Charlotte Vichery

Mélanie Poggi

Pierre Bonville

Thierry Gacoin

Isabelle Maurin

Post-synthesis annealing of coprecipitated CoFe2O4 nanoparticles in silica matrix

Keywords: magnetic nanoparticles, cobalt ferrite, annealing, magnetic anisotropy, canting, silica host matrix

Introduction

Spinel oxides are widely investigated in materials science because of their broad range of applications as magnetic materials, semiconductors, pigments, catalysts for water oxidation or in artificial photosynthesis cells. [START_REF] Nlebedimn | Effect of heat treatment on the magnetic and magnetoelastic properties of cobalt ferrite[END_REF][START_REF] Indra | Unification of catalytic water oxidation and oxygen reduction reactions: amorphous beat crystalline cobalt iron oxides[END_REF] Their design as nanoparticles has also opened perspectives in the field of medicine and biotechnologies. [START_REF] Tartaj | The preparation of magnetic nanoparticles for applications in biomedicine[END_REF][START_REF] Sharifi | Ferrite-based magnetic nanofluids used in hyperthermia applications[END_REF][START_REF] Kudr | Magnetic nanoparticles: from design and synthesis to real world applications[END_REF] Particles made of the ferrimagnetic binary oxides Fe 3 O 4 and -Fe 2 O 3 have been extensively studied for the development of novel or combined imaging/therapy approaches. One of the main issues related to size reduction is that the magnetic properties of the particles significantly deviate from those of their bulk counterparts. [START_REF] Batlle | Finite-size effects in fine particles: magnetic and transport properties[END_REF] Many studies have emphasized the role of a surface shell made of weakly coupled and misaligned spins, which leads to a substantial decrease of saturation magnetization. [START_REF] Millan | Surface effects in maghemite nanoparticles[END_REF] Canted or misaligned spins may also be found in the core of the particles because of the presence of structural defects, either point defects (impurities or vacancies) [START_REF] Mørup | Spin-canting and transverse relaxation at surfaces and in the interior of ferrimagnetic particles[END_REF][START_REF] Morales | Spin canting due to structural disorder in maghemite[END_REF] or extended defects (twins, antiphase or grain boundaries) that may be related to a poor crystallinity due to the low temperatures of elaboration or to the nucleation and growth processes. [START_REF] Luigjes | Diverging geometric and magnetic size distributions of iron oxide nanocrystals[END_REF][START_REF] Levy | Correlating magneto-structural properties to hyperthermia performance of highly monodisperse iron oxide nanoparticles prepared by a seeded-growth route[END_REF] For the specific case of ternary MFe 2 O 4 nanoparticles (with M a divalent metal ion), the inversion degree, which characterizes the cationic distribution over the interstitial sites of the AB 2 O 4 spinel structure, is also of importance. A and B here stand for tetrahedral and octahedral cation sites in a cubic close packing of oxygen atoms. The inversion degree (x) is defined as the fraction of tetrahedral A sites occupied by trivalent ions:

(M 1-x Fe x ) A [M x Fe 2-x ] B O 4 . If Fe 3+ ions occupy only octahedral sites, the ferrite is called normal (x = 0), whereas if Fe 3+ ions occupy equally tetrahedral and octahedral sites, the ferrite is named inverse (x = 1). In nanoparticles, the inversion degree can significantly deviate from that of the bulk materials. [START_REF] Gyergyek | Influence of synthesis method on structural and magnetic properties of cobalt ferrite nanoparticles[END_REF] It may also vary depending on the synthesis protocol [START_REF] Da Silva | Magnetic studies of CoFe 2 O 4 /SiO 2 aerogel and xerogel nanocomposites[END_REF][START_REF] Chinnasamy | Synthesis of size-controlled cobalt ferrite particles with high coercivity and squareness ratio[END_REF][START_REF] Peddis | Spin-glass-like freezing and enhanced magnetization in ultra-small CoFe 2 O 4 nanoparticles[END_REF] and the thermal history of the sample. [START_REF] Sawatzky | Mössbauer Study of Several Ferrimagnetic Spinels[END_REF][START_REF] Guire | The cooling rate dependance of cation distributions in CoFe 2 O 4[END_REF][START_REF] Wang | Effects of heat treatment and magnetoannealing on nanocrystalline Co-ferrite powders[END_REF] In the case of cobalt ferrite (CoFe 2 O 4 ) studied for high frequency or stress sensing applications, [START_REF] Suzuki | Structure and magnetic properties of epitaxial spinel ferrite thin films[END_REF][START_REF] Wang | High-coercivity Co-ferrite thin films on (100)-SiO 2 substrate[END_REF][START_REF] Nlebedim | Structural, magnetic, and magnetoelastic properties of magnesium substituted cobalt ferrite[END_REF] the inversion degree will both affect magnetization values, as the A and B sublattices are antiferromagnetically coupled, and the anisotropy energy. [START_REF] Zhang | Anisotropy and Faraday effect in Co spinel ferrite films[END_REF] In addition, the strength of the magnetic exchange interaction is also expected to vary [START_REF] Sawatzky | Mössbauer Study of Several Ferrimagnetic Spinels[END_REF] and, as a consequence, the canting angles of the magnetic moments for ions located in A and B sites. [START_REF] Peddis | Cationic distribution and spin canting in CoFe 2 O 4 nanoparticles[END_REF] The primary goal of this study is to assess the influence of annealing treatments on the magnetic properties of 11 nm CoFe 2 O 4 nanoparticles prepared by coprecipitation at room temperature. Such an investigation is of importance to establish a clear comparison with synthesis routes involving high temperatures such as autocombustion of metal salts or decomposition of organometallic precursors. In that latter case, formation of nanoparticles by thermolysis of the Co and Fe precursors in high-boiling point solvents typically occurs at about 200-300°C. As the saturation magnetization (M s ) and magnetic anisotropy constant (K) show a marked dependence on particle size, [START_REF] Cullity | Introduction to magnetic materials[END_REF] we used a so-called protected annealing process [START_REF] Maurin | A protected annealing process for the production of high quality colloidal oxide nanoparticles with optimized physical properties[END_REF][START_REF] Vichery | Influence of protected annealing on the magnetic properties of γ-Fe 2 O 3 nanoparticles[END_REF] to avoid any coarsening of the particles during heating and investigate the impact of heat treatments on M s , K and canting angle values at fixed particle size.

Materials and methods

An aqueous dispersion of cobalt ferrite nanoparticles was prepared by coprecipitation of

Co 2+ and Fe 3+ salts in alkaline medium. Fe(NO 3 ) 3 .9H 2 O and Co(NO 3 ) 2 .6H 2 O powders were first dissolved in 70 mL of HCl at 0.33 mol.L -1 , with a 2 to 1 (Fe/Co) atomic ratio and a cobalt salt concentration of 0.14 mol.L -1 . Potassium hydroxide (32 mL, 10 mol.L -1 ) was quickly poured into this acidic solution under vigorous stirring. The slurry was agitated during 30 min and then heated up to reflux for 2 h. The resulting cobalt ferrite particles were washed with distilled water and finally dispersed by peptization in 30 mL of nitric acid at pH 2. The solution was sonicated during 2 min, centrifuged at 11,400 g for 20 min, and the supernatant recovered in order to lower the size polydispersity. CoFe 2 O 4 particles were then embedded in a sol-gel SiO 2 matrix. 11 mL of a solution of tetraethoxysilane (TEOS) in ethanol (2.25 mol.L -1 ) were first hydrolyzed by adding 2.25 mL of HCl solution at pH 1.15.

The resulting mixture was heated for 1 h at 60°C. 0.5 mL (respectively 3.8 mL) of the CoFe 2 O 4 colloid were added to the sol leading, after a drying step at 100°C, to a composite materials with a Co/Si atomic ratio of 0.004 (respectively 0.03). These atomic ratios were derived from Inductively Coupled Plasma Atomic Emission Spectroscopy. The CoFe 2 O 4 /SiO 2 powders were subsequently heated in air at different temperatures (400-1000°C) for 3 hours. All heating and cooling stages were carried out with a sweep rate of 4°C.min -1 .

Powder x-ray diffraction patterns were recorded using a PANalytical X'Pert diffractometer equipped with a Cu Kα anode (λ = 1.5418 Å) and a rear-side graphite monochromator. Peak positions were extracted using the Fit subroutine of the Fullprof suite of programs. [START_REF] Rodriguez-Carvajal | Recent advances in magnetic structure determination by neutron powder diffraction[END_REF] The lattice constant was determined by least-square refinement of the interspacing distances, after correction for instrumental shifts using a silicon internal calibrant. The structural coherence length was evaluated through Pseudo-Voigt peak profile analysis, [START_REF] Langford | Accuracy in Powder Diffraction II[END_REF] after correction for the instrumental resolution function. The crystallinity, morphology and size distribution of the particles were also investigated by transmission electron microscopy (TEM) using a Philips CM30 microscope operating at 300 kV. Magnetization measurements were performed with a SQUID magnetometer (Cryogenic Ltd SX600, H max = 5 T) for magnetization measurements between 5 and 300 K, a SQUID magnetometer (Quantum Design MPMS-5) equipped with an oven for measurements between 300 and 800 K and a Vibrating Sample Magnetometer equipped with a 14 T magnet (Cryogenic Ltd) for measurements at high magnetic fields. Samples were mounted in polycarbonate capsules with the powder immobilized by paraffin wax. For high-temperature measurements, the powder samples were wrapped into a piece of aluminum foil. Magnetic data were systematically corrected for the diamagnetic contributions of the silica matrix, wax and polycarbonate capsule or Al foil. Due to dehydration of the silica matrix and of the ferrite particles during annealing, the fraction of magnetic material in each sample was derived from the iron content measured by ICP-AES. Note that the Fe/Co atomic ratio is close to 2 for the three samples (2.02(4) for the crude particles; 2.01(5) and 1.95 (5) for the low and high particle loading in the composite samples, respectively). A Mössbauer spectrometer, operating in constant acceleration mode with a 57 Co:Rh γ-ray source and a 7 T magnetic field, was used to record absorption spectra in transmission geometry.

Results and discussion

A representative image of the size distribution of the CoFe 2 O 4 colloid determined by TEM is displayed in Figure 1a. The particles are rather polydispersed in size, with a diameter in the range 3 -22 nm. Fit of the size histogram to a log-normal function yields a mean diameter value (d m ) of 11.5 nm and a standard deviation (σ d ) of 0.5 (Figure 1b). As reported in a previous work related to the annealing of maghemite particles in silica matrix, [START_REF] Vichery | Influence of protected annealing on the magnetic properties of γ-Fe 2 O 3 nanoparticles[END_REF] no flocculation nor aggregation were observed after addition of the magnetic colloid into the silica sol, leading to homogeneous CoFe 2 O 4 /SiO 2 composite powders after gelation and drying (see Figure 1c). 2). These Bragg reflections correspond to the two most intense peaks of fayalite (α-Fe 2 SiO 4 , JCPDS 01-070-1861). The presence of this impurity phase is already observed after annealing at 800°C, but its amount is much reduced. This parasitic reaction was not reported in the case of -Fe 2 O 3 nanoparticles produced by a similar coprecipitation route and dispersed in silica. [START_REF] Vichery | Influence of protected annealing on the magnetic properties of γ-Fe 2 O 3 nanoparticles[END_REF] Therefore, the α-(Fe 1-x Co x ) 2 SiO 4 impurity phase could result from a higher surface reactivity of the CoFe 2 O 4 particles. An alternative explanation would be the presence of free ions due to partial dissolution and release of Co 2+ and Fe 2+ in acidic conditions, [START_REF] Soler | Aging Investigation of Cobalt Ferrite Nanoparticles in Low pH Magnetic Fluid[END_REF][START_REF] Sileo | Correlation of reactivity with structural factors in a series of Fe(II) substituted cobalt ferrites[END_REF] such as those used for TEOS hydrolysis. The lattice parameter (a) and coherence length (L c ) values are summarized in Table 1 for crude particles and for particles dispersed in silica. The coherence length of the assynthesized particles is about 9 nm, i.e. close to the mean particle size derived from the TEM observations. This value is relatively constant upon thermal treatment for particles embedded in silica, whereas L c is doubled between 100 and 800°C for the crude particles. This result confirms the homogeneous distribution of particles within the SiO 2 binder and the efficiency of this matrix to prevent grain growth through sintering of the initial particles. Similar results were reported for -Fe 2 O 3 nanoparticles. [START_REF] Vichery | Influence of protected annealing on the magnetic properties of γ-Fe 2 O 3 nanoparticles[END_REF][START_REF] Chanéac | Magnetic iron oxide-silica nanocomposites. Synthesis and characterization[END_REF] For the samples dried at 100°C, the a-parameter values are significantly smaller than the ones expected from the Poix's model: a = 8.410 Å for normal (x = 0) and a = 8.385 Å for inverse (x = 1) CoFe 2 O 4 spinel oxide. 32 Such a reduced a-parameter is commonly reported for cobalt ferrite nanoparticles, [START_REF] Peddis | Cationic distribution and spin canting in CoFe 2 O 4 nanoparticles[END_REF][START_REF] Soler | Aging Investigation of Cobalt Ferrite Nanoparticles in Low pH Magnetic Fluid[END_REF][START_REF] Nordhei | Nanophase cobalt, nickel and zinc ferrites: synchrotron XAS study on the crystallite size dependence of metal distribution[END_REF] and should be assigned to the low temperature of elaboration rather than to an impact of size or surface energy terms. Indeed, the lattice constant of both crude and embedded particles shows a similar evolution upon heating (see Table 1), while there is grain growth for the former (surface/volume ratio divided by 2 from the increase in the L c value) and almost no size change for the latter. The a-value of 8.37 Å may result from the presence of strain in the crystal structure but the nature of the crystalline defects is probably important to determine the sense of variation of the a-parameter, as strain generated by ball milling was found to increase the lattice constant in the work of Liu and Ding. [START_REF] Liu | Strain-induced high coercivity in CoFe 2 O 4 powders[END_REF] Here, only heat treatments above 800°C allow recovering the lattice constant of bulk . [START_REF] Soler | Aging Investigation of Cobalt Ferrite Nanoparticles in Low pH Magnetic Fluid[END_REF] To some extent, this lattice expansion may be related to an improved crystallinity.

Indeed, the increase of the coherence length observed for both crude and embedded particles definitely proves that atomic diffusion in the volume and at the surface is effective above 800°C. This higher crystallinity is also confirmed by the high-resolution TEM images shown in Figures 3a and3b, as the particles annealed at 800°C appear clearly more facetted than the pristine ones. Note however that post-annealing is not equivalent to direct synthesis in To get quantitative information on the inversion degree, Mössbauer absorption spectra were recorded at 4.2 K with a 7 T magnetic field applied parallel to the γ-ray propagation direction. Measurements were performed on crude particles heat treated at 100 and 800°C

(Figure 4). Note that the use of crude particles permits not only to obtain better resolved spectra but also to avoid the contribution of the fayalite impurity phase. The as-obtained spectra were fitted using two sextets, corresponding to the contribution of iron in tetrahedral (A) and in octahedral (B) sites. The relative weight of the two subspectra (P A /P B ) allows for the calculation of the inversion degree x, which refers to the fraction of Co 2+ ions in octahedral environment. The isomer shift value, with respect to -Fe, is equal to 0.26 mm/s for iron in A sites and 0.38 mm/s for iron in B sites, in agreement with a trivalent state.

Values of the P A /P B ratio, inversion degree (x) and hyperfine fields (H hf ) for Fe in A and B sites are listed in Table 2. For the as-synthesized particles, the inversion degree is close to 0.67, i.e. representative of a random distribution of Co and Fe in the cationic sites of the spinel structure (Table 2). This value is consistent with what was previously found for low-temperature synthesis routes, [START_REF] Gyergyek | Influence of synthesis method on structural and magnetic properties of cobalt ferrite nanoparticles[END_REF][START_REF] Ferreira | Structural and morphological characterization of FeCo 2 O 4 and CoFe 2 O 4 spinels prepared by a coprecipitation method[END_REF][START_REF] Carta | A Structural and Magnetic Investigation of the Inversion Degree in Ferrite Nanocrystals MFe 2 O 4 (M = Mn, Co, Ni)[END_REF][START_REF] Concas | Inversion degree and saturation magnetization of different nanocrystalline cobalt ferrites[END_REF] for which we anticipate kinetically-controlled cation incorporation.

Displacement of Co 2+ ions from A to B sites is expected upon heating, because of their higher stabilization in octahedral coordination. However, the inversion degree is found to hardly increase upon heating up to 800°C, in fair agreement with earlier works. [START_REF] Carta | A Structural and Magnetic Investigation of the Inversion Degree in Ferrite Nanocrystals MFe 2 O 4 (M = Mn, Co, Ni)[END_REF][START_REF] Carta | X-ray Absorption Investigation of the Formation of Cobalt Ferrite Nanoparticles in an Aerogel Silica Matrix[END_REF] Additional measurements performed with the 7 T magnetic field applied perpendicular to the γ-ray propagation direction confirmed the absence of large changes in inversion, with x values of 0.61, 0.60, 0.64 and 0.66 (± 0.04) for samples heat treated at 100, 400, 600 and 800°C, respectively. Note that all samples were cooled down to room temperature at 4°C/min after heating. Hence, the smooth change of x values with the annealing temperature would account for some quenching of a high-temperature disordered state as reported in previous works, [START_REF] Sawatzky | Mössbauer Study of Several Ferrimagnetic Spinels[END_REF][START_REF] Guire | The cooling rate dependance of cation distributions in CoFe 2 O 4[END_REF][START_REF] Wang | Effects of heat treatment and magnetoannealing on nanocrystalline Co-ferrite powders[END_REF] but rather reflects the absence of large cationic rearrangements due to the limited temperature range. Entropy-favored randomization would actually be expected by heating above 900-1000°C. [START_REF] Guire | The cooling rate dependance of cation distributions in CoFe 2 O 4[END_REF] In the next section, we discuss the influence of post-synthesis annealing on the magnetic properties of these coprecipitated CoFe 2 O 4 nanoparticles. Samples were first characterized by hysteresis cycles measured in the blocked state, with magnetization (M) expressed per gram of CoFe 2 O 4 . M(H) loops were recorded at 10 K for the dilute composite samples (Co/Si = 0.004), so that the influence of interparticle interactions can be neglected as a first approximation. [START_REF] Maurin | A protected annealing process for the production of high quality colloidal oxide nanoparticles with optimized physical properties[END_REF] Note that samples heat treated up to 600°C will be mostly considered in the following to ensure that the observed evolutions are not related to the presence of fayalite, which is antiferromagnetic at 10 K. [START_REF] Belley | The magnetic properties of natural and synthetic (Fe x Mg 1-x ) 2 SiO 4 olivines[END_REF] We observed that the coercive field value (μ 0 H c ) changes significantly upon annealing (Figure 5). μ 0 H c equals 1.0, 1.2 and 1.6 T for the particles heat treated at 100, 400 and 600°C, respectively (Table 3). A similar evolution is reported for the crude particles. A modified Stoner-Wohlfarth model predicts that the coercive field value of an assembly of non-interacting and randomly oriented single-domain particles with cubic anisotropy can be expressed as: [START_REF] Cullity | Introduction to magnetic materials[END_REF][START_REF] Garcia-Otero | Influence of temperature on the coercive field of non-interacting fine magnetic particles[END_REF]  

                4 3 0 25 4 1 96 . 0 ) ( KV T k M K T H B s c  Eq. 1
where k B is the Boltzmann constant. As a result, for particles of fixed size, μ 0 H c is inversely proportional to the saturation magnetization (M s ), and increases when the anisotropy constant (K) increases. A modification of M s and/or K should thus be responsible for the large changes in H c reported in the present study, as the protected annealing process did not lead to a substantial increase of the particle size. To assess the evolution of M s at 10 K, additional magnetization measurements were carried out using a VSM magnetometer equipped with a 14 T magnet. The corresponding curves are plotted in the inset of Figure 5. The saturation field corresponds to about 14 T, irrespective of the annealing temperature. The saturation magnetization values of the different samples, hereafter taken as the magnetization values at 14 T, are reported in Table 3. We observed that M s (10K) continuously decreases when increasing the annealing temperature.

Measurements performed under 11 T between 5 and 275 K show that this decreased magnetization is actually effective over the whole temperature range (see the M s (265 K) values in Table 3 for instance). This evolution may thus explain part of the changes in coercivity.

From Eq. 1, one can derive the value of the magnetic anisotropy constant at 10 K using the experimental M s and μ 0 H c values and the mean volume of the particles. The corresponding K values are 0.9, 1.0 and 1.2 (± 0.1) × 10 7 erg/cm 3 for samples heat-treated 100, 400 and 600°C, respectively. We should thus also expect an increase in magnetic anisotropy upon annealing. This finding was experimentally confirmed by Zero Field Cooled (ZFC) magnetization measurements performed on a magnetometer equipped with a furnace because of the high anisotropy of cobalt ferrite (K ~ 2×10 7 erg/cm 3 at zero Kelvin for bulk CoFe 2 O 4 ), [START_REF] Shenker | Magnetic Anisotropy of Cobalt Ferrite (Co 1.01 Fe 2.00 O 3.62 ) and Nickel Cobalt Ferrite[END_REF] which leads to blocking temperatures higher than room temperature. Samples were first heated up to 575 or 675 K (depending on their annealing temperature), and then cooled down to room temperature under zero magnetic field. Magnetization was then measured on heating, under a magnetic field of 25 G. The composite sample dried at 100°C

has not been measured this way, as heating above 375-400 K (i.e., in the superparamagnetic state) could have induce some modification of the magnetic properties. Figure 6 shows that the maximum of the ZFC magnetization curves indeed increases upon annealing, indicating an increase of the blocking temperature (T B ) and of the magnetic anisotropy constant (K.V = 100k B .T B for cubic anisotropy). This enhanced anisotropy is probably mostly representative of an increased inversion degree (Table 2). Indeed, the anisotropy of Co 2+ ions is much larger in octahedral than in tetrahedral environment, [START_REF] Zhang | Anisotropy and Faraday effect in Co spinel ferrite films[END_REF] so that small changes in inversion can lead to substantial modifications of the effective anisotropy constant. In addition, as the particle size exceeds 10 nm, the impact of the surface on the magnetic properties is fairly weak, [START_REF] Artus | Size-dependent magnetic properties of CoFe 2 O 4 nanoparticles prepared in polyol[END_REF][START_REF] Tronc | Mössbauer investigation of the - -Fe 2 O 3 transformation in small particles[END_REF][START_REF] Tronc | Surface-related properties of -Fe 2 O 3 nanoparticles[END_REF] and modification of the surface anisotropy (K s ) due to deshydroxylation, as reported for 7 nm -Fe 2 O 3 nanoparticles, [START_REF] Vichery | Influence of protected annealing on the magnetic properties of γ-Fe 2 O 3 nanoparticles[END_REF] should have a minor contribution to the observed changes.

An increased inversion degree is also expected to decrease the saturation magnetization value because of the smaller magnetic moment of Co 2+ (3 μ B ) compared to Fe 3+ (5 μ B ).

However, the small changes in inversion degree, from 0.64 (before) to 0.66 (after annealing at 800°C), cannot explain a decrease by ~ 8 to 15% of the M s values at 265 K or 10 K, respectively (Table 3). Another important insight may be gained by considering the evolution of the canting angles upon annealing. Indeed, misaligned spins are expected in the volume of the particles, [START_REF] Cannas | Magnetic properties of cobalt ferrite-silica nanocomposites prepared by a sol-gel autocombustion technique[END_REF] because the presence of both Co and Fe in the two cationic sites give rise to multiple exchange interaction values and spin frustration. [START_REF] Sawatzky | Mössbauer Study of Several Ferrimagnetic Spinels[END_REF][START_REF] Peddis | Cationic distribution and spin canting in CoFe 2 O 4 nanoparticles[END_REF] Canting angle values can be extracted for iron from fits of the in-field Mössbauer absorption spectra because the relative intensity I of the pair of 2 nd and 5 th lines depends on the canting angle θ.

In the parallel configuration (with the field direction parallel to that of the  beam), it writes: I = 4sin 2 (θ)/(1+cos 2 (θ)). [START_REF] Cannas | Magnetic properties of cobalt ferrite-silica nanocomposites prepared by a sol-gel autocombustion technique[END_REF] In the absence of canting, the 2 d and 5 th lines are extinct. These canting angles are representative of a static canting between the magnetic moments in tetrahedral (A) or octahedral (B) site and the direction of the external field. As shown in Table 2, canting is present in both sites in agreement with other works, [START_REF] Peddis | Cationic distribution and spin canting in CoFe 2 O 4 nanoparticles[END_REF][START_REF] Ferreira | Structural and morphological characterization of FeCo 2 O 4 and CoFe 2 O 4 spinels prepared by a coprecipitation method[END_REF] with rather large  angles. The   and   angles slightly increase after annealing at 800°C, also contributing to the magnetization decrease.

Note that these canting angle values of 22° are comparable to those reported for 2.5 nm (Co 0.24 Fe 0.76 )[Co 0.76 Fe 1.24 ]O 4 particles, that were attributed to a surface contribution. [START_REF] Artus | Size-dependent magnetic properties of CoFe 2 O 4 nanoparticles prepared in polyol[END_REF] Indeed, in this later work, an increase of the particle size to 6 nm was found to decrease the canting angles from 25 to 17°. For our 11 nm coprecipitated particles, we expect the canting to be representative of a volume contribution mostly influenced by the spatial distribution of Co and Fe ions. The decreased inversion in our samples (0.67 vs. 0.76 in ref 35) is expected to give rise to a wider distribution of environments and presumably more canting that could explain the  values reported in this work. Moreover, the linewidths corresponding to Fe 3+ in tetrahedral coordination ( A ) tend to decrease with heating and the opposite trend is observed for Fe 3+ in octahedral sites ( B ), [START_REF]Mössbauer spectra were fitted using two sextets for A and B contributions, and three different linewidths  for each pair of lines[END_REF] reflecting different evolutions in the hyperfine environment of the two ions. The decreased  A values probably account for structural changes as  A is not severely impacted by the nature of the neighbors as already stressed by Sawatsky et al., [START_REF] Sawatzky | Mössbauer Study of Several Ferrimagnetic Spinels[END_REF][START_REF] Sawatzky | Cation Distributions in Octahedral and Tetrahedral Sites of the Ferrimagnetic Spinel CoFe 2 O 4[END_REF] and thus by inversion. In contrast, the exchange interaction for Fe 3+ in B site is strongly weakened when its next neighbor in A site changes from Fe 3+ to Co 2+ . As a consequence,  B is more sensitive to the local environment and its increase could result from an enhanced chemical disorder.

Modification of the magnetic properties of nanoferrites with annealing is complex, [START_REF] Beji | Annealing effect on the magnetic properties of polyol-made Ni-Zn ferrite nanoparticles[END_REF] with opposite evolutions reported by different authors and/or strong correlations with a particle size increase. [START_REF] Chiu | Tunable coercivity of CoFe 2 O 4 nanoparticles via thermal annealing treatment[END_REF][START_REF] Fabris | Effects of postdeposition heat treatment on the structural and magnetic properties of CoFe 2 O 4 nanoparticles produced by pulsed laser deposition[END_REF] Our results using a protected annealing approach allows to draw more definite conclusions. The variation of the a-cubic parameter upon heating, as well as the increased coherence length and the morphological changes observed by TEM evidence an enhanced crystallinity and definitely prove atomic diffusion at or above 800°C. However, inversion between Co and Fe must be restricted to exchanges between nearest neighbors and not over long distances. The presence of vacancies on the octahedral cationic sites would have favored a rearrangement at low temperatures, presumably 250-300°C according to ref.

51, but in the case of vacancy-free CoFe 2 O 4 , inversion requires heating above 900°C. [START_REF] Ferreira | Structural and morphological characterization of FeCo 2 O 4 and CoFe 2 O 4 spinels prepared by a coprecipitation method[END_REF] Two interpretations can be proposed to account for the increased canting and the opposite evolutions of the Mössbauer linewidths upon annealing. The first one involves local strain relaxations, in part related to inversion because of the differences in ionic radius for Co 2+ and Fe 3+ species. These structural changes tend to homogenize the environment around Fe 3+ in A site and thus decrease the width of the distribution in hyperfine fields, with hardly any change in the canting angle   . On the other side, magnetic exchanges for Fe 3+ in B site are more sensitive to the nature of the neighbors in A site. The increased Mössbauer linewidths after thermal treatment reflect an enhanced magnetic disorder and thus of frustration, which leads to an increased canting in B site. The evolution of  B would then mostly account for the change in inversion.

The alternative explanation considers gradients of the Co/Fe atomic ratio in the volume of the particles as mentioned in ref 23 for citrate autocombustion methods. For aqueous coprecipitation, a former study has emphasized the fact that the kinetics for Co 2+ insertion in magnetite is slower than that of Fe 2+ or that the leaching of Co 2+ in acid is much faster, leading to cobalt incorporation twice smaller than expected and large quantities of Co adsorbed at the surface of the iron oxide. [START_REF] Vichery | Introduction of cobalt ions in γ-Fe 2 O 3 nanoparticles by direct coprecipitation or postsynthesis adsorption: dopant localization and magnetic anisotropy[END_REF] Another work related to the coprecipitation of Fe 2+ , Fe 3+ and Co 2+ from hydroxides suggested the formation of a heterogenous phase and intergrowth of microdomains with slightly distinct compositions from the study of diffraction line broadening. [START_REF] Ferreira | Structural and morphological characterization of FeCo 2 O 4 and CoFe 2 O 4 spinels prepared by a coprecipitation method[END_REF] Here, the Co/Fe ratio is maintained from the precursors to the solid phase, so that separation in rich and poor cobalt regions might occur. Such segregation would explain the  B increase with annealing because of partial homogenization upon heating. Indeed, FeCo 2 O 4 is referred to as a normal spinel [START_REF] Lotgering | On the ferrimagnetism of some sulphides and oxides[END_REF] and Co preferentially occupy octahedral cationic sites in maghemite, so that homogenization will induce changes in the nature of the nearest neighbors for Fe 3+ ions in B site as it goes from pure Co (or Fe) to mixed (Fe,Co) environment. However, both the diffraction coherence length which is not very small with respect to the particle sizes and the magnitude of the canting angles suggest that heterogeneities are less pronounced than in citrate autocombustion routes (22° vs. 40°

for the  values in ref. 23). Comparison with the works of Artus [START_REF] Artus | Size-dependent magnetic properties of CoFe 2 O 4 nanoparticles prepared in polyol[END_REF] and Baaziz [START_REF] Baaziz | Tuning of synthesis conditions by thermal decomposition toward core-shell Co x Fe 1-x O@Co y Fe 3-y O 4 and CoFe 2 O 4 nanoparticles with spherical and cubic shapes[END_REF] shows that the design of new synthetic strategies to favor high crystallinity or to circumvent differences in reactivity between the Fe and Co precursors is more efficient that post-synthesis annealing to get homogenous hyperfine environments and less canting.

Conclusions

Post-synthesis heat treatments have been performed on  11 nm sized cobalt ferrite nanoparticles produced by coprecipitation in water and dispersed in a sol-gel silica matrix.

This protected annealing process allowed increasing their crystallinity while preventing them from aggregation and growth. The presence of this matrix also limits the impact of inter-particle interactions on magnetic evolutions upon heating. We reported a displacement of cobalt ions from tetrahedral to octahedral sites, with an inversion degree that slightly increases from 0.64 after synthesis to 0.66 after annealing at 800°C. Treatments at higher temperatures were found to be limited by side reaction with the host silica matrix. A rather large increase of the coercive field value was observed, from 1.0 to 1.6 Tesla after annealing. This evolution results from the combination of a slight increase in magnetic anisotropy, due to the increased inversion, and of a decrease in saturation magnetization, because of the change in inversion and of an increased canting. This study highlights that improving the crystallinity of cobalt ferrite nanoparticles does not necessarily lead to an increase in saturation magnetization or a decreased canting in the volume of the particles: for

Fig. 1 .

 1 Fig. 1. (a) TEM micrograph of the crude CoFe 2 O 4 particles; (b) histogram of the particle size distribution (statistics over ca. 800 particles) where the solid line is a fit to a log-normal function; (c) TEM micrograph of the concentrate CoFe 2 O 4 /SiO 2 composite sample, Co/Si = 0.03.

Fig. 2 .

 2 Fig. 2. X-ray diffraction profiles ( = 1.5418 Å) of CoFe 2 O 4 nanoparticles embedded in silica (Co/Si = 0.03) dried at 100°C and annealed at selected temperatures. The peaks related to the Si calibrant are indicated by open circles and the ones corresponding to fayalite by arrows. Indexation refers to the m Fd 3 space group of cobalt ferrite.

CoFe 2 O

 2 4 (a = 8.392 Å, JCPDS 022-1086 card), similarly to what was reported by Soler et al.

  high boiling point solvent as CoFe 2 O 4 nanoparticles produced at 150 °C by forced hydrolysis in polyol were found exhibit high crystallinity and bulk a-values right after synthesis.[START_REF] Artus | Size-dependent magnetic properties of CoFe 2 O 4 nanoparticles prepared in polyol[END_REF]

Fig. 3 .

 3 Fig. 3. High-resolution TEM images of the CoFe 2 O 4 particles before (a) and after annealing at 800°C (b), which show the facetted character of the particles after heat treatment.

Fig. 4 .

 4 Fig.4.57 Fe Mössbauer absorption spectra recorded at 4.2 K under a 7 T magnetic field for crude CoFe 2 O 4 particles heat treated at 100 and 800°C. The magnetic field is applied parallel to the γ-ray propagation direction. The dashed and dotted black lines correspond to the contribution of iron in tetrahedral and in octahedral sites, respectively. The bold grey solid lines refer to the fitted spectra.

Fig. 5 .

 5 Fig. 5. Magnetization at 10 K as a function of magnetic field for CoFe 2 O 4 nanoparticles embedded in silica (Co/Si = 0.004) and annealed at selected temperatures. In inset, the high-magnetic field part of the M(H) curves obtained by VSM measurements.

Fig. 6 .

 6 Fig. 6. Zero Field Cooled magnetization measurements under 25 G for CoFe 2 O 4 nanoparticles embedded in silica (Co/Si = 0.004) and annealed at 400 and 600°C.

CoFe 2 O 4 ,

 24 these two parameters are mostly influenced by inversion, and annealing temperatures below 1000°C does not allow for a large redistribution of Fe and Co in the cationic sites.

Table 1

 1 Lattice parameter (a) and coherence length (L c ) values for CoFe 2 O 4 particles embedded in silica (CS, Co/Si = 0.03) and for crude particles (C), after annealing at selected temperatures. the error bar on the a-parameter is mostly representative of instrumental line shifts, whereas the standard deviation on L c derives from the fitting.

	However, L	c only increases from

i

Table 2

 2 Mössbauer spectroscopy results for crude CoFe 2 O 4 particles heat-treated at 100 and 800°C.

		P A /P B	x	H hf (A)	H hf (B)	θ(A)	θ(B)
		(± 0.02)	(± 0.04)	(T)	(T)	(± 2°)	(± 1°)
	100°C	0.47	0.64	51.2	53.0	21	23
	800°C	0.49	0.66	51.2	53.6	24	27

Table 3

 3 Magnetic characterization of CoFe 2 O 4 nanoparticles embedded in silica and annealed at selected temperatures.

		M s (10 K) a (± 5 emu/ g CoFe2O4 )	M s (265 K) b (± 2 emu/ g CoFe2O4 )	μ 0 H c (10 K) c (± 0.05 T)	d (± 1 K) T peak
	100°C	146	49	1.00	-
	400°C	139	48	1.20	367
	600°C	125	45	1.60	390

a magnetization value under 14 T, b magnetization value under 11 T, c coercive field at 10 K, d temperature of the maximum of the ZFC curve.

Acknowledgements

The authors thank P. Berthet for valuable discussion and assistance with the hightemperature magnetic measurements. P.E. Coulon from the Laboratoire des Solides Irradiés (CNRS/Ecole Polytechnique) is also acknowledged for TEM observations. The research described here has been supported by Triangle de la Physique (contract 2008-051T).


Xue Yang

Michel Giorgi

Hakim Karoui

Didier Gigmes

Virginie Hornebecq

Olivier Ouari

Anthony Kermagoret

David Bardelang

crystal transformation affording photochromic 3D MORF crystals

Metal

-Organic Framework (MOF) type crystals in which rigid, viologen-based pillars are surrounded by cucurbit[7]uril (CB[7]) macrocycles are described. These Metal-Organic Rotaxane Frameworks (MORF) are obtained by desolvation of a crystal precursor featuring a network already near-optimal toward 3D Metal-Ligand reticulation and show reversible photochromism. 2

Predicting the arrangement of molecules in the solid state has remained a tremendous challenge 1 until recently when several design principles have arisen toward getting hybrid, metalligand coordination polymers. 2 While the crystallization of organic compounds often relies on weak supramolecular interactions, 3 the impact of which remains difficult to establish compared to packing effects, the construction of coordination polymers relies on the formation of, comparatively stronger, metal-ligand bonds. In this context, the family of Metal-Organic Framework (MOF) crystals 4 has largely developed over time to become a mature research field where much of the activity is centred on (i) tuning the void space often available after material activation and (ii) using it for example for gas storage, 5 liquid separation, 6 water capture 7 or controlled drug release. 8 One subfield of MOF research is focused on arranging entangled structures along the pillars of the frameworks. Doing so, the pillars can possess a rotaxane architecture, 9 affording Metal-Organic Rotaxane Frameworks (MORF). 10 Recent advances include the pre-organization of rotaxanes or pseudo-rotaxanes in the form of a thread carrying carboxylic acids and a ring, often a crown-ether, before crystal growth with relevant metal ions. 11 Dynamics studies showed possible ring rotation 12 and translation 13 along the pillars of the crystals with temperature. Another strategy relied on the pre-synthesis of a highly porous MOF crystal carrying functional groups amenable for subsequent linkage of rotaxanes in the pores of the crystals. 14 Ring movements were shown to operate with the right stimulus. Among the different rings available for MORF construction, the recent family of macrocycles cucurbit[n]urils (CB[n]) presents several advantages. 15 MORF crystals featuring CB [6] as the ring were introduced in 1997 in the form of 1D-MORF crystals. 16 A little later, 2D-17 and 3D-versions 18 were reported before a revival of this chemistry in recent years. 19 But these examples relied on the recognition properties of CB [6] toward a di-cationic guest flanked with two chelate ligands. To the best of our knowledge, there are no examples of CB [7] based 3D-MORF. On the other hand, another subfield of the CB[n] chemistry has focused on viologens and derivatives 20 due to the good recognition of this motif by CB [7]. 21 Owing to the responsiveness of the viologen function to various stimuli (light, chemicals, redox, temperature), 22 we hypothesized that a relevant viologen•CB [7] pseudo-rotaxane (Fig. 1) could be organized with the right metal ion into 3D-MORF crystals, and show stimuli-responsive properties. Following initial work about silver 23 and pH 24 responsiveness of a CB [7]-viologen pseudo-rotaxane, we describe herein the preparation of Zn-based 3D-MORF crystals featuring CB [7] and rigid pillars derived from a viologen core (Fig. 1). The synthesis of the pillar compound P-V-P (chlorine salt) is described in supporting information. 1 H NMR showed about quantitative binding at 1 equiv of CB [7]. With 2 equiv CB [7], stations P are occupied and so a 1:1 stoichiometry was selected. As already reported for similar compounds, 23 P-V-P binds CB [7] with a relatively good affinity, Ka ≈ 3.8 × 10 5 M -1 as checked by 1 H NMR (competition with 1-butyl-3-methyl-imidazolium chloride, Fig. S1, supporting information). A strong affinity between the host and the guest was an important requisite to warrant homogeneous crystal growth, incorporating the rings in a reliable way all along the procedure. A screening of >40 experimental conditions, varying the nature of the metal ion (Cu 2+ , Fe 2+ or Zn 2+ ), the concentration of host and guest, the host:guest ratio (between 1.04 and 1.20), and the ratio between water and DMF (Table S2, supporting information), allowed to identify a few cases for which single-crystals could be obtained. A few set of conditions was suitable for crystal growth with zinc, P-V-P and CB [7]. At 5 mM P-V-P, 1.2 equiv CB [7] and 6.4 equiv Zn(NO3)2, 6 H2O in a water/DMF mixture (8/2 vol%), single crystals were obtained reproducibly (see supporting information) and analysed by X-ray diffraction at low temperature (Fig. 2, left). A network featuring Zinc atoms with P-V-P surrounded by CB [7] was obtained, but the Zn nodes are also coordinated by three water molecules (one was disordered), hence preventing total metal-ligand reticulation. The basic unit is composed of a zinc ion connected by two P-V-P•CB [7] complexes and three water molecules. However, upon coming back to room temperature overnight in the diffractometer, the structure changed to a 3D-MORF arrangement showing full reticulation of the metal-ligand framework (Fig. 2, right). The crystals now belong to the 𝐼4 ̅ 2𝑑 tetragonal space group with unit cell parameters a = 32.6797(2) Å and c = 13.5625(2) Å. The zinc nodes are no longer carrying water molecules which disappeared from the framework suggesting a slow drying under air stream overnight. The other striking feature of this structure is that, as for the structure determined at 125 K, all the P-V-P molecules are surrounded by CB [7] according to a rotaxane topology. A careful examination of the former structure (125 K) showed that each component of the future network was already arranged in a relevant fashion so that the energetic cost of reorganization to metal-ligand coordination is presumably limited. The environment around the zinc nodes changed from penta-coordinated (two carboxylic acid oxygen atoms and three water oxygen atoms) to tetracoordinated (four carboxylic acid oxygen atoms). However, interpenetration also occurred, as commonly observed for frameworks with large void spaces. The crystal structure at 293 K shows 5-fold interpenetration (Fig. 3a) but there is still enough space in the form of large corrugated channels (Fig. 3b, left) of approximate dimensions ~ 1.2 × 1.8 nm. The channels are filled with CB [7] macrocycles that are threaded along the P-V-P pillars of the framework (Fig. 3b, right). It seems CB [7] is able to reduce the level of interpenetration, passing from 13-fold (Cd-PVP MOF) 25 to 5-fold. Isothermal thermogravimetric analysis (isothermal TGA) was performed to determine on which timescale the crystals corresponding to the low-temperature form are dehydrated. At room temperature, the Zn•P-V-P•CB [7] crystals are dehydrated and transformed to the 3D-MORF structure after a few hours (Fig. S2, supporting information). Powder X-ray diffraction was measured on a batch of dehydrated (3D-MORF) crystals. Please do not adjust margins Please do not adjust margins Reflections from the experimental diffractogram match generally well with those of the theoretical one derived from the single-crystal structure (Fig. S3, supporting information). To our knowledge, we are not aware of previous reports featuring CB [7] 3D-MORF or CB [7]-viologen MORF crystals. The colour of the 3D-MORF crystals changed when exposed to sunlight or UV light. Placed in a Rayonet© photochemical reactor ( = 305 nm), the crystals become green. Actually, MOFs containing viologen ligands present interesting colouration properties induced via light, 26 heat, 27 or pressure. 28 These properties are assigned to short distances between electron donor/acceptor functions, favouring electron transfer to the viologen. 29 The induced cationic radical viologens are stabilized in the MOF structure 30 and colour the crystals from green to blue. In this work, we have observed a similar colour change of the MORF crystals under UV-irradiation, from pale yellow to green, with a sharp absorption band centred at 454 nm for the yellow crystals and two broad bands with maximum at 730 and 668 nm associated with a shoulder at 602 nm for the green crystals (Fig. 4). 25, 28a Please do not adjust margins

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The colour change is reversible (timescale of minutes after switching the UV light source off), and reversible photoinduced electron transfer is the favoured explanation now. However, oxygen diffusion inside the crystals and viologen oxidation back to the 2+ charge state cannot be ruled out. 31 EPR spectra recorded before and after photoirradiation at 305 nm showed respectively no, and one signal at g ≈ 2.0032, indeed suggesting that reduction of the viologen pillars is involved in the colour change (Fig. S4, supporting information). 25,32 Previous reports suggest that the carboxylic acid functions are the electron donors in photoswitchable MOFs, 25-26, 26d, 28c, 33 but more investigations are needed to ascertain the nature of the electron donor in this case.

Here we have shown that 3D-MORF type crystals can be grown using CB [7] as ring molecules and a viologen derivative as the pillars. With a modest screening of experimental conditions, we have found suitable experimental parameters favouring crystal growth of a precursor of the designed MOF structure. Gentle dehydration enabled zinc nodes to fully connect to the pillars, affording the targeted 3D-MORF crystals featuring CB [7] molecules around the pillars. The presence of viologen fragments endowed the crystals with photochromism, which could open interesting perspectives in inkless printing 34 or transient writing/erasing for data encryption. 35 Aix-Marseille University and CNRS are acknowledged for continuous support. This project has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No713750. Also, it has been carried out with the financial support of the Regional Council of Provence-Alpes-

Fig. 1 .

 1 Fig. 1. Structures of (a) guest compound P-V-P and of CB[7], followed by the procedure (b) for single crystals preparation.

  a. Aix Marseille Univ, CNRS, ICR, Marseille, France b. Aix Marseille Univ, CNRS, Centrale Marseille, FSCM, Marseille, France c. Aix Marseille Univ, CNRS, MADIREL, Marseille, France † Footnotes relating to the title and/or authors should appear here. Electronic Supplementary Information (ESI) available: [details of any supplementary information available should be included here]. See DOI: 10.1039/x0xx00000x

Fig. 2 .

 2 Fig. 2. Single-crystal-to-single-crystal transformation of stacked dimetallic-Host-Guest dimers (left) toward the 3D-MORF structure. The pseudo-diamondoid network and reticulated one are shown in a) (CB[7] removed for clarity) and in b) with CB[7] (only one network is shown, for interpenetration, see text).

Fig. 3 .

 3 Fig. 3. 5-fold interpenetration of the Zn•P-V-P•CB[7] 3D-MORF (a) without (left) and with (right) CB[7]. Another view (b) of the interpenetrated framework without (left) and with (right) CB[7] along the c crystallographic axis.

Fig. 4 .

 4 Fig. 4. Solid UV-vis spectra before and after photoirradiation with UV source (inset, photos of a 3D-MORF crystal, respectively before and after irradiation)

  Côte d'Azur and with the financial support of the A*MIDEX (n° ANR-11-IDEX-0001-02), funded by the Investissements d'Avenir project funded by the French Government, managed by the French National Research Agency (ANR). Dr. Thomas Devic is also acknowledged for helpful discussions. CCDC numbers of the two crystal structures: 1950817 (293K), 1950818 (125K).

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

Conflicts of interest

There are no conflicts of interest to declare.

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Mónica Arias
email: moarias@gmail.com

Marianne Elias

Christine Andraud

Serge Berthier

Doris Gomez

Transparency improves concealment in cryptically coloured moths

Keywords: Predation, communication, vision, butterfly, camouflage

Predation is a ubiquitous and strong selective pressure on living organisms. Transparency is a predation defence widespread in water but rare on land. Some Lepidoptera display transparent patches combined with already cryptic opaque patches. A recent study showed that transparency reduced detectability of aposematic prey with conspicuous patches. However, whether transparency has any effect at reducing detectability of already cryptic prey is still unknown. We conducted field predation experiments with free avian predators where we monitored and compared survival of a fully opaque grey artificial form (cryptic), a form including transparent windows and a wingless artificial butterfly body. Survival of the transparent forms was similar to that of wingless bodies and higher than that of fully opaque forms, suggesting a reduction of detectability conferred by transparency. This is the first evidence that transparency decreases detectability in cryptic terrestrial prey. Future studies should explore the organisation of transparent and opaque patches in animals and their interplay on survival, as well as the costs and other potential benefits associated to transparency on land.

Introduction

Predation is ubiquitous and exerts a strong selection on living organisms. Often, prey sport cryptic colour patterns that reduce detectability by visual predators, rendering prey hardly distinguishable from their background. Crypsis is achieved if colour patterns represent random samples of background colouration [START_REF] Endler | An overview of the relationships between mimicry and crypsis[END_REF]. This is challenging, as backgrounds are often complex combinations of elements that can move and that vary in colour and pattern [START_REF] Ruxton | Avoiding attack[END_REF]. Background matching is efficient only if sufficient number of aspects perceived by predators (e.g., colour, brightness, polarization) are matched [START_REF] Endler | A predator's view of animal color patterns[END_REF][START_REF] Ruxton | Avoiding attack[END_REF]. Moreover, perception of prey coloration can vary between predators with different visual systems as suggested by the different colours displayed by the dwarf chameleon Bradypodion transvaalense when hiding from snakes or from birds [START_REF] Stuart-Fox | Camouflage and colour change: antipredator responses to bird and snake predators across multiple populations in a dwarf chameleon[END_REF]. Given the intimate dependence between prey survival and background colouration, cryptic colourations constrain prey movements, and potentially hinder "risky" activities such as foraging, which are associated to an increase in predation risk [START_REF] Stamp | On the cryptic side of life: being unapparent to enemies and the consequences for foraging and growth of caterpillars[END_REF][START_REF] Bernays | Feeding by lepidopteran larvae is dangerous[END_REF][START_REF] Gotthard | Increased risk of predation as a cost of high growth rate: an experimental test in a butterfly[END_REF]. By contrast, dynamic colour changes [START_REF] Hanlon | Crypsis, conspicuousness, mimicry and polyphenism as antipredator defences of foraging octopuses on Indo-Pacific coral reefs, with a method of quantifying crypsis from video tapes[END_REF] or transparency [START_REF] Johnsen | Hide and seek in the open sea: pelagic camouflage and visual countermeasures[END_REF] can free prey from background dependency, improve survival in visually heterogeneous environments, and foster vital activities such as foraging. Notably, transparency can minimize detectability against virtually any background [START_REF] Johnsen | Hide and seek in the open sea: pelagic camouflage and visual countermeasures[END_REF].

Transparency maximises light transmission, minimising reflection and absorption at all angles and for all wavelengths seen by predators. Transparency is rare on land, with the notable exception of insect wings. Among insects, Lepidoptera (moths and butterflies) typically have opaque wings covered by coloured scales involved in intraspecific communication [START_REF] Jiggins | Mimicry and the evolution of premating isolation in Heliconius melpomene Linnaeus[END_REF], and antipredator defences such as aposematism (i.e. advertisement of unpalatability, [START_REF] Mallet | Individual selection, kin selection, and the shifting balance in the evolution of warning colours: the evidence from butterflies[END_REF], masquerade (i.e. imitation of inedible objects, [START_REF] Suzuki | Gradual and contingent evolutionary emergence of leaf mimicry in butterfly wing patterns[END_REF] and camouflage (Stevens & Cuthill, 2006). Yet, wing transparency has evolved independently in multiple Lepidoptera families often in combination with opaque elements. By comparing detection of four real species of aposematic butterflies by predators, [START_REF] Arias | Transparency reduces predator detection in mimetic clearwing butterflies[END_REF] recently showed that even if all offered high visual contrast and were conspicuous to predators, fully opaque species were more detectable than species with transparent elements. In this case, transparency may help reducing detection in the first place, but if detected, butterflies may benefit from advertising their unpalatability with highly contrasting patches [START_REF] Mcclure | Why has transparency evolved in aposematic butterflies? Insights from the largest radiation of aposematic butterflies, the Ithomiini[END_REF]. However, in many species, transparency has evolved in already cryptic butterflies, as in the Neotropical moth Neocarnegia basirei (Saturniidae) or the Malaysian Carriola ecnomoda (Erebidae), where transparent wing areas are surrounded by brownish patches frequent in their visual background. Whether transparency can decrease detectability of already cryptically coloured prey remains unknown. We here test for the first time whether transparency decreases detectability on already cryptic terrestrial prey, by conducting field predation experiments by free avian predators and using artificial moths.

Materials and Methods

Field experiments

We performed predation experiments in May 2018 in southern France, in La Rouvière forest, (43.65°N, 3.64°E) for one 1-week session and at the Montpellier zoo (43.64°N, 3.87°E) for the subsequent two 1-week sessions. According to the local bird-watcher website www.faune-lr.org insectivorous species reported for the zoo and its surroundings are more numerous. However, predator communities are rather similar between sites in spring (Table S2). Great tits (Parus major) and blue tits (Cyanistes caeruleus), predators of artificial prey in previous similar studies [START_REF] Rowland | Can't tell the caterpillars from the trees: countershading enhances survival in a woodland[END_REF][START_REF] Stevens | Conspicuousness, not eye mimicry, makes "eyespots" effective antipredator signals[END_REF], were seen every morning while monitoring artificial prey at both locations. Additionally, diurnal insectivorous birds such as the Eurasian jay (Garrulus glandarius), the common chaffinch (Fringilla coelebs), the golden oriole (Oriolus oriolus) and the European robin (Erithacus rubecula) have also been seen in both localities. We followed the previously validated protocol [START_REF] Rowland | Can't tell the caterpillars from the trees: countershading enhances survival in a woodland[END_REF][START_REF] Stevens | Conspicuousness, not eye mimicry, makes "eyespots" effective antipredator signals[END_REF] for monitoring artificial prey survival from predation by bird communities. Artificial prey (body and wings) were pinned on green oak Quercus ilex tree trunks (>10cm in diameter, with few or no moos cover), every 10m in the forest cover. To minimise ant attacks, artificial prey were one centimetre away from the trunk. Additionally, this space was covered by 0.4 cm of Vaseline and 0.6 cm of sticky double-faced transparent tape.

Vaseline and tape could be seen from the side, but not when facing the artificial moth. We randomly placed artificial moths with edible body, and three types of wings: fully opaque grey wings (C form), wings with grey contour and large transparent windows (T form), and no wings (B form) as a control of body attractiveness (Fig. S1). Prey were disposed vertically to the ground and mostly facing north to reduce direct sunlight reflection. We monitored prey survival once per day for the following four consecutive days after placing them on trunks, and removed them afterwards.

Artificial moths

As in other similar experiments [START_REF] Cuthill | Disruptive coloration and background pattern matching[END_REF]Stevens et al., 2006[START_REF] Stevens | Conspicuousness, not eye mimicry, makes "eyespots" effective antipredator signals[END_REF], artificial moths consisted of paper wings and an edible body, made of flour and lard in this case. Triangular shaped moths (triangle 25x36mm, surface of 450mm²) did not mimic any real local species, but resembled a generic resting moth (examples in Fig. S1). We designed moths that bore a constant crypsis level by displaying poor visual contrast (chromatic and achromatic) against the average trunk colouration of the highly abundant green oaks.

First, we took reflectance spectra of green oak trunk colouration (Fig. S2) and laminated grey paper. We then computed colour and brightness contrasts between paper and trunk as perceived by birds, by using Vorobyev and Osorio's discriminability model [START_REF] Vorobyev | Receptor noise as a determinant of colour thresholds[END_REF]. This model reconstructs the difference in colour and brightness between two colour patches (here butterfly and trunk), as seen by a predator under a given light environment. Contrasts are expressed in JNDs (just noticeable differences) and 1 JND is commonly assumed as the threshold below which two colours are indistinguishable. We found that Grey155 (i. e., in a RGB scale (Red, Green, Blue) from 0 (black) to 255 (white), R=G=B=155), printed with a HP officejet pro 6230 printer on Canson® sketch paper, was chromatically indistinguishable but lighter than oak trunks (Table S1), and was chosen as it allowed us testing transparency as a crypsis enhancer on moths bearing a colour that conferred the same imperfect level of crypsis to all artificial prey (see ESM for contrast calculation details). We built the "T" form by cutting two triangular windows (total area of 234 mm²) in the laminated grey triangle, and putting a transparent film (3M for inkjet, chosen for its high transparency even in the UV range see ESM, Fig S2 ) underneath the remaining parts. On top of moth wings, we added an artificial body made from pastry dough (428g flour, 250g lard, and 36g water, following Carrol & Sherratt ( 2013)), dyed grey by mixing yellow, red and blue food dyes (spectrum in Fig. S2, contrast values in Table S1).

Such malleable mixture allowed us to register and distinguish marks made by bird beaks from insect jaws. We finally computed the visual contrasts produced in the eyes of bird predators (for details see ESM extended materials and methods): C was cryptic (chromatic contrast < 1JND, achromatic contrast ≤ 1.64 JND) and more conspicuous than T and B forms (Table S1).

Data collection and analysis

We hereafter report all measures, conditions and data exclusions of our experiment, as well as how we determined our sample sizes. As it has been done in previous similar studies, prey were considered as attacked only when artificial moths showed V-shaped or U-shaped marks on their body, or when a prey was missing without signals of invertebrate attacks (i.e. no body scraps left on wings or around the butterfly on the trunk, [START_REF] Carroll | A direct comparison of the effectiveness of two anti-predator strategies under field conditions[END_REF][START_REF] Hossie | Body size affects the evolution of eyespots in caterpillars[END_REF]. We monitored hundreds of prey models within a few hours and decided as a group whether marks we observed on these models represented valid attacks. Because the observers were aware of the hypothesis, there was the opportunity for bias in the interpretation of marks on models. However, we believe this bias was minimized because we made decisions as a group, most marks were unambiguously attributable or not attributable to birds, and we were focused on the more difficult task of finding models in the field and thus were distracted from the hypothesis when assessing marks on models. We removed all remains of artificial moths attacked by birds, but replaced them when attacked by invertebrates or fully missing. Non-attacked prey were considered as censored data. We started with a sample size of 100 prey items per treatment, as reported on similar studies [START_REF] Schaefer | Disruptive coloration provides camouflage independent of background matching[END_REF][START_REF] Stevens | Predator perception and the interrelation between different forms of protective coloration[END_REF]. We increased the experimental effort until statistical clarity was reached while keeping similar sample sizes per treatment (see [START_REF] Amrhein | Inferential Statistics as Descriptive Statistics: There Is No Replication Crisis if We Don't Expect Replication[END_REF][START_REF] Dushoff | I can see clearly now: reinterpreting statistical significance[END_REF] for discussion on statistical clarity versus statistical significance). One hundred and fifty artificial moths placed in a frequently visited zone of the zoo were excluded from the experiment dataset. In this zone too many moths were completely removed (body, wing, and pin) and it was impossible to determine whether birds as magpies or humans took them. Therefore, all artificial prey from this zone (those that disappeared and those that had not) were removed from the analyses. We analysed prey survival using Cox proportional hazard regression [START_REF] Cox | Models and life-tables regression[END_REF], with type of prey, week and their interaction as explanatory variables. By including "week", the first contrast tests for time and place (by comparing week 1 at La Rouvière, and weeks 2 and 3 at the zoo), while the second contrast test for 'time' at the zoo (Table 1). Overall significance was measured using a Wald test. Statistical analyses were performed in R (R Foundation for Statistical Computing, 2014) using survival package [START_REF] Therneau | Survival: Survival analysis, including penalised likelihood[END_REF].

Results

492 artificial moths including 165 C moths, 163 T moths and 169 B moths were analysed. 70 of these moths were attacked (predation rate: 14.08%, see Fig. S3 for an example of an attacked prey).

Survival strongly differed between forms (Wald test =24.35, df = 8, p = 0.002): wingless bodies and butterflies with transparent windows were similarly attacked (z = 1.51, p = 0.13) and both were less attacked than opaque butterflies (z = 3.98, p < 0.001, Fig. 1, Table 1). Differences between attacks registered at La Rouvière (where 151 moths were displayed on week 1) and attacks at the zoo appeared small (hazard ratio = 0.96) and statistically unclear (pooling weeks 2 and 3 together, z = -0.04, p = 0.71, Table 1). At the zoo, more attacks were registered on week 2 (with 154 analysed moths and closer to blue and great tit reproduction peak) than on week 3 (with 192 analysed moths, z = 0.55, p = 0.003). No statistically clear interaction between prey form and time or prey form and place was detected (Table 1).

Discussion

Using artificial prey mimicking resting moths with and without transparent elements, exposed to a natural community of avian predators, we show for the first time that transparency confers survival benefits in already cryptically-coloured terrestrial prey. Transparent butterflies were attacked as little as wingless bodies and less than opaque butterflies, suggesting that transparent windows reduce detection. This study is the first to investigate the benefit value of transparency in cryptic terrestrial prey, and to experimentally isolate the effect of transparency from other aspects (as patch colour or patch size). Whether the position and the size of transparent windows, as well as the intrinsic optical properties of the transparent surface (levels of transmission and reflection briefly explored by [START_REF] Arias | Transparency reduces predator detection in mimetic clearwing butterflies[END_REF] and [START_REF] Mcclure | Why has transparency evolved in aposematic butterflies? Insights from the largest radiation of aposematic butterflies, the Ithomiini[END_REF]) and its interaction with the ambient light [START_REF] Johnsen | The physical basis of transparency in biological tissue: ultrastructure and the minimization of light scattering[END_REF] influence transparency efficiency also remains untested for terrestrial prey.

Although repeatedly proven useful to evaluate predation pressure in the wild [START_REF] Cuthill | Disruptive coloration and background pattern matching[END_REF]Stevens et al., 2006;[START_REF] Merrill | Disruptive ecological selection on a mating cue[END_REF][START_REF] Carroll | A direct comparison of the effectiveness of two anti-predator strategies under field conditions[END_REF], artificial prey experiments as ours have several limitations [START_REF] Irschick | Field experiments, introductions, and experimental evolution[END_REF]. Those include the lack of predator species identification, sampling a subset of all possible interactions between prey and predators (i.e. prey detection but not attack, attack of the same prey by different predators), and excluding prey movement, a feature that can facilitate detectability by predators. Camera traps have been useful to get more information on artificial prey predation [START_REF] Akcali | Evaluating the utility of camera traps in field studies of predation[END_REF]. However, they have provided little information when studying artificial insect predation so far [START_REF] Low | Determining predator identity from attack marks left in model caterpillars: guidelines for best practice[END_REF][START_REF] Ho | Attack risk for butterflies changes with eyespot number and size[END_REF], most likely because camera traps are less efficient at capturing birds than other predators such as mammals [START_REF] Ho | Attack risk for butterflies changes with eyespot number and size[END_REF][START_REF] Akcali | Evaluating the utility of camera traps in field studies of predation[END_REF]. Other options as DNA analyses on preyed items could be of better help at identifying avian predator species in future studies [START_REF] Rößler | The future of clay model studies[END_REF].

Crypsis can incur costs related to thermoregulation [START_REF] Carrascal | Behavioral thermoregulation by treecreepers: trade-off between saving energy and reducing crypsis[END_REF], intraspecific communication [START_REF] Dunham | The communicative cost of crypsis in a hermit crab Pagurus marshi[END_REF], and, more importantly, mobility [START_REF] Stamp | On the cryptic side of life: being unapparent to enemies and the consequences for foraging and growth of caterpillars[END_REF][START_REF] Ruxton | Avoiding attack[END_REF], thereby hindering foraging and looking for mates. While costs of transparency in terms of thermoregulation and communication have been unexplored so far, transparency can potentially reduce detectability in virtually all backgrounds, reducing the mobility costs associated to crypsis, and enlarging habitat exploitation as reported for the transparent form of the Hippolyte obliquimanus shrimp [START_REF] Duarte | Shape, colour plasticity, and habitat use indicate morph-specific camouflage strategies in a marine shrimp[END_REF]. However, if camouflage is maximal when including transparency and offers additional benefits in terms of mobility, the low representation of transparency in land, especially in Lepidoptera, is puzzling. As it has been hypothesised for benthic habitats, transparency may be more costly than pigmentation [START_REF] Johnsen | Hidden in Plain Sight: The Ecology and Physiology of Organismal Transparency[END_REF]. In Lepidoptera, scales are involved in several physiological adaptations (communication, water repellency, thermoregulation) [START_REF] Miaoulis | Butterfly thin films serve as solar collectors[END_REF][START_REF] Jiggins | Mimicry and the evolution of premating isolation in Heliconius melpomene Linnaeus[END_REF][START_REF] Wanasekara | Role of surface roughness on wettability and coefficient of restitution in butterfly wings[END_REF].

Whether transparent wings may incur communication, hydrophobic or thermal costs remains to be studied to better understand the costs associated with the evolution of transparency on land and explain its rarity.

Figure 1 .

 1 Figure 1. Survival of artificial prey with (T) transparent elements on their wings, (B) bodies without

Table 1 .

 1 Cox regression analyses for attacks on artificial butterflies.Explanatory variables are: form (fully coloured 'C', with transparent windows 'T', and wingless bodies 'B'), week (w1, w2, w3), place (Rouvière and zoo) and their interactions. Notice that the experiment was performed only at Rouvière on w1 and only at the zoo on w2 and w3. Therefore, the contrast " Rouvière w1 < Zoo w2 & w3 " tests both time and place simultaneously. z corresponds to the values from the Wald z test used to test for factor significance, Exp. Coeff. to exponentiated coefficients or hazard ratios and SE to standard errors. Symbols: ** p < 0.01, *** p < 0.001.

	Coefficient Exp. Coef SE	z	p

Acknowledgments

We thank Amandine Aullo, Tiffanie Kortenhoff, Mireia Kohler, Baptiste Laulan, Manon Duty, Samuel Perret and Pablo Giovanni for their major contribution to prey elaboration and fieldwork, and Montpellier zoo for their support. We thank Johanna Mappes and Tom Sherratt for useful comments on the experimental set-up. We thank Christophe de Franceschi for his help at identifying local avian predators.

Funding

ANR program (ANR-16-CE02-0012) and Human Frontier Science Program grant (RGP 0014/2016).

Competing interests

Authors have no conflict of interest to declare.

Ethical statement

No animal was intentionally harmed during this experiment and most artificial prey were found and collected, avoiding leaving wastes in the forest.