Bin Ni

Hui Chen

Mengxue Zhang

Patrick Keller
email: patrick.keller@curie.fr

Michael Tatoulian
email: michael.tatoulian@chimieparistech.psl.eu

Min-Hui Li
email: min-hui.li@chimieparistech.psl.eu

Thermo-mechanical and photo-luminescence properties of microactuators made of liquid crystal elastomers with cyano-oligo(pphenylene vinylene) crosslinking bridges †

Information (ESI) available: Synthetic routes, MS and NMR spectra of β-cyano-OPV; DSC curves of β-cyano-OPV; Fluorescence images of β-cyano-OPV powders. Photo-isomerization of β-cyano-OPV in solution and the stability of its isomers. Effect of the photo-isomerization on the optical properties. Complemental data of thermomechanical and photo-luminescence properties and DSC curves of LCE micropillars with different contents of β-cyano-OPV. Complemental data of factors that might affect the PL intensity of LCE micropillars.

Introduction

Liquid crystal elastomers (LCEs) are a type of smart materials, which have attracted increasing research interests in recent years owing to their potential applications in smart sensors 1 , high-frequency oscillators 2,3 , microfluidic device 4,5 , gas separation membranes 6 , artificial muscles 7,8 , and so on. LCEs can be seen as liquid crystalline polymers (LCP) with slightly crosslinked polymer chains, which show both the anisotropic orientation characteristic of liquid crystals and the rubbery elasticity of polymer networks 9 . The molecular organization in the different phases has a great influence on the properties of the LCEs. In the LC phase, the conformation of the macromolecular backbone is coupled with the orientational order of the mesogen units. For example, in the nematic phase, the orientated mesogens force the polymer chains to stretch along the direction of the orientation [10][11][12] . However, when the transition from nematic to isotropic phase takes place, the orientational order is lost; consequently, the polymer chains can go back to the usual random coil conformation. This conformational change induced by nematic to isotropic phase transition causes the macroscopic shape change of LCEs. Meanwhile, thanks to the crosslinked network, the orientational order can be restored to its original state, which endows LCEs with the reversible shape-memory properties. Moreover, the shape variation of LCEs can be produced by using various stimuli, such as light illumination, heating, electric and magnetic fields etc., giving the opportunity to develop thermo-responsive [13][14][15][16][17][18][19][20][21] , photoresponsive [22][23][24][25][26][27][28][29] , and electro-responsive [30][31][32][33] smart LCEs.

Luminescent smart materials responsive to external stimulus, like mechanical stress [34][35][36] or temperature 37 , have gained increased attention since they might have applications in sensitive sensors 38 , organic light emitting diodes (OLEDs) 35 and security printing 39 . Weder 40,41 introduced dye molecules, cyano-p-phenylene vinylene (cyano-OPV) derivatives, as the dopant compound into linear low-density polyethylene (LLDPE) to make stress-sensitive luminescent materials. The tensile deformation of the LLDPE materials forced the cyano-OPV molecules to change from the "excimer" state to the "monomer" state to realize the chromic properties. This kind of deformation-induced fluorescence color change could be used as strain sensors in polymer objects. In a previous study, one of us 42 mixed similar cyano-OPV molecule (1,4-bis(αcyano-4-methoxystyryl)benzene) as the dopant with a nematic LCE. Upon the thermomechanical shape change of LCE, they observed, however, only a diminution of the fluorescence This journal is © The Royal Society of Chemistry 20xx

Please do not adjust margins Please do not adjust margins intensity during the contraction of the LCE. As a matter of fact, in the systems where shape changes co-exist with temperature variations and phase transitions, other physical phenomena could also affect the PL intensity variations. In particular, the influence of phase transitions was already reported in ceramic 43 , gels 44 , and liquid crystal 45 . G. Meier 45 et al discovered that the quantum yield of fluorescence of a dye dissolved in a LC mesophase changed dramatically at the phase transition from the nematic to the isotropic phase.

Following our previous work on fluorescent LCEs 42 , we prepared here new nematic LCEs by introducing the fluorescent cyano-OPV dye(Scheme 1, β-cyano-OPV) as the crosslinker which was thus connected chemically to the LCEs. The fluorescent nematic LCEs were prepared in the form of micropillars with different crosslinking degrees and different contents of dye crosslinker cyano-OPV in the whole concentration of crosslinkers. Those new micropillars gave us the opportunity to study in detail the relationships between the PL properties and each potential factor influencing the PL intensity, such as temperature change, contraction and extension, and phase transition. The monodomain LCE micropillars were fabricated using β-cyano-OPV ((((1Z,1'Z)-1,4phenylenebis(1-cyanoethene-2,1-diyl))bis(4,1phenylene))bis(oxy))bis(hexane-6,1-diyl) bis(2-methylacrylate), (see Scheme 1) as the dye crosslinker associated with a flexible and non-fluorescent crosslinker and MA444 ((4methacryloyloxy)butyl 2,5-di-(4′-butyloxybenzoyloxy)benzoate, see Scheme 2) as the nematic monomer, by the previously described magnetic alignment method 14 . Then the PL intensity variations and the thermomechanical properties of these micropillars were investigated. It was found that the phase transition from nematic to isotropic of the LCEs played the major role in the PL intensity variations.

Results and discussion

Synthesis of the dye crosslinker and preparation of LCE micropillars

We synthesized the dye crosslinker β-cyano-OPV according to the Scheme 1. The details of the syntheses and characterization are described in the Electronic Supplementary Information(ESI).

Scheme 1. Synthetic route of the dye crosslinker β-cyano-OPV (β-cyano-OPV).

The phase behaviour of the β-cyano-OPV was studied by differential scanning calorimetry (DSC) and polarizing optical microscopy (POM). The β-cyano-OPV exhibits two endothermic transitions at 130 °C and 146 °C according to DSC measurement (Figure S3 in SI). A complementary analysis by POM helped to identify the phase transition at 130°C as the crystal-to-nematic phase transition, and the one at 146°C as the nematic-to-isotropic phase transition.

Then the micron-sized LCE pillars were fabricated by photopolymerization under magnetic field alignment as previously described 14 , using the mixtures of LC monomer (MA444), photo-initiator (2-benzyl-2-(dimethylamino)-4'morpholinobutyrophenone) and a flexible crosslinker (1,6hexanediol dimethacrylate) (see Scheme 2) together with the dye crosslinker (β-cyano-OPV). The monomer MA444 was synthesized according to the procedure already published. The phase sequence of MA444 are Cr 83 Iso on heating and Iso79 N 60 Cr on cooling 46 . The concentration of the photoinitiator was kept at 2 mol%, while various concentrations of crosslinkers were used. First on keeping the whole crosslinkers concentration at 10 mol%, the concentration of the β-cyano-OPV was varied from 0.5 mol% to 5 mol% in order to study the effect of the dye concentration. Then on keeping the concentration of β-cyano-OPV at 1 mol%, LCEs with different whole crosslinker concentrations (10 mol% and 35 mol%) were prepared in order to compare PL properties of slightly and highly crosslinked LCEs. The phase transition of each mixture for micropillar preparation was checked beforehand to ensure that the photo-polymerization occurred in aligned nematic phase.

Scheme 2. Chemical structures of the monomer MA444, the photo-initiator (Irgacure 369) and the flexible crosslinker.

Fluorescence properties of the dye crosslinker (β-cyano-OPV)

The PL properties of the cyano-OPV derivatives are sensitive to their "environment", for example in solution or in solidstate (amorphous or crystalline) as reported by Weder et al 41,42 . The authors linked this sensitivity to the existence of "monomer state" versus "excimer state" for the dye molecules when they were "dispersed" versus "aggregated". Therefore, it is important to characterize the fluorescence properties of the β-cyano-OPV in different environments, i.e., in solution, in the LCE micropillars and in pure state.

Fig 1 shows the PL excitation and emission spectra of the βcyano-OPV in different environments. Fluorescence excitation spectra of the β-cyano-OPV, both in dichloromethane solution and in the micropillar, are presented in Fig 1a and1b, respectively. The maximum excitation is around 396 nm in solution, while in the micropillar, the excitation curve broadens and red-shifts with a maximum around 420 nm. As Please do not adjust margins Please do not adjust margins for the fluorescence emission of the β-cyano-OPV, Fig 1c and1d show very similar emission spectra with maximum around 460 -490 nm in the CH2Cl2 solution and in the micropillars. This observation indicates a similar molecular organization of the dye molecules both in solution and within the elastomer. Since the β-cyano-OPV is well dissolved in the dichloromethane, the dye crosslinker is in the monomeric state (unimer) in solution. We can then deduce that the dye crosslinker is also in the monomeric state in the LCE micropillars. Finally, we checked the fluorescence emission of the pure β-cyano-OPV in the solid state. As shown in Fig 1e, a sharp emission peak was observed at 515 nm, together with a shoulder emission at 550 nm, which is a typical emission spectrum for the excimer emission as previously ascribed in the literature 41 . With naked eyes, we observed that the βcyano-OPV in solid state exhibited a strong green-yellow fluorescence under UV light irradiation (Fig S4). This fluorescence was associated with the presence of excimers as described by Weder et al 41 . The sharp contrast between the emission spectra of the dye crosslinker in pure solid state and in LCE micropillars confirmed again that the dye crosslinker molecules are in the monomeric state both in the LCE micropillars. The thermally-induced actuation properties of the micropillars were investigated by measuring the micropillars lengths at different temperatures. Before this investigation, we first checked the thermo-stability of the LCE by TGA (see Figure S11C). The TGA showed that the LCE was very stable in the temperature range of our investigation (< 200°C). Micropillars were dispersed in low viscosity silicone oil on a clean glass slide in order to avoid friction between the actuators and the glass surface. The glass slide was kept on a heating stage under the optical microscope. Four different micropillars were prepared with various contents of the dye crosslinker (0.5 mol %, 1 mol %, 2 mol %, 5 mol % respectively) on keeping the whole crosslinkers concentration constant (10 mol%). As an example, the deformation of the micropillars with 1 mol % of dye crosslinker as a function of temperature is presented in In addition, the influence of the β-cyano-OPV content on the deformation properties was also investigated. As shown in Fig 4a, the contraction behaviours kept the same, but their extent L/Liso decreased as the content of the dye crosslinker increased. This observation indicated probably the order parameters of the LC alignment decreased with the increase of β-cyano-OPV content in the crosslinking bridges. As a matter of fact, during the photopolymerization to prepare the LCE, the β-cyano-OPV dye could also absorb the UV light to undergo photoisomerizations (see ESI Section 3, Fig S5). The initial isomer Z/Z was rod-like and compatible to the nematic order, however the isomers Z/E and E/E produced under UV illumination had bent form and should play the role of impurities in the nematic system. Therefore, the order parameters of the LC alignment should decrease with the increase of content of isomers Z/E and E/E, which were proportional to the starting content of β-cyano-OPV crosslinker. To evaluate the content of possible isomers in the micropillars and their thermal stability, we used the β-cyano-OPV dye in solution in CDCl3 or DMSO-D6 (concentration = 10 -4 mol/L) as model system, which was irradiated during different time until 30 min and then analysed by 1 H-NMR. Note that 30 min is the duration of photopolymerization. The details are presented in the ESI (Section 3). As shown in Table S1 and Fig S5 andS6, the study by 1 H NMR spectra indicated that the isomerization took place in β-cyano-OPV in solution after 30 min of UV irradiation. However, after keeping the irradiated βcyano-OPV solution at room temperature for 30 days in dark or heating up to 110 o C for 10 min in dark, there is nearly no change in the positions and intensities of the 1 H NMR signals. That means that the isomerized structures are thermally stable. We extrapolated this conclusion to the possible isomers in the LCE micropillars. (We believe this is reasonable because the molecular mobility is lower in LCE than in solution). This indirectly indicated that the possible isomer structures existing in the LCE micropillars will not be affected during the thermally-induced deformation process. This was actually

Analyses of factors that might affect the PL intensity of LCE micropillars

The potential factors which might affect the PL intensity variations during the heating and cooling processes were explored. Firstly, during the photopolymerization to prepare the LCE, the β-cyano-OPV dye could also absorb the UV light to undergo a photoisomerization as already discussed above and ESI (section 3). Nevertheless, we have shown that the isomerized structures are thermally stable. This indicated that the isomer structures in the LCE micropillars will not be affected during the thermally-induced deformation process. Thus, the observed PL intensity variations do not come from a phenomenon related to a change in the isomerization ratio.

Photo-bleaching is another phenomenon that could induce the decrease of PL intensity. It is related to the photochemical destruction of the fluorophore, which, if present, might also prohibit to perform long observations of our samples by fluorescence microscopy of our samples. We In order to study the fluorescence sensitivity to a simple temperature variation, we specially fabricated new micropillars containing 1 mol% β-cyano-OPV but a high concentration of whole crosslinkers (35 mol% in total) which was photopolymerized in the isotropic phase. These pillars were not monodomain with aligned mesogens and were densely crosslinked, so that they would not exhibit any thermomechanical effect since they are some kind of polydomain thermosets. The POM of the highly crosslinked micropillars showed a very weak birefringence, as compared to the lightly crosslinked micropillars (Fig S18 ). Moreover, only one broad peak was observed in the DSC curve (Fig S19 ), which was ascribed to the glass-to-isotropic phase transition. The PL intensity and the size of these highly crosslinked micropillars were monitored during a heating-cooling cycling process, from The "natural" decrease of the PL intensity with the temperature can be then deducted from the global variation traced in the Figure 6, leading to the results presented in the Fig 9 . The PL intensity variations depend now only on the deformation process and on the phase transition. In order to further study the relative influences of the deformation process and the nematic-to-isotropic phase transition on the PL intensity variation, we built a new experiment. We studied the evolution of the PL intensity as a function of the temperature for an unpolymerized mixture containing the LC monomer, the flexible crosslinker and the βcyano-OPV crosslinker, but without the photoinitiator. The observed PL intensity variations will reflect only the evolution connected with the phase transition (nematic-to-isotropic). As shown in the Fig 10a, the PL intensity decreased with the temperature increase. In the Fig 10b, the PL intensity variation for the mixture and its DSC curve are grouped, demonstrating that the sharp decrease in PL intensity is concomitant with the nematic-to-isotropic phase transition at 70-80°C. We also checked out that the PL intensity of the pure β-cyano-OPV in the solid state was not sensitive to temperature in the studied range (see Fig S21). We could conclude that the PL intensity variation observed for the monomers mixture was caused by the phase transition.

At last, the evolution of the emission spectra with the concentration of β-cyano-OPV crosslinker in micropillars was investigated. As shown in Fig 11a, when the concentration of the dye was 0.5 mol%, the maximum emission spectra had peak value at 476 nm, while for a 5 mol% concentration, the emission peak had a limited red shift to 492 nm. Meanwhile, the variation of the emission spectra with the temperature change was also explored to check the state of the β-cyano-OPV molecules during the deformation process. As seen in Fig 11b, the PL intensity decreased with the temperature increasing. However, the emission spectra did not show any shift and curve shape change up to the isotropic temperature, which means that the β-cyano-OPV molecules always remained in the "monomer state" during the deformation process. Similar results were obtained for the micropillars with different β-cyano-OPV concentrations (Fig S22). This might be linked with the relative small contraction observed for the LCE micropillars, which prevented the dye molecules to come close enough to each other to build effective aggregates and to behave as excimers. After these careful analyses of factors that might affect the PL intensity of LCE micropillars, we can conclude that the PL intensity variation observed for the micropillars was caused by the phase transition and the temperature change. The N-I phase transition was the major reason of the abrupt decrease of PL intensity of LCE micropillars during thermal-mechanical deformation, while the temperature increase caused only a Please do not adjust margins Please do not adjust margins smooth decrease of PL intensity. This is comprehensible from the principle of fluorescence. A part of the energy of the excited state of monomeric β-cyano-OPV was annihilated by radiation, leading to the fluorescence. Upon heating and especially in isotropic phase above TNI, the β-cyano-OPV molecule gained more freedom of inter-and intra-molecular motions. These molecular motions deactivated more efficiently its excitons in non-radiative manners. Consequently, the part of radiative channel to decay the energy of the excited state became less efficient, and then the fluorescence emission of the micropillars was weakened.

Conclusion

We prepared new nematic LCE micropillars by including a fluorescent dye (β-cyano-OPV) in the crosslinker. The quantum yield of the fluorescent LCE was 85% ± 10% as measured by using the integrating sphere method. The potential factors which might affect the photoluminescence intensity during the heating and cooling cycling process were investigated in detail. The nematic-to-isotropic phase transition of the LCE micropillars has the greatest effect on the PL intensity variations. Meanwhile, during the deformation process, no obvious shift of the fluorescence emission spectra could be detected. We concluded that the β-cyano-OPV dye molecules stayed in the monomeric state during the deformation process of LCE micropillars with the contraction (expansion) ratio L/Liso of 1.25. The study of fluorescent LCEs presenting higher contraction ratio are in progress in order to get excimers and shift of the fluorescence emission spectra. The present work might provide a potential protocol to develop LCE fluorescent sensors and might have potential application in dynamic display device.

Experimental section

Characterization

1H NMR and 13C NMR spectra were recorded on Bruker Avance 400 MHz spectrometer at 297 K. Deuterated chloroform (CDCl3) was used as the solvent and TMS (tetramethylsilane) was used as the internal reference. Calorimetric measurements of LC monomers and elastomers were performed using a Perkin Elmer DSC7 instrument. The reference cell was kept empty and the sample cell was filled with polymer sample (5 to 10 mg). Samples were scanned over a temperature range between 25 o C and 120 o C with a scanning rate of 10 o C/min. Thermogravimetric analysis (TGA) of the elastomer containing β-cyano-OPV crosslinker was carried out on SDT Q600 TA instrument. The sample (5-10 mg) was measured under a nitrogen atmosphere over the temperature range of 25 to 700 °C with a heating rate of 20 °C/min. The fluorescence emission spectroscopy characterization was carried out on a FluoroMax spectrofluorometer. The fluorescence quantum yield was measured on spectrofluorometer Fluorolog FL3-221 from Horiba Jobin-Yvon, equipped with a Horiba Jobin-Yvon integration sphere accessory. PL dyes samples were dissolved in the dichloromethane (DCM). Fluorescence emission spectroscopy of the LCE micropillars was carried out on the same FluoroMax spectrofluorometer. The LCE micropillars were dispersed in low viscosity silicone oil in a 0.2 cm thickness quartz cell. UV-Vis spectra were obtained with a PerkinElmer Lambda 800 instrument. The PL dye was dissolved in dichloromethane (DCM) to obtain the spectra in solution. The PL intensity variations were measured by fluorescence microscopy. Samples were observed directly via a Leica DMIL LED Fluo inverted microscope equipped with a Linkam heating stage. The excitation light was supplied by a Cool LED system (pE-300 white, Leica). For the UV light excitation, the fluorescence filter cube of DAPI was used (excitation wavelength between 325-375 nm, emission wavelength between 435-485 nm). For the blue light excitation, the fluorescence filter cube of GFP was used (excitation wavelength between 450-490 nm, emission wavelength between 500-550 nm). Polarizing microscopy (POM) observations were obtained on a Motic BA310POL equipped with a Motic CCD camera, and a Mettler FP 80 hot stage.

Fabrication of the micropillars

Poly(dimethylsiloxane) (PDMS) molds were prepared via photo-lithography and soft-lithography steps 47 The concentration of the photoinitiator was kept at 2 mol%, while various ratios of monomer/flexible-crosslinker/OPV-crosslinker have been tested.

The micron-sized LCE pillars were fabricated as previously described 14 . The mixture of LC monomer, flexible and fluorescent crosslinkers, and photo-initiator were deposited on a glass coverslip placed atop a permanent magnet (~1 T NdFeB rare earth magnet) in a heating stage. Then the mixture was heated up to its isotropic phase (100 o C). A soft PDMS mold was gently pressed onto the melted mixture to force the mixture to fill the inner structure of the mold. The monomers mixture was then cooled down to its nematic phase (60 o C) at a slow rate (1 o C/ min). During the cooling process, the magnetic field aligns the nematic phase with the nematic director pointing along the long axis of the pillars. During the process, an oxygen-free environment was kept by placing the setup in a zip-lock bag flushed with a slow stream of argon. A 365 nm UV light source (30mW/cm 2 ) was used to initiate the photopolymerization for 30 minutes. After the sample was cooled down to room temperature, the PDMS mold was peeled off to leave a regular array of micropillars. A sharp razor blade was used to untie the micropillars from the glass This journal is © The Royal Society of Chemistry 20xx

Please do not adjust margins Please do not adjust margins surface. The free micropillars were suspended in low viscosity silicone oil.

PL intensity measurement of the micropillars

The relative PL intensity variations for the micropillars were measured using Image J software. Keeping the same exposition time (40 ms), gain (2%), Gamma (1) parameters of the fluorescence microscope, the fluorescence images at different temperatures were recorded. The captured fluorescence images were imported in Image J, and changed to an 8-bit grayscale mode. Recording the sum of grayscale values of a fluorescent micropillar in its whole area gave us the relative fluorescence intensity of a micropillar. Ten micropillars were analysed for each experiment to give the average value.

Fig. 1

 1 Fig. 1 The PL excitation and emission spectra of the β-cyano-OPV in different environments. (a) The excitation spectrum in CH2Cl2 solution (10 -5 mol/L). (b) The excitation spectrum in micropillars. (c) The emission spectrum in CH2Cl2 solution under the excitation light of 396 nm. (d) The emission spectrum in micropillars under with the excitation light of 420 nm. (e) The emission spectrum in the pure solid state with the excitation light of 420 nm. Thermomechanical properties of the LCE micropillars After the photopolymerization, optical microscopy, scanning electronic microscopy (SEM) and fluorescence microscopy were used to observe the regular array of micropillars and separated micropillars. As shown in Fig 2a, the optical microscopy image showed the regular array of micropillars on a large scale, demonstrating that the method was suitable for preparation of micropillars with a high efficiency and accuracy. SEM image of the micropillars array is presented in Fig 2b, which further confirms the structures. Fig 2c and 2d show the separated micropillars under POM without and with crossed polarizers, respectively. No obvious defects could be observed in the long axis of the micropillars in the Fig 2d, indicative of a relatively good alignment of the nematic mesophase. The regular array of micropillars and the separated micropillars were also observed by fluorescence

Fig. 2

 2 Fig. 2 Images of the LCE micropillars containing the β-cyano-OPV as crosslinker. (a): Optical microscopic image of the micropillars. (b): SEM image of the micropillars. (c): Separated micropillars observed under POM without crossed polarizers. (d): Separated micropillars under POM with crossed polarizers. (e) and (f): Fluorescence image of the micropillars excited by UV light of 365 nm. (g) and (h): Fluorescence image of the micropillars excited by blue light of 450 nm.

  Fig 3 (the others are presented in Fig S9 and S10). Fig 3a shows the thermally-induced spontaneous shape deformation behavior of the micropillars during a heating and cooling cycle between 25 o C and 100 °C. Upon heating at 100 o C, the length of micropillars reduced around 30%. Upon cooling, the length of micropillars recovered to their original value. Then we selected 10 separated micropillars to measure their lengths along the longitudinal direction when the temperature was varied from 25 o C to 100 o C and from 100°C back to 25 o C. The contraction and relaxation were characterized by L/ Liso as a function of temperature as shown in Fig 3b, where Liso is the minimum length of the micropillar in its isotropic state and L is the length of the micropillar at any given temperature. Contraction during the heating process was observed in the temperature range 70-85 o C, which corresponds to the nematic-isotropic transition temperature (TNI) of the LCE samples (Fig S11). It is clear that the thermomechanical deformation mainly occurs at TNI of the LCEs. On cooling back to 25 o C, pillars expanded and recovered the initial shape and This journal is © The Royal Society of Chemistry 20xx Please do not adjust margins Please do not adjust margins size. All these observations demonstrated that the micropillars have a fully reversible shape memory property.

Fig. 3

 3 Fig. 3 (a) The optical images of micropillars containing 1 mol % dye crosslinker at different temperatures. (b) The contraction and relaxation represented by L/Liso along the long axis of these micropillars at heating and cooling process, 10 micropillars being analyzed. Liso and L are the length of the micropillar in its isotropic state and at any given temperature, respectively.

Fig. 4 Fig. 5

 45 Fig. 4 (a) The deformation properties of the micropillars with different concentrations of β-cyano-OPV dye crosslinker. (b) The reversible thermal deformation of these micropillars within 10 heating and cooling cycles between 25 o C and 100 o C. approved by the repeating cycles of the thermally-induced deformation as shown in Fig 4b. The contraction and recovery cycling processes were carried out from 25 o C (in nematic phase) to 100 o C (in isotropic phase) and back to 25 o C repeatedly for ten times. The thermally-induced deformation properties remained stable, which demonstrates the thermomechanical stability of these micropillars.

Fig. 6

 6 Fig. 6 The variation of fluorescence intensity and the ratio of contraction of the micropillars containing 1 mol% β-cyano-OPV crosslinker, as a function of temperature during the heating and cooling process. (a) under UV light excitation at 365 nm. (b) under blue light excitation at 450 nm.

  have recorded the PL intensities of the micropillars containing 1 mol% βcyano-OPV at 25 o C as a function of illumination time until 65 min under UV light (the 365 nm) and under blue light (450 nm) with the same intensities as those used in other experiments presented in this work. The data presented in Fig 7 show that PL intensity remained stable during more than 1h of illumination. This result suggests that the photo-bleaching can be ignored in our experiments.

Fig 7 .

 7 Fig 7. The PL intensity evolution of micropillars containing 1 mol% β-cyano-OPV at 25 o C under the continuous 365 nm UV light and 450 nm blue light illumination.

  25 o C to 115 o C. Firstly these micropillars did not change shape and size (Fig S20). Then, as shown in Fig 8, the PL intensity decrease and recovery during the heating and cooling cycle were smooth without abrupt change with temperature increasing under UV or blue light excitations (more changes were observed under blue light excitation). Thus, a simple effect of temperature cannot explain the sharp PL intensity variation for the LCE micropillars, as reported in Fig 6. 6 | J. Name., 2012, 00, 1-3 This journal is © The Royal Society of Chemistry 20xx Please do not adjust margins Please do not adjust margins

Fig. 8

 8 Fig. 8 The PL intensity variation with temperature increasing for the highly crosslinked micropillars containing 1 mol% β-cyano-OPV crosslinker under UV light excitations (a) and under blue light excitation (b).

Fig. 9

 9 Fig. 9 The PL intensity variation after removing the pure temperature effect during the heating and cooling process under the UV light excitation (a) and under the blue light excitation (b).

Fig. 10 (

 10 Fig. 10 (a) The fluorescence emission spectra evolution with temperature for the LC monomers mixture containing 1 mol % β-cyano-OPV crosslinker under excitation light at the 420 nm. (b) The PL intensity variation at λem = 481 nm and the DSC curve of the unpolymerized mixture upon heating.

Fig. 11 (

 11 Fig. 11 (a) The fluorescence emission spectra of the micropillars with different content of β-cyano-OPV. (b) The thermally-induced PL intensity variation for micropillars containing 1 mol% β-cyano-OPV.

  . Multiple mixtures were prepared from the monomer MA444 (dimethacrylate) and the β-cyano-OPV crosslinker ((((1Z,1'Z)-1,4-phenylenebis(1-cyanoethene-2,1-diyl))bis(4,1phenylene))bis(oxy))bis(hexane-6,1-diyl) bis(2-methylacrylate)

This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 3

This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 5

Acknowledgements

This work has received the support from the French National Research Agency (ANR-16-CE29-0028) and from the "Institut Pierre-Gilles de Gennes" (IPGG, laboratoire d'excellence, "Investissements d'avenir" programs ANR-10-IDEX-0001-02 PSL, ANR10-LABX-31 and ANR-10-EQPX-34). Bin NI gratefully acknowledges the China Scholarship Council for funding his PhD scholarship. We thank Guylaine Ducouret, Mohamed Hanafi and Rémi Métivier for their help in TGA analysis and quantum yield measurement.

Conflicts of interest

There are no conflicts to declare.


Xinfeng Tao

Hui Chen

Sylvain Trépout

Jiayu Cen

Jun Ling

Min-Hui Li

Polymersomes with aggregation-induced emission based on amphiphilic block copolypeptoids

come

Nanoparticles composed of amphiphilic copolymers have attracted extensive attention in the past decades due to their ordered nanostructures with various morphologies, including spherical micelles, cylindrical micelles and vesicles, etc., which can be potentially used in drug delivery and bioimaging systems. 1 Among them, polymer vesicles, also known as polymersomes, are cell-mimicking hollow spheres with a polymer bilayer membrane enclosing an aqueous volume in its interior. They are more stable and robust than lipid vesicles (liposomes) and have high encapsulation capacity for both hydrophilic and hydrophobic molecules. 2,3 Polypeptoids are a class of poly(amino acid)s suitable for the polymersome preparation with the advantages of excellent biocompatibility, degradability, and low cytotoxicity. 4 Polypeptoids have a protein-mimicking structure with Nsubstituted glycine as repeating units but lack intramolecular and intermolecular hydrogen bonding along the amino acid backbone. Therefore, polypeptoids show better solubility, thermal processability, cell permeability and proteolytic stability compared to polypeptides. 4 Polypeptoids are usually synthesized by ring-opening polymerization (ROP) of Nsubstituted glycine N-carboxyanhydride (NNCA). 4 Recently, we developed an efficient alternative way to synthesize welldefined polypeptoids using of a more stable monomer Nsubstituted glycine N-thiocarboxyanhydride (NNTA). 5 Nanoparticles based on polypeptoids have been widely investigated. 6 Luxenhofer and co-worker prepared worm-like micelles and polymersomes by using amphiphilic block copolypeptoids containing hydrophilic polysarcosine (PSar) block and hydrophobic polypeptoid block with butyl, pentyl, benzyl or phenethyl side groups. 7 We reported previously that PSar-block-poly(ε-caprolactone)-block-PSar (PSar-b-PCL-b-PSar) formed unilamellar sheets, worm-like cylinders, and multilamellar polymersomes under different conditions. 8 Spherical micelles formed in water by poly(N-ethylglycine)-

block-poly[(N-propargylglycine)-r-(N-decylglycine)]

(PNEG-b-P(NPgG-r-NDG)) were studied by Zhang et al. 9 They used the hydrophobic core of micelles to encapsulate Doxorubicin (DOX) for further application of controlled release of DOX. 9 Sun et al. synthesized different PEG-block-polypeptoid which selfassembled into two-dimensional nanosheets. 10,11 All the above reported nanoparticles are based on polypeptoids with normal aliphatic or aromatic side chains. Nevertheless, functional side chains are often expected in various applications, for example, for the preparation of stimuliresponsive nanoparticles in controlled release or of fluorescent nanoparticles in bioimaging. In a recent paper, we developed oxidation-responsive polymersomes based on copolypeptoids, poly(N-3-(methylthio)propyl glycine)-block-PSar (PMeSPG-b-PSar), containing thioether side-chains on PMeSPG. 12 However, this method has some limitation, i.e., we need to develop a specific route of synthesis for each functional monomer, which is tedious and time-consuming. In this study, we propose to use a functionalizable poly(N-allylglycine) (PNAG) as hydrophobic block, which is an excellent platform for modification through thiol-ene reaction to get functional polypeptoids. 13 PNAG-b-PSar was synthesized using benzylamine-initiated ROP of Nallylglycine NTA (NAG-NTA) and sarcosine NTA (Sar-NTA). To highlight their ability to be functionalized in side-chains and to self-assemble into functional polymersomes, we attached the tetraphenylethylene (TPE), [14][15][16][17][18] the emblematic luminogen with aggregation-induced emission (AIE), to PNAG side chains through postpolymerization modification (Fig. 1A). Polymersomes and micelles with AIE properties were obtained by using nanoprecipitation method. Cytotoxicity test showed good biocompatibility of these nanoparticles. We believe these AIE polymersomes based on polypeptoids will have potential application in bio-imaging and drug delivery.

We first synthesized Sar-NTA and the novel monomer NAG-NTA through a phosgene-free method (Fig. S1-S6, ESI). 5 Homopolymerization of NAG-NTA initiated by benzylamine in acetonitrile at 60 °C was then studied (Table S1, ESI). The wellcontrolled characteristic of NAG-NTA polymerization based on single-site initiation by benzylamine was supported by MALDI-ToF mass spectra, 1 H NMR spectra and SEC traces (see ESI and Fig. S8-S10 for details). PNAG can be modified easily through thiol-ene radical addition reaction with mercaptan, e.g. mercaptoacetic acid, mercaptoethanol, and benzyl mercaptan (Scheme S5, ESI). The 1 H NMR spectra (Fig. S9, ESI) indicated the quantitative transformation of allyl groups to the selected functional groups.

PNAG-b-PSar with three different molecular weights were then synthesized by sequential feeding of different amounts of NAG-NTA and Sar-NTA (Fig. 1A). The successful synthesis of PNAG-b-PSar was proved by 1 1D and Fig. S12) and SEC traces (Fig. S13). Their molecular weights (MW) and distributions (Đ) are summarized in Table S2. The hydrophilic weight ratios (fPSar) of P(TPE-NAG)-b-PSar are 18%, 30% and 38%, respectively. P(TPE-NAG)25-b-PSar46 was chosen as an example to study the AIE properties of copolypeptoids. We found it exhibited obvious AIE fluorescence when dispersed in water owing to the restriction of intramolecular rotation (RIM) of TPE moieties in the aggregated state (Fig. 2A and discussion in ESI). 18 Moreover, we were also interested in the morphology of aggregates formed in this experiment. The dispersion in DMF/H2O mixture with fw = 99% was selected for DLS and cryo-EM analysis. Fig. 2B shows the size distribution of aggregates weighed by scattering intensity and by number. There are two populations of nanoparticles, where the smaller one at about 56 nm of average hydrodynamic diameter is predominant and a few of big particles with diameters > 1000 nm co-exist. Fig. 2C andD show the nanostructures observed by cryo-EM, which confirm the results of DLS. Many nanoparticles with diameters < 50 nm were observed. Note that hydrophilic chains located on the membrane coronas (exterior and interior sides) of the polymersomes are flexible and swollen in water, which do not constitute enough density contrast, relative to vitreous ice at the sample grid, to be visible in cryo-EM. [START_REF] Michler | Electron Microscopy of Polymers[END_REF] Therefore, it is normal that the sizes measured by cryo-EM are smaller than the Please do not adjust margins Please do not adjust margins hydrodynamic diameters. Meanwhile, a few of vesicles (Fig. 2D, Fig. S15) are also observed, and some of them have very big size, which correspond effectively to the second population of particles in DLS curves. These observations showed that the copolypeptoid P(TPE-NAG)25-b-PSar46 had the tendency to form vesicular structures. However, well defined polymersomes were not obtained probably because of the quick addition of large quantity of water.

Then, we used a nanoprecipitation method with DMF/water as co-solvents and a slow water addition process to selfassemble P(TPE-NAG)-b-PSar in a more controlled way. Typically, P(TPE-NAG)-b-PSar was first dissolved in 1 mL DMF with a concentration of 2 mg/mL, and deionized water was added then very slowly (2.5 μL/min) with slight shaking. The slow rate of water addition could avoid the early freezing of nanostructure and keep the self-assembly dynamic as long as possible. When the water content was increased to around 70% in volume, the dispersions were dialyzed against deionized water to remove DMF and get P(TPE-NAG)-b-PSar selfassemblies in pure water. Table S2 summarizes hydrodynamic diameters of all nanoparticles and membrane thicknesses of the polymersomes. For P(TPE-NAG)10-b-PSar53 (fPsar = 38%), micelles with a diameter around 26 nm are mainly observed by cryo-EM (Fig. 3G, Fig. S16) and TEM (Fig. S17). DLS intensity profile shows a bimodal distribution (Fig. 3A) with the hydrodynamic diameters of 29 nm and 312 nm, respectively. When there are several populations of nanoparticles, it is difficult to quantify the statistics with only one kind of size profile in DLS because the intensity profile privileges the big particles and the number profile privileges the small particles. Therefore, both intensityweighed and number-weighed DLS curves are given here (Fig. 3A and3D). The smaller one corresponds to the micelles, while the larger one (very few in number as shown in number profile of DLS) may attribute to a few of aggregations (clusters) of micelles (Fig. 3G and Fig. S16). When the fPsar decreases to 30% (P(TPE-NAG)15-b-PSar54), both micelles and polymersomes were observed by cryo-EM (Fig. 3H and Fig. S18) and TEM (Fig. S19), whose diameters are 26 nm and 357 nm, respectively. DLS profile of P(TPE-NAG)15-b-PSar54 nanoparticles are double peaks with the hydrodynamic diameters of 38 nm and 203 nm (Fig. 3B), which is consistent with the TEM results. Note that the polymersomes were minor population as shown in DLS number profile and connected or fused polymersomes were observed in some cases (Fig. S18). In the case of PNAG25-b-PSar46 (fPsar =18%), only polymersomes are formed according to cryo-EM images (Fig. 3I and Fig. S20), with two distributions of sizes: one around a Z-average diameter of 804 nm, and another around a Z-average diameter of 136 nm as measured by DLS (Fig. 3C and3F). As for the sizes and size distributions, the results obtained from cryo-EM and DLS (Table S2) are coherent to some extent.

With the increase of the hydrophobic P(TPE-NAG) chain length and the decrease of the hydrophilic ratio fPsar (here the length of hydrophilic part PSar is similar for three copolypeptoids), the nanostructures transform from micelles to vesicles and their average sizes become larger. These tendencies can be empirically explained by the increase of the packing parameter p = v/al (v is the hydrophobic volume, a is the optimal interfacial area and l is the length of the hydrophobic block normal to the interface) when increasing hydrophobic block MW, as already reported previously. 15,[START_REF] Blanazs | [END_REF]21 According to the cryo-EM images, the thickness of the hydrophobic part of membrane is measured as to be 8 ± 1 nm and 10 ± 1 nm for P(TPE-NAG)15-b-PSar54 and P(TPE-NAG)25-b-PSar46 polymersomes, respectively. It is easy to understand that the longer the hydrophobic block leads to the thicker membrane. By using the MM2 energy minimization with the Chem3D software, the contour lengths of the hydrophobic part P(TPE-NAG)15 and P(TPE-NAG)25 are about 4.4 nm and 7.3 nm, respectively. Consequently, a perfectly-ordered tail-to-tail bilayer should have a membrane thickness of 8.8 nm and 14.3 nm, respectively. The membrane thickness measured by cryo-EM is lower than the double lengths of two extended P(TPE-NAG) chains. Therefore, the obtained polymersomes should be unilamellar vesicles, in which P(TPE-NAG) chains might not be totally extended or might be interdigitated between the two leaflets.

Then, we investigated the fluorescence of these well-defined nanostructures formed by P(TPE-NAG)-b-PSar. The DMF solution of P(TPE-NAG)-b-PSar is non-emissive while the aqueous solution of P(TPE-NAG)-b-PSar micelles and vesicles shows strong emission under 365 nm UV lamp (Fig. 4A) due to the RIM of TPE moieties in the core of polymer micelles and of polymersome membrane as expected. The emission spectra of nanoparticles of P(TPE-NAG)10-b-PSar53, P(TPE-NAG)15-b-PSar54 and P(TPE-NAG)25-b-PSar46 are shown in Fig. 4B with maximal emission wavelengths at 478 nm, 474 nm and 471 nm, respectively, the excitation wavelength being set as 370 nm that corresponded to the maximal wavelength in the excitation spectrum (Fig. S21). Rather high quantum yields were measured as 26%, 29% and 34% for P(TPE-NAG Please do not adjust margins Please do not adjust margins using the integrating sphere method. The fluorescence intensity and quantum yield increase following the morphology evolution from micelles, micelles/polymersomes to polymersomes, which is attributed to the increasing packing density of polymer blocks within the nanoparticles and the increasing confinement of the AIEgens. 14 Finally, the biocompatibility of the nanoparticles was assessed by using MTT cell viability assay. 22

Fig 1 .

 1 Fig 1. (A) Synthesis of PNAG-b-PSar through block copolymerization of NAG-NTA and Sar-NTA and its postpolymerization modification by reacting with mercaptoacetic acid and TPE-C3OH successively to produce P(TPE-NAG)-b-PSar. 1 H NMR spectra of PNAG15-b-PSar54 (B), P(CA-NAG)15-b-PSar54 (C) and P(TPE-NAG)15-b-PSar54 (D) in DMSO-d6. (*: diethyl ether; **: water)

  H NMR spectra and SEC traces (see Fig. 1B, Fig. S11 and discussion in ESI). The compositions of the three block copolymers were PNAG10-b-PSar53, PNAG15-b-PSar54 and PNAG25-b-PSar46 as calculated from NMR data.

Fig. 2 .

 2 Fig. 2. (A) Photo-luminescence (PL) spectra of P(TPE-NAG)25-b-PSar46 in DMF/H2O mixtures with different water fractions (concentration: 2.2×10 -5 M, 0.4 mg/mL; excitation wavelength: 370 nm). The inserted photographs in (A) represent samples with different water fractions under UV light of 365 nm. (B) DLS profiles (Z-average with intensity and number average) of aggregates of P(TPE-NAG)25-b-PSar46 in DMF/H2O mixture with water fraction at 99%. (C) and (D) cryo-EM images of aggregates of P(TPE-NAG)25-b-PSar46 in DMF/H2O mixture with water fraction at 99%.

Fig. 3 .

 3 Fig. 3. DLS profiles (Z-average with intensity A-C and number average D-F) and cryo-EM images of nanoparticles in water formed by P(TPE-NAG)10-b-PSar53 (fPsar = 38%) (A, D and G), P(TPE-NAG)15-b-PSar54 (fPsar = 30%) (B, E and H) and P(TPE-NAG)25-b-PSar46 (fPsar = 18%) (C, F and I). The dark densities present at the center of the polymersomes are caused by the scattering of electrons due to the inherent thickness of the polymersome spheres.

  )10-b-PSar53, P(TPE-NAG)15b-PSar54 and P(TPE-NAG)25-b-PSar46 nanoparticles, respectively, This journal is © The Royal Society of Chemistry 20xx

Fig. 4 .

 4 Fig. 4. (A) Schematic diagram of self-assembly of P(TPE-NAG)-b-PSar in aqueous solution and the AIE phenomenon of the polymersome dispersion. (B) PL spectra of different nanoparticles (concentration: 0.4 mg/mL; excitation wavelength: 370 nm). (C) Relative cell viability of P(TPE-NAG)25-b-PSar46 polymersomes for human vein endothelial cells after 24 h incubation at a concentration of 23.7 μg/mL, 47.5 μg/mL, 95.0 μg/mL, 190 μg/mL, 380 μg/mL and 760 μg/mL, respectively.

  Human vein endothelial cells were incubated for 24 h with P(TPE-NAG)-b-PSar nanoparticles at different concentrations. All P(TPE-NAG)b-PSar micelles and polymersomes studied are non-cytotoxic (Fig. 4C, Fig. S22 and S23), indicating their excellent biocompatibility. The negligible cytotoxicity of P(TPE-NAG)-b-PSar polymersomes makes possible their applications in biorelated domains such as bio-imaging and drug delivery. In summary, N-allylglycine NTA was synthesized for the first time, whose corresponding polymer PNAG is an excellent platform for post-modification to produce different functional polypeptoids. Well-controlled polymerization of NAG-NTA was achieved by using primary amine as the initiator, and PNAGs with narrow distributions were produced. A series of diblock copolypeptoids PNAG-b-PSar with different chain lengths were synthesized by sequential feeding of NAG-NTA and Sar-NTA. TPE-functionalized P(TPE-NAG)10-b-PSar53 (fPsar = 38%), P(TPE-NAG)15-b-PSar54 (fPsar = 30%) and P(TPE-NAG)25-b-PSar46 (fPsar = 18%) were prepared by post-polymerization reaction. Selfassembling was performed by nanoprecipitation method using DMF/water as co-solvents. Micelles, micelles/polymersomes and polymersomes were obtained for P(TPE-NAG)10-b-PSar53, P(TPE-NAG)15-b-PSar54 and P(TPE-NAG)25-b-PSar46, respectively. All the nanoparticles show strong AIE fluorescence in aqueous solution. These AIE polymer micelles and AIE polymersomes made of block copolypeptoids are non-cytotoxic, which guarantee their safety in potential applications in bio-related field. Financial supports from French National Research Agency (ANR-16-CE29-0028) and National Natural Science Foundation

of China (21674091) are acknowledged. We thank Prof. Ben Zhong Tang (Hong Kong University of Science and Technology) for fruitful discussions. We thank Prof. Zhengwei Mao (Zhejiang University) for cytotoxicity analysis, and Dr. Chao Deng (Zhejiang University) for quantum yield measurement. The authors thank the PICT-Ibisa for providing access to the cryo-EM facility at Institut Curie in Orsay.

Conflicts of interest

There are no conflicts to declare.

Notes and references


Valerian Estragnat
email: valerian.estragnat@gmail.com

Laurence Volatier
email: laurence.volatier@entpe.fr

Sustainability of gastropod introduction for ecological engineering solution in infiltration basins: feeding strategy of V

Keywords: Trophic strategy, ecological engineering, biofilm, physiological state, gastropod, algae

The aim of this study was to test the sustainability of ecological engineering solutions based on the introduction of the gastropod V. viviparus in infiltration basins designed for groundwater recharge and clogged by benthic biofilms. This sustainability depends on the ability of gastropods to deal with variations in trophic resources throughout seasonal changes and so their feeding strategy. It is expected from literature that V. viviparus is a generalist species, well-adapted for ecological engineering approaches. With this objective, laboratory and field experiments were developed to measure the ability of gastropods to maintain energy body stores under several food sources and seasons, in relation with their physiological state. Our results showed that V. viviparus was not a strict generalist: it tended to be more efficient to constitute energy reserves when feeding on algae under laboratory conditions and its energy stores were positively correlated with primary productivity of the benthic biofilm in the field. Despite this higher efficiency of V. viviparus to produce energy reserves from algal resources, the survival and the levels of energy reserves measured on the field, even when trophic conditions were the harshest (low algal development), make this species a good candidate for ecological engineering approaches.

Introduction

Ecological engineering is a discipline aiming to use ecological theories to treat/ameliorate environmental issues.

Ecological engineering approaches are often based on the introduction/manipulation of species diversity to modify environmental systems. For example, introduction of piscivorous species has been proposed to reduce eutrophication problems in lakes by trophic cascade effects [START_REF] Carpenter | Cascading trophic interactions and lake productivity[END_REF]. However, the maintenance of ecological engineering solutions on the long-term needs to evaluate the ability of introduced species to deal with environmental changes. Then, knowing the ecological strategies of introduced species is crucial to evaluate the sustainability of ecological engineering solutions.

The identification of feeding strategies is fundamental to the understanding of many ecological processes such as the flow of energy within food webs, the spatial distribution of species and the evolution of competitive interactions in communities [START_REF] Bolnick | The ecology of individuals: incidence and implications of individual specialization[END_REF][START_REF] Holt | Bringing the Hutchinsonian niche into the 21st century: ecological and evolutionary perspectives[END_REF][START_REF] Poisot | A conceptual framework for the evolution of ecological specialisation[END_REF]. Theory distinguishes generalist and specialist strategies, even though organisms employ a range of feeding strategies between these two extremes [START_REF] Devictor | Defining and measuring ecological specialization[END_REF][START_REF] Futuyma | The Evolution of Ecological Specialization[END_REF][START_REF] Levins | Evolution in changing environments: some theoretical explorations[END_REF]. Species with generalist strategies have a higher ability to endure environmental changes than organisms with specialist strategies [START_REF] Clavel | Worldwide decline of specialist species: toward a global functional homogenization?[END_REF]. For example, [START_REF] Devictor | Functional biotic homogenization of bird communities in disturbed landscapes[END_REF] showed that landscape disturbance would lead to a dominance of generalist individuals in bird populations.

Recently, we measured a positive influence of freshwater gastropod introduction to limit excessive development of benthic biofilm and its impact on the hydraulic performance of infiltration basins designed for groundwater recharge [START_REF] Estragnat | Does the efficiency of grazer introduction to restore and preserve the hydraulic performance of infiltration basins depend on the physical and biological characteristics of the infiltration media?[END_REF][START_REF] Gette-Bouvarot | Ecological engineering approaches to improve hydraulic properties of infiltration basins designed for groundwater recharge[END_REF]. Experimentations were performed on biofilms developed under various environmental conditions (hydrological regime, nutrients fluxes, light availability) resulting in the formation of biofilms exhibiting contrasted structural and functional characteristics. However, all these experimentations were conducted during autumn season when chlorophyll-a concentrations are the highest (more than 10 µg of chlorophyll-a per g of dry sediment in [START_REF] Gette-Bouvarot | Ecological engineering approaches to improve hydraulic properties of infiltration basins designed for groundwater recharge[END_REF].

With seasonal changes and their effects on biofilm abundance, composition, primary production and metabolic balance (heterotrophs/phototrophs), the long-term sustainability of the introduction of the gastropod Viviparus viviparus (Linnaeus, 1758) needs to consider the feeding strategy of this organism under contrasting food sources availability. Recent findings demonstrated that V. viviparus was efficient to reduce clogging in infiltration basins characterized by variable quantities of food sources (bacterial-algal mat occurring on surface sediment, sedimentary biofilm and organic matter from surrounding vegetation) and suggested a low influence of food sources availability on gastropod feeding behaviour [START_REF] Estragnat | Does the efficiency of grazer introduction to restore and preserve the hydraulic performance of infiltration basins depend on the physical and biological characteristics of the infiltration media?[END_REF]. This supposition was consistent with the study of [START_REF] Jakubik | Food and feeding of Viviparus viviparus (L.)(Gastropoda) in dam reservoir and river habitats[END_REF] which reported that the mass and composition of foods ingested by V. viviparus mainly depended on the availability and abundance of food sources in the environment. This study, performed in lotic and lentic freshwater environments between 1995 and 2006, also indicated that V. viviparus could significantly feed on low-energetic food sources such as detritus. Indeed, detritus could represent 70 to 90% of the food mass ingested by the gastropod whatever the season or the habitat (river, reservoir, lake). All this information suggests that V. viviparus has a generalist feeding behaviour favouring the development of this species in contrasted trophic environments and thus making V. viviparus a good candidate for ecological engineering approaches. However, this hypothesis has to be tested by evaluating the ability of V. viviparus to maintain its physiological state under different food sources. Among the parameters that can be measured to assess physiological state, the reserve in triglycerides of an organism can be considered as a good proxy, because it is the major form of energy storage in gastropods [START_REF] Gust | Comprehensive biological effects of a complex field poly-metallic pollution gradient on the New Zealand mudsnail Potamopyrgus antipodarum (Gray)[END_REF]) and free lipids (mainly triglycerides) have been shown to be correlated with the growth rate of aquatic snails [START_REF] Hill | Food limitation of two lotic grazers: quantity, quality, and sizespecificity[END_REF].

To test the hypothesis that V. viviparus is a generalist species, we used a combination of laboratory and in situ experiments. First, laboratory microcosms were used to quantify to what extent different food sources affect reserve in triglycerides of gastropods. To this end, gastropods were fed ad libitum on three food sources: two sources found in natural environments (algal mat and sedimentary biofilm) and one control food used for gastropod breeding (Tetramin® goldfish flakes). We also measured the oxygen consumption rate to determine whether gastropod metabolism has been impacted by food source. Then, we used in situ enclosures within infiltration basin to track seasonal changes in triglyceride reserve of gastropods depending on various quality and quantity of available food resource. Under the hypothesis that V. viviparus is a generalist species, gastropods would have the same triglyceride concentrations whatever the season, i.e. the proportion of heterotrophic (sedimentary biofilm) and phototrophic (algal mat) benthic food sources.

Materials and Methods

Study site

The experiment was set up in the pumping well field of "Crépieux-Charmy" which provides drinking water for the metropolitan area of Lyon. Infiltration basins have been excavated to recharge the aquifer with surface water from the "Vieux Rhône" channel of the Rhône River. The field experiment was performed in a basin, in which the water column was maintained independently of the usual site management, during the entire course of the experiment. Sedimentary biofilm and algal mat used in laboratory experiment were also collected from this basin.

Model species

The studied species was V. viviparus, a prosobranch gastropod. Literature shows that V. viviparus are filter-feeders in phytoplankton-rich environments but they clearly switch to grazing behavior in environments characterized by low phytoplankton concentrations [START_REF] Cook | A ciliary feeding mechanism in Viviparus viviparus (L.)[END_REF][START_REF] Höckelmann | The respiration and filter-feeding rates of the snail Viviparus viviparus (Gastropoda) under simulated stream conditions[END_REF] as observed in our study site.

Indeed, the chlorophyll-a concentrations in the water column of the studied basin never exceeded 7 µg.L -1 whatever the season in 2016, 2017 and 2018 (data from Eau du Grand Lyon). Specimens were collected in a cut off channel of the Rhône River. Before laboratory and field experiments, gastropods were kept during six months in 20 litre glass aquariums (15 specimens per aquarium) under controlled laboratory conditions following the studies of [START_REF] Cook | A ciliary feeding mechanism in Viviparus viviparus (L.)[END_REF] and [START_REF] Tsvetkov | Acid phosphatase complex from the freshwater Snail Viviparus viviparus L. under standard conditions and intoxication by cadmium ions[END_REF]: water temperature (18°C), lightening condition (12:12 h light:dark regime), aerated water (ambient air bubbling) and regular water replacement (50% of the volume once a week). Animals were fed each week with Tetramin® goldfish flakes. These laboratory conditions were favourable as the survival of V. viviparus was more than 99 % after 3 months of acclimation. The gastropods used in this experimentation were mature and had total shell height comprised between 20 and 30 mm. We used a sex ratio of 2 males for 1 female in the experiments to fit with the sex ratio observed during animal collection. Although triglyceride concentrations often varied between males and females in aquatic invertebrate species, preliminary experiments showed that no difference in triglyceride concentrations was observed between males and females of V. viviparus (Online Resource 1).

Laboratory experiment Experimental design

The laboratory experiment was conducted in 36 glass aquariums (21 cm in length x 10 cm in depth x 20 cm in height). The experiment lasted six weeks under the same laboratory conditions than those used for animal maintenance before the experiments (see paragraph 2.2). We selected an experimental duration of 6 weeks to fit with the feeding experiments performed by [START_REF] Hill | Food limitation of two lotic grazers: quantity, quality, and sizespecificity[END_REF] on aquatic snails. The aquariums were filled with tap water previously dechlorinated by air bubbling during two days. One centimetre of sand collected from a terrestrial quarry having a grain size distribution comparable to those of sand in the field [START_REF] Gette-Bouvarot | Coupling hydraulic and biological measurements highlights the key influence of algal biofilm on infiltration basin performance[END_REF] and presenting low organic matter concentrations (Table 1) was placed in the bottom of each aquarium. Three gastropods previously maintained in dechlorinated water to clean their gut content were introduced in each aquarium. Then, a density of 142 individuals.m -2 was tested to be in the range of V. viviparus densities observed in the field [START_REF] Jakubik | Life strategies of Viviparidae (Gastropoda: Caenogastropoda: Architaenioglossa) in various aquatic habitats: Viviparus viviparus (Linnaeus, 1758) and V. contectus (Millet, 1813)[END_REF]. Three food treatments were applied and randomly distributed among the aquariums (12 replicated aquariums per food treatment):

(i)
Breeding food: Tetramin® goldfish flakes (the positive control)

(ii) Algal mat (predominantly phototrophic) collected within the studied basin by scraping the upper 5 mm layer of illuminated sediments (iii) Sedimentary biofilm (predominantly heterotrophic) collected in the studied basin by sampling a 5 mm layer of non-illuminated sediments (1 cm above the surface)

Natural food sources (algal mat and sedimentary biofilm) were freshly collected in the field at the start of the experiment and introduced in aquariums to cover the bottom sand layer. Additional samples of food sources were stored at 4°C to constitute a fresh stock for food replacement during the experiment. Food was added (flakes) or replaced (algal mat and sedimentary biofilms) ad libitum once a week (i.e., we observed that animals never consumed the totality of each food source during a week).

These different foods were characterized by their elemental composition in C, N and P (Table 1). Total organic carbon (TOC) and total nitrogen were measured with an elemental analyzer (FlashEA, Thermo Electron Corporation). Total phosphorus concentrations of foods were determined following the method of [START_REF] Murphy | A single-solution method for the determination of soluble phosphate in sea water[END_REF]. We measured the following decreasing gradient of nitrogen, organic carbon and phosphorus contents among the 3 food sources: breeding food > algal mat > sedimentary biofilm (Table 1). It is worth noting that the sand at the bottom of the aquariums was very poor in these elements (more than 15 fold lower in N, organic C and P than sedimentary biofilm) and cannot be considered as a valuable food source for gastropods.

Controls of physical and chemical conditions were performed in each aquarium every week. Temperature, pH, dissolved oxygen and electric conductivity were measured with a HQ40 multiparametric probe (HACH) and nutrient concentrations (N-NO2 -, N-NO3 -, N-NH4 + and P-PO4 3-) with a sequential analyser (SmartChem 200, AMS Alliance, Frépillon, France) using colorimetric methods [START_REF] Grasshoff | Methods of Seawater Analysis[END_REF]. Comparable physical and chemical conditions were measured in the aquariums during the course of the experiment with mean values of 18.6 ± 0.1 °C, 8.4 ± 0.1, 9.4 ± 0.1 mg.L -1 , 340 ± 20 µS.cm -1 , 0.14 ± 0.08 mg.L -1 , 2.67 ± 0.70 mg.L -1 , 0.11 ± 0.07 mg.L - 1 and 0.06 ± 0.03 mg.L -1 (mean ± standard deviation) for temperature, pH, dissolved oxygen, electric conductivity, N-NO2 -, N-NO3 -, N-NH4 + and P-PO4 3-, respectively.

Sampling

For each measured variable (triglyceride concentrations and oxygen consumption rates) and time of experiment (start, 2, 4 and 6 weeks), twelve individuals were used. Gastropods were analysed individually to obtain 12 replicates per date and treatment for each variable. Then, a total of 120 individuals (12 at the start and 108 during the experiment) were used for the laboratory experiment.

Measurements performed on V. viviparus

For oxygen consumption rates, the shells of the snails were gently cleaned before the measurements to remove biofilm development, to ensure that an overshoot in oxygen uptake due to biofilm metabolism did not affect the results. Then, each individual was placed in 250 mL watertight container filled with aerated dechlorinated water (about 9 mg.L -1 of dissolved oxygen) for 4 hours under dark conditions. Dissolved oxygen measurements were made with an inoLab Oxi 7310 (WTW, Xylem Analytics) at the start and the end of the incubation to determine the consumption of oxygen during the 4 hours. Oxygen consumption was expressed as consumption of dissolved oxygen per hour and per gram of dried mass.

Before triglyceride measurements, each individual was placed in a beaker filled with dechlorinated tap water to empty its digestive tract. Then, animals were frozen, lyophilised and homogenized with a ball mill. Triglycerides were extracted from 30 mg of homogenized tissues (from the whole organism) according to the methods of [START_REF] Barclay | Changes in lipid and protein during starvation and the moulting cycle in the tiger prawn, Penaeus esculentus Haswell[END_REF] and [START_REF] Elendt | Effects of starvation on growth, reproduction, survival and biochemical composition of Daphnia magna[END_REF], and then evaluated using specific test-combinations (Boehringer-Mannheim) (triglyceride levels were measured by determining glycerol released from triglycerides hydrolysis). All assays were performed in a recording spectrophotometer (Beckman DU-6) at 25°C. Enzymes, coenzymes and substrates used for enzymatic essays were purchased from Boehringer (Mannheim, Germany) and Sigma Co. (St Louis, USA). Triglyceride concentrations were expressed as mg of glycerol per g of dried mass.

Field experiment Experimental design and sampling

Experimentations were performed at different periods to test the trophic response of gastropods to contrasted biofilm characteristics at the water-sediment interface. Respectively, nutrient concentrations were inferior to 1.1 mg.L -1 , 24.9 µg.L -1 and 13.5 µg.L -1 for N-NOx, N-NH4 + and P-PO4 3-. pH, electric conductivity and dissolved oxygen ranged between 7.3 and 8.4, 298.7 and 351.7 µS.cm - 1 , and 8.2 and 12.7 mg.L -1 , respectively. weeks), for the structural characteristics of the sedimentary interface. In addition, two intact cores were collected for the functional characteristics of the sedimentary interface. Sampling was conducted with sufficient water level in the basin, to prevent any drying and disturbance of sediment and biofilm. All samples were then stored in a cool box during transport to the laboratory within 4 h.

Biological characteristics

The phototrophic biomass of the sediment biofilm was assessed by measuring chlorophyll-a content on frozen sediment by spectrophotometry using the SCOR-UNESCO method (UNESCO, 1966). The phototrophic biomasses were expressed as µg of chlorophyll-a per gram of dried sediment.

Total organic carbon (TOC) was measured on lyophilised sediment as an estimator of the sedimentary biofilm biomass. According to [START_REF] Mermillod-Blondin | Dynamics of dissolved organic carbon (DOC) through stormwater basins designed for groundwater recharge in urban area: Assessment of retention efficiency[END_REF], TOC was determined by high-temperature combustion of in situ pre-acidified dry samples (60°C, 48h) and subsequent measurement of CO2 by thermal conductometry using an elemental analyser (FlashEA, Thermo Electron Corporation). The TOC was expressed as the percentage of organic carbon in each sample.

The Benthic Trophic State Index (BTSI) was assessed using comparison between net photosynthetic activity (NPA) and respiration (RES). This index is a metabolism-based trophic index for comparing the ecological values of shallow-water sediment habitats [START_REF] Rizzo | A metabolism-based trophic index for comparing the ecological values of shallow-water sediment habitats[END_REF] and is divided in four classes: 0 = totally heterotrophic (NPA ≤ RES), 1 = net heterotrophic (RES < NPA ≤ 0), 2 = net phototrophic (0 < NPA ≤ |RES|) and 3 = highly autotrophic (|RES| < NPA). The net photosynthesis rates were obtained by measuring the oxygen production on fresh sediment under illumination (irradiance of 150 µmol.m -2 .s -1 ) and the respiration was obtained by measuring the oxygen uptake in the dark. For each sample, oxygen production and uptake were obtained at 15°C using 150 mL airtight glass bottle filled with filtrated water (0.22µm) from the infiltration basin. Oxygen production and uptake were measured with an inoLab Oxi 7310 (WTW, Xylem Analytics) at the start and the end of the incubation.

Oxygen production and uptake were expressed as consumption of dissolved oxygen per hour and per gram of dried organic matter.

Triglyceride analyses performed on gastropods

Triglyceride concentrations were measured at the start and the end of the experiment according to the method presented for the laboratory experiment (see section 2.3.4). At the start of the experiment, triglyceride concentrations of gastropods previously maintained in laboratory were 0.0038 ± 0.0016 (mean ± standard deviation) mg of glycerol per mg of dried mass.

Statistical analyses

For the laboratory experiment, the influence of the food treatment on triglyceride concentrations and oxygen consumption were assessed using two-way analyses of variance (ANOVA II), with treatment (food source) and time as fixed factors. Then, subsequent Tukey's tests were performed to determine which treatments differed.

Pearson's correlation tests were performed between the triglyceride concentrations at the end of the experiment and the three food elements that were measured (total organic carbon, total nitrogen and phosphorus).

For the field experiment, the influence of the period on triglyceride concentrations, TOC, chlorophyll-a concentrations and the BTSI were assessed using one-way analyses of variance (ANOVA I), with period as fixed factor. To evaluate the link between food sources and triglyceride concentration in gastropods, Pearson's correlation tests were performed between triglyceride concentrations measured at the end of the experiment and the three proxies of food source availability (TOC concentrations, chlorophyll-a concentrations and the BTSI).

For all variables, the normality and the homoscedasticity of the residues were tested using the Shapiro-Wilk's test and the Levene's test, respectively. All statistical analyses were performed using R software (R Development Core Team, 2008). Significance for statistical tests was accepted for a first species risk set at α < 0.05 considering a bilateral risk.

Results

Laboratory experiment

At the start of the experiment the triglyceride concentration of gastropods was 0.0027 ± 0.0010 (mean ± standard deviation) mg of glycerol per g of dry mass. Triglyceride concentrations increased from week 2 to week 6 in all food treatments (Figure 1.A, ANOVA II, "time" effect, F2,99 = 4.57, p = 0.013). A significant difference in triglyceride concentrations was measured among food treatments (ANOVA II, "treatment" effect, F2,99 = 17.98, p < 0.0001): triglyceride concentrations in gastropods were the highest with the breeding food and the lowest with sedimentary biofilm. Triglyceride concentrations for gastropods fed on algal mat were intermediate with concentration lower than those of gastropods fed with breeding food (Tukey's test, algal mat/breeding food differences, p < 0.001). Although the statistical test was not significant at 5%, gastropods fed with algal mat tend to have higher triglyceride concentrations than those fed with sedimentary biofilm (Tukey's test, algal mat/sedimentary biofilm differences, p = 0.08). No significant interaction between "time" and "treatment" effects on triglyceride concentrations was measured (ANOVA II, "treatment*time" effect, F4,99 = 2.28, p = 0.065).

At the start of the experiment, the oxygen consumption rate of gastropods was 0.35 ± 0.09 (mean ± standard deviation) mg of dissolved oxygen per hour and per gram of dry mass. Oxygen consumption rates significantly decreased from week 2 to week 6 (ANOVA II, "time" effect, F2,86 = 5.01, p = 0.009) but this decrease seemed more pronounced in the treatment with sedimentary biofilm than in other treatments (Figure 1.B). Despite no statistically significant differences among food treatments (ANOVA II, "treatment" effect, F2,86 = 2.23, p = 0.11), the oxygen consumption rates of gastropods fed on sedimentary biofilm tended to be lower than those measured in the two other treatments after 6 weeks of experiment (Figure 1.B). No significant interaction between "time"

and "treatment" effects on oxygen consumption rates was measured (ANOVA II, "treatment*time" effect, F4,86 = 0.86, p = 0.49).

The link between energy reserves of gastropods and the elemental compositions of food sources was clearly shown by the positive correlations obtained between triglyceride concentrations in gastropods measured after 6 weeks of experimentation and the concentrations of TOC (Pearson's correlation, R = 0.69, t = 5.61, df = 34, p < 0.0001), total nitrogen (Pearson's correlation, R = 0.70, t = 5.73, df = 34, p < 0.0001) and total phosphorus (Pearson's correlation, R = 0.70, t = 5.65, df = 34, p < 0.0001) in food sources.

Field experiment

The mean Chlorophyll-a and TOC concentrations of the sedimentary interface measured from 6 enclosures (based on one value per enclosure calculated from measures performed at the start and the end of the experiment) were significantly different among periods (Figure 2.A&B ANOVA I, "period" effect, F3,116 = 82.85 for the chlorophylla concentration and F3,116 = 30.10 for TOC concentration, p < 0.0001 for the two variables) with highest values measured in June 2017, which was the most favourable period for the development of phototrophic organisms.

Mean BTSI values were comprised between 2 and 3 (i.e. net phototrophic to highly phototrophic states) for all seasons at the exception of October 2016 during which BTSI values were lower (between 1 and 2) indicating variations between net heterotrophic to net phototrophic states (Figure 2.C, ANOVA I, "period" effect, F3,116 = 34.33, p < 0.0001).

Significant differences were observed among the periods for triglyceride concentrations in gastropods (Figure 3, ANOVA I, "period" effect, F3,116 = 17.83, p < 0.0001). Surprisingly, triglyceride concentrations were the lowest in October 2016 and June 2017, which were the most contrasted periods for chlorophyll-a concentrations at the sedimentary interface (Figure 2.A). Indeed, no positive correlation was observed between triglyceride concentrations in gastropods after 6 weeks of experimentation and chlorophyll-a concentrations at the interface (Figure 4.A; Pearson's correlation, R = -0.2, t = -2.22, df = 118, p = 0.03). We also did not detect a positive correlation between triglycerides concentrations in gastropods after 6 weeks of experimentation and TOC concentrations at the interface (Figure 4 B, Pearson's correlation, R = -0.11, t = -1.19, df = 118, p > 0.05). In contrast, the metabolic-based trophic status (BTSI) was positively correlated to triglyceride concentrations in gastropods after 6 weeks of experimentation (Figure 4 C, Pearson's correlation, R = 0.33, t = 3.87, df = 118, p < 0.001), suggesting a positive influence of the phototrophic primary production on the ability of gastropods to constitute energy reserves. From the comparison between field and laboratory experiments, it is also worth noting that triglyceride concentrations measured on gastropods enclosed in the field were around 2-fold higher than concentrations measured for gastropods feeding on algal mat and sedimentary biofilm in the laboratory experiment (see Figure 1.A).

Discussion

Our results provided evidences that V. viviparus could not be considered as a strict generalist in our experiments.

Indeed, V. viviparus tended to more efficiently constitute energy reserves on algal mat than on sedimentary biofilm in laboratory. In parallel, oxygen consumption rates of gastropods tended to be lower after feeding on sedimentary biofilms than on algal mats, indicating an effect of food type on the metabolism of V. viviparus. These results suggest a lower food consumption rate for V. viviparus feeding on sedimentary biofilm than on algal mat.

Moreover, the fact that triglyceride concentrations in gastropods measured in the field were positively correlated with BTSI suggested a positive influence of phototrophic primary production on the constitution of energy reserves in V. viviparus. Thus, both laboratory and field experiments showed that phototrophic food sources (algal mat), and most particularly active algal mat, were energetically more favourable for gastropods than heterotrophic food sources (sedimentary biofilm).

The laboratory experiment specifically highlighted that the efficiency of gastropods to constitute energy stores among food sources was positively correlated to food nutrient (C, N, P) concentrations. This relationship was most likely due to the equilibrium between the nutrient supplied by the food sources and the energy demand needed to acquire these nutrients: gastropods feeding on sedimentary biofilm had to ingest more food (in terms of mass) to acquire the same amount of nutrients compared to those feeding on algal mat. Previous studies reported an increase in the rate of food ingestion by aquatic grazers as a behavioral response to acquire energy from low-nutrient food sources (Fink and Von Elert, 2006;Flores et al., 2014). For example, [START_REF] Calow | The feeding strategies of two freshwater gastropods, Ancylus fluviatilis Müll. and Planorbis contortus Linn. (Pulmonata), in terms of ingestion rates and absorption efficiencies[END_REF] reported an inverse relationship between the ingestion rate of freshwater benthic organisms and food quality: animals compensated for less nutritive food by increasing their feeding rates. [START_REF] Cruz-Rivera | Macroalgal traits and the feeding and fitness of an herbivorous amphipod: the roles of selectivity, mixing, and compensation[END_REF] also observed that the marine amphipod Ampithoe longimana increased its food consumption as the total organic matter content in algae declined.

However, in the present laboratory experiment, the decrease in oxygen consumption measured on V. viviparus fed with sedimentary biofilms more likely suggests a physiological response rather than a behavioral feeding response of gastropods to a low-nutritive food source. More precisely, gasteropods tended to reduce their metabolic rates (i.e. to enter in a prolonged state of torpor) rather than increase their food acquisition to maintain their energy budget. The present study is the first to report this metabolic response of V. viviparus to low-nutrient food sources, although such response has already been reported for other aquatic invertebrates such as gammarids which can adjust their respiration rates to food quality [START_REF] Graça | Importance of fungi in the diet of Gammarus pulex and Asellus aquaticus I: feeding strategies[END_REF]. Moreover, our interpretation is consistent with the laboratory observations of [START_REF] Cook | A ciliary feeding mechanism in Viviparus viviparus (L.)[END_REF] who reported that V. viviparus can be inactive and partially buried within sediments during several days.

Field measurements showed that the triglyceride concentrations of gastropods were positively correlated with BTSI whereas it was not the case with chlorophyll-a concentrations. This result indicates that the photosynthetic activity of the algal biofilm (associated with BTSI) had more impact on the production of energy reserves in gastropods than the algal biomass of the biofilm. This interpretation is consistent with the fact that the photosynthetic activity of the biofilm positively influences the production of extracellular polymeric substances (EPS) which can be high nutritional compounds for invertebrates (e.g., [START_REF] Decho | Microbial exopolymer secretions in ocean environments : their role ( s ) in food webs and marine processes[END_REF][START_REF] Decho | Bacterial exopolymer utilization by a harpacticoid copepod: A methodology and results[END_REF].

Indeed, photosynthetic activities and algal growth have been particularly related to the secretion of EPS [START_REF] Staats | Oxygenic photosynthesis as driving process in exopolysaccharide production of benthic diatoms[END_REF][START_REF] Barranguet | Divergent composition of algal-bacterial biofilms developing under various external factors[END_REF]. In active biofilms, microbial cells secrete EPS which mainly consists of carbohydates and proteins [START_REF] Stewart | Characterization of extracellular polymeric substances (EPS) from periphyton using liquid chromatography-organic carbon detection-organic nitrogen detection (LC-OCD-OND)[END_REF][START_REF] Costa | Microbial extracellular polymeric substances: ecological function and impact on soil aggregation[END_REF]. For example, [START_REF] Fang | Characteristics of extracellular polymeric substances of phototrophic biofilms at different aquatic habitats[END_REF] measured a total content in EPS close to 13 mg/g of dried weight containing 67% of carbohydrates and 25% of protein (w:w) in a lake phototrophic biofilm. Depending on environmental conditions (light, nutrient concentrations), [START_REF] Bellinger | Composition of extracellular polymeric substances from periphyton assemblages in the florida everglades[END_REF] also reported that carbohydrate concentrations in EPS varied between 20 and 60% (w:w) in phototrophic biofilms from freshwater wetlands. Therefore, under the varying environmental conditions observed in our field study, BTSI was likely associated with the production of EPS which contributed to the nutritional quality of biofilms for algal grazers. As a consequence, we measured a positive correlation between the energy reserves of V. viviparus and the BTSI index.

The comparison of triglyceride concentrations in gastropods between field and laboratory experiments showed that stores in animals collected in the field were comparable to those measured in animals feeding on breeding food in the laboratory. In comparison, gastropods feeding on algal mat and sedimentary biofilm during the laboratory experiment exhibited 2-fold lower triglyceride concentrations. We suggest that these differences in gastropod triglyceride concentrations between laboratory (for algal mat and sedimentary biofilm) and field experiments were due to the possibility for animals to mix trophic resources in the field. V. viviparus is known to "bulldoze" the sediment surface by their crawling and feeding activities [START_REF] Estragnat | Does the efficiency of grazer introduction to restore and preserve the hydraulic performance of infiltration basins depend on the physical and biological characteristics of the infiltration media?[END_REF], disturbing the vertical organisation of phototrophic and heterotrophic sediment layers [START_REF] Cook | A ciliary feeding mechanism in Viviparus viviparus (L.)[END_REF]. Therefore, the non-selective ingestion of sediment by gastropods was associated with the ingestion of a mixture of algae, heterotrophic bacteria, fungi and detritus [START_REF] Jakubik | Diversity of mollusca in lowland river-lake system: lentic versus lotic patches[END_REF]. As shown by [START_REF] Groendahl | The effect of diet mixing on a nonselective herbivore[END_REF] in Limnea stagnalis, this mixing diet could have been crucial for V. viviparus to achieve a more balanced diet by acquiring all essential microelements (fatty acids or vitamins) they needed for growth. Then, simplified food sources (algal mat or sedimentary biofilm) supplied to gastropods in the laboratory might have limited the acquisition of resources by V. viviparus, and therefore the ability to synthetize energy stores such as triglycerides. Such conclusion was consistent with the observations of [START_REF] Jakubik | Food and feeding of Viviparus viviparus (L.)(Gastropoda) in dam reservoir and river habitats[END_REF] indicating that V. viviparus feed on varying food sources depending on their availability in the environment.

Although our results highlighted that V. viviparus was not a strict generalist in the laboratory, the potential use of multiple food sources and their availabilities during the year in the field allowed V. viviparus to maintain triglyceride reserves that were comparable to those obtained in the laboratory with breeding food (positive control).

Indeed, the comparison of food sources among periods in the field showed that all foods were quite available in infiltration basin with chlorophyll-a concentrations always higher than 4 µg per g of dry sediment and TOC concentrations varying between 0.5 and 0.7%. In these conditions, V. viviparus was able to survive and constitute energy reserves throughout the year, even when the trophic conditions were expected to be the harshest (i.e. low algal levels). In conclusion, the introduction of V. viviparus appears as sustainable ecological engineering solution to maintain hydraulic performance of infiltration basins.

Supplementary material

For

  each period, six in situ enclosures (see the description in[START_REF] Gette-Bouvarot | Ecological engineering approaches to improve hydraulic properties of infiltration basins designed for groundwater recharge[END_REF][START_REF] Estragnat | Does the efficiency of grazer introduction to restore and preserve the hydraulic performance of infiltration basins depend on the physical and biological characteristics of the infiltration media?[END_REF] with 5 V. viviparus were used in the infiltration basin. Briefly, enclosures consisted of stainless steel cylinders (internal diameter of 30 cm and height of 14 cm) firmly buried in the sediment at a depth of 11 cm and covered with nylon nets (0.5 cm mesh size) to avoid the escape of the gastropods. For data independence, different enclosures with different positions were used for each of the four periods. Ten individuals were used to assess the triglyceride concentrations at the beginning of each period. A total of 160 individuals were used for the field experiment. A density of 71 individuals.m -2 was introduced per enclosure to mimic natural densities observed in freshwater habitats. Measurements were performed at the start and at the end (6 weeks) of the experiment to determine biological structural characteristics (algal biomass and total organic carbon) and functional characteristics (net photosynthetic activity, respiration) of the sedimentary interface for each enclosure. The top layer (0-1 cm) of sediments was randomly sampled in each enclosure at the start of the experiment and after 6 weeks of experimentation by coring using cut syringes (internal diameter = 15 mm for structural characteristics and internal diameter = 20 mm for functional characteristics). Fifteen samples were collected, mixed and homogenized per enclosure to obtain representative one sediment sample for each enclosure and date (start and 6

FiguresFig. 1 Fig. 2 Fig. 3 Fig. 4 Figure 3 Figure 4

 123434 Figures

  hourly radiations which were favourable to the development of phototrophic organisms(June 2017 and April 2018), compared to the other periods(October 2016 and February 2017). Physical and chemical variables were measured to characterize each period. The photoperiod and the global hourly radiation have been obtained from Météo-France. Temperature and the water level were measured every 15 minutes with a water level logger Diver (Schlumberger Water Sciences, The Netherlands). Nutrient concentrations were weekly measured and obtained from filtered water (0.7 µm) conserved at 4°C before analysis within 24 h with sequential analyser (SmartChem 200, AMS Alliance, Frépillon, France) using colorimetric methods[START_REF] Grasshoff | Methods of Seawater Analysis[END_REF].Conductivity, dissolved oxygen and pH were also weekly measured with a Ponsel Odeon data logger and the associated probes (Aqualabo Contrôle, France). Photoperiods were10.6, 11.4, 15.8, and 14.4h for October 2016, February 2017, June 2017 and April 2018, respectively. Global hourly radiations were 33.0, 46.9, 96.0, and 77.5 joules.cm -² for October 2016, February 2017, June 2017 and April 2018, respectively. Temperatures were16, 11, 20, and 16°C for October 2016, February 2017, June 2017 and April 2018, respectively. The mean water levels varied among the periods between 90 and 120 cm.

The experiment consisted of four periods of 6 weeks in October 2016, February 2017, June 2017 and April 2018. Two periods were characterized by elevated photoperiods and global

Table Table 1

 Table Elemental composition (total nitrogen, total organic carbon and total phosphorus contents) of the three food sources and the sand used in the aquariums (mean ± standard deviation)

		%N	% organic C	%P
	Breeding food 6.5 ± 0.1	45.1 ± 0.85	0.74 ± 0.03
	Algal mat	1.5 ± 0.15	19.1 ± 0.19	0.3 ± 0.05
	Sedimentary
		0.25 ± 0.025	2.13 ± 0.24	0.037 ± 0.005
	biofilm
				0.0003	±
	Sand	0.013 ± 0.005	0.036 ± 0.004
				0.000015

Acknowledgements

We express our gratitude to Pauline Barbe and Mélissa Tenaille for their help during the collection of organisms and the experimentation, and to Laurent Simon and Félix Vallier who were involved in chemical analyses. Our thanks also go to Météo-France and Eau du Grand Lyon for the data they provided. This research was done on the Research Platform of Crépieux-Charmy (Plateforme de Recherche de Crépieux-Charmy) and received financial and technical support from the Lyon Metropole and Veolia Water (Eau du Grand Lyon).

Funding

This study was funded by the Lyon Metropole and Veolia Water (Eau du Grand Lyon).

Online Resource 1 Box-plots of the triglycerides concentrations in gastropods depending on the sex (n=107). Red crosses represent the means. No significant differences were observed (t-test, "sex" effect, t= -0.51, p-value=0.61)

Author's contributions

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Jean-Bastien Gambonnet, Laurence Volatier, Florian Mermillod-Blondin, Frédéric Hervant and Valerian Estragnat. The first draft of the manuscript was written by Valerian Estragnat, Florian Mermillod-Blondin and Laurence Volatier. All authors contributed critically to the drafts and gave final approval for publication.

Ethical approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.

Conflict of Interest

The authors declare that they have no conflict of interest.