Ph. D Sophie Gautron
email: sophie.gautron@inserm.fr

Trace amine-associated receptor 1 regulates central effects of MAO inhibitors: involvement of tyramine and glutamate

Trace amines constitute a group of endogenous molecules found in both invertebrates and vertebrates, bearing strong similarities with the monoamine neurotransmitters. The physiological significance of these molecules has long remained elusive, until the discovery in 2001 of a large subfamily of G-protein coupled receptors (GPCR) interacting with these compounds, the trace amine-associated receptors (TAAR) [START_REF] Borowsky | Trace amines: identification of a family of mammalian G protein-coupled receptors[END_REF][START_REF] Bunzow | Amphetamine, 3,4-methylenedioxymethamphetamine, lysergic acid diethylamide, and metabolites of the catecholamine neurotransmitters are agonists of a rat trace amine receptor[END_REF]. This major breakthrough spurred a regain of interest for trace amines and their role in the brain. TAAR1, the best-known member of this family, was found to interact with endogenous compounds including the trace amines p-tyramine, β-phenylethylamine, tryptamine and octopamine, the monoamines dopamine and serotonin, the thyroid hormone derivative 3-iodothyronamine (T1AM), as well as several psychoactive compounds like amphetamine [START_REF] Bunzow | Amphetamine, 3,4-methylenedioxymethamphetamine, lysergic acid diethylamide, and metabolites of the catecholamine neurotransmitters are agonists of a rat trace amine receptor[END_REF]. A profusion of studies using synthetic modulators including full and partial agonists and antagonists uncovered the importance of TAAR1 in central functions such as locomotor activity, attention and learning, emotional behavior, drug reward and wakefulness. This multifaceted role was comforted by genetic studies also suggesting its potential implication in schizophrenia, bipolar disorder and addiction (3).

In the current issue of Biological Psychiatry, Mantas et al. [START_REF] Mantas | TAAR1-Dependent and -Independent Actions of Tyramine in Interaction With Glutamate Underlie Central Effects of Monoamine Oxidase Inhibition[END_REF] investigate some of the processes at work in the effects of the monoamine oxidase (MAO) inhibitors in the brain and controlled by TAAR1.

In a first set of experiments, the effects of MAO inhibitors, either tranylcypromine (TCP) or a combination of clorgiline and rasagiline, were compared in WT mice and TAAR1 knockout mice. The systematic administration of these MAO inhibitors led to an increase in antidepressant-like response and in locomotor activity in normal mice. Remarkably, these increases were found significantly stronger in TAAR1 knockout than in WT control mice, indicating that TAAR1 activity negatively regulates the behavioral effects of MAO inhibitors.

Next, to obtain a bird's-eye view of the various perturbations evoked in the brain by chronic administration of TCP, the content of monoamines, diamines and various metabolites was examined by MALDI mass spectrometry (MS) imaging on mouse brain sections. Exploiting the data generated by this powerful approach, the authors pinned-down tyramine as a main culprit in effects of TCP.

Quantification of MALDI-MS imaging indicated that this trace amine was significantly enhanced after a chronic TCP treatment in the striatum of normal and TAAR1 knockout mice, and that this level after TCP was notably higher in TAAR1 knockout mice than in WT mice. Dopamine levels were also increased in this brain area after TCP, but remained unaffected by TAAR1 deletion, suggesting that in conditions of MAO inhibition TAAR1 exerts a negative control selectively on tyramine level.

Interestingly, this enhancement of TCP-induced tyramine levels by TAAR1 deletion was correlated with a similar enhancement of locomotor activity in the TAAR1 mutants.

The neural correlates of the effects of TCP treatment on locomotor activity were next investigated by monitoring expression of the mRNA encoding the immediate-early gene Arc, in the striatum (4). In this region, approximately half of the medium spiny neurons (MSN) population, characterized by the expression of dopamine 1 (D1)-like receptors, project to the substantia nigra (the so-called "direct pathway) and has been shown to control positively locomotor activity. In these experiments, TCP treatment was found to increase arc mRNA expression in the striatum, concomitantly with the increase in tyramine level and locomotor activity. This neural activation, which appears to be due at least in part to increased activation of the D1 (dynorphin+) MSN population, was further increased in the TAAR1 mice mutant, consistently with the observed increase in locomotor activity. TAAR1 and dopaminergic neurons share a complex relationship, and several studies have shown that TAAR1 activation influences dopamine neuron activity. Whether these effects are excitatory or inhibitory is a matter of debate, and might vary depending on the region examined and the experimental paradigm [START_REF] Yang | Dopamine Evokes a Trace Amine Receptor-dependent Inward Current that is Regulated by AMP Kinase in Substantia Nigra Dopamine Neurons[END_REF][START_REF] Lindemann | Trace amine-associated receptor 1 modulates dopaminergic activity[END_REF]. The observation of Arc overexpression in TAAR1 mice mutants(4) is consistent with the view that trace amines via TAAR1 activation tone down DA neuron excitability, thereby decreasing DA release in the striatum. These intriguing findings raised the question of how TAAR1 acts to modulate the nigrostriatal pathway activity. TAAR1 is expressed in monoaminergic circuits but also in brain areas containing glutamatergic neurons [START_REF] Borowsky | Trace amines: identification of a family of mammalian G protein-coupled receptors[END_REF][START_REF] Lindemann | Trace amine-associated receptor 1 modulates dopaminergic activity[END_REF]. Multiple excitatory afferents from various brain regions converge on the substantia nigra (SN), exerting important modulatory effects on dopamine neuron activity [START_REF] Prisco | Group I metabotropic glutamate receptors activate burst firing in rat midbrain dopaminergic neurons[END_REF], while SN dopaminergic neurons themselves are interspersed with glutamatergic neurons [START_REF] Morales | Glutamate neurons within the midbrain dopamine regions[END_REF]. Using the ß galactosidase reporter under the control of the TAAR1 promoter in TAAR1 knockout mice, Mantas et al (4) expand previous anatomical studies of TAAR1 expression by reporting yet unidentified expression sites in glutamatergic regions that send strong projections to the substantia nigra compacta (SNc) and VTA, such as BSTM, LHA, ZI and LPBD. These authors also provide original findings on how TAAR1 and tyramine influence glutamate release in the dopaminergic areas, in ventral midbrain. In a final series of experiments, they investigated using a biosensor-mediated electrochemistry technique the effects of TCP or tyramine on glutamate release in SNc in TAAR1 knockout mice and wild-type controls, using two paradigms. In anesthetized mice, local infusion of TCP in the SN area led to a decrease in glutamate release in this area, measured by FAST recording. This effect of TCP was mimicked by a local infusion of tyramine and appears to implicate TAAR1, since it was abolished in TAAR1 knockout mice and by local application of EPPTB, a TAAR1 antagonist. From these experiments, it could be concluded that TAAR1 was implicated in the negative modulation of glutamate release by TCP. Further reinforcing this demonstration, a similar effect was found using FAST recording in freely moving mice. These last experiments showed that glutamate release was increased by systemic TCP administration in TAAR1 KO mice but not in WT mice, correlated with an increase in locomotor activity. Although it is not clear from these experiments which glutamatergic neuronal populations may be implicated, and whether the glutamatergic terminals themselves within SNc express the TAAR1 protein, they reveal unequivocally the prevalent role of TAAR1 in the SNc in toning-down both glutamate release in this area and ambulation in anesthetized or awake animals, and favor a role of local tyramine. The contributions of the various glutamatergic circuits and populations potentially implicated in these effects of TAAR1 require further investigation. It is important to note that deregulation in glutamate transmission or in the activation of NMDA glutamate receptors was reported also for the cortex and striatum of TAAR1 mutant mice [START_REF] Gainetdinov | Trace Amines and Their Receptors[END_REF], suggesting an extended control of excitatory neurotransmission by TAAR1 in not only dopaminergic nuclei but also dopaminergic projection areas.

Apart from tyramine, MALDI-MSI imaging revealed the influence of TCP treatment on other amines such as histamine, taurine and the melatonin metabolite 6-hydroxymelatonin, whose levels seem also regulated by TAAR1 (4). Obviously, a host of information can be drawn from use of this powerful and innovative tool to examine the effects on other brain areas. Altogether, this original and comprehensive study enriches the knowledge on how TAAR1 acts to balance dopamine-related functions and glutamate signaling in the brain. Inasmuch MAO inhibitors may have not said their last word in the management of depression [START_REF] Suchting | Revisiting monoamine oxidase inhibitors for the treatment of depressive disorders: A systematic review and network meta-analysis[END_REF], the mechanisms disclosed also have a broader relevance for the understanding of the circuits implicated in the action of this class of antidepressants.

Acknowledgments and Disclosures

The author reports no biomedical financial interests or potential conflicts of interest.


Gonzague Agez

Chloé Bayon

Michel Mitov
email: mitov@cemes.fr

Multiwavelength micromirrors in the cuticle of scarab beetle Chrysina gloriosa

Keywords: Arthropod exoskeleton, Beetle cuticle, Chitin, Biological composite, Cholesteric liquid crystals, Structural color, Iridescence, Bio-inspiration, Electron microscopy, Modeling

Beetles from the genus Chrysina show vivid reflections from bright green to metallic silvergold as a consequence of the cholesteric liquid crystal organization of chitin molecules.

Particularly, the cuticle of Chrysina gloriosa exhibits green and silver stripes. By combining confocal microscopy and spectrophotometry, scanning electron microscopy and numerical simulations, the relationship between the reflectance and the structural parameters for both stripes at the micro-and nanoscales are established. Over the visible and near IR spectra, polygonal cells in tessellated green stripes behave as multiwavelength selective micro-mirrors

Introduction

Arthropod cuticles as multifunctional materials

Arthropod cuticles serve a number of fundamental functions; the exoskeletons support the body, resist mechanical loads, provide environmental protection and resistance to desiccation [START_REF] Neville | Biology of the Arthropod Cuticle[END_REF][START_REF] Vincent | Design and mechanical properties of insect cuticle[END_REF][START_REF] Sanchez | Biomimetism and bioinspiration as tools for the design of innovative materials and systems[END_REF]. Beyond these basic functions, the cuticles can also generate optical information with vivid structural colors [START_REF] Berthier | Iridescences[END_REF] or chemical information [START_REF]Insect hydrocarbons: Biology[END_REF]. These more advanced biological functions are of paramount importance in living organisms and understanding their physical properties will inform the creation of novel synthetic functional materials [START_REF] Wang | Stimuli-Directing Self-Organized 3D Liquid-Crystalline Nanostructures: From Materials Design to Photonic Applications[END_REF][START_REF] Guarín-Zapata | Shear wave filtering in naturally-occurring Bouligand structures[END_REF][START_REF] Grunenfelder | Bio-inspired impact-resistant composites[END_REF][START_REF] Wu | Extreme Optical Properties Tuned Through Phase Substitution in a Structurally Optimized Biological Photonic Polycrystal[END_REF][START_REF] Klocke | Material properties of photomechanical infrared receptors in pyrophilous Melanophila beetles and Aradus bugs[END_REF][START_REF] Parker | Biomimetics of photonic nanostructures[END_REF].

The cuticle of beetles is a composite material including chitin [START_REF] Neville | Biology of the Arthropod Cuticle[END_REF]. Owing to their chiral nature, chitin macromolecules may self-organize into a helicoidal structure and produce cholesteric liquid crystal (CLC) phase [START_REF] Neville | Scarabaeid Beetle Exocuticle as an Optical Analogue of Cholesteric Liquid Crystals[END_REF]. As a Bragg medium, the CLC phase may selectively reflect light; the reflection wavelength is directly proportional to the helicoidal Accepted version in: Acta Biomaterialia 2017, 48, 357-367. 3 pitch [13,[START_REF] Belyakov | Optics of chiral liquid crystals[END_REF]. At normal incidence, the reflected light is circularly polarized in accordance with the helix, whereas the transmitted light is polarized with the opposite direction. This reflection of circularly polarized light by beetle cuticles appears to be restricted to Scarabaeidae, occurring predominantly in subfamilies Rutelinae, Scarabaeinae and Cetoniidae [START_REF] Seago | Gold bugs and beyond: a review of iridescence and structural colour mechanisms in beetles (Coleoptera)[END_REF]. Several books and papers have reviewed the biological importance of iridescent colors in the insect world [START_REF] Berthier | Iridescences[END_REF][START_REF] Neville | Biology of fibrous composites[END_REF][START_REF] Lenau | Colours and Metallic Sheen in Beetle Shells-A Biomimetic Search for Material Structuring Principles Causing Light Interference[END_REF][START_REF] Srinivasarao | Yearbook of Science and Technology[END_REF][START_REF] Arwin | Chirality-induced polarization effects in the cuticle of scarab beetles: 100 years after Michelson[END_REF]. In comparison with pigment-based printing, structural colors like those of insect cuticles offer high brightness and saturation, lower cost, easier recycling, and higher reproduction fidelity and stability to applications in the following domains: security marking, information storage, high resolution and selectivity filtering, printing technologies, cosmetics, paints, coatings for vehicles, textile engineering [START_REF] Shen | Functional Polymer Coatings: Principles, Methods, and Applications[END_REF].

Background and literature review

A great number of insects utilize tessellated exocuticles, containing arrays in the form of bumps, pits, or other physical variations. Some representative examples are given by: Manuka beetles [21], June beetles [START_REF] Buhl | Biologically inspired optics: analog semiconductor model of the beetle exoskeleton[END_REF], Plusiotis boucardi [START_REF] Jewell | Circularly polarized colour reflection from helicoidal structures in the beetle Plusiotis boucardi[END_REF], Pyronata festiva [START_REF] Crowe | Which New Zealand Insect?[END_REF], Chlorophila obscuripennis [START_REF] Liu | Inconspicuous structural coloration in the elytra of beetles Chlorophila obscuripennis (Coleoptera)[END_REF], several tiger beetles of the genus Cicindela [START_REF] Seago | Gold bugs and beyond: a review of iridescence and structural colour mechanisms in beetles (Coleoptera)[END_REF][START_REF] Schultz | The Ultrastructure of the Epicuticular Interference Reflectors of Tiger Beetles (Cicindela)[END_REF], Calidea panaethiopica [START_REF] Vigneron | Spectral sideband produced by a hemispherical concave multilayer on the African shield-bug Calidea panaethiopica (Scutelleridae)[END_REF], Chrysochroa fulgidissima [START_REF] Stavenga | Polarized iridescence of the multilayered elytra of the Japanese jewel beetle Chrysochroa fulgidissima[END_REF], Chrysina aurora [START_REF] Hernández-Jiménez | Qualitative correlation Accepted version in[END_REF] or Anomala dimidata [START_REF] Wilts | Natural helicoidal structures: Morphology, self-assembly and optical properties[END_REF]. The cuticle of these animals constitutes a remarkable example of evolution providing a solution to conflicting requests, such as the selectivity of light reflection in a narrow wavelength band and diffusion over a wide angular range. "Jewel scarabs", beetles from the genus Chrysina, exhibit vivid light reflection in a variety of colors from bright green to metallic silver-gold, indicating both selective and broadband reflection [START_REF] Goldstein | Polarization properties of Scarabaeidae[END_REF][32][START_REF] Azofeifa | A quantitative assessment approach of feasible optical mechanisms contributing to structural color of golden-like Chrysina aurigans scarab beetles[END_REF][START_REF] Vargas | Broadening of effective photonic band gaps in biological chiral structures: From intrinsic narrow band gaps to broad band reflection spectra[END_REF]. One of the most spectacular Chrysina species is C. gloriosa (Figure 1a), which can be found from southwestern North America to Central America in pine, pine-oak and juniper forests. C. gloriosa cuticle reflects left-handed circularly polarized light. It includes green and silver stripes which alternate (Figure 1b). This patterning is well-matched to the habitat of C. gloriosa, as the foliage of the juniper tree is green with white resin flecks [START_REF] Thomas | Reflections on golden scarabs[END_REF]. The green stripes feature a polygonal texture which can be observed in bright field unpolarized microscopy (Figure 1c). Each polygonal cell contains a bright yellow core with orange edges surrounded by a dark-green region, with a brighter green rim delimiting the cells. these observations, a lens-like structure was suggested [36], comparable to the patterns, discovered by Bouligand, in the tubercle of crabs [START_REF] Bouligand | Sur l'existence de « pseudomorphoses cholestériques » chez divers organismes vivants[END_REF].

(ii) The reflectance exhibits, at normal incidence, a broad halo from 500 to 600 nm with two peaks at 530 nm (green) and 580 nm (yellow) [START_REF] Goldstein | Polarization properties of Scarabaeidae[END_REF]. (iv) Fernandez del Rio et al. investigated the polarizing properties of the cuticle by using Mueller-matrix spectroscopic ellipsometry [START_REF] Del Rio | Polarizing properties and structural characteristics of the cuticle of the scarab beetle Chrysina gloriosa[END_REF][START_REF] Del Rio | Polarization of light reflected from Chrysina gloriosa under various illuminations[END_REF]. The reflectance was studied under illumination with various polarizations and the dependence of the polarizing properties of the cuticle with the polarization sensitivity of the detector was also characterized [START_REF] Del Rio | Polarization of light reflected from Chrysina gloriosa under various illuminations[END_REF]. The roughness of the green stripe surface was determined to be the source of light scattering [START_REF] Del Rio | Polarizing properties and structural characteristics of the cuticle of the scarab beetle Chrysina gloriosa[END_REF].

Objectives of the study

As a prerequisite to producing bio-inspired artificial replicas from C. gloriosa cuticle, a detailed understanding of structural regularities and irregularities in the cuticle at the nanoscale and, concomitantly, the related optical response is essential. Although the cholesteric texture in the cuticle plane [36,[START_REF] Sharma | Structural origin of circularly polarized iridescence in jeweled beetles[END_REF] or transversely [36,[START_REF] Sharma | Structural origin of circularly polarized iridescence in jeweled beetles[END_REF][START_REF] Del Rio | Polarizing properties and structural characteristics of the cuticle of the scarab beetle Chrysina gloriosa[END_REF], and the polarization properties of the cuticle surface [START_REF] Del Rio | Polarizing properties and structural characteristics of the cuticle of the scarab beetle Chrysina gloriosa[END_REF][START_REF] Del Rio | Polarization of light reflected from Chrysina gloriosa under various illuminations[END_REF] have been investigated, limited knowledge on all the structural and optical parameters necessary for determining the optical response and its origin is available and only phenomenological arguments have been given in literature. The relationship between reflectance and the structural parameters in the bulk of the material, i.e. pitch distribution and orientation of the helicoidal axis; the reflective properties of polygonal cells at multiple scales-from 25 m for a set of four cells down to submicronic areas in the inner core of a single cell; the spatial distribution of cell reflectance with wavelength; its relationship to the cholesteric periodicity as measured in cross-sections observed by scanning electron microscopy (SEM); and its correlation to the simulated optical response of the distinct cuticle areas have not been reported. Besides, in the context of artificial replicas, we show that the polygonal cells, structurally and optically, are not equivalent to focal conic domains found in open synthetic cholesteric films as it is believed in literature: the cuticle has concave cells whereas the artificial films have convex cells.

Materials and methods

1. Sample

The analyzed areas of Chrysina gloriosa cuticle specimen correspond to the elytra (wing covers). A cuticle sample, including green and silver stripes, was removed by using a razor blade. A diamond knife of an ultramicrotome (UltraCut S from Reichert) was then used at room temperature to cut the sample in a transverse direction by aiming for a smooth surface.

Optical micrographs

Details of the elytral surface were photographed with an Olympus BX51 stereomicroscope equipped with an Olympus DP73 digital camera.

SEM

JSM-6490 Scanning electron microscope from JEOL was used (secondary electron imaging mode at 20 kV under normal conditions).

TEM

TEM investigations were performed using a Philips CM30 microscope operating at 300 kV under normal conditions. This means that the dose of electrons received by the specimen is higher than the critical dose, deleting the diffraction contrast, so the images are produced by thickness diffusion contrast subsequent to irradiation. The contrast was enhanced by means of a slight defocusing [START_REF] Pierron | Cholesteric textures observed by transmission electron microscopy in diffraction contrast[END_REF][42][START_REF] Boudet | Glassy cholesteric structure: thickness variation induced by electron radiation in transmission electron microscopy investigated by atomic force microscopy[END_REF].

Reflectance mapping

We used a spectrometer equipped with a standard confocal microscope (XploRA from Horiba) to scan an area of four adjacent polygonal cells. The reflectance baseline was realized by using a flat silver mirror located at the conjugate focal plane of the detector pinhole. 35% of reflectance in the scale bar means the intensity of the light reflected by the structure was 35% of the light reflected by a flat silver mirror. The reflectance spectrum was recorded at every point of the area with a step of 0.5 µm. An XY map contains 49x49 pixels so the scanning area of a map is 24x24µm². One XY reflectance map is related to 2400 reflection spectra. The acquisition was made with a x50 objective: one pixel of the XY map integrated information inside a voxel (volumetric element) with 0.5x0.5x2 µm 3 dimensions.

Simulations methods

To demonstrate the basic role of the cholesteric Bragg grating with variable periodicity from visible to NIR on the wavelength-tunability of a micromirror, numerical simulations were performed by taking into account the Bragg modulation of the index. Pitch gradient, distribution of the orientation of the helicoidal axis, spatial resolution and optical indices matched the experimental features. Simulations were performed by using the finite-difference time-domain (FDTD) method of the Meep software package [START_REF] Oskooi | MEEP: A flexible free-software package for electromagnetic simulations by the FDTD method[END_REF]. This electromagnetic simulation software uses Maxwell's equations and were run over time within a finite computational region. Computational modeling by the FDTD method provides an accurate prediction of the optical response in structure-gradient LC structures [START_REF] Hwang | Computational modeling of the propagation of light through liquid crystals containing twist disclinations based on the finite-difference time-domain method[END_REF]. The topic of complex pitch gradients present in beetle cuticles was recently addressed by Aguilar Gutierrez et al. via a geometric model and a computational visualization tool [START_REF] Aguilar Gutierrez | Structure characterisation method for ideal and nonideal twisted plywoods[END_REF][START_REF] Aguilar Gutierrez | Chiral graded structures in biological plywoods and in the beetle cuticle[END_REF]. The computational grid resolution was 40 pixels per μm. The 2D box size is 1200 pixels along the direction of light propagation and 800 pixels along the transverse direction, corresponding to two adjacent polygons. We imposed absorbing boundary conditions (the present approach utilizes a setup where the computational cell is surrounded with a medium that absorbs light without any reflection). We set the ordinary and extraordinary indices to 1.59 and 1.68 from data published by Caveney [START_REF] Caveney | Cuticle reflectivity and optical activity in scarab beetles: the role of uric acid[END_REF]. Since propagative and contra-propagative waves interfere, it was challenging to separate incident and reflected waves. To overcome this problem, light pulses were used. The incident wave was not monochromatic; a Gaussian beam as a wave train was used. The polarization was linear and orthogonal to the XZ plane. After a transient regime, 400 images of the electric-field energy density E*.D/2 were recorded during 200 periods. The energy density from both the forward and backward (due to Bragg reflection) fields were recorded. This quantity oscillates with a pulsation equal to 2ω. The color scale is proportional to the intensity.

Accepted version in: Acta Biomaterialia 2017, 48, 357-367. 9

Experimental results

Cuticle structure

The cuticle of insects consists of three layers (from the exterior, inwards): the epicuticle (thin and transparent waxy layer), the exocuticle and the endocuticle. The critical cholesteric organization of chitin molecules is present in both the exo-and endocuticle. The SEM investigation of cross-sections of C. gloriosa cuticle demonstrates the typical cholesteric fingerprint texture, with alternate dark and bright lines (Figure 2). The helicoidal axis is perpendicular to the lines and the distance between two consecutive lines of equal contrast is related to the half-pitch of the helices. The distance between two consecutive lines of equal contrast is related to the helicoidal pitch, while the helicoidal axis is perpendicular to the lines. The formation of the exocuticle and endocuticle is usually separated in time by moulting and is recorded structurally as the emergence line.

Green stripe

Inside a green stripe, the fingerprint texture stands as a network of nested arcs in the exocuticle (Figure 2a). This demonstrates that, inside a polygonal cell, the orientation of the helicoidal axis is not constant but, in fact, changing between regions. The curvature of the lines is attenuated in the lowest part of the exocuticle and progressively disappears in the endocuticle. The fingerprint texture in the endocuticle appears as less regular (lines are interrupted) and grainy, similar to the texture observed in the endocuticle of Homarus americanus [START_REF] Raabe | The crustacean exoskeleton as an example of a structurally and mechanically graded biological nanocomposite material[END_REF]. The endocuticle of arthropods is often characterized by a much coarser texture. This texture change, related to a change in the stacking density of cholesteric pseudolayers [50], is accompanied by an abrupt mechanical discontinuity; in the endocuticle, both stiffness and hardness are much smaller than in the exocuticle [START_REF] Raabe | The crustacean exoskeleton as an example of a structurally and mechanically graded biological nanocomposite material[END_REF]. The cholesteric structure presents an increasing pitch from the exo-to the endocuticle. The fingerprint texture exhibits a non-monotonic profile with three distinct regions (Figure 3) as measured along the optical axis marked in in Figure 2a. Each region is associated with a specific optical response: (i) the exocuticle consists of a quasi-constant pitch (~400 nm) to a depth of 6 m, followed by (ii) a slight pitch variation from 400 to 700 nm to a depth of 10-11 m, structurally producing color in the visible spectrum; (iii) a more drastic pitch increase, from 700 to 1100 nm, relating to the end of the visible and into the near IR (NIR) spectrum, is observed in the endocuticle, deeper than 11 m from the surface. Diffuse reflectance-when an incident ray is reflected at many angles, rather than at one angle as in the case of specular reflection-arises in green stripes from a combination of this continuous pitch gradient, with the irregularity of the polygon-studded exocuticle surface below the flat transparent wax layer. The angle-independent selective wavelength reflectance of C. gloriosa arises from these irregularities in the cholesteric region of the cuticle, in contrast to the single-pitch or graded-pitch cholesteric iridescent reflectors found in some insect cuticles where the reflected color changes with the viewing angle. Reflecting a consistent green color and intensity independently of viewing angle may presumably enhance the camouflage function in green foliage.

In the endocuticle, a cholesteric grating with a large-pitch gradient (lowest part in Figure 2a) might serve to avoid overheating. Such thermoregulation seems to be offered to many arthropods-insects and beach-dwelling crabs-whose cuticle exhibits a pitch gradient in the IR range [START_REF] Mitov | Cholesteric liquid crystals with a broad light reflection band[END_REF]. Dorsal coloration has consequences for thermoregulation in diurnal beetles; niche differentiation associated with thermoregulation is documented in tiger beetles in Ref.

[52] and references therein.

In summary, the green stripe consists of a reflector in the exocuticle to display information-color, polarization [START_REF] Del Rio | Polarizing properties and structural characteristics of the cuticle of the scarab beetle Chrysina gloriosa[END_REF][START_REF] Del Rio | Polarization of light reflected from Chrysina gloriosa under various illuminations[END_REF]-in the visible spectrum, and a NIR reflector in the endocuticle supposed to regulate temperature. To manage electromagnetic interactions between the body of the arthropods (cuticle or wings) and the outer world, nature has developed structures adapted to different requirements. It is interesting to draw a parallel between the present cuticle structure and the structure found in chameleon skin [START_REF] Teyssier | Photonic crystals cause active colour change in chameleons[END_REF], by keeping in mind that this parallel has obviously strong limitations since both "coverings" have huge differences in chemical composition and structure. Chameleons have two superimposed populations of iridophores with different morphologies and functions: the upper multilayer is responsible for structural color change through the tuning of nanocrystal spacing in a triangular lattice, whereas the deeper population of cells broadly reflects light in the NIR range. This combination allows some species of chameleons to exhibit dramatic camouflage while moderating the thermal consequences of solar radiation. The combined presence of both reflecting structural elements and mobile absorbing pigments likely allows chameleons to adjust their body temperature in a larger set of conditions than if only pigments or photonic crystals were present.

Silver stripe

The exocuticle in the silver stripes is characterized by pitch increasing with depth (Figure 2b). However, in a singular region at the junction between the exo-and the endocuticle, the pitch is clearly smaller. Such a peculiar region was observed in the cuticle of the insect Tenebrio and reported as the emergence line [START_REF] Caveney | Juvenile hormone and wound modelling of Tenebrio cuticle architecture[END_REF]. The formation of the exocuticle and endocuticle is usually separated in time by ecdysis (moulting), recorded structurally as the emergence line [START_REF] Neville | Biology of the Arthropod Cuticle[END_REF]. Silver stripes are essentially chirped mirrors, i.e. broadband reflectors.

Because the reflected light is directional (i.e. parallel incoming light is reflected into parallel rays), and not scattered, specular reflection is produced, and a metallic appearance with a mirror finish is the result. In silver metallic beetles, the reflector often reflects a broader spectrum than the visible; the human eye is therefore not capable of detecting iridescence.

The bright glare from silver stripes might temporarily blind a potential predator, enabling C. gloriosa beetle to escape [START_REF] Hinton | Some recent work on the colours of insects and their likely significance[END_REF]. It has been postulated that the mirror-like nature of the exocuticle could enable a beetle to match whatever environment it finds itself in; the shiny surface would mirror the green color of its environment and, during the dry season, the beetle could hide among the same leaves when they are brown [START_REF] Hinton | Some recent work on the colours of insects and their likely significance[END_REF]. However, this hypothesis has been disputed by other authors who emphasize that gold and silver species are also found in evergreen tropical cloud forests, indicating separate evolutionary driving forces [START_REF] Thomas | Reflections on golden scarabs[END_REF].

While the cholesteric gratings of both the green and the silver stripes are of comparable scales, the presence of the polygonal patterning in the green stripes serves as an important differentiator. The pitch variations are not strictly superimposable, as it will be shown below, because the pitch has to adapt to the curvature of pseudo-layers in a green stripe. The tessellated surface texture in green stripes contrasts with the flatness of specular reflection in silver stripes and as well as imparts an angle distribution relative to the orientation of the helicoidal axis.

Mapping of light reflection in green stripes

This section presents a local and wavelength-dependent analysis of cholesteric reflections by the tessellated cuticle (Figure 4). An area of four polygonal cells was analyzed via a spectrometer mounted to a confocal microscope (inset of Figure 4). The reflection maps of various wavelengths, from blue (475 nm) to NIR (975 nm), with a narrow wavelength increment of 25 nm is shown. The microscope focus was set to the surface, i.e. the air-wax interface. Intense reflections occur along the sides of the polygons and manifest as donut-like patterns from [475-500] nm to [525-550] nm; this illustrates why the sides of polygons appear green in the optical image (Figure 1c or inset of Figure 4). The donuts extinguish in the 550-575 nm map. After transitional patterns into the next three spectral regions, a bright central spot-at the intersection between the focus plane and the optical axis as marked in Figure 2a-appears in the 650-675 nm range. This spot corresponds to the polygon center and the reflection intensifies into the next two channels, up to 725 nm. The yellow (respectively red) part of the visible spectrum is selectively reflected by the center (respectively sides) of polygons, which explains why a yellow spot with an orange-red contour is visible in the optical image. The reflection of these spots then decreases from the 725-750 nm range. After transitional patterns in between visible and NIR spectra, from 750 to 825 nm, reflection patterns occurring in rims remain visible until the end of the investigation at 975 nm. The In summary: (i) the reflectance is spatially sensitive to wavelength changes, the highest levels of reflectance come from different parts of a polygon-from the sides in the lower (green) end of the visible spectrum and longer wavelengths (orange) from the center; (ii) in the NIR spectrum, from 825 to 975 nm, the variation in reflectance consists of a mean level change weakly sensitive to the patterning. This result is consistent with the structure (Figure 2a) since the cholesteric pseudo-layers in the endocuticle, which generate the NIR reflections, appear continuously parallel; the orientation of the helicoidal axis changes slightly and this deeper part of the cuticle is not sharply tessellated, and thus the reflection pattern is weakly structured. In §3.1.1, we suggested that the endocuticle served as a broadband reflector in the IR spectrum for possible thermoregulation purposes. Additionally, it would be worthwhile to further investigate if crepuscular and nocturnal insects, such as C. gloriosa [START_REF] Young | Notes on the Habits of Plusiotis gloriosa Le conte (Scarabaeidae)[END_REF], use NIR or IR wavelengths for navigation or intra-species communication [START_REF] Hernández-Jiménez | Qualitative correlation Accepted version in[END_REF]. Finally, many green insects show a high NIR reflectance close to that of their habitat foliage [START_REF] Mielewczik | Near-infrared (NIR)-reflectance in insects-Phenetic studies of 181 species[END_REF]; this property could be used by C. gloriosa beetles as camouflage against nocturnal predators capable of detecting IR light.

More generally, the wavelength-sensitivity of the reflection pattern-occurring essentially in the visible spectrum-would benefit from being related to the spectral sensitivity of eyes of C. gloriosa conspecifics as well as predators. For example, different species of Coleoptera are known to show mid-green wavelength sensitivity [START_REF] Lin | A comparative-study on the color-vision of 4 coleopteran insects[END_REF][START_REF] Briscoe | The evolution of color vision in insects[END_REF][START_REF] Dacke | A specialized dorsal rim area for polarized light detection in the compound eye of the scarab beetle Pachysoma striatum[END_REF]. A link might exist between the reflection spectra and the peak sensitivities of the photoreceptors of conspecifics [START_REF] Jewell | Circularly polarized colour reflection from helicoidal structures in the beetle Plusiotis boucardi[END_REF].

Alternatively, the wavelength-dependent patterns could simply be a by-product of light interaction with the chiral nested-arc network (Figure 2a).

Numerical simulations

To calculate the propagation of light across the cuticle, numerical replicas were generated.

Based on the structures observed by SEM, the reflected light response was simulated using the FDTD (finite difference time domain) method and Meep software (see §2.6), and the results were compared to the experimental observations for green and silver stripes.

Accepted version in: Acta Biomaterialia 2017, 48, 357-367.

.1. Green stripe

A transverse graded-pitch grating (Figure 5), occurring in concave layers, was generated from the experimental image. The structure is illuminated with a plane wave Gaussian pulse (centered at 0 with a spectral width 0/0=0.1); the reflected pulse propagating backward is isolated from the forward input beam. Simulated cross-sectional maps of light reflection, accompanied by experimental maps as charted in the cuticle plane, are displayed for four different incident wavelengths in Figure 6. For comparison purposes, the experimental image was situated in a plane perpendicular to the simulated view and through the imaging plane . The simulated grating is superimposed onto the numerically calculated energy density of the electric field. When the wavelength of incident light is equal to 525, 650, 800 and 1000 nm, the basic patterns, donut, bright spot, diffuse spot and continuum background, respectively, found in Figure 4 are produced. The four images are snapshots at different times, tmax, chosen when the backward pulse intensity is maximum in the plane . A small tmax means that the corresponding light is reflected by the pseudo-layers nearest to the surface and, consequently, the forward pulse has propagated over a shorter distance. Moreover, the lack of intensity in the forward pulse indicates which part the polygonal cell (center, side or rim) is responsible for the corresponding reflected light.

The simulations show that the bright central spots at 650 nm are induced by the central part of the very first concave pseudo-layers of the exocuticle at quasi-normal incidence. In that spectral range, it is confirmed that the concave polygon behaves as a spherical micromirror.

The donuts at 525 nm are produced by the sides with oblique incidence resulting in off-axis reflection with a spectral blue-shift. At 800 nm, the diffuse spots are the consequence of a reflection by the lower part of the exocuticle whereas in the IR spectral range the flat structure of the endocuticle is responsible for the continuum background observed at 1000 nm.

There is good agreement between the simulated and experimental maps, confirming the wavelength dependence of the diffuse reflectance and the distribution of the reflected light in the different parts of polygonal cells.

White stripe

Since the silver stripes are not patterned, it is relevant to describe their reflectance properties by using spectral, rather than spatial, maps. Figure 7a shows the variation of the periodicity (distance between two lines of same contrast, i.e. the half-pitch p/2) with the z depth in the cuticle thickness, and Figure 7b shows the related SEM micrograph of the 16-m thick cuticle layer. The graph highlights the presence of a trough in the periodicity variation at z ~ 11 m. We allot this trough to the emergence line (see §3.1.2). Figures 7c and7e are, respectively, the experimental and simulated reflection spectra when the trough is taken into account; the spectrum in Figure 7e was generated from the simulated grating in Figure 7d.

Accepted version in: Acta Biomaterialia 2017, 48, 357-367. 20 The agreement between spectra is good, with broadband reflection occurring from 450 to 1000 nm and exhibiting ripples. The presence of a ripple structure superimposed onto a broadband plateau is also present in the spectrum of the cuticle of the Chrysina aurigans beetle [START_REF] Hernández-Jiménez | Qualitative correlation Accepted version in[END_REF][START_REF] Libby | Light reflection by the Accepted version in[END_REF], where it was interpreted as the consequence of defects in the twisted structure.

These defects are fluctuations in the azimuthal angle, specifying the of the chitin fibrils at a depth z, modulating the reflection spectrum [START_REF] Libby | Light reflection by the Accepted version in[END_REF]. In the case of C. gloriosa, the site of defects is related to the emergence line. Ripples can be attributed to longitudinal interferences as a consequence of the existence of this singular region between exo-and endocuticles. To demonstrate, we simulated the reflection spectrum from a grating exhibiting a continuous pitch gradient without the trough (Figure 7f). The result (Figure 7g) shows a strong attenuation of ripples, and the agreement with the experimental spectrum (Figure 7c) is diminished.

Returning to the pitch variation in the green stripe (Figure 3): region 2 marks an interruption in the variation of increasing pitch and appears to be an intermediate variation; it falls in the thickness range in which the emergence line is expected and region 2 could correspond to the structural features of its presence. The morphology of the emergence line is different (not visible at first glance in Figure 2a vs. a group of few lines of smaller periodicity visible in Figure 2b) and the pitch variation is less abrupt (slow slope in Figure 3 vs. trough in Figure 7a), as a consequence of the nested-arc structure.

Comparison artificial vs. biological structures

In the context of the fabrication of advanced functional materials inspired from C. gloriosa micromirrors, we compare the physical properties of polygonal textures as found in synthetic CLCs and the C. gloriosa cuticle in light of present results.

In 1972, Bouligand studied, via optical microscopy, the geometry and defects of polygonal textures found in mixtures of MBBA (methoxybenzylidene butylaniline; nematic LC) with cholesterol benzoate or Canada balsam as a chiral dopant [START_REF] Bouligand | Recherches sur les textures des états mésomorphes: Les champs polygonaux dans les cholestériques[END_REF]. More recent investigations have used oligomeric films to investigate polygonal textures by AFM (Atomic Force Microscopy) [START_REF] Bunning | Cholesteric liquid crystals: Image contrast in the TEM[END_REF][START_REF] Meister | The anchoring of a cholesteric liquid crystal at the free surface[END_REF][START_REF] Meister | Structure of the cholesteric focal conic domains at the free surface[END_REF][START_REF] Agez | Color selectivity lent to a cholesteric liquid crystal by monitoring interface-induced deformations[END_REF][START_REF] Bitar | Cholesteric liquid crystal self-organization of gold nanoparticles[END_REF][START_REF] Bobrovsky | Features of Double-Spiral 'Valley-Hills' Surface Topography Formation in Photochromic Cholesteric Oligomer-Based Films and Their Changes Under Polarized Light Action[END_REF][START_REF] Bobrovsky | Atomic force microscopy study of surface topography of films of cholesteric oligomer-and polymer-based mixtures with photovariable helix pitch[END_REF][START_REF] Sinitsyna | Peculiarities and mechanism of surface topography changes in photochromic cholesteric oligomer-based films[END_REF][START_REF] Bobrovsky | AFM study of laser-induced crater formation in films of azobenzene-containing photochromic nematic polymer and cholesteric mixture[END_REF][START_REF] Sinitsyna | Surface Relief Changes in Cholesteric Cyclosiloxane Oligomer Films at Different Temperatures[END_REF], TEM [START_REF] Agez | Color selectivity lent to a cholesteric liquid crystal by monitoring interface-induced deformations[END_REF][START_REF] Bitar | Cholesteric liquid crystal self-organization of gold nanoparticles[END_REF][START_REF] Bobrovsky | Atomic force microscopy study of surface topography of films of cholesteric oligomer-and polymer-based mixtures with photovariable helix pitch[END_REF] and SEM [START_REF] Agez | Color selectivity lent to a cholesteric liquid crystal by monitoring interface-induced deformations[END_REF]. These oligomers consisted of cyclic siloxane chains to which two types of side chains are attached via aliphatic spacers (achiral mesogen and chiral cholesterol-bearing mesogen) [START_REF] Agez | Cholesteric liquid crystalline materials with a dual circularly polarized light reflection band fixed at room temperature[END_REF]. The CLC phase appeared between 180-210°C (clearing temperature range) and 40-50°C (glass-transition temperature range), with a helicoidal pitch of 370 nm and a left-handed structure. The mean position of the reflection band was 560 nm-centered inside the 500-600 nm range assigned to the green stripe of C. gloriosa cuticle [START_REF] Goldstein | Polarization properties of Scarabaeidae[END_REF]. More details, including the transmittance spectrum of the sample, are in Ref. [START_REF] Agez | Color selectivity lent to a cholesteric liquid crystal by monitoring interface-induced deformations[END_REF].

In the present study, thin films of varying thickness were coated at 140°C onto a plain glass plate and covered with another glass plate. The upper plate was then removed after quenching, when the film had solidified. The spontaneous nucleation and growth of the polygonal texture from the air interface promptly occurred in the viscous state (at 140°C in our study) and gave rise to an array of polygons with various sizes as shown in Figure 8a. The material was then rapidly quenched to room temperature and the cholesteric organization, producing photonic properties, was stored inside a solid film. In 1996, Meister et al. showed, by TEM, that the nested arc patterns found close to the free surface in a single polygon cut in a direction oblique to the helicoidal axis [START_REF] Meister | The anchoring of a cholesteric liquid crystal at the free surface[END_REF]. In 2011, by combining SEM and TEM, Agez et al. provided the 3D representation of polygons with the required connections between polygons [START_REF] Agez | Color selectivity lent to a cholesteric liquid crystal by monitoring interface-induced deformations[END_REF]. Figure 8b shows a TEM image of neighboring polygons in a direction transverse to the film plane. Sharma et al. [START_REF] Sharma | Structural origin of circularly polarized iridescence in jeweled beetles[END_REF] described the polygonal cells of C. gloriosa as completely analogous to the focal conic domains found in Ref. [START_REF] Meister | The anchoring of a cholesteric liquid crystal at the free surface[END_REF]. Agez et al. [START_REF] Agez | Color selectivity lent to a cholesteric liquid crystal by monitoring interface-induced deformations[END_REF] and Bayon et al. [START_REF] Bayon | Wavelength-tunable light shaping with cholesteric liquid crystal microlenses[END_REF] have mentioned analogies between the polygonal textures found in the C. gloriosa cuticle and in the same oligomer film. In fact, the present investigation highlights fundamental differences.

(i) The most important differentiator is that the nested arced patterns inside a cholesteric polygon are convex for the synthetic film (Figure 8b), while they are concave for the biological material (Figure 2a). (ii) The cuticle surface is quite flat due to the presence of the wax layer which fills the pits in concave mirrors whereas the convex polygons in the synthetic film exhibits a small relief in the 20-100 nm range as shown by AFM [START_REF] Agez | Color selectivity lent to a cholesteric liquid crystal by monitoring interface-induced deformations[END_REF]. (iii) The anchoring of molecules is hybrid in the synthetic film (homeotropic anchoring close the air interface vs. planar anchoring close to the glass substrate) and it is planar close to the waxexocuticle and the endocuticle-epidermis interfaces. As a consequence, lines in the fingerprint texture connect perpendicularly to the air interface and in parallel to the glass interface in the synthetic film whereas they remain parallel to both interfaces in the cuticle. (iv) The synthetic film presents no pitch gradient when a pitch gradient from visible to NIR spectrum is present in the cuticle.

By investigating the light transmission of oligomer films, the wavelength-tunable microlens properties of polygons [START_REF] Bayon | Wavelength-tunable light shaping with cholesteric liquid crystal microlenses[END_REF] and the role of the microlens size on the intensity and the focal length [START_REF] Bayon | Size-effect of oligomeric cholesteric liquid-crystal microlenses on the optical specifications[END_REF] have been demonstrated. Here we supplement these studies, focused on Accepted version in: Acta Biomaterialia 2017, 48, 357-367. [START_REF] Liu | Inconspicuous structural coloration in the elytra of beetles Chlorophila obscuripennis (Coleoptera)[END_REF] transmission, with the reflective properties to draw comparisons with the behavior found in C. gloriosa cuticle (Figure 4). Figure 9 shows the reflection maps for five wavelength ranges from blue to red. The microscope was focused onto the surface, i.e. the air-LC interface. The main result is that the minimum intensity is located at the center of synthetic polygons for all ranges, whereas a bright central spot was allotted to the polygon center in gloriosa cuticle.

Reflection behaviors are thus antagonistic, as a consequence of the basic difference in the orientation of cholesteric pseudo-layers: convex in the oligomer film vs. concave in the cuticle. Concave micromirrors must therefore be targeted under the strategy of fabrication of biomimetic optical components.

Potential applications

The reflection properties of the polygon arrays in the green stripes of C. gloriosa cuticles may serve as an inspiration for the fabrication of wavelength-tunable micromirrors. Each micromirror could serve as a wavelength-selective spatial light modulator, optimized for small form-factor applications and with highly flexible light patterns. Such micromirrors are desirable for fiber optics or optical switching [START_REF] Fabert | Temporal synchronization and spectral combining of pulses from fiber lasers Q-switched by independent MEMS micro-mirrors[END_REF][START_REF] Shin | Wavelength-tunable thulium-doped single mode fiber laser based on the digitally programmable micro-mirror array[END_REF][START_REF] Huang | Bio-inspired wide-angle broad-spectrum cylindrical lens based on reflections from micro-mirror array on a cylindrical elastomeric membrane[END_REF], where micromirror technology will be essential for increasing the speed of communication. For example, when information needs to be re-routed to a personal computer. In an optical network, the transmission process is as follows: the switching station converts the incoming beam into a digital signal, sends it to the computer, then converts it back into a light beam. This task would become much easier if, instead of having to convert the signal twice, the signal may be focused onto a micromirror, automatically sending it to another fibre which is connected to the correct destination. By utilizing many of these micro-mirrors in sequence, the incoming signals can be re-routed to their destination without ever having to be converted into a digital signal, saving considerable time, and producing a higher network data transmission speed. This type of optical packet switching and routing technologies promises to provide a more efficient source of power and footprint scaling with increased router capacity.

Conclusions and summary

With its green stripes, Chrysina gloriosa simultaneously realizes the selective reflection of multiple wavelength ranges at normal incidence and the generation of diffusive reflections at a wide angular range. These seemingly conflicting requirements are fulfilled by combining, on a curved surface, appropriate regions of regularity and irregularity at different length scales: regularity with a set of polygonal cells, including a maximized fill factor, and a continuous pitch gradient inside an individual cell; irregularity with the tessellated texture and a non-monotonic orientation of the helicoidal axis inside the exocuticular part of an individual cell, forming a concave micro-mirror. In summary, the elaborate structure of C. gloriosa The present contribution may learn about the fabrication and design of photonic devices.

The light reflection by C. gloriosa cuticles, with a variety of wavelengths and spatial patterns, may inspire the fabrication of wavelength-specific micromirrors and photonic crystals.

Fig. 1 .

 1 Fig. 1. (a) Chrysina gloriosa beetle (this dry specimen was used in this study). (b) Interface

(

  iii) In 2009, Sharma et al. confirmed the polygonal patterns with concentric rings by fluorescence confocal microscopy [38].

Fig. 2 .

 2 Fig. 2. SEM images of cross-sections in (a) green and (b) silver stripes. In a green stripe and

Fig. 3 .

 3 Fig. 3. Periodicity (half-pitch) in the fingerprint texture of a green stripe (like in Figure 2a) as

  Accepted version in: Acta Biomaterialia 2017,48, 357-367. 15 main feature of this sequence (from 825 nm) is that the reflection continuously increases as the spectrum moves into the NIR.

Fig. 4 .

 4 Fig. 4. (Top left:) The experimental setup: confocal microscope coupled to a spectrometer in

17

 17

4

 4

Fig. 5 .

 5 Fig. 5. Transverse network generated for numerical simulations in direct relation with the

Fig. 6 .

 6 Fig. 6. Numerical simulations vs. experiment for four wavelengths. For each case is

Fig. 7 .

 7 Fig. 7. Experimental and simulated reflection spectra in a silver stripe in relationship with the

Fig. 8 .

 8 Fig. 8. Polygonal texture in a synthetic oligomer film. Thickness = 12.5 m. Annealing time =

Fig. 9 .

 9 Fig. 9. Mapping of reflected intensity of polygonal texture from a synthetic oligomer film at

  environment and, during the dry season, the beetle could hide among the same leaves when they are brown. Survival: the bright glare from silver stripes might temporarily blind a potential predator, enabling C. gloriosa beetles to escape. Navigation if it is proved that a crepuscular and nocturnal insect like C. gloriosa use NIR or IR wavelengths for such a purpose. Studying biological structures such as the tessellated structure of the C. gloriosa cuticle at the nanometer and micrometer scales may thus provide valuable insight into how such phenomena could be exploited in the living world.

Acknowledgements

We are grateful to Dr. E. Hanelt from Wacker-Chemie GmbH (Munich, Germany) for providing us with CLC oligomers. We acknowledge Mr. C. Deshayes for his technical assistance in SEM investigations.

cuticles displays several optical phenomena: [START_REF] Neville | Biology of the Arthropod Cuticle[END_REF] the green stripe serves as a wavelengthselective (green) optical diffuser due to the set of polygons arranged on a curved surface; [START_REF] Vincent | Design and mechanical properties of insect cuticle[END_REF] the green stripe is a wavelength-selective mirror with, strikingly, reflection domains that may sometimes be delimited with net frontiers, whereas the cholesteric structure is regular and continuous from domain to domain; (3) the silver stripe plays the role of a flat metallic reflector operating over the visible spectrum and into the NIR spectrum; (4) the endocuticular part of both stripes are characterized by a broad reflection in the NIR-IR spectrum due to a graded-pitch CLC structure with no tessellated texture.

Previous literature has inferred that the polygonal cells of Chrysina gloriosa were structurally and optically analogous to the focal conic domains formed spontaneously on the free surface of a CLC film, on the basis of similarities between optical textures. It has been possible to correct this inaccurate hypothesis by addressing the topic with confocal microscopy and spectrophotometry, scanning and transmission electron microscopy, and numerical simulations. The main conclusion on this issue is that the cuticle has concave cells whereas the artificial films have convex cells.

We hypothesized several functions: Conspecific or intra-species communication: the green stripe displays information (color, polarization) in the visible spectrum and NIR spectrum; the wavelength-sensitivity of the reflection pattern, occurring essentially in the visible spectrum, may convey this information by being related to the spectral sensitivity of eyes of C. gloriosa conspecifics (and predators). Thermoregulation: NIR reflector may regulate temperature.

Camouflage against diurnal predators (green diffuse reflection) or nocturnal predators capable of detecting IR light (since many green insects show a high NIR reflectance close to that of their habitat foliage); in addition, the shiny surface would mirror the green color of its


Nathalie Sermondade

Emmanuelle Benaloun

Isabelle Berthaut

Emilie Moreau

Marie Prades

Alix Béranger

Nathalie Chabbert-Buffet

Nicolaï Johnson

Rachel Lévy

Dr Charlotte Dupont
email: charlotte.dupont@aphp.fr

Authors Affiliations

PROOF-READING AND REPRINT Correspondence

Reproductive functions and fertility preservation in transgender women: a French case series

Keywords: Fertility preservation, sperm cryopreservation, semen parameters, transidentity, transgender woman

Question

The reproductive potential of transgender people may be impaired by gender-affirming hormone treatment (GAHT) and is obviously suppressed by gender-affirming surgery involving bilateral orchiectomy. The evolution of medical support for transgender people has made fertility preservation strategies possible. Fertility preservation in transgender women mainly relies on sperm cryopreservation. There are few studies on this subject, and the sample sizes are small. Consequently, is fertility preservation procedure feasible and effective in trans women?

Design

In this retrospective study, the management of fertility preservation in transgender women referred to our centre for sperm cryopreservation was reported, and trans women's semen parameters were compared with sperm donors' semen parameters.

Results

Ninety-six per cent of transgender women who had not started treatment benefitted from sperm cryopreservation, compared to 80% of those who attempted a therapeutic window and 50% of those receiving hormonal treatment at the time of sperm collection. No major impairment of semen parameters was observed in transgender women who had not started GAHT compared to sperm donors. However, even though the frequency of oligozoospermia was not different, two transgender women presented azoospermia. Some transgender women who had started GAHT could benefit from sperm freezing. None of them was treated with GnRH analogues.

Conclusions

Parenthood strategies for transgender people have long been ignored. Nevertheless, this issue is important to consider, especially since medical treatments and surgeries may be undertaken in adolescents or very young adults. Fertility preservation should ideally be offered prior to initiation of GAHT.

INTRODUCTION

Trans identity is defined as a gender identity different from the sex assigned at birth. The evolution of transgender people generally corresponds to a physical transition that may involve several stages, including changes in physical appearance and clothing style, as well as medical and surgical interventions. Medical treatments include hormone therapy that suppresses the sexual characteristics of the original gender, usually combined with genderaffirming hormone treatment (GAHT), which induces the evolution of physical characteristics towards the gender with which the individual identifies [START_REF] Tangpricha | Oestrogen and anti-androgen therapy for transgender women[END_REF]. For transgender women, hormonal treatment generally consists of the administration of hormones (anti-androgens and progestogens with or without oestrogens) that allow feminisation. Genderaffirming surgery, although not systematic, is also a therapeutic option.

The reproductive potential of transgender people is suppressed by gender-affirming surgery involving bilateral orchiectomy, and it can also be impaired by hormonal treatments.

Although the consequences likely vary widely based on the treatment strategy [START_REF] Schneider | Testicular Functions and Clinical Characterization of Patients with Gender Dysphoria (GD) Undergoing Sex Reassignment Surgery (SRS)[END_REF], testicular histology of transgender women shows a major decrease in or even a lack of spermatogenesis after initiation of GAHT [START_REF] Schneider | Andrology of male-to-female transsexuals: influence of cross-sex hormone therapy on testicular function[END_REF]. Moreover, there is little research on the potential recovery of normal spermatogenesis during a therapeutic window, and discontinuing GAHT is usually difficult for transgender people to consider because it is viewed as a step backwards. Hence, offering fertility preservation prior to the initiation of hormonal treatment is of particular importance. Numerous scientific organisations, including the American Society of Reproductive Medicine (ASRM), have proposed recommendations concerning information about the potential impact of treatments on reproductive functions and about fertility preservation techniques in a trans identity context (W-PATH, Ethics Committee of the American Society for Reproductive, 2015).

The main method of fertility preservation for transgender women is sperm cryopreservation. There are few studies on transgender women who have benefitted from sperm cryopreservation, and the sample sizes are small [START_REF] Baram | Fertility preservation for transgender adolescents and young adults: a systematic review[END_REF]. A significant alteration in semen parameters was observed in transgender women taking GAHT, as well as a high risk of azoospermia [START_REF] Adeleye | Semen Parameters Among Transgender Women With a History of Hormonal Treatment[END_REF]. However, a recent retrospective study showed that the sperm parameters of transgender women before beginning GAHT were significantly lower compared to WHO data from the general population and nearly 10% of them presented with azoospermia [START_REF] De Nie | Access to fertility services by transgender persons: an Ethics Committee opinion[END_REF].

Although the need to inform patients about the effect of the transition on fertility and the options for fertility preservation has been emphasised, the suspected sperm alterations could complicate and limit the efficiency of such procedures. In this study, we aimed to evaluate the feasibility and effectiveness of the fertility preservation procedure in transgender women by comparing their semen parameters with those of a population of healthy sperm donors and by reporting the results of the cryopreservation.

MATERIALS AND METHODS

Patient selection

This is a retrospective study of transgender women who contacted the Assisted Reproductive Technology (ART) centre at Tenon University Hospital, Paris, between 2018 and 2020. They contacted the ART centre to get information about fertility preservation and, in most cases, to benefit from sperm cryoconservation. Most of the patients were referred by their physicians (endocrinologists or general practitioners). A medical prescription was necessary for them to benefit from sperm freezing. Patients were offered medical and psychological consultations, followed, if necessary, by an appointment for sperm collection and freezing.

Data collection

The health care path of transgender women after the first contact in our centre was fully recorded, including attendance to the different appointments, such as consultations and sperm freezing. Data about age, hormonal treatment, semen parameters and sperm cryoconservation were collected. The patients were classified into three groups based on GAHT intake: no history of hormonal medication (NHM), previous hormonal medication (PHM) and current hormonal medication (HM).

Clinical and biological data about sperm donors recruited in our public centre between 2018 and 2020 were also collected in order to compare semen parameters between the groups.

The sperm donors (SD) were healthy men aged 18 to 44 years old, with or without children.

All sperm donors presented normal semen parameters.

Our study protocol was approved by a local ethics committee (IRB CLEA-2020-109) on 17 th April 2020.

Sperm parameters analysis

Semen samples were collected following masturbation into a sterile plastic cup in the laboratory. After 30 minutes of liquefaction at room temperature, conventional semen parameters (semen volume and sperm concentration and motility) were evaluated according to WHO guidelines (WHO, 2010). Sperm morphology was assessed using David's criteria [START_REF] Auger | Another look at human sperm morphology[END_REF]. Oligozoospermia, or a decrease in total sperm count, is defined as a total of fewer than 39 million spermatozoa in the ejaculate according to WHO guidelines (WHO, 2010).

Only semen parameters from the first sperm collection were considered for statistical analyses. All samples from patients and sperm donors were collected at the same centre and analysed under the same conditions.

Sperm freezing

The semen samples of trans women and sperm donors were frozen according to the same standardised protocol. The semen samples were diluted with cryoprotectant medium (SpermFreez TM , FertiProNV, Belgium) and distributed into straws (CBS TM High Security Sperm 0.3ml Straw, CryoBioSystem, Group IMV Technologies). They were frozen in liquid nitrogen vapours using an automatic freezer (Nano-Digitcool, CryoBioSystem, Group IMV Technologies). The straws were then plunged into liquid nitrogen and stored in nitrogen tanks.

Freezing tolerance was evaluated after one straw was thawed. Motility and sperm concentration were analysed, and the total number of progressive motile spermatozoa per straw (NMSPS) was calculated. A possible assisted reproductive technology strategy was defined according to NMSPS as follows: straws containing less than one million progressive motile sperm were considered usable for in vitro fertilisation with (ICSI) or without (IVF) intracytoplasmic sperm injection, and straws containing more than one million progressive motile sperm were considered usable for intrauterine insemination (IUI).

Statistical analysis

Semen parameters were compared between transgender women who had not started GAHT and sperm donors, as well as between transgender women who had started GAHT and those who had not. The number of sperm collection appointments was also compared across the three groups of trans women.

Variables are presented as mean ± standard error of measurement (SEM) for quantitative variables and as a percentage for qualitative variables. Quantitative variables were analysed using an independent t-test or Wilcoxon-Mann-Whitney test when appropriate and Fisher's exact test for qualitative variables. All statistical analyses were performed using Prism 6 Software (GraphPad Software Inc., La Jolla, CA, USA), and p < 0.05 was considered significant.

RESULTS

Description of the population

Between June 2018 and November 2020, 118 fertility preservation counselling appointments were proposed. Twenty-two patients cancelled the appointment and 96 patients were seen in medical and psychological appointments. Among them, 83 attempted at least one sperm collection. One patient experienced sperm collection failure, nine presented with azoospermia and 73 could benefit from sperm cryopreservation (Figure 1).

Among the 82 patients for whom sperm parameters could be evaluated, 65 patients had not started GAHT (NHM), five patients declared they had stopped treatment three to six months before sperm collection (PHM) and twelve patients were still on hormonal medication (HM) (Table 1).

Comparison between trans women with no history of GAHT (NHM) and sperm donors

The transgender women were significantly younger than the sperm donors (23.9 ± 0.6;

(NHM), 27.2 ± 2.5 (PHM), 30.8 ± 3.1 (HM) vs 35.1 ± 1.0 (SD); p<0.01). The main semen parameters, including semen volume, sperm concentration, progressive motility and vitality, were not statistically different between transgender women and sperm donors (Table 1).

However, normal sperm morphology was significantly lower in transgender women than in sperm donors (p=0.004). Although two transgender women presented with azoospermia, the transgender women did not display a higher frequency of oligozoospermia compared to the sperm donors (Table 1).

Comparison between trans women with no history of GAHT (NHM) and trans women with a history of GAHT (PHM and HM)

The patients with current hormonal medication (HM) were significantly older than the women with no history of GAHT (NHM) (p<0.01). All semen parameters (volume, concentration, motility and morphology) were significantly altered in patients with current hormonal medication in comparison with those who never had hormonal treatment (p=0.04, p<0.01, p=0.01, p<0.01, respectively). The finding of oligozoospermia and azoospermia was also more frequent (p<0.01) (Table 1). No differences were observed in transgender women who stopped hormonal medication before sperm cryopreservation, but the number of patients included in the study was low.

Results of trans women's sperm cryopreservation

Ninety-seven per cent of women who had not started treatment benefitted from sperm cryopreservation, compared to 80% of those who attempted a therapeutic window and 50% of those receiving hormonal treatment at the time of sperm collection (table 2). The total number of progressive motile spermatozoa per straw (NMSPS) was not significantly different across the three groups, but progressive motility after thawing was reduced in women under GAHT compared to women with no history of hormonal medication (p=0.03). Possible ART strategies (IUI vs IVF/ICSI) were not different between groups.

The majority of the transgender women who had not started treatment (76.2%) and those who were under hormonal treatment (66.7%) visited the centre only once for semen collection and freezing, while most patients who attempted a therapeutic window (75.0%) had to visit at least twice.

The patients under GAHT therapy who could benefit from sperm cryopreservation were using oestrogens combined with progesterone (n=5) or oestrogens combined with spironolactone (n=1). None of the patients taking cyproterone acetate (alone or associated with oestrogens) (n=6) could benefit from sperm cryopreservation due to azoospermia.

DISCUSSION

This study represents the first large French case series of transgender women referred for sperm cryopreservation for fertility preservation purposes. No major impairment of semen parameters was observed in transgender women who had not started GAHT compared to sperm donors. We observed an increase in morphological abnormalities in transgender women, but the clinical consequences are likely irrelevant [START_REF] Gatimel | Sperm morphology: assessment, pathophysiology, clinical relevance, and state of the art in 2017[END_REF]. Moreover, even though the frequency of oligozoospermia did not seem different, two transgender women presented with azoospermia in our case series of 65, corresponding to an unexpectedly high prevalence.

These findings are in line with previous publications suggesting that trans women had slightly poorer sperm parameters than cisgender men [START_REF] Li | Sperm cryopreservation of transgender individuals: trends and findings in the past decade[END_REF] or young fathers [START_REF] Marsh | Low total motile sperm in transgender women seeking hormone therapy[END_REF] or significantly decreased sperm parameters compared to WHO data from the general population [START_REF] De Nie | Access to fertility services by transgender persons: an Ethics Committee opinion[END_REF]. The observed alterations may have been caused by an increase in scrotal temperature due to tight clothing or the tucking technique that hides the penis and testes [START_REF] Thonneau | Occupational heat exposure and male fertility: a review[END_REF]. A decrease in the frequency or even the absence of ejaculation could also cause decreased sperm production [START_REF] Alawaqi | Sexual abstinence and sperm quality[END_REF]. Some of the transgender women who had started treatment could benefit from sperm freezing. None of them were treated with cyproterone acetate, a treatment that led to azoospermia in 100% of the cases in our series. Although the literature on the subject remains scarce, a significant alteration in semen parameters was previously observed in transgender women treated with GAHT, as well as a high risk of azoospermia [START_REF] Adeleye | Semen Parameters Among Transgender Women With a History of Hormonal Treatment[END_REF]. In that study, the only patient with normal semen parameters was supplemented with oestrogens only [START_REF] Adeleye | Semen Parameters Among Transgender Women With a History of Hormonal Treatment[END_REF]. However, even if sperm production is maintained, there are concerns regarding the potential impact of hormonal treatments on the quality of the spermatozoa, such as epigenetic marks [START_REF] Semet | The impact of drugs on male fertility: a review[END_REF], as well as the safety of utilisation in terms of embryo development and child health. Some of the transgender women included in our study discontinued their hormonal treatments during a therapeutic window of three to six months for fertility preservation purposes, either by their own decision or following medical advice. Our results suggest that the cyproterone acetate effects on semen parameters are not completely reversible. Many questions about the reversibility of GAHT remain, and the required duration of a therapeutic window for the recovery of normal spermatogenesis is unknown. Although it is likely to be at least three months (one complete cycle of spermatogenesis) [START_REF] Barnard | Fertility Preservation Outcomes in Adolescent and Young Adult Feminizing Transgender Patients[END_REF], it would probably depend on the duration, dose and nature of the hormones, as well as on individual factors. In a prior study, patients underwent a therapeutic window of three to six months and exhibited slightly poorer semen parameters than transgender women who had never taken hormones [START_REF] Adeleye | Semen Parameters Among Transgender Women With a History of Hormonal Treatment[END_REF]. In a different case, an absence of sperm production was described four months after treatment interruption [START_REF] Barnard | Fertility Preservation Outcomes in Adolescent and Young Adult Feminizing Transgender Patients[END_REF], suggesting that a complete reversal of GAHT-related semen impairment cannot be guaranteed. Moreover, a therapeutic window could be difficult to consider for people who have been in treatment for a long time because discontinuation of the treatment can lead to significant physical and psychological changes.

Hence, offering fertility preservation prior to treatment initiation is of particular importance.

However, transgender patients may experience difficulties in accessing fertility preservation procedures. Fertility preservation for transgender patients is not equally available in all countries and regions, and it has been reported that information about fertility preservation remains unsystematic [START_REF] Vyas | Access, barriers, and decisional regret in pursuit of fertility preservation among transgender and gender-diverse individuals[END_REF]. Health professionals need more comprehensive information in order to provide more information to patients and health care facilities.

Moreover, although the costs of fertility preservation strategies are covered by national health insurance in France, and all patients have equal access to care, the cost of sperm banking can constitute a barrier in many other countries.

Parenthood strategies for transgender people have long been ignored. Nevertheless, this issue is important to consider, especially since medical treatments and surgeries may be undertaken in adolescents or very young adults [START_REF] Rafferty | Ensuring Comprehensive Care and Support for Transgender and Gender-Diverse Children and Adolescents[END_REF]. Although transgender women's fertility was not initially a priority, the recent increase in literature reflects a growing interest in this issue. In particular, some studies relying on questionnaires reveal that information about reproductive functions and fertility preservation opportunities is more and more systematic during the transition process [START_REF] Baram | Fertility preservation for transgender adolescents and young adults: a systematic review[END_REF]. The majority of transgender men and women interviewed stated that they wanted to become parents, but few of them actually benefitted from fertility preservation techniques [START_REF] Riggs | Fertility preservation decision making amongst Australian transgender and non-binary adults[END_REF], Chen et al., 2019[START_REF] Segev-Becker | Children and Adolescents with Gender Dysphoria in Israel: Increasing Referral and Fertility Preservation Rates[END_REF]. This may be due to several factors. Fertility preservation procedures are sometimes responsible for a delay in treatment initiation. The cost may also be a hindrance in countries where patients have to pay for fertility preservation procedures. The possibilities of further use of cryopreserved gametes, depending on sexual orientation and the possibility of a partner carrying a pregnancy, may also play a role in the decision. Finally, it is also reported that transgender patients are sometimes not particularly attached to biological parenthood and would be open to alternative strategies such as adoption (Chen et al., 2019).

Frozen-thawed sperm can be used for intrauterine insemination (IUI) or in vitro fertilisation (IVF) with or without microinjection (ICSI). Very few cases of the utilisation of frozen sperm samples have been reported in the literature. In 2014, a child was born after IUI was performed with cryopreserved sperm in a couple consisting of a transgender woman and a cisgender woman (Wierckx et al., 2012). In 2017, a live birth was achieved following IVF using cryopreserved spermatozoa [START_REF] Broughton | Factors Affecting Fertility Decision-Making Among Transgender Adolescents and Young Adults[END_REF], as well as an ongoing pregnancy following IVF with ICSI [START_REF] Jones | Fertility preservation in transgender patients[END_REF]. French legislation does not allow the use of cryopreserved spermatozoa once the civil status change is official. To date, no request for cryopreserved sperm use has been made in our centre.

This study presents inherent limitations due to its retrospective design. However, it represents the first French case series from a reference centre for transgender care, and the number of patients included is relatively high compared to most published studies. Moreover, our studied population includes transgender women who had not started GAHT as well as transgender women who had started GAHT with and without a therapeutic window. Lastly, we were able to compare the semen parameters of these three groups with the semen parameters of a reference group of healthy sperm donors.

CONCLUSION

Although further use of cryopreserved gametes remains uncertain and will depend on current regulations in various countries, the cryopreservation of gametes represents an important step in global care for transgender people. Our research shows that it is feasible and effective to provide fertility preservation for trans women through sperm cryopreservation.

When performed before the introduction of hormonal therapy, sperm parameters seemed slightly altered compared to those of healthy sperm donors. In this situation, one or two appointments were sufficient in most cases to obtain satisfactory results with a reasonable number of usable straws. Our results reveal that information about fertility preservation options should be provided early during the transition in order to facilitate optimal care and avoid the need to resort to a therapeutic window. The spread of this information will rely on networking between practitioners in endocrinology, surgery, gynaecology, reproductive biology, psychiatry and psychology.

Aknowledgements:

Séda Kiliboz, Manel Ichou, Nadine Proust, Clément Lebulanger, Tapa Kaba, Hasret Kiliboz, Nadine Comper who were involved in patients care.