflorescence scents of A. maculatum and to scent released by cow dung. Specifically, we asked (1) which of the many compounds recorded from this plant species elicit antennal responses in inflorescence visitors,

(2) whether antennal responses differ among insect species, and between sexes within species, and (3) how many of the EAD-active scent compounds are shared between A. maculatum and cow dung. The study overall aimed to identify potential scent candidates responsible for attracting and deceiving different pollinators of A. maculatum.

Material & Methods

Insect sampling

During 2017-2020, we obtained insects at two natural sites of A. maculatum, one located in Salzburg (Austria, 47°46'59"N 13°04'30"E) and the other in Marktschellenberg (Southern Germany, 47°41'05"N 13°03'30"E). In 2017 and 2018, containers (5 L microboxes, Model: TP5000 + TPD5000-18.5 cm × 18.5 cm × 19.1 cm; Combiness nv, Nevele, Belgium) filled with fresh cow dung (c. 2 L) were offered to insects for three consecutive days as oviposition substrate at the Salzburg site (April-October). Afterwards, the containers were brought to the lab. Once psychodids and sphaerocerids started to hatch in the boxes, the flies were transferred to a small outdoor flight cage (60 cm × 60 cm × 60 cm; BugDorm, Talchung, Taiwan), in which they were offered different breeding substrates (soil, leaf litter, mushrooms, cow or horse dung). Insects reproduced in the cage and were available for electrophysiological measurements for four to six weeks, depending on the species. In spring 2019 and 2020, all insects used for measurements were obtained by bagging A. maculatum individuals with mesh bags at both sites in the morning of the second day of anthesis, prior to the release of the trapped insect visitors. Once released by the plant and trapped in the bag, the flies were transferred to a flight cage and bred as described above.

Floral scent collection, electrophysiological analyses (GC-EAD), and identification of EAD-active compounds

To acquire solvent scent samples for electroantennographic analyses, we collected inflorescence scent of A. maculatum and volatiles released by cow dung using dynamic headspace methods, following [START_REF] Heiduk | Ceropegia sandersonii mimics attacked honeybees to attract kleptoparasitic flies for pollination[END_REF][START_REF] Lukas | Flowers of European pear release common and uncommon volatiles that can be detected by honey bee pollinators[END_REF] . Plant volatile samples were collected from a total of eight populations (see Supplementary information Table S1), covering most of the observed scent diversity [START_REF] Gfrerer | Floral scents of a deceptive plant are hyperdiverse and under populationspecific phenotypic selection[END_REF] , whereas dung volatiles were obtained from dung samples (fresh or 1-day old) used for the rearing of flies (see above). Each inflorescence was enclosed in a plastic oven bag (c. 30 cm × 12 cm; Toppits, Melitta, Germany) on the first day of anthesis between 17:30 and 20:00, when scent emission is strongest [START_REF] Gfrerer | Floral scents of a deceptive plant are hyperdiverse and under populationspecific phenotypic selection[END_REF][START_REF] Marotz-Clausen | Incomplete synchrony of inflorescence scent and temperature patterns in Arum maculatum L. (Araceae)[END_REF] . C. 60 ml dung was placed into 250 ml glass jars, covered with a plastic oven bag (see above). Volatiles were collected on adsorbent tubes (length: 8 cm, diameter: 2 mm), filled with a mixture of Tenax-TA (mesh 60-80) and Carbotrap B (mesh 20-40; 10 mg each; both Supelco, Germany), that were inserted through small holes into the headspace of the inflorescence and dung each. Samples were collected for 0.5-1.5 h with a flow of 100 mL min -1 , generated by a battery-operated vacuum pump (rotary vane pump G12/01 EB, Gardner Denver Austria GmbH, Vienna, Austria). Due to the thermogenic activity of the inflorescence [START_REF] Marotz-Clausen | Incomplete synchrony of inflorescence scent and temperature patterns in Arum maculatum L. (Araceae)[END_REF][START_REF] Bermadinger-Stabentheiner | Dynamics of thermogenesis and structure of epidermal tissues in inflorescences of Arum maculatum[END_REF] , we partly opened the plastic oven bag at the top to avoid strong condensation of water inside the bag. Samples were eluted from each adsorbent tube using 80-100 µL acetone (SupraSolv, Merck KgaA, Germany; following [START_REF] Lukas | Flowers of European pear release common and uncommon volatiles that can be detected by honey bee pollinators[END_REF][START_REF] Dötterl | Qualitative and quantitative analyses of flower scent in Silene latifolia[END_REF] ).

Then, samples were pooled per population (plant) or per age (dung, i.e., fresh or 1-day-old) to be used for the physiological measurements. To confirm physiological responses to specific compounds, we recorded antennal responses to mixtures of synthetic compounds for a subset of compounds found in A. maculatum (Table 1, Supplementary information Table S1).

Electrophysiological measurements were performed with a gas chromatograph (Agilent 7890A, Santa Clara, California, USA) equipped with a flame ionization detector (FID) and an electroantennographic detection system (GC-EAD) [START_REF] Heiduk | Ceropegia sandersonii mimics attacked honeybees to attract kleptoparasitic flies for pollination[END_REF][START_REF] Lukas | Flowers of European pear release common and uncommon volatiles that can be detected by honey bee pollinators[END_REF] . One microliter of a solvent scent sample was injected in splitless mode (250 °C), with hydrogen as the carrier gas (column flow: 3 mL min -1 ). During the period of testing (2017-2020), the GC was equipped with three different columns. In 2017, it was a ZB-5 fused silica column (5% phenyl polysiloxane; 30 × 0.32 mm, 0.25 µm film thickness; Phenomenex, Torrance, CA, USA), which was replaced in 2018 by a chiral fused silica capillary (30 m × 0.23 mm I.D.), coated with a 0.23 μm film of 0.4% heptakis (2,3-di-O-methyl-6-O-tert-butyldimethylsilyl)-βcyclodextrin (DIME-β-CD) (30%) in SE-52 (70%), the same as described in [START_REF] Dötterl | Linalool and lilac aldehyde/alcohol in flower scents. Electrophysiological detection of lilac aldehyde stereoisomers by a moth[END_REF] and [START_REF] Brandt | Subtle chemical variations with strong ecological significance: stereoselective responses of male orchid bees to stereoisomers of carvone epoxide[END_REF] . From mid-2019 onwards, the GC was equipped with another DIME-β-CD chiral column (MEGA-DEX DMT Beta SE, 30 m × 0.25 mm ID, 0.23 µm film thickness, MEGA S.r.l., Legnano, Italy). The end of each column was split into two capillaries by a μFlow splitter (Gerstel, Mühlheim, Germany), with nitrogen (N2) as make-up gas (flow rate 25 mL min -1 ). One of the capillaries (2 m × 0.15 µm inner diameter) led to the FID and the other (1 m × 0.2 µm inner diameter) to the EAD setup. The EAD was set up by a transfer line, heated at 220 °C, and a 2channel USB acquisition controller (Syntech, Kirchzarten, Germany). The outlet of the EAD was placed in a cleaned, humidified airflow, directed onto the mounted antenna. Prior to measurements, each fly was anaesthetised with CO2, and the head and last antennomere (apical-tip) were removed. Subsequently, the head and one randomly selected antenna were each connected to a glass micropipette electrode, filled with 95% insect Ringer's solution (8.0 g L -1 NaCl, 0.4 g L -1 KCl, 4.0 g L -1 CaCl2) and 5% Tween 20 (Sigma Aldrich, Vienna, Austria), and connected to silver wires. The recording electrode was attached to the tip of the antenna, while the reference electrode was connected to the caudal side of the head [START_REF] Heiduk | Ceropegia sandersonii mimics attacked honeybees to attract kleptoparasitic flies for pollination[END_REF][START_REF] Zito | Floral volatiles in a sapromyiophilous plant and their importance in attracting house fly pollinators[END_REF] .

Solvent scent samples of A. maculatum were tested on the antennae of five Diptera species: the Sphaeroceridae Coproica ferruginata STENH. (one female) and four Psychodidae species, i.e., Psychoda phalaenoides (12 females and eight males), P. zetterstedti (two females and two males), P. trinodulosa (one male), and P. cinerea BANKS (two females, all Psychodidae) (Table 1, Supplementary information Table S1). All these fly species are visitors of A. maculatum 24,25;27 , except for P. cinerea, which is a pollinator of A. hygrophilum and A. italicum [START_REF] Gibernau | Pollination in the genus Aruma review[END_REF] , with the latter sharing several floral compounds with A. maculatum [START_REF] Chartier | Geographical variations of odour and pollinators, and test for local adaptation by reciprocal transplant of two European Arum species[END_REF][START_REF] Diaz | A comparison of the pollination ecology of Arum maculatum and Arum italicum in England[END_REF] .

Two additional female Psychoda individuals (collected directly from A. maculatum) could not be determined to species level, as their abdomen were too damaged, and might belong to the four above mentioned species or (an)other species.

For identification of EAD-active compounds, scent samples were run on a gas chromatograph/mass spectrometer (GC/MS, model QP2010 Ultra EI, Shimadzu, Tokyo, Japan), equipped with either a non-chiral ZB-5 column (in 2017; see above) or a chiral column (2018-2020; MEGA-DEX DMT Beta SE, see above). Helium was used as carrier gas (flow: 3 mL min -1 ) and samples (injection volume: 1 µL) were run with a split ratio of 1:1 [START_REF] Heiduk | Ceropegia sandersonii mimics attacked honeybees to attract kleptoparasitic flies for pollination[END_REF][START_REF] Lukas | Flowers of European pear release common and uncommon volatiles that can be detected by honey bee pollinators[END_REF] .

Obtained data were handled using GCMSolution v.4.41 (Shimadzu Corporation, Kyoto, Japan). We tentatively identified components by comparison of Kováts' retention indices [START_REF] Kováts | Über den Retentionsindex und seine Verwendung zur Aufstellung einer Polaritätsskala für Lösungsmittel[END_REF] (KRIs; based on commercially available n-alkanes C7-C20) and mass spectra available in the libraries of Adams [START_REF] Dougherty | Behavioural and electrophysiological responses of the phlebotomine sandfly Lutzomyia longipalpis (Diptera: Psychodidae) when exposed to canid host odour kairomones[END_REF] , FFNSC 2, Wiley9, NIST11, and ESSENTIAL OILS (available in MassFinder 3, Hochmuth Scientific Consulting, Hamburg, Germany). The identity of some of the components was verified by authentic reference standards, available in the collection of the Plant Ecology Lab of Salzburg University (see Table S1). Compounds were classified as inflorescence-specific or as vegetative compounds, according to Gfrerer et al. [START_REF] Gfrerer | Floral scents of a deceptive plant are hyperdiverse and under populationspecific phenotypic selection[END_REF] . Absolute amounts of compounds tested in the GC/EAD measurements (Table 1) were quantified by injecting known amounts of various aliphatics and terpenoids and the resulting mean peak areas were used for quantification [START_REF] Gfrerer | Floral scents of a deceptive plant are hyperdiverse and under populationspecific phenotypic selection[END_REF] .

Results

Across all tested dipterans, we found a total of 78 volatile organic compounds (VOCs) from A. maculatum (together c. 88% of the relative inflorescence scent emission of this species 5 ) that were electroantennographically active. The majority of these compounds were inflorescence-specific, but five of them were vegetative compounds [i.e., (Z)-3-hexenyl acetate, nonanal, limonene, linalool, α-pinene; Table 1]. Overall, 55 of the EAD-active VOCs could be (tentatively) identified (Table 1). They represented several chemical classes, including monoterpenoids (n=25 VOCs), aliphatic compounds (15), sesquiterpenoids (eight), irregular terpenoids (two), nitrogen-bearing compounds (three), and aromatic compounds (two). Seven of these VOCs elicited antennal responses in all tested insect species: the monoterpenoid 2,6-dimethylocta-2,6-diene (isomer 2), the aliphatic compound 1-octen-3-ol, the sesquiterpene (E)-β-caryophyllene, the nitrogen-containing component indole, the aromatic components methyl salicylate and p-cresol, and the unknown UNK1415 (Table 1, Fig. 2). All other VOCs elicited responses only in a subset of insect species.

On average, 38 scent compounds yielded a response per species (and sex), with a minimum of 13 volatiles in male P. trinodulosa (Psychodidae; one individual tested on one scent sample), and a maximum of 60 VOCs in female P. phalaenoides (12 individuals tested on nine scent samples). When considering both sexes of P. phalaenoides (total of 20 individuals tested on 16 scent samples) 61 VOCs were EAD-active, and in both sexes of P. zetterstedti 49 VOCs (four individuals on eight scent samples; Table 1, Supplementary information Table S1). For the female C. ferruginata (Sphaeroceridae; one individual tested on three samples) 36 VOCs resulted in an antennal response. Notably, the aliphatic compound 2-nonanone was perceived by all psychodid species but did not elicit signals in C. ferruginata, even though this compound was present in all three scent samples tested on the latter species.

In contrast, the monoterpene γ-terpinene was EAD-active only in C. ferruginata, but not in the two psychodid species tested on this compound, i.e., P. phalaenoides and P. sp. Some compounds elicited specific responses in certain Psychoda taxa. For instance, the aliphatic compound nonanal induced responses in P. phalaenoides and P. zetterstedti, but not in P. cinerea and P. trinodulosa. The nitrogen-bearing compound skatole was EAD-active in P. phalaenoides and P. cinerea, but not in P. zetterstedti and P. sp. (Table 1). The sesquiterpene α-humulene resulted in responses in most individuals of P. zetterstedti and in a few individuals of P. phalaenoides, but not in P. cinerea and P. sp. A number of the unknown volatiles (e.g., UNK883, UNK1394, UNK1492) elicited responses in P. phalaenoides and P. zetterstedti, but not in P. cinerea.

For P. phalaenoides and P. zetterstedti, the two species for which we tested male and female individuals, the analyses revealed some sex-specific responses. In P. phalaenoides, 24 compounds were EAD-active in both sexes, but one compound elicited responses only in females (unknown UNK1030; n=12 individuals). In P. zetterstedti, 21 compounds were EADactive in both sexes, but four [2,6-dimethylocta-2,6-diene isomer 1, (Z)-β-ocimene, αcopaene, 6-methyl-5-hepten-2-one] and two [(+)-α-citronellene, unknown UNK1378/81] compounds elicited responses only in females (n=2) and males (n=2), respectively.

Of the 78 EAD-active floral compounds, 18 were also physiologically active in cow dung samples (Fig. 2, Table 1), representing all different chemical classes recorded in A. A few of the compounds recorded as physiologically active in the antennae of Psychoda spp. (1-octen-3-ol, butanoic acid, α-pinene, and α-terpinene) were also found to elicit electrophysiological responses in females of the phlebotomine sandfly Lutzomyia longipalpis (Psychodidae, Diptera) [START_REF] Dougherty | Behavioural and electrophysiological responses of the phlebotomine sandfly Lutzomyia longipalpis (Diptera: Psychodidae) when exposed to canid host odour kairomones[END_REF][START_REF] Sant'ana | Electroantennographic responses of the Lutzomyia (Lutzomyia) longipalpis (Lutz & Neiva) (Diptera: Psychodidae) to 1-octen-3-ol[END_REF] , the only other psychodid used so far for physiological measurements in the olfactory circuitry. The sandfly, which was tested on faeces from vertebrates and canid host odours, additionally responded to several other volatiles that do not occur in A. maculatum (including different isomers of monoterpenoids), but we provide the first evidence that psychodids are able to perceive sesquiterpenoids. Physiological measurements on antennae of Sphaeroceridae were not available before our measurements, and thus, our study overall increases the knowledge about the peripheral olfactory circuitry of psychodids and Sphaeroceridae.

Among the EAD-active volatiles recorded in the present study are the most abundant inflorescence scents of A. maculatum included (i.e., indole, p-cresol, (+)-β-citronellene, 2heptanone, 3,7-dimethyloct-2-ene, UNK1415, [START_REF] Gfrerer | Floral scents of a deceptive plant are hyperdiverse and under populationspecific phenotypic selection[END_REF][START_REF] Kite | The floral odour of Arum maculatum[END_REF][START_REF] Chartier | Geographical variations of odour and pollinators, and test for local adaptation by reciprocal transplant of two European Arum species[END_REF][START_REF] Marotz-Clausen | Incomplete synchrony of inflorescence scent and temperature patterns in Arum maculatum L. (Araceae)[END_REF][START_REF] Szenteczki | Spatial and temporal heterogeneity in pollinator communities maintains within-species floral odour variation[END_REF][START_REF] Diaz | A comparison of the pollination ecology of Arum maculatum and Arum italicum in England[END_REF] ), but also numerous compounds emitted only in small relative amounts by the plant (e.g., 3-octanone, 2,6-dimethyl-7-octen-2ol; Fig. 2). Several of these abundant and less abundant compounds are also released from cow dung (Table 1, Fig. 2) or other breeding/mating sites of the tested insect species. These are, for instance, p-cresol, 2-heptanone, terpinen-4-ol, α-citronellene, and 2,6-dimethylocta-2,6-diene (cow dung; Table 1, [START_REF] Sládeček | Succession of dunginhabiting beetles and flies reflects the succession of dung-emitted volatile compounds[END_REF] ), 2-octanone (horse dung [START_REF] Dormont | Innate olfactory preferences in dung beetles[END_REF] ), β-citronellene and α-humulene (both cow and horse dung [START_REF] Dormont | Innate olfactory preferences in dung beetles[END_REF][START_REF] Sládeček | Succession of dunginhabiting beetles and flies reflects the succession of dung-emitted volatile compounds[END_REF][START_REF] Johnson | Convergent evolution of carrion and faecal scent mimicry in flypollinated angiosperm flowers and a stinkhorn fungus[END_REF] ), or 3-octanone and (E)-2-octen-1-ol (fungi e.g., [START_REF] Thakeow | Antennal and behavioral responses of Cis boleti to fungal odor of Trametes gibbosa[END_REF] ). Some of the EAD-active compounds have not been detected in ours or others' scent samples from (cow) dung and have not been described elsewhere. We speculate that those volatiles are released from various other, likely differently scented breeding and/or mating substrates of A. maculatum pollinators, such as mud-flats or leaf litter. It has been suggested that the hyperdiverse floral scent of A. maculatum might result from the imitation of various differently scented breeding substrates [START_REF] Gfrerer | Floral scents of a deceptive plant are hyperdiverse and under populationspecific phenotypic selection[END_REF] . The sensitivity of the tested flies to a variety of different compounds would support this idea, although other possible roles for those volatiles cannot be excluded (e.g., repellence of florivores [START_REF] Junker | Floral scents repel facultative flower visitors, but attract obligate ones[END_REF] , defence against pathogens [START_REF] Junker | Volatile organic compound mediated interactions at the plantmicrobe interface[END_REF] ).

Altogether five inflorescence scent compounds of A. maculatum have previously been reported as attractive to psychodids. In detail, indole and p-cresol, together with α-humulene or 2-heptanone, were found to attract female P. phalaenoides [START_REF] Scheven | MS Untersuchungen des Appendixduftes blühender Pflanzen von Arum maculatum L. und Arum italicum MILLER; Nachweis der attraktiven Wirkung der Duftbestandteile Indol, Humulen und p-Kresol auf Psychoda phalaenoides L[END_REF] and Psychoda spp [START_REF] Kite | Inflorescence odours and pollinators of Arum and Amorphophallus (Araceae)[END_REF] . In Kite et al. [START_REF] Kite | Inflorescence odours and pollinators of Arum and Amorphophallus (Araceae)[END_REF] , the attracted psychodids have not been identified to the species level; hence, it is unknown whether they are flower visitors of A. maculatum or not. A synthetic mixture of skatole, indole, and p-cresol, together with VOCs not occurring in A. maculatum (geranyl acetone, dihydro-, and β-ionone), was shown to attract psychodid and sphaerocerid pollinators of Typhonium eliosurum, a dung-mimicking aroid endemic to Australia [START_REF] Sayers | Dung mimicry in Typhonium (Araceae): explaining floral trait and pollinator divergence in a widespread species complex and a rare sister species[END_REF] . In preliminary bioassays in the field, we tested the above five compounds, using the same composition and concentration as released by the inflorescences of A. maculatum. Yet those volatiles did not attract psychodid or sphaerocerid flies. This suggests that other, not yet tested scent compounds (additionally) contribute to pollinator attraction in A. maculatum. Potential candidates are other odours known also from cow or horse dung (e.g., 2,6-dimethylocta-2,6diene, unknown UNK1415; Table 1) or compounds known from other breeding substrates (e.g., fungi: 3-octanone e.g., [START_REF] Thakeow | Antennal and behavioral responses of Cis boleti to fungal odor of Trametes gibbosa[END_REF] ). The unknown UNK1415, one of the main scent compounds of A. maculatum [START_REF] Gfrerer | Floral scents of a deceptive plant are hyperdiverse and under populationspecific phenotypic selection[END_REF] , yielded antennal responses in all insect taxa and in nearly all individuals tested in the present study. Interestingly, this unknown volatile is possibly identical to unknown "RI 1531" found in T. eliosurum [START_REF] Sayers | Dung mimicry in Typhonium (Araceae): explaining floral trait and pollinator divergence in a widespread species complex and a rare sister species[END_REF] , as both volatiles have the same mass spectra (Supplementary information Fig. S1).

Our study shows that some of the antennal responses to scent differ among insect species, and some also between males and females within species. This finding is in agreement with results obtained by physiological measurements in other insects [START_REF] Jhumur | Electrophysiological and behavioural responses of mosquitoes to volatiles of Silene otites (Caryophyllaceae)[END_REF][START_REF] Abraham | Behavioral and antennal responses of Drosophila suzukii (Diptera: Drosophilidae) to volatiles from fruit extracts[END_REF] . Some of the species-level effects described in the present study might have been influenced by sex-specific effects, because for some species (P. cinerea and P. trinodulosa) we only tested males or females. Hence, differences in antennal responses among these species need to be interpreted with caution. Nonetheless, species-and sex-specific differences in the peripheral olfactory circuitry of insects can result in different behaviours e.g., [START_REF] Stökl | Scent variation and hybridization cause the displacement of a sexually deceptive orchid species[END_REF][START_REF] Salamanca | From laboratory to field: electro-antennographic and behavioral responsiveness of two insect predators to methyl salicylate[END_REF] . Interestingly, antennae of P. cinerea, the only non-pollinating species of A. maculatum (but of other Arum spp.) we tested in this study, did not respond to some abundant compounds emitted by A. maculatum (e.g., UNK1394, UNK1492), which otherwise elicited responses in the other pollinating species tested (e.g., P. phalaenoides and P. zetterstedti). The lack of antennal sensitivity to (some of) those compounds might explain why P. cinerea does not visit A. maculatum, while close relatives including P. phalaenoides and P. zetterstedti are important pollinators [START_REF] Espíndola | Variation in the proportion of flower visitors of Arum maculatum along its distributional range in relation with community-based climatic niche analyses[END_REF][START_REF] Laina | Local insect availability partly explains geographical differences in floral visitor assemblages of Arum maculatum L. (Araceae)[END_REF] .

Conclusions

Until now, it has been not known which (and how many) volatile compounds of the complex floral scent of Arum maculatum can be perceived by its floral visitors. Our study identified 78 physiologically active compounds from hundreds of potentially behaviourally active VOCs, which is still a rather high number the psychodid and sphaerocerid flies are sensitive to. Our results thus provide a basis for future studies that aim to understand the floral volatiles of A. maculatum involved in the chemical attraction and deception of its pollinators, and which VOCs guide the flies to their breeding/mating substrates. Some of the EAD-active VOCs (4terpinenol, α-terpinene, 2-heptanol, 2-nonanol, UNK1503) have recently been shown to be under phenotypic selection in A. maculatum [START_REF] Gfrerer | Floral scents of a deceptive plant are hyperdiverse and under populationspecific phenotypic selection[END_REF] . These compounds and those EAD-active ones shared with the pollinators' breeding substrates (e.g., UNK1415, 3-octanone) are the most promising candidates for future behavioural assays. As the tested Diptera species (Psychodidae, Sphaeroceridae) are also known pollinators of other (similarly-scented) species of Arum (e.g., A. italicum, A. concinnatum) as well as other species/genera of Araceae (e.g., Typhonium eliosurum, [START_REF] Sayers | Dung mimicry in Typhonium (Araceae): explaining floral trait and pollinator divergence in a widespread species complex and a rare sister species[END_REF][START_REF] Urru | Pollination strategies in Cretan Arum lilies[END_REF][START_REF] Revel | Investigating the relationship between pollination strategies and the size-advantage model in zoophilous plants using the reproductive biology of Arum cylindraceum and other European Arum species as case studies[END_REF] ), our study should also help to elucidate the chemical interactions between these plants and their fly pollinators. Future research is now needed to test the behavioural function of physiologically active floral volatiles, which is crucial for a better understanding of olfactory cues mediating plant-animal interactions in general, and in sapromyiophilous species, in particular. Coproica ferruginata (Sphaeroceridae; violet) to scent samples of A) inflorescences of Arum maculatum and B) cow dung (fresh and 1-day old). EAD-active compounds (see also Table S1); S identity was confirmed by synthetic standard; L in leaf blanks see 5 ; m/z = mass-to-charge ratio in decreasing order of abundance; ‡ coeluted on non-chiral ZB-5 column; ‡ ‡ coeluted on chiral columns.

Figure legends

  maculatum. More specifically, six of the seven VOCs that elicited antennal responses in all tested insect species were among those 18 compounds [i.e., 2,6-dimethylocta-2,6-diene isomer 1, 1-octen-3-ol, (E)-β-caryophyllene, indole, p-cresol, and UNK1415; Fig.2]. Among those 18 were also VOCs that elicited different responses among fly families (2-nonanone), fly species (nonanal, skatole), and within species between sexes [i.e., 2,6-dimethylocta-2,6diene isomer 1, 6-methyl-5-hepten-2-one, (E)-β-caryophyllene].DiscussionOur study is the first to identify electroantennographically active compounds in Psychoda spp. (Psychodidae) and a Sphaeroceridae (Coproica ferruginata). It shows that these insect visitors of deceptive Arum maculatum are sensitive to a high number of the plants' inflorescence scent compounds. The EAD-active compounds identified represent various chemical classes, including mono-and sesquiterpenoids, aliphatic, aromatic, nitrogen-bearing, and unknown compounds. Antennal responses differed among insect species and between sexes within species. More than a fifth of the physiologically active scent compounds were also released by cow dung, linking insect breeding/mating sites, floral VOCs of A. maculatum, and its floral visitors.

Fig. 1 .

 1 Fig. 1. A flowering individual of Arum maculatum in a natural habitat. (Photo E. Gfrerer; Salzburg, Austria).

Fig. 2 .

 2 Fig. 2. Representative physiological responses (gas chromatography coupled to electroantennographic detection) of a female Psychoda sp. (Psychodidae; red) and a female

1): ( 1 )

 1 UNK883; (2) hexanal; (3) 1-hexanol; (4) 3,7-dimethyloct-1-ene; (5) (+)-α-citronellene; (6) (+)-β-citronellene; (7) 3,7-dimethyl-2-octene isomer 1; (8) 3,7-dimethyl-2-octene isomer 2; (9) 2,6-dimethyl-2,6-octadiene isomer 2; (10) 6-methyl-5-hepten-2-one; (11) UNK1030; (12) octanal; (13) p-cymene; (14) 6-methyl-5-heptene-2-ol; (15) 1-octen-3-ol; (16) 2methylbutanoic acid; (17) 2-nonanone; (18) nonanal; (19) 2,6-dimethyl-7-octen-2-ol; (20) (E)-2-octen-1-ol; (21) (S)-linalool; (22) 2-decanone; (23) UNK1135; (24) decanal; (25) pcresol; (26) 3,7-dimethyl-1-octanol; (27) α-copaene; (28) UNK1378/81; (29) UNK1394; (30) UNK1415; (31) indole; (32) UNK1503/UNK1492; (33) UNK 1658. Numbers in bold (top line) are VOCs eliciting signals in A) and B); numbers in italics (bottom line) are VOCs that do not occur in A. maculatum. FID1 A. maculatum, population Josefiau; FID2 A. maculatum, population Murnau; FID3 cow dung fresh; FID4 cow dung1-day-old. See Supplementary Table S1 for detailed population information. All samples shown were run on a chiral fused silica capillary column (30% DIME-β-CD in 70% SE-52, see methods) and each measurement lasted either 8 (Coproica ferruginata) or 10 min (Psychoda sp.).

Table 1 .

 1 Inflorescence scents of Arum maculatum that elicited electroantennographic responses in Psychoda phalaenoides, P. zetterstedti, P. cinerea, P. trinodulosa, P. sp. (Psychodidae) and Coproica ferruginata (Sphaeroceridae). Compounds are sorted according to chemical classes, and within those alphabetically. The volatiles printed in bold were also detected in cow dung. In parentheses, the numbers of individuals tested (n) and runs performed per fly species and sex are denoted. Superscript values indicate the amounts (mean±SD) of volatiles tested in the GC/EAD measurements on different fly species and Levels of EAD-activity: ++++ (EAD-active in >80% of runs with samples that contained this compound), +++ (80-50%), ++ (50-25%), + (>25%), -(VOC did not elicit a signal, even though the same or a lower amount elicited a signal in other sexes or species), NP (VOC not present in scent samples tested). * The physiological activity was confirmed by a synthetic standard (see Supplementary information Table

	sexes.
	Species	P. phalaenoides	P. zetterstedti	P. cinerea P. trinodulosa	P. sp.	C. ferruginata
	Sex	♀	♂	♀	♂	♀	♂	♀	♀
	# individuals, # runs	(n=12, 33)	(n=8, 14)	(n=2, 6)	(n=2, 4)	(n=2, 6)	(n=1, 1)	(n=2, 8)	(n=1, 3)
	Chemical class / compound
	Aliphatic compounds
	Butanoic acid S	++++ 0.6±1.2	NP	NP	NP	NP	NP	++++ 0.7±1.6	NP
	Decanal S	++ 1.9±1.1	+ 1.3±0.9	++++ 0.5±0.8 NP	+++ 1.0±1.4	+++ 3.4	++++ 1.3±1.2	++++ 1.4±1.6
	2-Decanone	++++ 0.4±0.5	NP	++++	NP	+++ 0.3±0.5	NP	++++ 0.3±0.3	++++ 0.4±0.7
	2-Heptanol* S	++ 5.8±10	+++ 8.9±11	++ 2.1±3.6	++ 6.6±9.0	+++ 20.5±27	NP	++ 4.4±7.1	+++ 18.7±27
	2-Heptanone* S	++ 14.8±17	+ 22.2±22	+++ 24.5±27	++++ 17.6±18	+++ 28.7±35	NP	-15±13	+ 28.8±33
	1-Hexanol S	NP	NP	NP	NP	NP	NP	NP	+++ 0.2±0.2
	(Z)-3-Hexenyl acetate S,L	+++ 0.5±0.9	NP	NP	NP	++ 0.5±1.1	NP	NP	NP
	Nonanal S,L	++ 1.3±1.6	NP	++ 0.8±0.7	NP	-0.6±0.9	-2.6	++ 0.5±1.0	++ 0.3±0.5
	2-Nonanol S	++ 0.4±0.7	NP	+++ 1.5±2.6	NP	++ 3.5±4.7	NP	+ 0.3±0.5	+++ 3.1±4.9
	2-Nonanone* ,S	++ 2.1±1.6	++ 3.0±2.7	+++ 3.9±3.7	++ 2.7±1.9	+++ 5.4±6.7	NP	+++ 1.9±1.7	-5.16±6.5
	2-Octanone S	+ 0.6±0.6	NP	-0.5±0.3	NP	NP	NP	+ 0.5±0.7	NP
	3-Octanone S	++ 2.4±2.0	NP	++++ 0.3±0.4 ++++ 4.0±1.6	+++ 3.7±4.8	NP	++ 3.1±4.4	++++ 5.7±5.5
	1-Octen-3-ol* ,S	+++ 41.9±31	+++ 32.9±18	+++ 1.71±2.0	+++ 54.1±29	+++ 119±130	+++	+++ 27.5±28	++++ 111±132
	(E)-2-Octen-1-ol S	+ 0.3±0.5	NP	NP	++++ 0.4±0.6	+++ 4.0±8.4	NP	NP	++++ 6.7±10
	2-Undecene S	++ 5.2±15	NP	NP	NP	NP	NP	NP	NP
	Aromatic compounds
	p-Cresol* ,S	+++ 56.4±45	+++ 39.6±12	++ 13.4±9.7	++ 92.0±53	+++ 41.9±49	+++ 0.2	++ 41.6±38	++++ 52.2±67
	Methyl salicylate S	++ 1.9±1.4	+ 1.1±1.1	++++ 2.1±1.2	++++ 2.2±1.9	+++ 1.6±2.1	+++ 0.8	++++ 1.3±1.7	++++ 1.4±2.4
	Irregular terpene
	6-Methylhept-5-ene-2-ol	++ 2.3±1.2	NP	NP	NP	NP	NP	+++ 1.5±1.7	NP
	6-Methylhept-5-ene-2-one S	++ 2.8±1.4	NP	-1.8±1.5	++++ 2.5±1.6	-1.0±1.3	NP	++ 1.9±2.2	++++ 1.3±1.4
	Monoterpenoids			NP				NP
	(+)-α-Citronellene* ,S	+++ 9.2±6.2	NP	++ 2.4±3.5	-14.6±1.5	+++ 6.9±5.1	NP	+++ 6.3±4.2	++ 9.6±3.9

Acknowledgements

We are grateful to RNDr. Jindřich Roháček for identifying the tested Sphaeroceridae individual to the species level. We would further like to thank Dr. Irmgard Schäffler and Dr. Roman Fuchs for methodological support, Prof. Dr. Robert R. Junker for the microboxes, and family Bruckbauer (Lienbacher farm) for continuously providing breeding substrates for insect rearing. We are also thankful to members of the Plant Ecology research group at the University of Salzburg for their helpful comments on an earlier version of the manuscript, and to two anonymous reviewers for their suggestions that improved the final manuscript. This study was funded by a grant from the Austrian Science Fund (FWF; P30175-B29) to ACH, HPC, and SD (PI).

Author contributions

MG, ACH, HPC and SD designed the study. EG and DL collected the scent samples. DL and RW identified the Psychodidae to the species level. EG reared the insects, performed all measurements, analysed all data, and wrote the first draft of the manuscript. All authors contributed to the final manuscript and approved the submitted version.

Additional Information

Data availability

All data that support the findings of this study are included in this published article (and its supplementary information files).

Competing interests

The authors declare no competing interests.

Experimental research and field studies on plants All samplings were carried out in compliance with the current laws of the respective countries