Alexandre Barras

Félix Sauvage

Inès De Hoon

Kevin Braeckmans

Dawei Hua

Gaëtan Buvat

Juan C Fraire

Christophe Lethien

J Sebag

Michael Harrington

Amar Abderrahmani

Rabah Boukherroub

Stefaan De Smedt
email: stefaan.desmedt@ugent.be

Sabine Szunerits
email: sabine.szunerits@univ-lille.fr

Carbon quantum dots as a dual platform for the inhibition and light-based destruction of collagen fibers: implications for the treatment of eye floaters

Keywords: Vitreous floaters, carbon quantum dots (CQDs), collagen fibers, pulsed-laser, ophthalmology, fibrillation

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Introduction

Opacities in the vitreous body are one of the most common causes of severe impairment in ocular vision. Vitreous opacities are characterized by floating objects inside the vitreous body, which can result from several origins including myopia, infection, diabetic retinopathy, aging and eye injury. Vitreous is a highly hydrated gel-like structure (>98% water), which is maintained by a diluted network of long, thin collagen fibrils. Individual collagen fibrils are normally spaced apart by hyaluronan, an anionic, non-sulfated glycosaminoglycan (polysaccharide), which attracts water and generates a swelling pressure that inflates the gel.

Vitreous collagen fibrils are heterotypic in composition, containing type IX collagen being regularly distributed along the fibril surfaces; 1 rope-like fibrils of collagen types II surrounding a central core of hybrid V/XI, with type II collagen predominating. 2 Type IX collagen has chondroitin sulfate glycosaminoglycan chains attached to it which extend away from the fibril surfaces and space apart the collagen fibrils, thereby preventing fibril aggregation. 3 Vitreous opacities and the degeneration of the vitreous body result from several interconnected mechanisms including the progressive cross-linking of individual collagen fibrils and their aggregation, and finally, liquefaction. 4 Liquefaction is a physicochemical degenerative change that disrupts the homogeneity of the gel vitreous and is characterized by the dissociation of hyaluronan from collagen. Thus, vitreous progressively liquefies as the collagen fibril aggregates and HA dissociates from collagen drawing water with it to form liquid vitreous.

Collagen becomes concentrated in some areas of the gel, while other parts of the vitreous body are devoid of collagen fibrils forming liquid compartments. 5 Another proposed mechanism for vitreous liquefaction is that collagen fibrils are fragmented. 6 It is believed that type IX collagen shields type II collagen and prevents fusion between fibrils. 3 Loss of type IX collagen results in type II collagen becoming increasingly exposed, predisposing to fusion of adjacent fibrils on contact. In both cases, when the aggregates are sufficiently large, they are visible as vitreous opacities resulting in degradation of contrast sensitivity function 4,7 with a negative impact on the quality of life. 8,9 Other mechanisms of vitreous liquefication can be considered, including enzymatic liquefaction and oxidative stress-induced liquefaction. 10 The treatment of vitreous opacification relies on its origin. However, in case of treatment failure, the only curative treatment for removing floaters remains vitrectomy (i.e. a surgical removal of vitreous through small openings in the pars plana) 11,12 or by Nd:YAG laser treatment. 13 However, treatment with the Nd:YAG laser has relatively little efficacy 14 and sometimes can worsen the eye symptoms. In addition, if the opacities are located too close to the retina, for safety reasons, laser treatment cannot be achieved. There is a true medical need for searching for safe and non-invasive methods for treating vitreous floaters. Also, non-surgical therapies have been under development, but, pharmacologic vitreolysis has not been designed to address vitreous opacities. 10,15,16 More recently, the use of light absorbing gold nanoparticles (Au NPs) was proposed by some of us as an alternative solution. 17 When coated with hyaluronic acid, after injection in vitreous ex vivo, these particles diffuse through vitreous and cluster on the collagenous opacities. Thanks to their photothermal properties, Au NPs heat up in an ultrafast manner when illuminated by a nanosecond pulsed-laser; this leads to the evaporation of the water surrounding the Au NPs and the formation of vapor nanobubbles (VNBs). We showed that VNBs collapsing at the surface of the vitreous opacities provide sufficient mechanical force to ablate them. Importantly, the combined use of the Au NPs and nanosecond laser pulses allowed destruction of the opacities at a laser energy ∼1000 times lower than typically used in YAG laser therapy. Of note is that pulsed lasers are commonly used in ophthalmology for the treatment of cataract and in corneal surgery.

Carbon-based nanostructures, such as carbon quantum dots (CQDs), offer promising therapeutic potential as they can engage with biological molecules. 18 These nanostructures have proven to be excellent drug carrier due to its good biocompatibility. 19 The easy of introducing functional groups has enabled their application as medical countermeasure to enveloped viruses, including next to herpes simplex virus type 1 20 human cornaiviruses. The intrinsic fluoresce properties make them well adapted for bio-imaging but allows as well its use in biosensing platforms. 18 One of their applications is the treatment of eye infections with highly positively charged CQDs. 21 CQDs have also been explored for the treatment of corneal neovascularization 22 and to improve retinal angiography. 23,24 Motivated by the reported ability of CQDs to prevent human insulin fibrillation, [25][26][27][28] and the potential of CQDs in regulating the aggregation behavior of human islet amyloid polypeptide (hIAPP) 29 and by their good light absorbing properties over a wide wavelength spectrum, we aim to investigate (i) the potential of CQDs to inhibit the formation of collagen aggregates and (ii) the combined use of CQDs and pulsed laser light to destroy collagen fibers and vitreous opacities through the generation of VNBs (Figure 1).

Results and discussion

One-step microwave-assisted synthesis of aminated and carboxylated fluorescent carbon dots

We have shown previously that a large panel of differently functionalized CQDs can be produced by hydrothermal carbonization of different organic precursors. 30 Hydrothermal techniques lead to highly fluorescent CQDs, but long reaction times and high temperature conditions using a close Teflon-line reactor lead too often to a lack of reproducibility and replicability. Alternatively, microwave-assisted synthesis of CQDs is a more reproducible and easier approach due to the on-line monitoring of temperature and pressure in the reaction vessels. 18,31 To this end, a rapid synthesis of carboxylic acid-decorated CQDs (CQD-1) using glucosamine as a starting material and an amino acid (β-alanine) as surface a passivating agent was investigated. 32 Aminated CQDs (CQD-2) were prepared in a comparable manner from glucosamine hydrochloride and ethylenediamine as a passivating agent (Figure 2a). 33 To remove larger precipitates, the CQDs suspension was centrifuged and then dialyzed against water with a final yield of 0.8% and 3.4% for CQD-1 and CQD-2, respectively. CQD-1 exhibit a spherical shape with a primary average diameter of 20.8 ± 6.6 nm (Table 1), as determined by AFM (Figure 2b). CQD-1 form a "bunch of grapes" in solution and their size reaches up to 50-350 nm in good accordance with the hydrodynamic size recorded by DLS. CQD-2 display a spherical shape with an average diameter of 16.8 ± 6.7 nm (Table 1), as determined by AFM (Figure 2b). Transmission electron microscopy (TEM) observations reveals that the CQDs were almost spherical with an average particle size of ~26 and ~19 nm, respectively, in a good concordance with sizes determined by AFM (Figure S1). The amorphous nature of the CQDs was concluded by the absence of clear lattice fringes as well as any clear pattern in the selected area electron diffraction in the TEM microscopy (data not shown). The chemical composition of the carbon dots was assessed by X-ray photoelectron spectroscopy (XPS, see survey in SI, Figure S2). The C1s high resolution XPS spectra of the CQDs depict different carbon features (Figure 2c). In the case of CQD-1, the high-resolution spectrum of the C1s reveals the presence of fives peaks ascribed to C=C (283. eV (graphite-like structure N-C3). Both CQDs have a variety of polar groups (hydroxyl, carbonyl, carboxyl, amine) present on their surface. The presence of N-C3 moieties indicates that some nitrogen atoms were incorporated into the carbon framework. The oxygen content in the CQD-1 and CQD-2 was determined to be 22.8 at. % and 14.4 at. %, respectively. Higher nitrogen content (13.7 at. %) was found in CQD-2 in comparison with CQD-1 (5.7 at. %). Furthermore, the presence of primary amino groups was validated by the ninhydrin reaction test (see SI, Figure S3), which allowed estimating a surface coverage of 0.24 ± 0.2 mmol g -1 .

The chemical composition of the CQDs was further investigated by Fourier transform infrared (FT-IR) spectroscopy (Figure 2e). The FTIR spectrum of CQD-2 comprises a broad band between 3415-3425 cm -1 attributed to the O-H/N-H stretching vibrations, while the C-H stretching and bending vibrations are located at 2932 and at 1384 cm -1 , respectively. The band at 1636 cm -1 is due to C=N and C=C bonds of the aromatic structure, and C=O stretching vibration (-NHCO-). Compared to CQD-2, the more intense peaks at 1083 and 1041 cm -1 in the FTIR spectrum of CQD-1 are associated with C-O stretching of either the residual carbohydrate or β-alanine. The sharp peak at 1710 cm -1 is assigned to the asymmetric stretching vibration of carboxylic acid moieties. 34 Typical Raman spectra of CQD-1 and CQD-2 are seen in Figure 2f. The observed peaks are typical of sp 2 carbon materials at 1580 cm -1 (G-band) and 1378 cm -1 (D band), indicating that the material is ordered and graphitic in nature. The fact that the D band is rather broad points towards a disordered structure, keeping in mind that the D-peak is not related to disorder but indirectly to the size of the sp 2 coordinating ring domains. The ID/IG ratio is 1.94 for CQD-1 and 1.56 for CQD-2 suggesting large surface disorder and amorphous nature. 35 Zeta potential analysis is a source of information in terms of surface functionalities (Figure S4a). CQD-1 display a negative zeta potential value (= -24.0 ± 1.6 mV) in pure milli-Q water.

The negative surface charge is due to an abundance of deprotonated carboxyl groups on its surface. In contrast, CQD-2 exhibits a positive surface charge with  = +32.5 ± 0.8 mV, underlining the presence of NH3 + groups. Due to the presence of hydrophilic groups, CQDs were readily dispersible at high concentrations (> 10 mg mL -1 ) in pure water or PBS. The dispersion of 250 µg mL -1 CQDs (Figure S4b) in pure water and Dulbecco's Modified Eagle's medium (DMEM) is excellent for both particles. In PBS, the colloidal stability of CQD-2 is reduced and a precipitate is formed after 1 h incubation at 37 °C. The CQDs absorb in the short-wavelength region with a maximum at around 220 nm due to ππ* transition of C=C and C=N carbon cores with a weak extending tail in the visible region (Figure 3a). An additional shoulder at approximatively 280 nm is attributed to π-π* transition of different sp 2 domains. 36 The absorption shoulder at around 306 nm in CQD-1 is due to n-π* transitions of C=O. This contribution is decreased in CQD-2 due to the high content of ethylenediamine; 37 the strong absorption extending from 350 to 600 nm is related to surface passivation of CQDs-2 with ethylenediamine. 38 As expected, CQD-1 and CQD-2 reveal photoluminescence characteristic (Figure 3b) with a maximum fluorescence intensity at 450 nm under 380 nm excitation. 39 The fluorescence emission is wavelength-dependent (Figure 3c,3d). CQD-1 display a broad (full width at half maximum of around 100 nm) and intense photon emission, while CQD-2 exhibit a lower fluorescence. Increasing the excitation wavelength to 500 nm results in a red-shift of the emission band to 590 nm for CQD-1 and 580 nm for CQD-2, with the fluorescence emission intensity of CQD-1 being somehow higher than that of CQDs-2. Using quinine sulfate as reference with a quantum yield of 0.54, the fluorescent quantum yields of CDQ-1 and CQD-2 varied between 0.008 and 0.002, respectively (Table 1, Figure S5). The long storage stability at 4 °C and effects of parameters such as prolonged UV exposure of CQDs were studied. The fluorescence intensity of CQD-1 had no obvious decrease (14%) after being stored 2-years at 4 °C (Figure S6). The fluorescence intensity of CQD-2 had higher decrease (48%). In contrast,

Photothermal properties of CQD-1 and CQD-2

The photothermal properties of CQD-1 and CQD-2 (50 µg mL -1 ) were determined upon irradiation the aqueous solutions with a laser at 810 nm for 15 min. Figure 4 indicates that in both cases a temperature plateau is reached after 10 min, reaching about 50°C for a laser power of 1 W cm -2 and 66 °C for 2 W cm -2 . However, the same temperature trend is observed with PBS solution, indicating poor photothermal properties of these CQDs under these experimental conditions. upon laser irradiation at 810 nm laser at two different power densities: 1 W cm -² (a) and 2 W cm -² (b).

Metabolic activity and cellular uptake of CQDs

The cell toxicity of CQDs-1 and CQDs-2 was established on ARPE-19 and MIO-M1 cell lines after 8 h and 24 h incubation (Figure 5). The CQDs toxicity was evaluated using the resazurin assay, based on the conversion of non-fluorescent dye to a fluorescent molecule by mitochondrial and cytosolic enzymes. All CQDs are non-toxic to ARPE-19 and MIO-M1 cells until 100 µg mL -1 for 8 h incubation. For longer incubation times, the metabolic activities decrease from 25 µg mL -1 onwards.

Furthermore, the cell toxicity of CQDs-1 and CQDs-2 was established on HeLa and U-87 MG cell lines (Figure S7a). All CQDs are non-toxic to HeLa and U-87 MG cells until 100 µg mL - 1 for both incubation times. HeLa and U-87 MG cells were incubated with CQDs at 100 µg mL - 1 during 24 h and then, nuclei were stained with Hoechst 33258, a fluorescent dye for labeling DNA in fluorescence microcopy (Figure S7b). The green fluorescence, which is attributed to the CQDs, is homogeneously distributed in the cytoplasm after 24 h when incubated at 37 °C, which confirms the adsorption and internalization of positively charged CQDs-2 inside the cells, while there is almost no fluorescence for CQDs-1.

Inhibition of type I collagen aggregation with CQD-1 and CQD-2

Fibril formation of type I collagen has been widely studied by some research teams. [40][41][42][43][44][45][46] Therefore, type I collagen was investigated in this present work to assess to what extent CQDs may inhibit its fibrillation. As explained in the experimental section, fibril formation of type I collagen was initiated by incubating collagen I solution at 37 °C. The change in the optical density of the sample at 313 nm as a function of time was recorded (Figure 6a). After a lag time of about 2 min, a collagen self-assembly growth phase was observed, reaching a plateau after about 3 min which is in line with other reports. 45 AFM imaging (Figure 6b) confirmed the formation of collagen fibers, with a typical diameter of ~150 nm.

The presence of a low amount of negatively charged CQD-1 (< 100 µg mL -1 ) mainly influences the lag time i.e. the onset of collagen fibrillation. Through AFM imaging aggregate formation is observed in the presence of 100 µg mL -1 of CQD-1, with fibers with a diameter of 40-50 nm, much smaller compared with control collagen fibers. A higher CQD-1 concentration of 250 µg mL -1 resulted in complete inhibition of the collagen fibrillation. In contract, the presence of a low amount of positively charged CQD-2 induced an acceleration of type I collagen fibril formation (Figure 6a). The AFM image of collagen in the presence of 100 µg mL -1 CQD-2 (Figure 6b) indicates a quasi-complete inhibition of fibrillation with isolated aggregates of 1.5

µm in length and a diameter of ∼50 nm. Note that a complete inhibition of collagen fibrillation was already observed at a concentration of 150 µg mL -1 .

At low CQDs concentration, some collagen fibril assembly remains. This indicates that the inhibitory effect of CQDs on the nucleation step and intermolecular interactions is concentration dependent. CQDs inhibits fibrillation in a mechanism involving an endothermic reaction along with electrostatic and hydrophobic interactions between neighboring molecules.

Hydrogen bonds are believed to be the most stabilizing forces prevailing in these proteins. It is, thus, hypothesized that the hydrogen bonds may be formed between carbonyl and carboxylic acid groups present on the surface of CQD-1 and amine and hydroxyl groups of type I collagen. 47 This might alter the interactive forces between collagen molecules. The main differences between both type of particles is their surface charge and functionalization. Note that physiochemical properties of nanoparticles such as size and surface charge, have been considered as critical factors for their antifibrotic properties. 48,49 (a)

T im e / m in ) in the absence and presence of 100 µg mL -1 CQD-1 and CQD-2.

C Q D -2 0 µ g m L - 1 1 0 µ g m L - 1 5 0 µ g m L - 1 1 5 0 µ g m L - 1 2 5 0 µ g m L - 1 1 0 0 µ g m L - 1 (b)

Destruction of type I collagen fibers under pulsed-laser irradiation in the presence of

CQDs

We have recently shown that Au NPs, able to trigger the formation of vapor nanobubbles (VNBs) under pulsed-laser irradiation, adhere to type I collagen fibers and can destroy vitreous opacities. 17 Though, different to CQDs, Au NPs do not seem to interfere with collagen fibrillation (Figure S8). The destruction of the type I collagen fibers/vitreous opacities is believed to be mechanical: collapsing VNBs induce a mechanical force which breaks down the fibers. To investigate whether a similar effect could be observed with CQD-1 and CQD-2, type I collagen fibers were mixed with CQD-1 or CQD-2 and irradiated with a pulsed-laser at different fluences and wavelengths. Dark field microscopy was used to visualize the collagen fibers before and after laser irradiation.

As seen in Figure 7a, applying light fluences of 1.1 and 4.5 J/cm² and laser irradiation wavelengths of 488, 561 and 650 nm, without the use of CQDs, did not destroy the type I collagen fibers. Irradiation of the collagen fibers in the presence of CQD-1 showed the same result: the collagen fibers remained intact (Figure 7b). This contrasts to the use of CQD-2 (Figure 7c). Light pulses of <3 ns at 488 and 561 nm (4.5 J cm -2 ) clearly destroyed the collagen fibers. Using a higher wavelength of 650 nm did no longer allow the complete destruction of the fibers, even if 100 pulses were applied (Figure 7c). This could be expected as CQD-2 hardly absorbs at 650 nm (Figure 3a). Given that the 488 nm laser light might be (more) harmful for ocular tissues (if compared to 561 nm light), we selected 561 nm to continue our experiments on human opacities. While we find that the pulsed-laser irradiation in the presence of CQD-2 totally destroys the aggregates, the same approach is inefficient using CQD-1. This difference may rely on the surface charge of the two nanoparticles, which is negative for CQD-1 and positive for CQD-2

(Table 1). The difference in charges could lower the interaction of CQD-1 with the collagen fibers, when compared to CQD-2. Temperature effects could be also involved. However, CQDs show no photothermal activity (Figure 4). In spite one can expect higher photothermal effects with gold nanoparticles, no differences in terms of fluence was observed for fiber destruction: in both cases fluence of 4.5 J/cm² was required for efficient fiber demolition. This can be explained by the relatively small size of both AuNPs and CQDs which cannot trigger formation of vapor nanobubbles in solution but only when they form clusters or in situation where the distance between particles is reduced (as it is likely the case when both AuNPs or CQDs are bound to fibers).

However, inhibition of the fibrillation with both types of CQDs clearly suggests that they can both interact with collagen molecules (Figure 7a).

Assessment of the mobility of CQDs in vitreous by fluorescence recovery after photobleaching (FRAP)

After intravitreal injection, CQDs must keep their mobility to diffuse and their capacity to bind to the target, the vitreous opacities. Although we expected that CQDs, due to their relatively small size (around 10 nm), could diffuse in the bovine vitreous, 50 we decided to perform FRAP experiments to ensure they were mobile and could therefore reach the opacities after intravitreal injection. Indeed, while CQDs are positively charged and the vitreous net charge is negative, one can expect that they will interact with the glycosaminoglycans and collagen network of the vitreous thus forming aggregates. FRAP is a technique allowing to measure the diffusion coefficients of fluorescent molecules or nanoparticles. Briefly, a laser of strong intensity is applied on the sample and induces its photobleaching. The diffusion of the surrounding (fluorescent) molecules (or particles) into the bleached area can be measured and observed by a progressive increase of fluorescence intensity. Recovery curves can, therefore, be obtained from which diffusion coefficients can be calculated.

As observed in confocal images (Figure 8a), fluorescence recovery curves in water and in vitreous (Figure 8b andc) indicate that a complete fluorescence recovery occurs in the bleached area. This observation attests that cationic CQDs can diffuse both in water and in bovine vitreous. Their diffusion coefficients in water and vitreous are comparable (31.4 +/-7.4

µm²/s and 32.9 +/-13.2 µm²/s, respectively). Our observation suggests that, despite a positive charge, CQDs keep their mobility in vitreous. The diffusion of CQDs can be due to their small size (around 10 nm) allowing diffusion through the meshes of the collagen network whose size are comprised between 500 and 1000 nm. 50 This result also indicates that, after intravitreal injection, CQD-2 can sufficiently diffuse to bind the opacities.

Destruction of human vitreous opacities treated with CQD-2 under pulsed-laser irradiation

Type I collagen fibers were previously used to attest the efficacy of their destruction after treatment with CQD-2 and upon pulsed-laser irradiation. Since the collagen composition, size and shape of vitreous opacities differ from type I collagen fibers, we therefore decided to test our concept on real opacities from human vitreous obtained from patients after vitrectomy. The first observation is that, without treatment with CQDs, no destruction or change in opacities could be observed after 50 pulses at 561 nm using a fluence of 4.5 J/cm², suggesting the laser itself cannot destroy the opacities. However, as shown in Figure 9, when vitreous samples were mixed with CQD-2 (0.4 mg mL -1 ) a clear destruction of the opacities could be observed after 50 pulses (561 nm; 4.5 J/cm²). This result is in line with previous observations from some of us at a same wavelength and fluence using 10 nm hyaluronan-coated Au NPs and suggests a similar efficacy. 17

Conclusion

In this work, we have shown that cationic CQDs could inhibit the fibrillation of type I collagen in vitro and destroy type I collagen fibers and human vitreous opacities through the generation of light-induced VNBs at a low fluence (4.5 J/cm²). While negatively charged CQDs can inhibit collagen fibrillation, they were unable to destroy the collagen fibers at the studied fluences and wavelengths. Interestingly, while Au NPs can generate VNBs and have been reported to destroy vitreous opacities, they did not impact collagen fibrillation. CQDs can promote the destruction of vitreous opacities in a similar manner (fluence, number of pulses) with a capacity to inhibit collagen fibrillation. Thanks to this dual effect (i.e. inhibition and destruction), cationic CQDs become attractive therapeutic tools for the treatment of vitreous opacities by avoiding or postponing their reformation after laser treatment.

Taken together, our results suggest that this dual effect provided by CQDs could be further explored in the context of fibrotic diseases and more generally for diseases involving protein aggregation. solution was heated under pressure for 5 min from room temperature to 200 °C and then maintained for 10 min at 200 °C under stirring (1200 rpm). The resulting brown solution was centrifuged at +4 °C (10000 g, 10 min) in order to remove big particles. The supernatant was dialyzed against water during 24 h (SpectraPor RC membranes, 1 kDa) and then kept (or lyophilized) to yield a brown solution.

Supporting Information Available

Synthesis of hyalorunic acid-functionalized gold nanoparticles (AuNPs)

The synthesis and hyaluronic acid (HA)-coated AuNPs (10 nm) was performed in house, as previously reported. 17 Briefly, HAuCl4 (in water) and sodium ascorbate (in water) were mixed at equimolar ratios (concentration=0.2 mM, final volume=100 mL) under rapid stirring for at measurements. The final concentration of the AuNPs was estimated from the extinction of the dispersions as measured at the maximal extinction wavelength (Ext) and the theoretical extinction cross section σEXT (cm 2 ), as previously reported. 17

Primary amine quantification using a modified Kaiser test

The amount of primary amine groups on the CQDs was calculated using a spectrophotometric assay based on the modified Kaiser test with the following modifications. 51 Briefly, CQDs-2 were diluted to 0.1 mg mL -1 in milli-Q water. To this solution was added 50 µL of phenol solution (40 g in 10 mL EtOH), 50 µL of potassium cyanide (2 mL of KCN solution -65 mg in 100 mL water -in 100 mL pyridine) and 50 µL of ninhydrin solution (2.5 g in 50 mL EtOH).

The mixture was heated at 100 °C during 10 min, cooled down on ice and further diluted with 1 mL of ethanolic solution (EtOH/water=60/40). The absorption of the mixture was determined in triplicate at 570 nm using a Perkin Elmer Lambda UV/Vis 950 dual-beam spectrophotometer in 10 mm quartz cuvette. Benzylamine solution was used from 10 to 100 µM to generate the calibration curve.

Characterizations

Atomic Force Microscopy (AFM): AFM images were recorded in tapping mode on a Dimension TM 3100 (Digital Instruments) in air at a scan rate of 1 Hz, and with an antimony doped n-type silicon cantilever (resistivity: 0.01 -0.025 Ohm.cm, frequency resonance ranging from 130 to 250 kHzwith a constant stiffness k ~ 48 N.m -1 ). Samples were diluted by 15 with filtered PBS, deposited on freshly cleaved mica substrates, dried in air and then rinsed three times with water. Data were analyzed with a Gwyddion software (2.56).

Transmission electron microscopy (TEM):

The morphology and sizes of CQDs were characterized using FEI Tecnai G2-20 microscope.

UV-visible measurements: Absorption spectra were recorded from 200 to 800 nm using a Perkin Elmer Lambda UV/Vis 950 dual-beam spectrophotometer in 10 mm quartz cuvette. CQDs were diluted to 20 µg mL -1 in PBS (pH 7.4, 0.01 M, Gibco®).

Raman spectroscopy: Raman spectroscopy measurements were performed on a LabRam HR Micro-Raman system (Horiba Jobin Yvon) using a 473 nm laser diode as excitation source.

CQDs were deposited on a clean silicon wafer. The visible light is focused by a 100× objective.

The scattered light is collected from 100 to 4000 cm -1 by the same objective in backscattering configuration, dispersed by a 1800 mm focal length monochromator and detected by a CCD camera.

Fourier transform infrared (FT-IR) spectroscopy: FT-IR analyses were performed on Thermo Fisher Scientific Inc. Nicolet 8700, in the frequency range between 650 and 4000 cm -1 with a resolution of 6 cm -1 . Dried CQDs (around 1 mg) were mixed with potassium bromide powder (200 mg) in an agar mortar. The mixture was pressed into a pellet under 7 tons of load for 10 min, and the spectrum was recorded immediately. An average of 64 scans was carried out for each sample.

X-ray photoelectron spectroscopy (XPS): XPS spectra were recorded on a VG Escalab 220XL spectrometer from Vacuum Generators featuring a monochromatic Al Kα X-ray source (1486.6 eV) and a spherical energy analyzer operated in the CAE (constant analyzer energy) mode (CAE = 100 eV for survey spectra and CAE = 40 eV for high-resolution spectra), using the electromagnetic lens mode. The angle between the incident X-rays and the analyzer is 58° and the detection angle of the photoelectrons is 30°.

DLS measurements:

The average hydrodynamic diameter and the zeta-potential were recorded on a Zetasizer® Nano ZS (Malvern Instruments S.A., Worcestershire, UK). All the batches were diluted to 200 µg mL -1 in milli-Q water and analyzed in triplicate at 20 °C. The zeta potential of CQDs in milli-Q water (200 µg mL -1 ) was measured at 20 °C using a folded zeta capillary cell (DTS1070). Experiments consisted of 15 runs per measurement with an applied voltage of 150 V.

Fluorescence measurements: Emission and excitation fluorescence spectra were recorded between 200-900 nm using a Xenius XC spectrofluorometer (Safas S.A., Monaco).

Fluorescence measurements of CQDs were performed in PBS (pH 7.4, 0.01 M, Gibco®) at 20 µg mL -1 (excitation and emission bandwidth=10 nm, step=1 nm, PMT voltage=460 V, ultrascan). For quantum yield measurements, fluorescence emissions were recorded from 350-900 nm with an excitation wavelength of 350 nm (excitation and emission bandwidth=10 nm, step=1 nm, PMT voltage=260 V, ultrascan). Integrated fluorescence emissions were calculated from 365 to 680 nm.

Measurement of the photothermal properties

The photothermal properties of 50 µg mL -1 solutions of CQDs in PBS (pH 7.4, 0.01 M, Gibco®)

were determined by using a continuous laser beam at 810 nm (Gbox model, Fournier Medical Solution) at two power densities (1 and 2 W cm -2 ). The temperature changes were recorded by an infrared camera (FLIR A655sc, IR lens: f=24.6mm and 25°) and an image analysis software FLIR ResearchIR Max4.

Biological evaluations

Cell viability assay

The ARPE-19 spontaneously immortalized cell line of human retinal pigment epithelium, the MIO-M1 Müller progenitor cell line, the HeLa cell line derived from cervical carcinoma from a 31-year-old female (ECACC 93021013, Sigma Aldrich Chimie S.a.r.l, St. Quentin Fallavier, France) and the U-87 MG cell line derived from a malignant glioma (ECACC 89081402, Sigma Aldrich Chimie S.a.r.l, St. Quentin Fallavier, France) were cultured and maintained in Dulbecco's Modified Eagle's medium (DMEM, Gibco®) high glucose with 2 mM Glutamine, DMEM/F12 media with 2 mM Glutamine and DMEM supplemented with 10% fetal bovine serum (FBS, Gibco®) and 1% penicillin-streptomycin (Gibco®), respectively, in a humidified incubator at 37 °C and 5% CO2. Cells were seeded at a density of 10 4 cells/well in a 96-well plate and grown for 24 h before assay. The culture medium was replaced with a fresh medium that contains the CQDs from 0 to 400 µg mL -1 . After 24 h, the old medium was aspirated and cells were washed with PBS. The cell viability was evaluated using resazurin cell viability assay.

Briefly, 100 µL of the resazurin solution (11 µg mL -1 ) in appropriate media were added to each well and the plate was incubated for 4 h in the humidified incubator. The fluorescence emission of each well was measured at 593 nm (20-nm bandwidth) with an excitation at 554 nm (18-nm bandwidth) using a Cytation™ 5 Cell Imaging Multi-Mode Reader (BioTek Instruments SAS, France). Each condition was replicated three times and the mean fluorescence value of nonexposed cells was taken as 100% cellular viability.

Cell imaging

HeLa and U-87 MG cells were seeded in a 96-well plate at a density of 10 4 cells/well and grown for 24 h before assay. The CQDs solution were diluted in DMEM/10% FBS to a final concentration of 400 µg mL -1 . After 24-hours incubation at 37 °C, the cell monolayers were washed three times with PBS and then stained with Hoechst 33258 (5 µg mL -1 ) in PBS in the dark for 5 min. The plates were directly observed using a Cytation™ 5 Cell Imaging Multi-Mode Reader (BioTek Instruments SAS, France) equipped with a 20x objective (Plan Fluorite WD 6.7 NA 0.45). The fluorescence signal was acquired using excitation and emission filter sets for DAPI (exc. 377/50 nm and em. 447/60 nm) and for GFP (exc. 469/35 nm and em.

525/39 nm). All the images were further analyzed by Gen5iPlus 3.04 Imaging Software.

Collagen fibril formation

Collagen I from rat tail (A10483-01, Gibco®) was dissolved in PBS (pH 7.4, 0.01 M, Gibco®) at 0 °C (ice bath) to obtain a final concentration of 0.3 mg mL -1 . CQDs at different concentrations were added. Subsequently, the collagen/CQDs solution was homogenized and incubated without stirring during 1 h at 37 °C. The fibrillation process was followed by measuring the absorbance/turbidity of the solutions at 313 nm by using a NanoDrop TM One C spectrophotometer (Thermo Scientific). The baseline correction was done just before to start each experiment (t = 0 min).

Photoablation of collagen fibers and human vitreous opacities

Photoablation experiments were performed on collagen fibers in water and human vitreous containing opacities. Collagen fibers (0.02 mg mL -1 ) were mixed with CQDs in water (0.4 mg mL -1 ) and allowed to equilibrate for 30 min before the experiment. After thawing the human vitreous samples, they were directly mixed with an equal volume of a dispersion of CQDs (typically 0.4 mg mL -1 in water). The samples were then allowed to equilibrate for 30 min at room temperature prior to applying (nanosecond) laser pulses.

First, dark-field microscopy imaging was performed to locate and align the nanosecond laser on the collagen fibers or human vitreous opacities in the sample. To generate vapor nanobubbles, 7 ns laser pulses were used. The wavelengths of the laser light were 488 nm, 561 nm or 650 nm.

A beam expander (#GBE05-A, Thorlabs) combined with an iris diaphragm (#D37SZ, Thorlabs) was used to adjust the diameter of the laser beam to 150 μm. The laser pulse energy was monitored by an energy meter (J-25MB-HE&LE, Energy Max-USB/RS sensors, Coherent) synchronized with the pulsed laser. bleached zone by fitting the data points to appropriate equations. 52 For FRAP experiments in water, 5 µL of a water solution of CQD-2 (2.2 mg mL -1 ) was applied between a microscope slide and a coverslip which were separated by a 120 µm-thick double adhesive spacer (Secure-Seal Spacer; Molecular Probes, Leiden, The Netherlands). For FRAP experiments in bovine vitreous, bovine vitreous was carefully removed from the eye ball (to avoid undesired liquefaction) and 400 µL were carefully cut and transferred into a glassbottomed dish. 40 µL of a solution of CQD-2 (2.2 mg mL -1 ) were then injected in the vitreous using a 1 mL syringe equipped with a 21.5G needle and allowed to equilibrate at room temperature for 30 min before performing FRAP experiments.

Diffusion experiments in water and bovine vitreous by photobleaching

Figure 1 .

 1 Figure 1. Carbon quantum dots (CQDs) to inhibit the formation of collagen aggregates (top) and to destroy collagen aggregates by light induced VNBs (bottom).

  8 eV), C-C/C-H (285.0 eV), C-N/C-O (286.8 eV), C=O (287.9 eV) and -O-C=O (289.2 eV). The presence of carbonyl and carboxylic functions is additionally validated by the presence of a band at 530.7 eV (C=O) in the O1s high resolution spectrum, next to 531.5 eV (C-O). The N1s high resolution spectrum can be curve-fitted with bands at 398.3 eV (pyridinic C=N-C), 399.1 eV (pyrrolic N-H) and 400.3 eV (graphite-like structure N-C3). In case of CQD-2 (Figure 2d), the high-resolution spectrum of the C1s can be deconvoluted with fives peaks assigned to C=C (283.9 eV), C-C/C-H (285.0 eV), C-N/C-O (286.1 eV), C=O (287.1 eV) and -O-C=O (288.4 eV). The presence of oxygen functions is also corroborated by the presence of the band at 530.3 eV (C=O) in the O1s high resolution spectrum, next to 531.6 eV (C-O). The N1s high resolution spectrum shows comparable bands as CQDs-1: 398.3 eV (pyridinic C=N-C), 399.2 eV (pyrrolic N-H) and 400.9

Figure 2 :

 2 Figure 2: (a) Schematic representation of the synthesis of CQD-1 and CQD-2. (b) AFM images of CQD-1 and CQD-2 together with size distribution histograms. (c) XPS analysis of CQD-1: C1s, O1s and N1s. (d) XPS analysis of CQD-2: C1s, O1s and N1s. (e) FT-IR spectra of CQD-1 (red) and CQD-2 (blue). (f) Raman spectra of CQD-1 (red) and CQDs-2 (blue).

the normalized fluorescence intensity before and after 2 -Figure 3 :

 23 Figure 3: (a) UV-vis spectra of CQD-1 and CQD-2 at 20 µg mL -1 in PBS (pH 7.4, 0.01 M). (b) Normalized emission (exc. at 378 nm) and normalized excitation (em. 453 nm) spectra of CDQ-1; normalized emission (exc. at 382 nm) and normalized excitation (em. 452 nm) spectra of CQD-2. (c) Emission spectra of CQD-1 at progressively increasing excitation wavelengths from 280 to 500 nm. (d) Emission spectra of CQD-2 at progressively increasing excitation wavelengths from 280 to 500 nm.

Figure 4 :

 4 Figure 4: Photothermal properties of CQDs. Temperature profiles of CQDs (50 µg mL -1 )

Figure 5 :

 5 Figure 5: Metabolic activity of CQDs. Metabolic activity of ARPE-19 and MIO-M1 cells treated with CQDs: cells were grown in 96-well plates (10 4 cells/well) with 100 µL of culture medium containing increasing concentration of CQDs for 8 h (left) and 24 h (right). The results, expressed as percentage of viability, are the mean value of two independent experiments with each treatment performed in triplicate. Negative control: without CQDs.

Figure 6 :

 6 Figure 6: Collagen I fibril formation and inhibition by CQDs. (a) Absorption (at 313 nm) over time of a collagen I solution (0.3 mg mL -1 ) incubated at 37 °C in the absence and presence of increasing concentrations of CQD-1 and CQD-2; (b) AFM images of collagen I (0.3 mg mL - 1 ) in the absence and presence of 100 µg mL -1 CQD-1 and CQD-2.

Figure 7 :

 7 Figure 7: Dark-field microscopy images of water-dispersed type I collagen fibers. (a) Before and after light illumination with lasers of different intensities and wavelengths (488, 561 and 650 nm). (b) Before and after light illumination with lasers of different intensities and wavelengths (488, 561 and 650 nm) in the presence of negatively charged CQD-1 (0.4 mg mL - 1 ). (c) Before and after light illumination with lasers of different intensities and wavelengths (488, 561 and 650 nm) in the presence of positively CQD-2 (0.4 mg mL -1 ). Scale bar: 100 μm. The yellow dotted squares/rectangles indicate the position of the targeted fiber. Dotted circles indicate the position of the laser beam.

Figure 8 :

 8 Figure 8: Diffusion of cationic CQDs. (a) Confocal microscopy images of CQDs (2.2 mg mL - 1 ) before, during (t=0s) and 130s after photobleaching in water and in bovine vitreous. In water (b) and in vitreous (c) fluorescence of recovery as a function of time occurs in the bleaching area indicating that the cationic CQDs have the ability to diffuse both in water and in bovine vitreous. Scale bar = 100 µm.

Figure 9 :

 9 Figure 9: Dark-field microscopy images of human vitreous opacities: Before and after light illumination (20 and 50 pulses) with laser (561 nm, 4.5 J cm -2 ) in absence or in presence of CQDs-2 (0.4 mg mL -1 ). Scale bar (for bottom row): 100 μm. The yellow dotted squares/rectangles indicate the position of the targeted fiber. Dotted circles indicate the position of the laser beam.

Figure S1 :Figure S2 :

 S1S2 Figure S1: TEM images of CQD-1 (a) and CQD-2 (b) Figure S2: XPS high resolution spectra of CQD-1 and CQD-2.

Figure S3 :

 S3 Figure S3: Kaiser test for the determination of the presence of primary amine groups: (a) Calibration curve (benzylamine solution from 10 to 100 µM). (b) UV-vis absorption spectra of CQD-2, heated at 100 °C during 10 min, cooled down on ice and further diluted with 1 mL of ethanolic solution (EtOH/water=60:40).

Figure S4 :

 S4 Figure S4: (a) Zeta potential analysis. (b) Photographs of CQDs suspensions (250 µg mL -1 ) after 1 h incubation at 37 °C in water (W), PBS (0.01 M, P) and Dulbecco's Modified Eagle's medium (M).

Figure S5 :

 S5 Figure S5: The relationship between the integrated fluorescence intensity and the absorbance of quinine sulfate (QS), CQD-1 and CQD-2.

Figure S6 :

 S6 Figure S6: Optical stability of CQD-1 (red) and CQD-2 (blue) in various conditions: Fluorescence emission after 2-years, normalized fluorescence emission after 2-years and relative fluorescence emission of CQDs solution (20 µg mL -1 in PBS) during UV light irradiation at 360 nm (10 mW cm -2 ) during 3 hours.

Figure S7 :

 S7 Figure S7: Metabolic activity and CQDs uptake: (a) Metabolic activity of HeLa and U-87 MG cells treated with CQDs: cells were grown in 96-well plates (10 4 cells/well) with 100 µL of culture medium containing increasing concentration of CQDs for 8 h (left) and 24 h (right). The results, expressed as percentage of viability, are the mean value of two independent experiments with each treatment performed in triplicate. Negative control: without CQDs. (b) Fluorescence microscopy of HeLa and U-87 MG cells treated with 400 µg mL -1 of CQDs for 24 h The blue signal corresponds to the nuclei stained with Hoechst 33258, while the green signal is attributed to CQDs. Scale bars = 100 µm.

Figure S8 :

 S8 Figure S8: Fibril formation study of type I collagen using turbidity data: UV-vis absorption evolution of collagen (0.3 mg mL -1 ) at 313 nm in PBS buffer (pH 7.4) at 37 °C under static condition (blue) and in the presence of increasing concentrations of gold nanoparticles (AuNPs) (10 11 -10 13 AuNPs mL -1 ).

  least 1 h. The formed AuNPs were washed by centrifugation (10 min at 13000 g), resuspended in an aqueous solution of HA (2-4 pM), and kept under rotation overnight. HA-coated AuNPs were finally washed by centrifugation, resuspended in water, and stored for further use. The synthetized NPs were characterized by DLS and zeta potential (Zetasizer Nano, Malvern)

Fluorescence

  recovery after photobleaching (FRAP) experiments were performed to determine the mobility of the fluorescent CQDs in water and bovine vitreous. FRAP experiments were performed using a confocal scanning laser microscope (Nikon A1R) equipped with a 10× lens (CFI Plan Apochromat; Nikon, Badhoevedorp, The Netherlands). In a FRAP experiment, fluorescent nanoparticles in a (vitreous) sample are irreversibly bleached in a small area (100 µm) by an intense laser beam. The surrounding particles (i.e. outside of the bleached zone) will diffuse into the photobleached area, leading to a fluorescence recovery over time. Diffusion coefficients (D) can be derived from the rate and amount of recovery of fluorescence in the

Table 1 :

 1 Physico-chemical properties of CQDs.

	CQD-1	CQD-2

Acknowledgements


Adrien Le Navenant

Corinne Brouchoud

Yvan Capowiez

Professor Magali Rault
email: magali.rault@univ-avignon.fr

Adrien Le

Séverine Suchail

Carlo Duso
email: carlo.duso@unipd.it

Michelle Fountain
email: michelle.fountain@emr.ac.uk

Vincent P Jones
email: vpjones@wsu.edu

Herman Helsen
email: herman.helsen@wur.nl

Daniel Cormier
email: daniel.cormier@irda.qc.ca

Professor Jay Gan Co

Professor Jay Gan

How lasting are the

Keywords: European earwigs, energy reserves, biometry, pesticides, pest management strategy European earwigs; energy reserves, biometry, pesticides, pest management strategy

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

HIGHLIGHTS

 The long-lasting effects of pesticide treatments in orchards on earwigs are unknown  Morphometric traits are the lowest in earwigs sampled in conventional orchards  Females appeared to fair better under pesticide pressure to ensure reproduction  Management strategy did not impact earwig growth according to Dyar's rules  Under relaxed pesticide pressure during development energy reserves increased *Highlights (for review : 3 to 5 bullet points (maximum 85 characters including spaces per bullet point)

HIGHLIGHTS

 The long-lasting effects of pesticide treatments in orchards on earwigs are unknown  Morphometric traits are the lowest in earwigs sampled in conventional orchards  Females appeared to fair better under pesticide pressure to ensure reproduction  Management strategy did not impact earwig growth according to Dyar's rules  Under relaxed pesticide pressure during development energy reserves increased

INTRODUCTION

Agricultural production needs to increase to meet the needs of a growing world population. Doing so in ways that do not compromise environmental integrity and public health is still a great challenge [START_REF] Gorbach | Antimicrobial use in animal feed-time to stop[END_REF][START_REF] Tilman | Agricultural sustainability and intensive production practices[END_REF]. However, intensive production systems and widespread use of pesticides to control crop pests still dominate, with adverse side effects on the environment [START_REF] Köhler | Wildlife Ecotoxicology of Pesticides: Can We Track Effects to the Population Level and Beyond?[END_REF]. Pesticides have an impact at all levels of biological organization from neurotoxicity, immunotoxicity, metabolism to behavior, which could induce cascade events at higher levels [START_REF] Köhler | Wildlife Ecotoxicology of Pesticides: Can We Track Effects to the Population Level and Beyond?[END_REF]. Such practices reduce biodiversity, affect ecosystem functioning and the production of goods and services by non-target organisms [START_REF] Mace | Biodiversity and ecosystem services: a multilayered relationship[END_REF]. This is particularly the case in apple orchards which are highly productive crops with, an average of 30 pesticide applications per year in Provence, South-Eastern France [START_REF] Sauphanor | Protection phytosanitaire et biodiversité en agriculture biologique[END_REF][START_REF] Mazzia | The impact of management strategies in apple orchards on the structural and functional diversity of epigeal spiders[END_REF]. Among these treatments, organophosphorus (OP), neonicotinoid and synthetic pyrethroid compounds are the most used insecticides in Integrated Pest Management (IPM) strategies. Today, there is general agreement among the public and growers, that crop protection should rely less on the use of synthetic pesticides and more on agro-ecology principles [START_REF] Marliac | Impact of crop protection strategies on natural enemies in organic apple production[END_REF][START_REF] Jones | From planning to execution to the future: An overview of a concerted effort to enhance biological control in apple, pear, and walnut orchards in the western U.S. Biological Control, Enhancing biological control in western orchards: a five-year project 102[END_REF]. Among these principles, biological control through the utilization and enhancement of natural enemies is of prime importance [START_REF] Beers | Nontarget effects of orchard pesticides on natural enemies: Lessons from the field and laboratory[END_REF]. In order to improve the delivery of ecosystem services, we need to identify the effects of factors that could explain the variability in the response of arthropod communities involved in such services.

Among those factors, pesticides, beside their lethal effect, induce sublethal physiological or behavioral effects, which may cause long-term effects on the population dynamics of natural enemies, and consequently on pest regulation [START_REF] Biondi | Do biopesticides affect the demographic traits of a parasitoid wasp and its biocontrol services through sublethal effects?[END_REF].

The European earwig, Forficula auricularia L. (Dermaptera: Forficulidae), is a natural enemy in apple orchards. They are effective generalist predators involved in the regulation of pests such as aphids, leaf rollers and psyllids [START_REF] Dib | Effect of codling moth exclusion nets on the rosy apple aphid, Dysaphis plantaginea, and its control by natural enemies[END_REF][START_REF] Dib | Predation potency and intraguild interactions between generalist (Forficula auricularia) and specialist (Episyrphus balteatus) predators of the rosy apple aphid (Dysaphis plantaginea)[END_REF][START_REF] Orpet | Field evidence and grower perceptions on the roles of an omnivore, European earwig, in apple orchards[END_REF]. The abundance and diversity of earwigs could be influenced either by practices such as tillage and hedge quality [START_REF] Sharley | The effects of soil tillage on beneficial invertebrates within the vineyard[END_REF][START_REF] Moerkens | Natural and human causes of earwig mortality during winter: temperature, parasitoids and soil tillage: Earwig mortality during winter[END_REF] or by pesticides use (French-Constant and Vickerman, 1985;Malagnoux et al., 2015a). Moreover, their ease of capture and identification makes them an interesting potential bioindicator of the possible detrimental effects of orchard management. Exposure to sublethal concentrations of pesticides may affect F. auricularia behavior. Diflubenzuron has been shown to be highly toxic to F. auricularia, affecting its weight, mobility and predatory activity [START_REF] Sauphanor | Side effects of diflubenzuron on a pear psylla predator: Forficula auricularia[END_REF]. More recently, Malagnoux et al. (2015b) reported that Spinosad (used in organic orchards), acetamiprid (neonicotinoid) and chlorpyrifos-ethyl (organophosphorus) have significant negative effects, on the predatory behavior of adult F. auricularia earwigs.

Beside direct impacts, long term pesticide exposure is responsible for inducing tolerance to OP insecticides in F. auricularia [START_REF] Navenant | Metabolic mechanisms and acetylcholinesterase sensitivity involved in tolerance to chlorpyrifos-ethyl in the earwig Forficula auricularia[END_REF]. Insecticide tolerance and/or resistance can be associated with fitness costs [START_REF] Rivero | Energetic Cost of Insecticide Resistance in Culex pipiens Mosquitoes[END_REF], and this explains why susceptible genotypes increase in frequency when insecticide pressure is relaxed [START_REF] Foster | Insecticide resistance[END_REF].

Resistance to OPs insecticides was observed to lead to fitness costs in the dipteran Culex pipiens (Culicidae), with an impact on predation activity [START_REF] Berticat | Insecticide resistance genes confer a predation cost on mosquitoes, Culex pipiens[END_REF], mating [START_REF] Berticat | Insecticide resistance genes induce a mating competition cost in Culex pipiens mosquitoes[END_REF], or fecundity and development [START_REF] Berticat | Costs and benefits of multiple resistance to insecticides for Culex quinquefasciatus mosquitoes[END_REF] in resistant strains. The diversion of the energy flow is the consequence of trade-offs, resulting in the reallocation of energetic resources from primary functions (growth, reproduction) to maintain secondary functions (overproduction of detoxifying enzymes). Such a reallocation of energy reserves induces a negative impact on life history traits [START_REF] Ribeiro | Energy storage systems for advanced power applications[END_REF][START_REF] Da Silva | Developmental stress by diflubenzuron in Haematobia irritans (L.)(Diptera: Muscidae)[END_REF]. In insects, life history traits have been shown to be linked to various environmental changes such as habitat loss (Öckinger et al., 2010), climate change [START_REF] Musolin | Insects in a warmer world: ecological, physiological and life-history responses of true bugs (Heteroptera) to climate change[END_REF] and agricultural management strategies [START_REF] Kliot | Fitness costs associated with insecticide resistance[END_REF].

Glycogen and the lipid triacylglycerol are the predominant energy reserves in animal cells. Both are stored in the fat bodies distributed throughout the body of the insect with triglycerides representing more than 50% of their dry weight [START_REF] Arrese | Insect Fat Body: Energy, Metabolism, and Regulation[END_REF]. Because glycogen can be quickly hydrolyzed to glucose in the hemolymph of insects, its content may vary depending on physiological status and/or environmental conditions [START_REF] Anand | Age-dependent changes of fat body stores and the regulation of fat body lipid synthesis and mobilisation by adipokinetic hormone in the last larval instar of the cricket, Gryllus bimaculatus[END_REF][START_REF] Chowanski | Cold induced changes in lipid, protein and carbohydrate levels in the tropical insect Gromphadorhina coquereliana[END_REF]. Triacylglycerol also provides energy during diapause for organism development and to sustain flight activity [START_REF] Van Der Horst | Insect adipokinetic hormones: release and integration of flight energy metabolism[END_REF]. Proteins produce energy through catabolism and are essential in tissue building, repair and protection. All of these reserves are used for growth, maintenance and reproduction [START_REF] Arrese | Insect Fat Body: Energy, Metabolism, and Regulation[END_REF].

Recently, we reported a severe depletion in energy reserves associated with lower weights and lower values for morphometric parameters in the earwig F. auricularia sampled in IPM orchards compared to those sampled in organic apple orchards [START_REF] Suchail | An exploratory study of energy reserves and biometry as potential tools for assessing the effects of pest management strategies on the earwig, Forficula auricularia[END_REF]. However, further points remain to be addressed to accurately assess the impact of pesticide pressure on F. auricularia. Firstly, IPM management covers a wide range of practices [START_REF] Barzman | Eight principles of integrated pest management[END_REF][START_REF] Caffi | Milticriteria evaluation of innovative IPM systems in pome fruit in Europe[END_REF][START_REF] Dumont | Transitions in agriculture: three frameworks highlighting coexistence between a new agroecological configuration and an old, organic and conventional configuration of vegetable production in Wallonia (Belgium)[END_REF] and little is known concerning the physiological effect in either the earwigs subjected to such a gradient of practices or in their progeny. Do all the IPM management approaches used in apple orchards have effects that impair energy reserves and morphometric sizes? Secondly, following energy reserve depletion, are the earwigs able to rapidly restore their own energy reserves once the pesticide treatment period is over (e.g. in autumn)? And would these impacts have a visible effect on the next generation? In this study, we aimed to assess the potential recovery of energetic reserves, modification of body weight and morphometric parameters, in two succeeding generations of earwigs with sex distinction. The organisms from the first generation were collected in late October in organic and IPM orchards with different levels of pesticide use. The next generation of these earwigs was obtained under a laboratory-rearing program, in the absence of pesticide exposure.

MATERIALS AND METHODS

Chemical compounds

Sodium chloride, Tris-HCL, trichloroacetic acid, ethanol, oyster glycogen, glucose, sodium acetate, amyloglucosidase from Aspergillus niger, phosphovanilline, sulfuric acid and bovine serum albumin were purchased from Sigma-Aldrich® (Saint Quentin-Fallavier, France). The glucose GOD-PAP kit was purchased from BioLabo SAS® (Maizy, France)

2.2.Characterization of crop management strategies in the apple orchards

The study was carried out in the South-East of France, where 24% of French apple orchards are concentrated (Agreste, 2019). To describe the diversity of protection management strategies within the orchards, the following characteristics were identified and recorded: pesticide usage (including microbial and classical insecticides) and the use of alternative strategies (presence of nets, sexual confusion). According to these characteristics, nine apple orchards were chosen in a 20 km circular region around Avignon (France) and were separated into three groups: organic, IPM and conventional orchards, with three orchards per group. Organic orchards were group one, where only "natural" pesticides are authorized. Those orchards were managed following organic production guidelines where no synthetic pesticides or fertilizers were used. The main pesticides used in organic apple production are copper and sulphur (fungicides), mineral oils as well as neem against aphids and Spinosad and microbial insecticides (granulovirus and Bacillus thuringiensis) against codling moth, Cydia pomonella L. (Lepidoptera: Tortricidae). The second group included three orchards classified as IPM where in addition to the use of sexual confusion, chemical insecticides are applied sparingly according to damage thresholds; the third group was composed of three orchards considered as conventional exhibiting higher rates of insecticide applications than regular IPM. In those orchards sexual confusion against codling moths was not used.

For each orchard, treatment calendars were recorded and analyzed to compute the overall treatment frequency index (TFI) as the total number of pesticide treatments per hectare with commercial products, weighted by the ratio of the dose used to the recommended dose [START_REF] Jørgensen | Denmarks action plant for pesticides -status and role of research[END_REF]. However, since organic farming uses different kinds of pesticides, the TFI was determined for insecticides depending on their composition, microbial and organic insecticides were pooled together and compared to synthetic ones.

where n is the total number of insecticides applied in one year in an orchard, ADi is the amount of each insecticide applied and HD is the recommended amount per hectare.

2.3.Earwig sampling and rearing

Male and female F. auricularia adults were sampled in October 2015 in the nine orchards using cardboard traps placed on apple tree trunks. Since the F. auricularia species is univoltine [START_REF] Dib | Report on the life history traits of the generalist predator Forficula auricularia (Dermaptera: Forficulidae) in organic apple orchards in southeastern France[END_REF], adults were of roughly the same age. Organisms were either directly stored at -20°C until biochemical and morphometric measurements were carried out (20 for each sex), or reared in an outdoor laboratory, under shelter and submitted to natural photoperiod, temperature and humidity, with no pesticide exposure. Twenty females per orchard were used for earwig breeding. All the individuals were fed ad libitum, regardless of the orchards group from which they were collected. Nymphs at the two last life-stages before reaching adulthood (N3 and N4), and adults (male and female) of the new generation (October 2016) were randomly chosen and withdrawn from the rearing. Ten earwigs per stage (N3, N4, female and male) were thus stored at -20°C for morphometric and reserve contents analysis.

2.4.Morphometric measurements

Frozen earwig specimen were slightly thawed to avoid any change in morphometric parameters, and dried. Then, they were weighed individually using a XT0120A Precisa® electronic precision balance with an accuracy of 0.01 mg. All morphometric measurements were carried out using an eyepiece micrometer on a Nikon® binocular microscope. Measurements were first recorded as a unit of the micrometer scale to the nearest 1 mm and were converted into millimeters (± 0.001 mm). Each earwig was positioned carefully to avoid bias in measurement and to achieve the same plan of view. A single person carried out all measurements to avoid user bias. For adults (males and females) and nymphs, we measured the maximum width of the prothorax, inter-eye and telson and the length of the left femur of the third pair [START_REF] Suchail | An exploratory study of energy reserves and biometry as potential tools for assessing the effects of pest management strategies on the earwig, Forficula auricularia[END_REF]. The growth of earwig nymphs was assessed through the increase in body weight, femur length and prothorax width by calculating the ratio between two successive life-stages (N4/N3 or adult/N4). Only males were used to calculate the ratio between adult life-stage and N4 to avoid errors due to the possible presence of premature eggs in the females.

Adults from apple orchards and from breeding were termed F0 or F1 earwigs, respectively.

2.5.Crude extract preparation and biochemical measurements

After the morphometric assessment, the whole earwig body was homogenized on ice in 10% (w/v) low-salt buffer containing 10 mM Tris-HCL (pH 7.3) and 10 mM NaCl and centrifuged for 10 min at 3000 x g according to [START_REF] Suchail | An exploratory study of energy reserves and biometry as potential tools for assessing the effects of pest management strategies on the earwig, Forficula auricularia[END_REF]. The supernatant was used for further glycogen, lipid and protein measurements.

To determine glycogen content, the method based on enzymatic hydrolysis of glycogen by amyloglucosidase (EC 3.2.1.3) was used (Parrou and Francois, 1997). Solution trichloroacetic 4% acid was added for deproteinization to 250 µL, 70 µL and 30 µL of crude extract (v/v) for adults, N4 and N3, respectively. The solution was centrifuged at 3000 x g for 1 min at 4 °C. Two volumes of 95% ethanol were added to precipitate glycogen that was present in the supernatant.

Glycogen was finally pelleted by centrifugation at 5000 x g for 5 min at 4 °C. Ethanol was removed and the pellet was dried for 2 h at room temperature. Once dried, the glycogen pellet was incubated for 2 h at 60 °C in 500 µL (adults) or 100 µL (nymphs) 0.2 M sodium acetate pH 5.2 containing 7 IU (International Units) of amyloglucosidase. After incubation, the solution was cooled on ice and the amount of glucose generated from glycogen was determined in each sample using the Glucose GOD-PAP method adapted to a 96-well microplate format. The Solution mixture containing 250 µL of Glucose GOD-POD and 25 µL of sample, was left to stand for 20 min at room temperature before glucose was detected by measuring the absorbance at 505 nm.

The amount of glucose was calculated from a standard curve (A505 = f[glucose]) containing pure glucose as a standard treated with the same conditions. Because the final values were included, the amount of glycogen was corrected for the glucose content in samples that were not incubated with amyloglucosidase.

Total lipids were determined according to [START_REF] Frings | Improved Determination of Total Serum Lipids by the Sulfo-Phospho-Vanillin Reaction[END_REF] adapted to a 96-well microplate format [START_REF] Suchail | An exploratory study of energy reserves and biometry as potential tools for assessing the effects of pest management strategies on the earwig, Forficula auricularia[END_REF]. Concentrated sulfuric acid (298 µL) was added to either 2 µL of sample or 2 µL of standard solution (olive oil). The solutions were homogenized and placed in water at 98 °C for 10 min and cooled on ice for 5 min. A 700 µL of phospho-vanillin reagent was added to each extract or lipid standard. After homogenization and incubation at 37 °C for 15 min, samples were cooled on ice and the absorbance was recorder at 540 nm.

The protein content was determined by the Lowry method modified according to [START_REF] Markwell | A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples[END_REF], with bovine serum albumin as a standard.

The energy reserve values were directly derived from a standard curve prepared with known concentrations of glycogen, lipid or serum albumin. All assays were conducted in triplicate and the above components were express as milligrams per gram of earwig.

Statistical analysis.

All data analyses were carried out using Software; R version 3.4.3 and RStudio version 1.1.423 (R Core Development Team, Vienna, Austria). The significance of all results was tested using permutation tests due to non-respect of normality and homoscedasticity. We used the 'lmPerm' package [START_REF] Wheeler | Package lmPerm[END_REF] and the function aovp combined with the function pairwise.perm.t.test from RVAideMemoir package. "Fdr" adjusted p-values were used due to the risk of multiple comparisons. ANOVA were used to test the effects of sex and management strategy (organic, IPM and conventional).

RESULTS

Pesticide applications

Only organic and microbial insecticides were applied in organic orchards with a TFI of about 6.6 compared to IPM (2) and conventional (1) orchards. Both IPM and conventional orchards used synthetic insecticides with an average TFI of 4.5 and 7.6, respectively. However, considering the total TFI, no significant differences were observed between management strategies (Figure 1).

Forficula auricularia body weight

No significant differences in the weight of adult males (p-value=0.965) or females (p-value=0.692) were observed among management strategies. However, regardless of the protection strategy factor, the earwig generation obtained after one year in laboratory conditions (F1) exhibited significant higher weights than the earwigs sampled in apple orchards (F0) (p-value<0.001; Figure 2). Whatever the orchard management or the generation of earwigs, females were significantly heavier (p-values<0.05) (49.91.7 mg (for F0) and 61.82.1 mg (F1)) than males (46.71.7 mg (F0) and 53.41.5 mg (F1)).

Morphometric traits and growth

Among the morphometric parameters measured, telson width was the only one that was significantly different between males and females (p-value<0,0001), either for earwigs sampled in orchards (F0) or for reared earwigs (F1). In both cases, males exhibited larger telson than females at about 37 % and 20 % for F0 and F1 adult's earwigs, respectively (Table 1). Orchards characteristics did not appear to affect telson width.

Femur length (and the width of prothorax, data not shown) was significantly different depending on the orchards groups from which earwigs were collected with lower values in conventional orchards (p-value<0.0001; Figure 3). No significant differences in these parameters were observed between the sexes in earwigs that were field sampled (p-values equivalent to 0.888, 0.058 and 0.995 for organic, IPM and conventional orchards, respectively, for femur length as an example). However, F0 earwigs had significant longer femurs (p-value<0,05) when collected from organic (2.206±0.135 mm) or IPM orchards (2.270±0.138 mm), compared to earwigs caught in conventional orchards (2.104±0.130 mm) (Figure 3). In the progeny earwigs (F1), we observed a homogenization in the femur length and prothorax width data for almost all the adult earwigs. The exception was the femur length of F1 females whose parents were previously sampled in conventional orchards, with these exhibiting a significantly smaller sized femur (p-value<0.0001, Table 2).

The management practices did not have an impact on earwig growth (Table 2). The mean ratio calculated between two successive life-stages for body weight was 2.02±0.76 between N3 and N4, and 2.62±0.40 between N4 and adults (Table 2). It is interesting to note that for all the earwigs, regardless of their origin, the growth of femur length and prothorax width obeyed Dyar's law with an average growth ratio of 1.43±0.07 and 1.33±0.09 from N3 to N4 respectively or 1.23±0.06 and 1.10±0.06 from N4 to adults, respectively.

Energy reserves

Energy reserves in the earwigs were found to depend on sex and management strategies. For the F0 earwigs, a higher lipid content was measured in females collected from organic orchards (p-values<0.0001). F0 females (IPM and conventional groups) and F0 male earwigs (all studied groups), contained the same amount of lipids (Figure 4A). Concerning glycogen contents, we observed a significant higher glycogen content for F0 females collected in conventional compared to organic and IPM orchards (p-value<0.05) 4C). Overall, males exhibited higher glycogen contents than females, with the highest significant value in those from organic orchards (p<0.0001) and the lowest in those from conventional orchards, which was also similar to females from conventional orchards (p-value=0.987; Figure 4C). No significant differences were observed for protein content (data not shown) with values close to 58 mg. g -1 of earwig, for both F0 and F1. A slight, but non-significant, reduction in protein content was measured in males sampled in orchards compared to those reared in the lab (p-value=0.053; data not shown).

Except for protein content, it is noteworthy that energy reserves in all the earwigs reared under our breeding program (generation F1) were higher than in their parents. Glycogen was approximately four times higher and lipids twice as high in F1 than F0 (4B, 4D). In addition, both glycogen and lipid contents reached similar levels in all F1regardless of F0 provenance.

DISCUSSION

In this study we aimed to highlight the potential benefit of an interruption in synthetic insecticide exposure on F. auricularia earwig parents as well as their progeny. Physiological, behavioral or morphological trade-offs occur in insects allowing them to adapt to environmental changes due to agricultural practices (pesticide usage and habitat loss). Such strategies are developed to minimize negative environmental effects but they are costly to the organism in terms of metabolic resources and energy demand [START_REF] Calow | Physiological costs of combating chemical toxicants: Ecological implications[END_REF]. Because an organism's energy resources are generally limited, agricultural management practices can lead to additional metabolic costs and modify energy reserves and/or morphometric parameters [START_REF] Congdon | Resource allocation-based life histories: A conceptual basis for studies of ecological toxicology[END_REF][START_REF] Yasmin | Effects of Pesticides on the Growth and Reproduction of Earthworm: A Review[END_REF]. Firstly, our results showed that there were no significant differences in weight for earwigs sampled from the orchards under different management practices in late October. The main difference observed for fresh weight was sex determined with females always being heavier than males, either for F0 or later for F1 earwigs. Our previous study reported contrasting results for measurements on earwigs sampled in the same orchards in July. In that latter study, earwigs from organic orchards were the heaviest and females were lighter than males [START_REF] Suchail | An exploratory study of energy reserves and biometry as potential tools for assessing the effects of pest management strategies on the earwig, Forficula auricularia[END_REF].

There are several explanations as to why time-specific and sex-specific differential responses could be observed. Firstly, earwigs are omnivores and can feed on several types of plant (leaves, flowers) and animal matter. Insecticides treatments stop at the end of August in apple orchards [START_REF] Sauphanor | Protection phytosanitaire et biodiversité en agriculture biologique[END_REF], thus from then the earwigs could return to their normal foraging behavior. Increased food availability and consumption could have contributed to body weight increases in earwigs from the conventional and IPM orchards which compensated for differences between management strategies. Secondly, the earwig life cycle and the sampling period (July in [START_REF] Suchail | An exploratory study of energy reserves and biometry as potential tools for assessing the effects of pest management strategies on the earwig, Forficula auricularia[END_REF] versus October in the present study) could account for sex-specific differential responses. According to [START_REF] Albouy | Dermaptères ou Perce-oreilles[END_REF] and [START_REF] Sauphanor | Side effects of diflubenzuron on a pear psylla predator: Forficula auricularia[END_REF], female earwigs start their oogenesis in late summer/early autumn and most were pregnant in October. A visual check during dissection confirmed that most of the females from both generations investigated (F0 and F1) were full of pre-mature eggs in October. Females also cease feeding between egg laying and egg hatching [START_REF] Kölliker | Benefits and costs of earwig (Forficula auricularia) family life[END_REF][START_REF] Meunier | One clutch or two clutches? Fitness correlates of coexisting alternative female life-histories in the European earwig[END_REF] and they continuously groom their eggs to prevent the growth of pathogenic fungi, a behavior crucial to reproductive success [START_REF] Boos | Maternal care provides antifungal protection to eggs in the European earwig[END_REF][START_REF] Diehl | Surrounding pathogens shape maternal egg care but not egg production in the European earwig[END_REF]. Over the course of winter, we can assume that females need to accumulate resources to sustain themselves and ensure their maternal care. In contrast, males live for a shorter time than females with a lifespan of eight compared to 18 months, respectively [START_REF] Ratz | The population determines whether and how life-history traits vary between reproductive events in an insect with maternal care[END_REF]. They invest most of their energy into mating and die in late winter or early spring [START_REF] Lamb | Life history and population characteristics of the European earwig Forficula auricularia (Dermaptera: Forficulidae), at Vancouver British Columbia[END_REF][START_REF] Albouy | Dermaptères ou Perce-oreilles[END_REF][START_REF] Dib | Report on the life history traits of the generalist predator Forficula auricularia (Dermaptera: Forficulidae) in organic apple orchards in southeastern France[END_REF]. This differential life-history trait could account for the sexspecific responses we observed in earwigs' body weight as well as energy reserves (see below). Interestingly, we observed that earwigs from IPM orchards displayed characteristics close to those of earwigs collected in organic orchards (regarding morphometric traits, and glycogen content), with the exception of lipid content in female earwigs. Earwigs sampled in conventional orchards had shorter femurs suggesting that a high historic level of insecticide exposure is needed to influence body shape of these organisms. These findings are consistent with a decrease in body size and elytron length seen for the carabidae, Calathus fuscipes, sampled in potato fields treated with the larvicide lambda-cyhalothrin [START_REF] Giglio | Impact of agrochemicals on non-target species: Calathus fuscipes Goeze 1777 (Coleoptera: Carabidae) as model[END_REF] and with previous analyses on earwigs [START_REF] Suchail | An exploratory study of energy reserves and biometry as potential tools for assessing the effects of pest management strategies on the earwig, Forficula auricularia[END_REF]. However, other studies failed to highlight changes in morphological size under insecticide exposure, as for C. pipiens mosquitoes [START_REF] Taskin | Seasonal dynamics of insecticide resistance, multiple resistance, and morphometric variation in field populations of Culex pipiens[END_REF] as an example.

Moreover, laboratory exposure to insecticides did not induce any morphometric variations in Drosophila melanogaster Meigen (Diptera: Drosophilidae) [START_REF] Marcus | Atrazine exposure affects longevity, development time and body size in drosophila melanogaster[END_REF]. These latter results, together with ours, point out the absence of a relationship between single insecticide exposure and insect morphometric traits. They also revealed that even if pesticides are mostly sprayed during periods when insects are developing, high levels of repeated pesticide exposure (such as those found in conventional orchards) are needed to shape morphological traits in adults.

It is noteworthy that we did not observe a legacy effect on second-generation earwigs since values for morphometric traits measured in F1 earwigs did not differ according to the insecticide exposure history of their parents. The absence of pesticide exposure and the unlimiting food provided during earwig breeding allowed them to reach about the same body size. These findings are in agreement with previous studies of the effect of exposure to Spinosad at the adult stage on the body size of offspring, in the yellow dung fly Scathophaga stercoraria L. (Diptera: Scathophagidae) [START_REF] Mahdjoub | Fitness consequences of the combined effects of veterinary and agricultural pesticides on a non-target insect[END_REF]. Another interesting point is a weak but significant decrease in telson width for F1 males compared to F0 males. This could be explained by captivity which provided a less hostile environment compared to the realistic environment conditions in orchards. The telson shape is generally related to forceps shape [START_REF] Albouy | Dermaptères ou Perce-oreilles[END_REF]. It is well known that male forceps are used as weapons in male contests for accessing to females, which also choose males based of their forceps length [START_REF] Rantala | Forceps size and immune function in the earwig Forficula auricularia L[END_REF]. Telson structure plays thus an important role in impact-resistance during contest, and such multifunctional structures of sexual ornaments can limit the development of other morphological traits [START_REF] Elmen | Costs and the diversification of exaggerated animal structures[END_REF]. We overall questioned whether the observed effects on the offspring are due to captivity and/or rearing conditions rather than the exposure history of their parents. Previous results pointed out that the development of macrolabic males (large earwigs with longer forceps) was not fully related to a favorable diet or rearing density [START_REF] Tomkins | Environmental and genetic determinants of the male forceps length dimorphism in the European earwig Forficula auricularia L[END_REF]. To disentangle which parameters are having the most impact on morphometric traits further experiments are needed with varying rearing conditions, diets, pesticide exposure and safety in captivity.

Energy reserves give an insight into rapid physiological responses. When F. auricularia were collected in July, both glycogen and lipid contents were lower in the earwigs from IPM apple orchards than from organic orchards [START_REF] Suchail | An exploratory study of energy reserves and biometry as potential tools for assessing the effects of pest management strategies on the earwig, Forficula auricularia[END_REF]. According to the treatment calendars, July is the peak of pesticide usage as they are applied from late March to late August [START_REF] Sauphanor | Protection phytosanitaire et biodiversité en agriculture biologique[END_REF][START_REF] Marliac | Impact of crop protection strategies on natural enemies in organic apple production[END_REF]. At this time, earwigs have to cope with pesticide exposure to survive. As a result, F. auricularia collected in conventional orchards, were shown to exhibit tolerance to chlorpyrifos-ethyl with higher constitutive enzymes activities involved in detoxication mechanisms such as Glutathione-S-transferases and carboxylesterases compared to earwigs from organic orchards (Le [START_REF] Navenant | Metabolic mechanisms and acetylcholinesterase sensitivity involved in tolerance to chlorpyrifos-ethyl in the earwig Forficula auricularia[END_REF]. These latter results highlight the ability of earwigs to develop physiological and biochemical modifications to adapt to high levels of pesticide exposure, which could be costly for the organisms.

The first interesting point observed in F0 earwigs collected in late October (i.e. two months after the last pesticide application) lies in the sex-specific energetic contents, which could be attributed to differential lifespans depending on sex, as specified above. The absence of significant differences in males either for lipid or glycogen contents contrasted with the lower lipid content observed in females sampled in conventional and IPM orchards. Females also contained more glycogen in conventional compared to other orchards. If we assume that females are able to quickly accumulate glycogen reserves to prepare their clutch (up to October), it appears that they could not make up the gap in lipid content. However, when a surplus of carbohydrates is available, pyruvate generated through glycolysis could be used to produce acetyl coenzyme A (acetyl-CoA), a central intermediate for lipid metabolism [START_REF] Wakil | Mechanism of fatty acid synthesis[END_REF]. Then, because lipid content was very low in females, we cannot exclude the possibility that lipids were burned at a faster rate than at which they were produced, resulting in stable lipid loss. In honey bees, mechanisms of stable lipid loss are known to depend either on behavioral maturation and novel regulatory processes [START_REF] Ament | Mechanisms of stable lipid loss in a social insect[END_REF] or on Nosema ceranae infection [START_REF] Li | Chronic Nosema ceranae infection inflicts comprehensive and persistent immunosuppression and accelerated lipid loss in host Apis mellifera honey bees[END_REF]. However, little is known about the molecular mechanisms involved in rapid body mass changes in insects. Establishing whether our observations were sued to stable lipid loss or an absence of lipogenesis will need further analysis. For example, in the parasitic wasp, the incorporation of radioactively marked glucose into lipids showed that lipogenesis did not occur at a sufficient level to increase lipid reserves and compensate for lipid use [START_REF] Giron | Lipogenesis in an adult parasitic wasp[END_REF].

To conclude, our results demonstrate that energy reserves, body weight and morphological parameters can be used to detect the effects of environmental stress in the European earwig, F. auricularia. They suggest that a relaxed period without pesticide exposure allows females to recover energy reserves to ensure reproduction and maternal care from egg-laying to the emergence of juveniles. Females appear to quickly adapt their energetic flow, from maintenance/tolerance to reproduction. We confirmed previous results concerning lower values for some morphometric traits of F0 earwigs collected in highly treated orchards compared to those collected in organic orchards. However, due to the rearing conditions (food given ad libitum), all the F1 earwigs exhibited higher body weights and energy reserve contents than their parents. In addition, all parameters in the F1 earwigs finally reached the same levels regardless of the historic provenance of their parents. In the future, metabolic pathways and specific enzymes involved in lipogenesis and glycogenesis, should be investigated to better understand the physiological and biochemical responses induced by pest management strategies on Forficula species and other important natural enemies. These findings open new research paths to explore the impact of management strategies on fecundity, maternal care and the success of egg hatching and nymph survival. They also pointed out the high variability of practices within IPM strategy and thus when possible TFI should be provided if we want to take this variability into account.

TABLES

Figure 1 :

 1 Figure 1: Treatment Frequency Index. Mean ± standard deviation of the Treatment Frequency

Figure 2 :

 2 Figure 2: F. auricularia body weight (g) depending on sex and generation (parents sampled from

Figure 3 :

 3 Figure 3: Femur length of adult European earwigs (F0) sampled from orchards of different

Figure 4 :

 4 Figure 4: Energy reserves for F0 earwigs collected in orchards (A) Lipid; (C) Glycogen; and for

  Figure 1

Table 1 :

 1 Telson width (mm) (mean  standard deviation) depending on sex, generation (parents sampled from orchards (F0) and their progeny resulting from laboratory rearing (F1) and management strategy in apple orchards. Significant differences between generation and sex are indicated by different letters (p-value<0.0001).

			Organic	IPM	Conventional	Mean
	F0	Male	2.743  0.261 a	2.787  0.218 a	2.749  0.229 a	2.762  0.236 a
		Female	2.003  0.130 c	2.046  0.129 c	1.991  0.112 c	2.018  0.126 c
	F1	Male	2.337  0.149 b	2.434  0.208 b	2.475  0.162 b	2.421  0.127 b
		Female	2.042  0.093 c	2.028  0.120 c	1.962  0.156 c	2.013  0.184 c

Table 2 :

 2 Femur length, prothorax width and ratio calculated between two successive instars for body weight, femur length and prothorax width in the European earwigs, Forficula auricularia resulted from laboratory rearing (F1). The ratio adult/N4 was calculated using the weight and size of the adult males to avoid errors due to the presence of premature eggs in the females. No significant differences were observed in adults depending on sex and/or management strategy, excepted for femur length (*** stands for p-value< 0.0001).

	Management strategy	Organic	IPM	Conventional	Mean  sd
		N4/N3	1.81  0.64	2.21  0.83	1.96  0.69	2.02  0.76
	Body weight
		F1 Adult/N4	3.08  0.47	2.65  0.44	2.23  0.30	2.62  0.40
		N4/N3	1.44  0.08	1.45  0.07	1.39  0.06	1.43  0.07
	Femur length	F1 Adult/N4	1.22  0.07	1.23  0.06	1.23  0.03	1.23  0.06
		F1 Male (mm)	2.115  0.117	2.165  0.104	2.134  0.059	2.142  0.098
		F1 Female (mm)	2.152  0.155	2.170  0.090	1.993  0.140***	-
		N4/N3	1.32  0.10	1.35  0.10	1.32  0.07	1.33  0,09
	Prothorax width	F1 Adult/N4	1.12  0.06	1.11  0.06	1.08  0.06	1.10  0,06
		F1 Adult (mm)	1.736  0.094	1.768  0.088	1.741  0.095	1.751  0.092

ACKNOWLEDGMENTS

The authors warmly thanked Chloé Malik, Barnabé Viala and Agathe Jobe for their useful technical assistance, Leigh Gebbie for English correction, and the Rovaltain Fundation for its financial support to the I-ResPect project. The authors also wish to thank Hazem Dib for valuable discussion and comments. Adrien Le Navenant is very grateful to the Federative Research Structure Tersys for his doctoral fellowship.

Figure 4

Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

*Declaration of Interest Statement