Maryam Hajji

Michele Cariello

Cameron Gilroy

Martin Kartau

Christopher D Syme

Affar Karimullah

Nikolaj Gadegaard

Aurélie Malfait

Patrice Woisel

Graeme Cooke

William J Peveler

Malcolm Kadodwala

A Chiral Quantum Metamaterial for Hypersensitive Biomolecule Detection

Keywords: superchirality, quantum dots, quantum metamaterials, chiral, plasmonics

Chiral biological and pharmaceutical molecules are analysed with phenomena which monitor their very weak differential interaction with circularly polarised light. This inherent weakness results in detection levels for chiral molecules which is inferior, by at least 6 orders of magnitude, to the single molecule level achieved by state-of-the-art chirally insensitive spectroscopic measurements.

Here we show a phenomenon based on chiral quantum metamaterials (CQM) which overcomes these intrinsic limits. Specifically, the emission from a quantum emitter, a semiconductor quantum dot (QD), selectively placed in a chiral nanocavity is strongly perturbed when individual biomolecules (here antibodies) are introduced into the cavity. The effect is extremely sensitive, with 6 molecules per nanocavity being easily detected. The phenomenon is attributed to the CQM being responsive to significant local changes in the optical density of states caused by the introduction of the biomolecule into the cavity. These local changes in the metamaterial electromagnetic environment, and hence the biomolecules, are invisible to "classical" light scattering based measurements. Given the extremely large effects reported, our work presages next generation technologies for rapid hypersensitive measurements with applications in nanometrology and bio-detection.

Introduction

Spectroscopic methods, based on the differential absorption / scattering of circularly polarised light (CPL) by chiral media, are routinely used for rapid characterisation and detection of the structure of biomaterials. [START_REF] Kelly | The Use of Circular Dichroism in the Investigation of Protein Structure and Function[END_REF][START_REF] Blanch | Raman Optical Activity of Biological Molecules[END_REF] The sensitivity of conventional chiroptical techniques, such as electronic or vibrational circular dichroism (CD / VCD), are inherently limited by the significant mismatch between the chiral length scales of CPL, equivalent to the wavelengths (for proteins typically 160-260 nm for CD and 5882 -6667 nm for VCD), and that of molecules (size range 1-10 nm). Consequently, the stereostructural discrimination capabilities of chiroptical methods cannot be exploited in applications, such has biosensing, which require ultra-sensitivity.

Chiroptical measurements can be enhanced using nanostructures to create near fields with enhanced intensity [START_REF] Langer | Present and Future of Surface-Enhanced Raman Scattering[END_REF][START_REF] Adato | In-situ Ultra-Sensitive Infrared Absorption Spectroscopy of Biomolecule Interactions in Real Time with Plasmonic Nanoantennas[END_REF][START_REF] Pour | Use of a Hydrogel Polymer for Reproducible Surface Enhanced Raman Optical Activity (SEROA)[END_REF][START_REF] Pour | Through-Space Transfer of Chiral Information Mediated by a Plasmonic Nanomaterial[END_REF][START_REF] Lieberman | Plasmon-Resonance-Enhanced Absorption and Circular Dichroism[END_REF] and / or enhanced chiral asymmetry. [START_REF] Mun | Electromagnetic Chirality: From Fundamentals to Nontraditional Chiroptical Phenomena[END_REF][START_REF] Hendry | Ultrasensitive Detection and Characterization of Biomolecules Using Superchiral Fields[END_REF][START_REF] Hendry | Chiral Electromagnetic Fields Generated by Arrays of Nanoslits[END_REF][START_REF] Garcia-Guirado | Enantiomer-Selective Molecular Sensing Using Racemic Nanoplasmonic Arrays[END_REF][START_REF] Garcia-Guirado | Enhanced Chiral Sensing with Dielectric Nanoresonators[END_REF][START_REF] Zhao | Chirality Detection of Enantiomers Using Twisted Optical Metamaterials[END_REF][START_REF] Xiao | All-Dielectric Chiral-Field-Enhanced Raman Optical Activity[END_REF][START_REF] Solomon | Fluorescence-Detected Circular Dichroism of a Chiral Molecular Monolayer with Dielectric Metasurfaces[END_REF][START_REF] Tullius | Superchiral Plasmonic Phase Sensitivity for Fingerprinting of Protein Interface Structure[END_REF][START_REF] Tang | Optical Chirality and Its Interaction with Matter[END_REF][START_REF] Tang | Enhanced Enantioselectivity in Excitation of Chiral Molecules by Superchiral Light[END_REF] Such near field enhanced chiroptical techniques still rely on monitoring very weak dichroic signals, but monolayer sensitivities ( femtomole) are achieved. However, even this level of sensitivity pales when compared to the zeptomole (1000 molecules)-single molecule / particle detection ranges that can be achieved using techniques based on labelling with bright, highly fluorescent species such as semiconductor quantum dots (QD). [START_REF] Michalet | Quantum Dots for Live Cells, in vivo[END_REF] In general fluorescent-based techniques are not structurally incisive, requiring the synthesis of target molecules labelled with two chromophores in specific positions. [START_REF] Feng | Multicolor Single-Molecule FRET for DNA and RNA Processes[END_REF] Consequently, if the detection sensitivity of QDs can be combined with the general structural incisiveness of chiroptical spectroscopy, one has the potential to probe the static and dynamic properties of biomaterials and interactions at  zeptomole level. This is a highly useful capability, which for instance, could be exploited as a discrimination mechanism in next generations sensing and metrology technologies.

We report an intriguing phenomenon which enables chiral detection at the  zeptomole level. The concept, Figure 1, relies on chiral quantum metamaterials (CQM), QD placed into a chiral nanocavity. Using a bio-immobilisation strategy the QD can be deposited with high spatial selectivity within the chiral nano-cavity. Luminescence from the QD is sensitive to changes in local optical density of states of the electromagnetic (EM) environment of a chiral nano-resonator caused by binding of biomolecules. Chiral detection is based on monitoring very large changes in the intense QD emission, which are clearly visible by eye in the emission spectra. Specifically, zeptomole detection is achieved with both a large change in absolute spectral intensity, and a significant modification in spectral line shape. The large effects reported in this study have a dual impact, they simplify experimental measurements, facilitating potential applications, and enable high effective detection limits.

Results / Discussion

Chiral Quantum Metamaterial (CQM).

Metamaterials are engineered structures the properties of which are governed to a great extent by the geometric arrangement of the constituent components. Incorporating an element, such as a QD, creates a material, referred to as a quantum metamaterial, the properties of which can only be described by a combination of Maxwell's equations and quantum mechanics. [START_REF] Solntsev | Metasurfaces for Quantum Photonics[END_REF] For a QD which is weakly coupled to a nanocavity, emission is dependent on the optical local density of states, and can be understood within the framework of the Purcell effect. [START_REF] Pound | Measurement of Magnetic Resonance Absorption by Nuclear Moments in a Solid[END_REF] This is the enhancement of the spontaneous emission from a quantum emitter when placed within a resonant cavity. In a quantum electrodynamics description, the Purcell effect arises from the modification, relative to free space, of the local optical density of states of the optical field due to the cavity environment. [START_REF] Xu | Quantum Plasmonics: New Opportunity in Fundamental and Applied Photonics[END_REF]

Optical and AFM characterisation of CQM substrates

The CQM used in this study are based on plasmonic Au metafilms consisting of a square periodic lattice, of periodicity 700 nm, of six-armed cavities, Figure 2 A, subsequently referred to as shurikens, which are either left (LH) or right-handed (RH). [START_REF] Karimullah | Disposable Plasmonics: Plastic Templated Plasmonic Metamaterials with Tunable Chirality[END_REF] When immersed in aqueous environments both plasmonic metafilm enantiomorphs display a resonance feature spanning 1.569 -1.797 eV, Figure 3 A,B, with a region of enhanced reflectivity at  1.708 eV. 25 LH and RH Au metafilms are functionalised first with a biotinylated thiol-polymer (5 nm thickness), that allows the specific binding of QDs through the strong biotin-streptavidin interaction, and then with a commercially produced semiconductor QD (Qdot TM 705, Thermofisher) which are conjugated to 5 -10 streptavidin protein units, Figure 2 B. No detectable electronic circular dichroism is observed over the range in which the QD has optical absorption. Thus, it can be inferred that the presence of the conjugated proteins does not significantly chirally perturb the electronic structure of the QD. The resonant region of the metafilms overlaps the emission from the QD (max = 705 nm (1.759 eV)).

The polymer layer reduces the QD interaction with the Au film, indeed the lifetime of QD is not significantly reduced by immobilisation (confirmed by lifetime measurements). The QD display a high level of spatial selectivity for binding to the arm regions, close to the tips, of the shuriken indentation, propose that this spatial selectivity arises because it maximises the contact of the QD with the polymer surfaces, providing a thermodynamic driver by maximising the level of biotinstreptavidin binding. On average over the whole of the surface, each arm of a shuriken has a bound QD within it; there are relatively few QD which are bound on the Au film between the nanostructures. It should be noted that the immobilisation of the QDs causes a small red shift ( 2 meV) of the plasmonic resonance of the metafilms. This is consistent with the low coverage,6 QD per shuriken, produced by the immobilisation procedure. The CQM are subsequently used to detect an antibody, a monoclonal mouse IgG, which has been produced against streptavidin, henceforth referred to as anti-strept. The antibody will bind to the streptavidin surrounding the QD, which is not already attached to the biotin head group of the polymer. Given the approximate size of IgG,  14 nm, combined with the steric constraints placed on the QD within the arm, it is proposed that  1 antibody binds to each QD. Consequently, on average each shuriken will bind 6 IgG molecules, which is  900 zeptomole over the whole 500 x 500 m array. Thus, within the field of view of the microscope this corresponds to the detection of 600 zeptomole. Optical rotatory dispersion (ORD) and reflectance spectra from the un-complexed and complexed CQM, show no significant differences, Figure 3A-C. Thus, demonstrating the insensitivity of light scattering measurements to the presence of zeptomole quantities of the biomolecules.

Luminescence spectra of un-complexed and complexed CQM

All luminescence spectra were excited with linearly polarised light, with detection at linearly polarisations which were either parallel or orthogonal to that of the excitation beam. Spectra were collected from the LH and RH CQM and for reference purposes data was also obtained from adjacent areas of unstructured Au surface functionalised with QDs. Spectra were collected prior to, and then immediately after, complexation with anti-strept. Spectra from un-complexed and complexed CQM are shown in Figure 4 and 5, while equivalents reference data from unstructured Au are shown in Figure 6. From an initial qualitative assessment of luminescence spectra there are some obvious patterns. The intensity of the emission from the CQM is much greater than that observed from QD immobilised on to the unstructured Au surface.

The most important pattern in the data is a clear asymmetry between emission from LH and RH CQM. There is significantly more emission from RH enantiomorph compared to the LH, and there are differences in line shapes. The level of asymmetry, in terms of both intensity and line shape decreases after complexation with anti-strept. Finally, the similar levels of emission detected for the parallel and orthogonal detection geometries indicate that there is a high level of depolarisation of the emitted light. This observation can be understood in terms of the relative timescales of emission and (rotational) motion of the immobilised QD. As with all biological interactions the biotin-streptavidin bond will be a dynamic process, with it constantly breaking and reforming, thus allowing for the effective (rotational) motion of the QD. This random motion will occur on a time scale which is relatively close to the s lifetimes of the emission of the QD. This will effectively smear out the linear polarisation of the incident excitation. Such depolarisation effects in emission from tumbling molecules in solution are well established. [START_REF] Jameson | Fluorescence Polarization/Anisotropy in Diagnostics and Imaging[END_REF] However, the level of circular polarisation from such a rotating emitter will not be impacted.

To provide a foundation for quantitative analysis the luminescence spectra have been processed in two distinct ways. Firstly, the total emission intensities from the spectra are used to derive a series of parameters. The depolarisation ratio () 123.

 = 𝐼 𝑂𝑟𝑡ℎ𝑜 𝐾 𝐼 𝑃𝑎𝑟𝑎 𝐾, (1)
The parameters , A and F display significant enantiomorphic dependencies which are modified on complexation with anti-strept. For the unstructured Au surface there is no significant change in  after complexation. However, this is not the case for the CQM, where there is a significant decrease (increase) in the  value for LH (RH) enantiomorphs after complexation. A similar behaviour is observed for the F parameter, with complexation resulting in an increase (decrease) in the F parameter for the LH (RH) enantiomorph. These enantiomorphic dependent changes in the enhancement parameter led to a decrease in the asymmetry between LH and RH which is reflected in a decrease in the A parameter after complexation.

In addition to deriving parameters from the total emission intensities, changes in the peak shapes are quantified using fitting with Lorentzian and Gaussian components. Fits are shown with the corresponding experimental data in Figure 456. Within the precision of the fits the same three components, albeit with different relative intensities can be used for all the spectra. The mean positions of the three components derived from fitting all luminescence spectra (see Supplementary information), and subsequently labelled ,  and , are 1.6770.001, 1.7540.002 and 1.8070.003 eV. The different relative contributions of the 3 components is the origin of the differing peak shapes between LH and RH enantiomorphs. For the un-complexed case,  and  components are the dominant contributions in the LH and RH structures, respectively. On complexing the CQM with anti-strept the  component becomes the largest contribution to both LH and the RH substrates. It is this change in the relative contribution of the  component that results in the modification in line shape of the emission from LH CQM on complexation with antistrept (Figure 45).

Luminescence of achiral QD references

For comparison, identical QD, which have not been conjugated to streptavidin and instead feature surface carboxylic acids, have been immobilised on to the metafilms using a chemical strategy (amide bond formation) which does not involve chiral molecules. Given the absence of any chiral molecular element these substrates will be subsequently referred to as "achiral QD references". It should be noted that there are significant differences in the position of the achiral QD, compared to that produced by the streptavidin-biotin strategy. The achiral QD is located much closer, 1 nm above, the Au surface. Crucially, the achiral QD immobilisation strategy is non spatially selective, as evidenced by AFM images. Based on the relative sizes of the luminescence from the unstructured Au surfaces the carbodiimide mediated amide formation strategy that immobilises the achiral QDs produces surface coverages of  1/3 of that of the biotin-streptavidin approach.

Luminescence spectra taken from LH and RH achiral QD reference substrates, with one from an unstructured area of Au for comparison, are shown in Figure 7. Values of , A and F, derived from these spectra are shown in Tables 123. There are substantial differences between the luminescence behaviours of the achiral QD and that of the CQMs. Principally, there are no significant asymmetries in the line shapes or intensities of the luminescence peak between LH and RH structures. All the spectra are fitted with single Gaussian peaks with similar widths, which are in turn not significantly different to those used to fit the streptavidin QD on unstructured Au. In addition to the lack of asymmetry, the achiral references substrates, including QDs on the unstructured Au, have no detectable emission in the orthogonal polarisation direction, implying a   0. The absence of significant depolarisation of the emitted light can be attributed to a significant reduction in lifetime due to an increase in non-radiative decay, caused by the QD being located closer to the Au surface. [START_REF] Waldeck | Nonradiative Damping of Molecular Electronic Excited-States by Metal Surfaces[END_REF] Absence of depolarisation implies that the lifetimes are now significantly shorter than the timescale of random motion of the QD.

Finally, the enhancement factor F is significantly reduced for the achiral QD, which we attribute to the non-spatially selective nature of the immobilisation strategy, producing a homogeneous random distribution of achiral QD across the metafilm.

Immobilisation using variations of the biotin-streptavidin strategy

The data from the achiral QD indicates the asymmetries in the luminescence data from the CQM is associated with the presence of the chiral protein layers. However, for completeness the possibility should be considered that the asymmetries in luminescence are derived from a non-chiral effect. Specifically, there is the possibility that they are caused by streptavidin-biotin based functionalisation strategy (i.e. more QD being immobilised on the LH than the RH shurikens). It should be pointed out, that this is not consistent with the AFM data which shows no evidence for significant differences in QD densities on LH and RH enantiomorphs. Also, disparities in QD density cannot account for the difference in line shape between LH and RH un-complexed CQM, and the effects of complexation, in particular the increase in the emission intensity from the LH substrate, with anti-strept. To further support the hypothesis that the asymmetries arise from the presence of the chiral proteins we have prepared substrates with two modified streptavidin-biotin immobilisation strategies that produce surface densities which are significantly higher (  10 xs) and lower ( 0.1 xs) than the standard procedure (see Supplementary Information). Both substrates display the same RH / LH asymmetry in intensity. Luminescence spectra from the substrates with higher QD density are qualitatively like those obtained from the un-complexed substrate. The lower coverage QD was obtained by coadsorption of QD with free streptavidin. Thus, the nanocavities contain proportionately more chiral molecules to QDs than either the high density or standard substrate. The luminescence data obtained from the low-density substrate are qualitatively similar to that from the complexed substrate. Supporting the observation that an increase in the amount of chiral material in the nanocavity decreases the RH / LH asymmetry.

Purcell versus Chiral Purcell Effect

The data presented provides clear evidence that emissions from the CQM are sensitive to the presence of chiral materials. The biomaterials cause an asymmetry, in terms of both intensity and line shape, in emission from LH and RH CQM. Counter intuitively the experimental data shows that the asymmetry decreases as the amount of chiral material increases. To rationalise the experimental observations, we now develop a discussion within a framework of the Purcell / chiral Purcell effect. This allows the luminescence data to be corelated to the properties of the electromagnetic (EM) environment.

When considered within a quantum electrodynamic framework, and with the application of Fermi's Golden Rule, spontaneous emission from excited states is dependent on the surrounding EM environment. Enhanced spontaneous emission can be engineered by increasing the optical density of states relative to that of the vacuum. For instance, by placing an emitter in a cavity with a resonant mode, spontaneous emission is enhanced, a phenomenon known as the Purcell effect. [START_REF] Pound | Measurement of Magnetic Resonance Absorption by Nuclear Moments in a Solid[END_REF] Alternatively, in the absence of a cavity, large densities of optical states generated in the vicinity of metallic nanostructures also cause enhanced emission. [START_REF] Maier | Plasmonic Field Enhancement and SERS in the Effective Mode Volume Picture[END_REF] In both cases the enhanced emission rates can be rationalised with a Purcell formalism. Where the enhancement in emission rate, called the Purcell factor (FP), is

𝐹 𝑃 = 3 4𝜋 2 ( 𝜆 0 𝑛 ) 3 ( 𝑄 𝑉 𝑃 ) (4)
where Q is the mode's quality factor, n the refractive index of the medium, 0 is the resonance wavelength, and VP is the effective mode volume,

𝑉 𝑃 = 𝑈 2𝑢 𝐸,𝑚𝑎𝑥 (5)
where U is the total energy and uE,max is the energy density. Q and 1/V can be considered to be the spectral and spatial energy density respectively of the resonant mode. [START_REF] Maier | Plasmonic Field Enhancement and SERS in the Effective Mode Volume Picture[END_REF] The Purcell formulism has been extended to chiral light matter interactions in optical resonators, where excited states are coupled to chiral optical states. [START_REF] Yoo | Chiral Light-Matter Interaction in Optical Resonators[END_REF] The critical parameter in this framework is the optical chirality density (C), which is equivalent to optical spin density. The C of an EM field [START_REF] Zhao | Chirality Detection of Enantiomers Using Twisted Optical Metamaterials[END_REF][START_REF] Lipkin | Existence of New Conservation Law in Electromagnetic Theory[END_REF][START_REF] Proskurin | Optical Chirality in Gyrotropic Media: Symmetry Approach[END_REF][START_REF] Bliokh | Characterizing Optical Chirality[END_REF] is defined as,

𝐶 = 1 2 (𝐃 • 𝐁 ̇ -𝐁 • 𝐃 ̇), ( 6
)
where 𝐃 is the displacement field, 𝐁 the magnetic induction and 𝐃 ̇ and 𝐁 ̇ are their respective timederivatives. C can be used to parameterise the chiral asymmetry of EM fields. [START_REF] Garcia-Guirado | Enhanced Chiral Sensing with Dielectric Nanoresonators[END_REF] C is a conserved quantity, [START_REF] Lipkin | Existence of New Conservation Law in Electromagnetic Theory[END_REF] like energy, and has been used in continuity expressions for the chiral (optical spin)

properties of light derived from Maxwell's equations. [START_REF] Kelly | Controlling the Symmetry of Inorganic Ionic Nanofilms with Optical Chirality[END_REF] The C of EM fields is a crucial parameter in understanding the chiral Purcell effect, since the enhancement it gives rise to, originates from an increase in the optical chirality density (spin density) in a chiral resonator relative to free space.

A chiral Purell factor (FC) that quantifies the enhancement of emission in a chiral system can be defined as,

𝐹 𝐶 = 1 4𝜋 2 ( 𝜆 0 𝑛 ) 3 ( 𝑄 𝑉 𝐶 ) (7)
where Vc is the chiral mode volume and represents the spatial confinement of chirality (or optical spin) of light, and 1/ Vc reflects the chiral energy density. VC is defined as

𝑉 𝐶 = 𝑈 𝑢 𝐶,𝑚𝑎𝑥 = - 𝜔𝑈 𝑐𝐶 𝑚𝑎𝑥 (8)
where U and uC,max are the total energy and maximum chiral energy density respectively. uC,mx = -cCmax/ where Cmax is the maximum optical chirality density.

The experimentally observed asymmetry in the enhanced emission from enantiomorphs of the CQM can potentially originate from either the Purcell or chiral Purcell effects. This is because the previously. [START_REF] Garcia-Guirado | Enantiomer-Selective Molecular Sensing Using Racemic Nanoplasmonic Arrays[END_REF]25,[START_REF] Abdulrahman | Induced Chirality through Electromagnetic Coupling between Chiral Molecular Layers and Plasmonic Nanostructures[END_REF][START_REF] Tullius | Superchiral" Spectroscopy: Detection of Protein Higher Order Hierarchical Structure with Chiral Plasmonic Nanostructures[END_REF][START_REF] Gilroy | Roles of Superchirality and Interference in Chiral Plasmonic Biodetection[END_REF] The chiral response of the dielectric layer is parameterised by a second rank tensor . For an isotropic layer xx = yy = zz, with all elements being zero if optical activity as an electric dipole -magnetic dipole (E1M1) origin. [START_REF] Garcia-Guirado | Enantiomer-Selective Molecular Sensing Using Racemic Nanoplasmonic Arrays[END_REF]25,[START_REF] Kelly | Chiral Plasmonic Fields Probe Structural Order of Biointerfaces[END_REF] In the current simulations a |𝜉| = 5 × 10 -4 , which is comparable to values used previously to mimic protein layers. [START_REF] Kelly | Chiral Plasmonic Fields Probe Structural Order of Biointerfaces[END_REF] it, show significant asymmetries, in terms of both line shape and intensity, between enantiomorphs.

In addition, only the spectra derived from the chiral Purcell effect display a change on complexation. However, there are clear inconsistencies between the modelled and experimental spectra. The line shapes of the modelled spectra are in poor agreement with experimental data.

However, the simulations do have three discernible peaks, consistent with the experimental data.

Also, although the simulations replicate the reduction in the intensity of signal from the RH enantiomorph on complexation, they do not show the concurrent increase in the LH. We propose that these discrepancies can be rationalised with the consideration of two factors. Firstly, the simple idealised model used in the simulations does not adequately replicate the structure of the "real" CQM. Specifically, the heterogeneity of the location of the QD within the shuriken structure, in terms of both lateral and vertical position, are not accounted for in the model. Also, the model accounts neither for the variability of QD morphology and size, nor the level of streptavidin conjugation. Secondly, within the precision of the models, spectra derived from VC and VP are of comparable magnitude. Consequently, the experimentally observed spectra could be a combination of both Purcell and chiral Purcell responses. Indeed, the contribution that each makes to the overall emission may be modified by complexation. This hypothesis is supported when one considers the behaviour of the complexed CQM. It would be expected that the complexed CQM would have a greater relative contribution from the Purcell effect than the uncomplexed case, as VC increases, and VP does not alter significantly for both enantiomorphs on complexation. This is consistent with both the line shape for the complexed case, which shows a level of similarity with that predicted by the Purcell model, and the reduction in the level of asymmetry between LH and RH enantiomorphs.

Despite the caveats outlined above, the numerical simulations do clearly demonstrate that only the contributions from the chiral Purcell effect can give rise to significant asymmetries in the luminescence behaviour when the chiral environments surrounding the QD is modified.

Consequently, allowing spectroscopic detection of chiral (bio)molecules at the  zeptomole level.

Conclusions

The combined experimental and numerical simulated data presented demonstrate that luminescence from QDs placed spatially selectively within a chiral nanocavity can detect the presence of chiral (bio)molecules at the zeptomole level. The phenomena can be understood within the framework of the chiral Purcell effect. Specifically, QD luminescence is an extremely sensitive probe of changes in the spatial confinement of local helicity caused by the modification of C induced by the introduction of single biomolecules into a region of space.

In the broadest sense, this work breaks the link between chiral spectroscopy and the measurement of very weak dichroic effects. It demonstrates the superior capabilities of techniques based on the measurement of local near, rather than far field properties for chiral sensing. Far field (chir)optical responses of nanostructures are correlated to the global EM near field environment. Consequently, molecules cannot be detected if they have concentrations too low to cause significant changes to the global EM environment. For instance, in this study the presence of antibodies did not cause detectable changes to either reflectance or chirally sensitive optical rotatory dispersion spectra measured in the far field. However, the introduction of even small amounts of material can result in significant local perturbations, even though these only result in a small net change in the global EM environment. Using CQM, where QD act as reporters of the EM environment, allow the large local changes to be detected. In the exemplar systems studied here this enabled the detection of  6 antibody molecules per nanostructure. Although, for completeness data for both LH ad RH structures were measured here, detection can be achieved by using just a single enantiomorph of a metamaterial. This contrast to the case for the isolated QD, the emission intensity from which is insensitive to the binding of the antibody. Consequently, with appropriate experimental design and instrumentation which would allow measurements on a single nanostructure, the effect would enable the detection and characterisation approaching single molecule detection. In this study the spatially selective placement of the QDs, relied on the streptavidin -biotin interaction, which limits potential targets for detection. If a more generic spatially selective immobilisation strategy was used, such as one based on nano-localised heating, [START_REF] Jack | Spatial Control of Chemical Processes on Nanostructures Through Nano-Localized Water Heating[END_REF] the necessity of using QD conjugated to proteins would be removed. This would enable a wide range of chiral materials to be detected.

Additionally, for extra flexibility the QDs could be substituted with an appropriate dye molecule if necessary. This study presages next generation nanometrology technologies for chiral detection with enhanced sensitivities, with applications in molecular diagnostics and nanometrology.

Methods / Experimental

Sample nanofabrication

The shuriken structure was fabricated on the Polycarbonate substrate using an injection moulding machine (ENGEL)was described in detail elsewhere. [START_REF] Gadegaard | Biomimetic Polymer Nanostructures by Injection Molding[END_REF] The pattern defined on the substrate had a depth of ~80nm and a length of 500nm from arm to arm. After fabrication, the slide was cleaned with IPA and dried with N2 gas. The cleaned slide was metal evaporated using Plassys MEB-400s

to the thickness of 100nm Au and then cleaned in an oxygen plasma asher.

Biotin-Streptavidin Functionalisation

A biotinylated thiol polymer (NIPAM) was synthesised as described below and diluted into PBS

Optical rotatory dispersion (ORD) and reflectivity measurements

A custom-made polarization microscope has been used to collect the linear optical measurement of our samples. [START_REF] Karimullah | Disposable Plasmonics: Plastic Templated Plasmonic Metamaterials with Tunable Chirality[END_REF] The set-up contains a tungsten halogen lamp (Thorlabs) Lightsource that propagates through a collimating lens, Glan-Thomson polariser (Thorlabs), 50:50 beamsplitter and a focused beam 10x objective lens (Olympus). The reflected beam propagates back from the second Glan-Thompson polarisers (Thorlabs) and captures by the spectrometer (Ocean Optics USB400). The sample was positioned and focused using a camera (Thorlabs DCC1645C) after the analyser. The Stokes methods are using to record the ORD spectra at four analyser angles (0°, ±45° and 90°) concerning the incident polarisation for both LH and RH pairs of samples.

Luminescence setup

Luminance measurement was carried out using a 404 nm laser diode with a 180-mA fixed current and maximum optical power output of 17 mW measured using a digital power meter. The two (linear) polarisers at the input after the laser diode were mounted to adjust the input power. The first polarizer was used to modify the input power and the second polarized to define the input polarization. The position of the sample and alignment of the laser on the desired point of the sample has been monitored using an optical camera. The luminance signals were captured using a CCD camera mounted on the top of the configuration. All components were rigidly mounted on an optical bench.

Numerical electromagnetic modelling

A commercial finite element package (COMSOL V5.6 Multiphysics software, Wave Optics module) has been used to simulate the intensity and chiral asymmetry of EM fields produced across the sample. Periodic boundary conditions have been imposed on the sides of the meta-film (i.e., equivalent to simulating a meta-film array). Reflections were minimized using a perfectly matched layer (PML) above and below the input and output ports.

Linearly polarised EM waves were performed at normal incidence onto the meta-film. The finiteelement method has been used to solve Maxwell's equations over a distinctive geometry which allows the optical chirality and E-field intensity to be measured in COMSOL. Additional 6

Quantum dots domains were placed on top of shuriken structure arms. The Quantum dots domains consist of a core structure surrounded with 5nm Protein shell were defined with a refractive index of 1.5. 40 The Protein domains were chiral with the optical chirality parameter of |𝜉| = 5 × 10 -4 .

Another 5nm shell has been added on top of the Protein shell as an antibody domain with the same refractive index of Protein. The optical chirality and E-field intensity of the protein layers were derived by integrating the geometrical model using the volume integration method ASSOCIATED CONTENT Supporting Information is available free of charge at

• A the optical set up of the luminescence spectrometer (Figure S1), the idealised model of the CQM used for numerical simulations (figure S2), spatial distribution of the C at a range of wavelength (figure S3), the parameters used in the fits of the luminescence data are tabulated, luminescence data from the two alternative QD deposition strategies, a description of the synthesis of the polymer used to create the SAM is given.

Figure 2 C

 2 and D. The level of spatial selectivity of the QDs is demonstrated by AFM images from ensembles of LH and RH shuriken structures, Figure 2 C. In figure 2D, higher resolution 3-D AFM images of QDs immobilised to LH and RH enantiomorphs are shown. We

  , Where K is LH or RH CQM, or the unstructured Au, and 𝐼 𝑂𝑟𝑡ℎ𝑜 𝐾 and 𝐼 𝑃𝑎𝑟𝑎 𝐾 are the total intensities of the emission in the parallel and orthogonal detection direction for references. From these intensities an asymmetry parameter A can also derived, increase in the level of emission from the CQM relative to the unstructured surface. Values of , A and F derived from un-complexed and complexed substrates are given in Tables

  presence of the chiral biomolecules in both the un-complexed and complexed CQM locally alter both |𝐸| and C. Consequently, given that from Figures3A and Bit is clear that complexation with anti-strept has a negligible effect on the Q of the resonances, modifications in the emission must be due to changes in either VP or Vc,.Numerical EM simulationsNumerical (finite element method) EM simulations have been performed to elucidate whether the Purcell or chiral Purcell effect is responsible for the asymmetric luminescence behaviour from uncomplexed / complexed CQM. The simulations are based on an idealised version of the real CQM structures shown in Figure2C. Although, in the real structure the QDs adopt a range of positions within the arms of the shurikens, for simplicity in the idealised model we have located them at the ends of the arms. In the model the QD is represented by a spherical achiral dielectric core (diameter 10 nm), which is surrounded by a chiral dielectric layer (thickness 5 nm) which represents the conjugated proteins. To replicate the complexed CQM an additional hemispherical chiral dielectric layer of 5 nm thickness is included, Figure8A. How chiral dielectric properties are accounted for in numerical simulations of chiral plasmonic systems has been described

  Reflectance spectra, Figure 8B, C, derived from simulations based on the idealised model are in good agreement with the experimental data, Figure 3, which provides validation of the robustness of the methodology. Using the data provided by the simulations, values of VP and Vc truncated to the protein layers, can be calculated for LH and RH enantiomorphs of the un-complexed / complexed CQM over the emission energy range, Figure 9A, B. By using these values in Equations 4 and 7, with other parameters either derived from experimental data or estimated from the literature, FP and Fc can be calculated. For simplicity the chiral asymmetry of n, typically  10 -3 , has been neglected. Subsequently, by multiplying FP and Fc with the Gaussian function used to fit the non-enhanced emission from the QDs on the unstructured Au surface, simulated emission spectra can be generated, Figure 10A, B. It is immediately apparent that only Vc, and the spectra derived from

  buffer (10x, pH 7.4) with the factor of 10:1 (PBS: Polymer) concentration. The substrate was left in 300 µL of NIPAM solution for 24 hours to allow to produce a self-assembled monolayer (SAM)on Au surface. The sample was rinsed with PBS and the optical rotatory dispersion (ORD) spectra and reflectivity were collected from LH and RH samples while immersed in both water and PBS. Qdot TM 705 Streptavidin Conjugate (Thermofisher) were deposited on sample and left for two hours, followed by rinsing in 0.1% NaOH/Tween (Sigma-Aldrich) which removes nonspecifically bound / weakly bound streptavidin. After preparing the sample, measurements were taken in the Photoluminescent (PL) measurement microscope to collect luminescence spectra using a linear polarisation in water and PBS. The antibody solution was added to the sample for two hours and rinsed with 0.1% NaOH/Tween (Sigma-Aldrich) to obtain PL spectra. The high coverage QD structures were produced by not doing the Tween surfactant wash. The QD / Strepavidin co-adsorbed structured were produced by using a solution with a 10:1 ratio by molar concentration of streptavidin to QDs.EDC-NHS functionalisation for achiral QD referenceThe metafilms were treated with 50 mM Carboxy-PEG-Thiol CT(PEG)12 (Thermo: 26133) and 50 mM Methyl-PEG-Thiol MT(PEG)4 (Thermo: 26132) (1:1, v/v) for 18 hours. The samples were rinsed with deionised water and dried with nitrogen. Subsequently the samples were treated with 180 mM EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) (Thermo: 22980) and 180 mM Sulfo-NHS (N-hydroxysulfosuccinimide) (Thermo: 25410) (1:1, v/v) for 15 minutes in MES (pH 6.0) buffer. Conjugation reagent solution was removed, the metafilms were rinsed with PBS (pH 7.4, Thermo: 10010023), and 800 nM solution of amine-terminated quantum dot (Qdot™ 705 ITK™ Amino (PEG) Quantum Dots) ) (solution applied for 60 minutes.)

Figure 1 .

 1 Figure 1. Graphical representation of the central concept of the current work. Luminescence from LH and RH CQM is highly sensitive to changes in optical helicity / chirality in local environment of the QD. This allows the introduction of  6 biomolecules per nanostructure to be detected.

Figure 2 .

 2 Figure 2. (A) The top and side views of the Au metafilms; (B) A graphic showing the structure of the QD; (C) AFM images of CQM (un-complexed), illustrating the spatially selective positioning of the QD for an area of 9 structures. To aid the eye some QD are highlighted with red circles; and (D) High resolution images of LH and RH CQM (complexed), which illustrate the distribution of sizes and morphologies of the QDs.

Figure 3 .

 3 Figure 3. Reflectance spectra from (A) LH (red) and (B) RH (Blue) CQM. Spectra for uncomplexed (solid) and complexed (dotted) CQMs. (C) Equivalent ORD spectra are shown. The data illustrated the negligible changes in spectra after complexation.

Figure 4 .

 4 Figure 4. Luminescence spectra (dotted lines) collected using the orthogonal detection geometry from LH and RH enantiomorphs of un-complexed (upper panels) and complexed (lower panels) CQMs. Corresponding fits (solid lines) to three Lorentzians components,  (red)  (green) and  (blue), are also shown.

Figure 5 .

 5 Figure 5. Luminescence spectra (dotted lines) collected using the parallel detection geometry from LH and RH enantiomorphs of un-complexed (upper panels) and complexed (lower panels) CQMs. Corresponding fits (solid lines) to three Lorentzians components,  (red)  (green) and  (blue), are also shown.

Figure 6 .

 6 Figure 6. Luminescence spectra (dotted lines) collected using the parallel and orthogonal detection geometries from un-complexed (upper panels) and complexed (lower panels) QDs immobilised on an unstructured Au film. Corresponding fits (solid orange lines) using a single Gaussian component.

Figure 7 .

 7 Figure 7. Luminescence spectra (doted black line) collected in a parallel detection geometry from achiral QDs immobilised using EDC functionalisation strategy. Spectra are shown for QDs

Figure 9 .

 9 Figure 9. (A) Chiral mode volumes as a functions photon energy for un-complexed and complexed CQMS, for LH (red) and RH (Blue) enantiomorphs. (B) Equivalent data as in (A) but for the effective mode volume for the Purcell effect.

Figure 10 .

 10 Figure 10. Simulated spectra (upper panels un-complexed, lower panels complexed) for LH (red) and RH (blue) enantiomorphs of CQMs derived from (A) Vc and (B) VP. identical vertical scales have been used in all four panels.

Table 1 .

 1 Values for the depolarisation () ratio for -un-complexed / complexed CQM and achiral

	Depolarisation	Ratio
	()

Table 2 .

 2 Values for the asymmetry factor (A) ratio for -un-complexed / complexed CQM and

	achiral reference are listed

Table 3 .

 3 Values for the asymmetry factor (F) ratio for -un-complexed / complexed CQM and achiral reference are listed

immobilised on (A) unstructured Au, (B) LH and (C) RH shurikens. Each spectrum is fitted to a single Gaussian component.

Funding Sources

Any funds used to support the research of the manuscript should be placed here (per journal style). . ACKNOWLEDGMENT The authors acknowledge financial support from the Engineering and Physical Sciences Research Council (EP/P00086X/1 and EP/M024423/1) Technical support from the James Watt Nanofabrication Centre (JWNC). CG's work was supported by the EPSRC CDT in Intelligent Sensing and Measurement, Grant Number EP/L016753/1. WJP acknowledges the University of Glasgow for an LKAS fellowship. MK acknowledges the Leverhulme Trust for the award of a Research Fellowship (RF-2019-02)

AUTHOR INFORMATION

Corresponding Author

*to whom correspondence should be addressed. Email: Malcolm.Kadodwala@glasgow.ac.uk

Author Contributions

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


Pascal Favrel
email: pascal.favrel@unicaen.fr

M P Dubos

B Bernay

J Pasquier

J Schwartz

B Lefranc

L Mouret

G Rivière

J Leprince

A Bondon

MP Dubos

Structural and functional characterization of an egg-laying hormone signaling system in a lophotrochozoan -The pacific oyster (Crassostrea gigas)

Keywords: Oyster, GPCR, egg-laying hormone, reproduction, stress Highlights

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

Introduction

The egg-laying hormones (ELHs) of gastropods were among the first neuropeptides to be functionally characterized in mollusks [START_REF] Chiu | Purification and primary structure of the neuropeptide egg-laying hormone of Aplysia californica[END_REF][START_REF] Ebberink | Purification and amino acid sequence of the ovulation neurohormone of Lymnaea stagnalis[END_REF]. They are synthesized as large precursor proteins that generate a set of neuropeptides in addition to ELHs and whose release controls and coordinates egg-laying and associated behavioral events in this class of mollusks [3][4][5]. With the development of transcriptomic and peptidomic resources for a variety of mollusks and annelids, it has become clear that orthologs of ELH neuropeptides exist in most lophotrochozoan species [START_REF] Conzelmann | The neuropeptide complement of the marine annelid Platynereis dumerilii[END_REF][START_REF] Jékely | Global view of the evolution and diversity of metazoan neuropeptide signaling[END_REF][START_REF] Mirabeau | Molecular evolution of peptidergic signaling systems in bilaterians[END_REF][START_REF] Salzet | Leech egg-layinglike hormone: Structure, neuronal distribution and phylogeny[END_REF][START_REF] Stewart | Neuropeptides encoded by the genomes of the Akoya pearl oyster Pinctata fucata and Pacific oyster Crassostrea gigas: a bioinformatic and peptidomic survey[END_REF][START_REF] Veenstra | Neurohormones and neuropeptides encoded by the genome of Lottia gigantea, with reference to other mollusks and insects[END_REF]. Because lophotrochozoan ELHs display structural homologies with ecdysozoan diuretic hormone 44 (DH44) and deuterostome corticotropin-releasing hormone (CRH) family members (CRH, urocortins1-3: Ucn1-3), the ancestor of these families of neuropeptides predated the split between the protostome and deuterostome lineages.

Both vertebrate CRH family peptides and ecdysozoan DH44 are key players of the physiological response or tolerance to stress [START_REF] Cannell | The corticotropin-releasing factor-like diuretic hormone 44 (DH44) and kinin neuropeptides modulate desiccation and starvation tolerance in Drosophila melanogaster[END_REF][START_REF] Deussing | The Corticotropin-Releasing Factor Family: Physiology of the Stress Response[END_REF] and are involved in the regulation of a variety of physiological functions including feedingassociated processes [START_REF] Cannell | The corticotropin-releasing factor-like diuretic hormone 44 (DH44) and kinin neuropeptides modulate desiccation and starvation tolerance in Drosophila melanogaster[END_REF][START_REF] Czimmer | Peripheral Corticotropin Releasing Factor Signaling Inhibits Gastric Emptying: Mechanisms of Action and Role in Stress-related Gastric Alterations of Motor Function[END_REF][START_REF] Dus | Nutrient Sensor in the Brain Directs the Action of the Brain-Gut Axis in Drosophila[END_REF][START_REF] Stengel | CRF and urocortin peptides as modulators of energy balance and feeding behavior during stress[END_REF][START_REF] Van Wielendaele | CRF-like diuretic hormone negatively affects both feeding and reproduction in the desert locust, Schistocerca gregaria[END_REF], immunity [START_REF] Lee | The role of diuretic hormones (DHs) and their receptors in Drosophila[END_REF]), reproduction (van Wielendaele et al., 2012), body fluid secretion in Drosophila [START_REF] Cabrero | The Dh gene of Drosophila melanogaster encodes a diuretic peptide that acts through cyclic AMP[END_REF] and the renal function [START_REF] Devetzis | The corticotropin releasing factor system in the kidney: perspectives for novel therapeutic intervention in nephrology[END_REF]. Among Lophotrochozoa, besides gastropod ELHs, only a first insight into the physiological role of ELH as an egg-laying (spawning) hormone had been provided so farin the Sydney rock oyster (Saccostrea glomerata) [START_REF] In | Reproductive neuropeptides that stimulate spawning in the Sydney Rock Oyster (Saccostrea glomerata)[END_REF].

Interestingly, a recent phylogenetic analysis showed that gastropod ELH sequences segregate apart from the clade including non-gastropod mollusk, annelid and nemertean counterparts [START_REF] De Oliveira | Extensive conservation of the proneuropeptide and peptide prohormone complement in mollusks[END_REF]. This singular clustering of gastropod ELHs raises the prospect that they could have acquired specialized functions in relation with reproduction more recently throughout the evolution of mollusks. To gain insights into the functional involvement and the evolution of ELH signaling in mollusks, the present study characterizes functionally an ELH receptor for the first time in a Lophotrochozoan, and investigates the specific tissue-dependent, temporal and environment-induced expression of the genes encoding the components of the ELH signaling system in a bivalve molluskthe oyster Crassostrea gigas.

2.

Material and methods.

Animals and tissue sampling

Two-year old adult oysters Crassostrea gigas (Thunberg 1793), purchased from a local farm (Normandy, France), were used for peptide characterization and transcription analyses. Adult tissues, mantle, mantle edges, gills, labial palps, digestive gland, gonad (mix of all stages), heart, adductor muscle and the visceral ganglia (VG) were carefully dissected-out.

A one-year sampling of animals was undertaken to collect gonads and VG at different stages of reproduction. Oysters were collected and their gonads and VG were immediately dissected.

VG and gonadal tissues were sampled for each individual, frozen in liquid nitrogen and stored at -80°C. A part of each gonad sample was also fixed for histological analysis. Gonadal development stage and sex were determined by histological methods according to the 4 stages previously described [START_REF] Rodet | Molecular cloning of a molluscan gonadotropin-releasing hormone receptor orthologue specifically expressed in the gonad[END_REF]: stage 0 corresponding to the resting undifferentiated stage with very few germinal stem cells; stage 1 corresponding to the gonial multiplication stage with poorly developed tubules surrounded by a large matrix of vesicular connective tissue (VTC); stage 2 corresponding to the maturation stage with tubule development and VCT starting to regress, with vitellogenesis occurring in females and all the cells of the germline can be observed in males (from spermatogonia to spermatozoa); stage 3 corresponding to sexual maturity with tubules full of mature germinal cells.

The effect of trophic conditions was investigated on one-year-old adult oysters reared in water tanks either in absence of food or in presence of Isochrysis galbana (clone T-Iso) maintained at a concentration of 6 million of cells/mL during 4 weeks. To study the influence of osmotic conditions, oysters were transferred from seawater (33‰) to brackish water (8‰) at 17°C by addition of distilled water once in the sea water tank (Acute stress) or by diluting the sea water with a continuous flow (0.5 Lh -1 ) of distilled water during 3 days (Mild stress). To prevent the closure of the shell, a wedge was inserted between the valves before applying the stress. VG were sampled and frozen after 12 h of incubation of the oysters in brackish water at the final salinity.

Peptide extraction: Fifteen VG, frozen and crushed in liquid nitrogen, were extracted in acetonitrile/water (90/10 v/v) 0.1% TFA for 30 minutes at 4°C. After centrifugation for 20 minutes at 15 000 g at 4°C, supernatant was concentrated on C18 Sep-Pak cartridges. Half of the recovered peptide sample was reduced with 100 mM DTT at 55 °C for 60 min and alkylated with 50 mM iodoacetamide at 55 °C for 45 min. Both mature or reduced/alkylated peptides were analyzed by mass spectrometry

For nano-LC fragmentation, peptide samples were first desalted and concentrated onto a µC18

Omix (Agilent) column before analysis. The chromatography step was performed on a NanoElute (Bruker Daltonics) ultra-high-pressure nano flow chromatography system.

Approximatively 200ng of each peptide sample were concentrated onto a C18 pepmap 100 (5mm x 300µm i.d.) precolumn (Thermo Scientific) and separated at 50°C onto a reversed phase Reprosil column (25cm x 75μm i.d.) packed with 1.6μm C18 coated porous silica beads (Ionopticks). Mobile phases consisted of 0.1% formic acid, 99.9% water (v/v) (A) and 0.1% formic acid in 99.9% ACN (v/v) (B). The nanoflow rate was set at 300 nL/min, and the gradient profile was as follows: from 2 to 15% B within 15 min, followed by an increase to 25% B within 15 min and further to 35% within 12 min, followed by a washing step at 95% B and reequilibration.

MS experiments were carried out on an TIMS-TOF pro mass spectrometer (Bruker Daltonics) with a modified nano electrospray ion source (CaptiveSpray, Bruker Daltonics). A 1400 spray voltage with a capillary temperature of 180°C was typically employed for ionizing. MS spectra were acquired in the positive mode in the mass range from 100 to 1700 m/z. Cragi-ELH1, Cragi-ELH1a, Cragi-ELH1b, Cragi-ELH1c, Cragi-ELH123-40, Cragi-ELH2, Cragi-ELH2a and Cragi-ELH2b were synthesized by Fmoc solid phase methodology on a Liberty microwave assisted automated peptide synthesizer (CEM, Saclay, France) using the standard manufacturer's procedures at 0.1 mmol scale as previously described [START_REF] Touchard | Venom Peptide Repertoire of the European Myrmicine Ant Manica rubida: Identification of Insecticidal Toxins[END_REF]. All Fmoc-amino acids (0.5 mmol, 5 eq.) were coupled on preloaded HMP resin (ELH1a and ELH1c) or Rink amide resin (ELH1, ELH1b, ELH123-40, ELH2, ELH2a and ELH2b), by in situ activation with HBTU (0.5 mmol, 5 eq.) and DIEA (1 mmol, 10 eq.) before Fmoc removal with 20% piperidine in DMF. After completion of the chain assembly, peptides were deprotected and cleaved from the resin by adding 10 mL of an ice-cold mixture of TFA/TIS/H2O (9.5:0.25:0.25, v/v/v) and agitating for 3 hours at room temperature. After precipitation in TBME followed by centrifugation (4500 rpm, 15 min), the crude peptides were purified by reversed-phase HPLC (Gilson, Villiers le Bel, France) on a 21.2 x 250 mm Jupiter C18 (5 µm, 300 Å) column (Phenomenex, Le Pecq, France) using a linear gradient (10-60% or 20-50% over 45 min) of acetonitrile/TFA (99.9:0.1) at a flow rate of 10 mL/min. Peptides were then characterized by MALDI-TOF mass spectrometry on a ultrafleXtreme (Bruker, Strasbourg, France) in the reflector mode using α-cyano-4-hydroxycinnamic acid as a matrix.

Analytical RP-HPLC, performed on a 4.6 x 250 mm Jupiter C18 (5 µm, 300 Å) column, indicated that the purity of the peptides was > 99.9%.

NMR and structure determination

NMR measurements

The nuclear magnetic resonance (NMR) samples contained 1-3 mM of peptide Cragi-ELH1a

or Cragi-ELH2 dissolved in water or in presence of deuterated dodecylphosphocholine (DPC) micelles (125 mM). All spectra were recorded on a Bruker Avance 500 spectrometer equipped with a 5 mm TCI cryoprobe (1H, 13C, 15N). Homonuclear 2-D spectra DQF-COSY, TOCSY and ROESY or NOESY were typically recorded using standard Bruker sequences in the phase sensitive mode using the States-TPPI method. NOESY or ROESY spectra were acquired with 8 scans, 8 spectra were summed leading to less t1 noise as recently reported [START_REF] Mo | A simple method for NMR t1 noise suppression[END_REF].

Typical spectra were acquired using matrices of 4096 x 320-600 zero filled in F1 to 2K x 1K after apodization with shifted sine-square multiplication in both domains. Spectra were processed with Topspin software (Bruker).

Structure calculations

with a 5 mm TCI cryoprobe (1H, 13C, 15N). Homonuclear 2-D spectra DQF-COSY, TOCSY and ROESY or 1 H chemical shifts were assigned according to classical sequential assignment procedure. NOE cross-peaks were integrated and assigned within the CcpNmr [START_REF] Vranken | The CCPN data model for NMR spectroscopy: Development of a software pipeline[END_REF] and NMR View software [START_REF] Johnson | Protein NMR Techniques. Using NMRView to Visualize and Analyze the NMR Spectra of Macromolecules[END_REF]. The volumes of NOE peaks between methylene pair protons were used as reference of 1.8 Å. Structure calculations were performed with AMBER 17 [START_REF] Case | The Amber biomolecular simulation programs[END_REF] in two stages: cooking and simulated annealing in explicit solvent. The cooking stage was performed at 1000 K to generate 100 initial random structures. Simulated annealing calculations were carried during 20 ps (20000 steps, 1 fs long). First, the temperature was risen quickly and was maintained at 1000 K for the first 5000 steps, then the system was cooled gradually from 1000 K to 100 K from step 5001 to 18000 and finally the temperature was brought to 0 K during the 2000 remaining steps. For the 3000 first steps, the force constant of the distance restraints was increased gradually from 2.0 kcal.mol -1 .Å to 20 kcal.mol -1 .Å. For the rest of the simulation (step 3001 to 20000), the force constant is kept at 20 kcal.mol -1 .Å.

The 20 lowest energy structures with no violations > 0.3 Å were considered as representative of the compound structure. All the dihedral angles ϕ and ψ belonged to the allowed regions of the Ramachandran plot. The representation and quantitative analysis were carried out using MOLMOL [START_REF] Koradi | MOLMOL: A program for display and analysis of macromolecular structures[END_REF].

2.5

In silico analyses Multiple sequence alignment was performed using Clustal Omega [START_REF] Sievers | Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega[END_REF].

Seaview [START_REF] Gouy | SeaView version 4: A multiplatform graphical user interface for sequence alignment and phylogenetic tree building[END_REF] was used for selecting the conserved protein regions and for manual correction of the alignment. PhyML was used for generating the trees. The reliability of the inferred trees was estimated by applying the bootstrap procedure with 1000 replications.

FigTree (http://tree.bio.ed.ac.uk/software/figtree/) was used to draw the tree.

2.6

Pharmacological characterization of Cragi-ELHR

Molecular cloning and transfection of mammalian cells

The CDS of Cragi-ELHR (XP_034310400.1) was amplified by PCR using the transcript specific sense primer (5′-CACCATGTATAACCTGACGGACTATC-3′) harboring a Kozak consensus sequence and the antisense primer (5'-TTACAACATTTTCTCCGATTCTAGTCC-3′). Ten nanograms of plasmid DNA (Pal 17.3 vector, Evrogen, Russia) from a C. gigas "all developmental stages and adult central nervous system" normalized cDNA library [START_REF] Fleury | Generation and analysis of a 29 , 745 unique Expressed Sequence Tags from the Pacific oyster (Crassostrea gigas) assembled into a publicly accessible database : the GigasDatabase[END_REF] was used as template. PCR was carried-out in a 50 µl reaction volume containing 1.5 mM MgCl2, 200 mM dNTPs, 1 mM each of the primer couples, 1.25 units of GoTaq® polymerase and the appropriate buffer (Promega, Madison, WI, USA) in nuclease-free water.

Samples were subjected to the following cycling parameters (95 °C, 2 min; 30 cycles of: 95 °C-45 s, 60°C-30 s, 72 °C-1 min, followed by 5 min at 72 °C).

The resulting PCR product was directionally cloned into the eukaryotic expression vector pTARGET (Promega) and the correct insertion confirmed by Sanger sequencing.

Human Embryonic Kidney (HEK293T) cells were transiently co-transfected with the Cragi-ELHR and pGloSensor™ -22F cAMP (Promega) constructs using Fugene HD (Promega)

according to the manufacturer's instructions. Cells for negative control experiments were transfected with an empty pcDNA3.1 and pGloSensor™ -22F cAMP plasmids.

cAMP luminescence assay

Cragi-ELHR transfected HEK293 T cells were incubated with Glosensor cAMP reagent (4% final concentration in the medium) (Promega) for 2 hours at room temperature prior to the injection of the candidate ligands. The peptides to be tested were diluted at 5 times their final concentration in 20mM HEPES in Hank's balanced salt solution pH 7.3 and distributed in a 96well plate. 25µL of the different peptide solutions were injected in the wells containing the cells. cAMP luminescence response was measured for 30 min after injection using a FLEX station 3 (Molecular Devices) at room temperature. Data were analyzed using SoftMax Pro (Molecular Devices). Candidate peptide ligands were first tested in triplicate at a final concentration of 10 -5 M then at different peptide concentrations. Concentration-response measurements of activating ligands were conducted in triplicate and for at least three independent experiments.

2.7

Reverse transcription and quantitative PCR RT-qPCR analysis was performed using the CFX96™ Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). Total RNA was isolated from adult tissues using Tri-Reagent (Sigma-Aldrich) according to the manufacturer's instructions. Recovered RNA was then further purified on Nucleospin RNAII columns (Macherey-Nagel). After treatment for 20 min at 37 °C with 1 U of DNase I (Sigma) to prevent genomic DNA contamination, 1 μg of total RNA was reverse transcribed using 1 μg of random hexanucleotide primers (Promega), 0.5 mM dNTPs and 200 U MMuLV Reverse Transcriptase (Promega) at 37 °C for 1 h in the appropriate buffer. (5'-ACCACCCTGGTGAGATCAAG-3') and QA-Cg-EF1α

(5'-ACGACGATCGCATTTCTCTT-3') transcript (BAD15289) was carried out to normalize the expression data of the analyzed transcripts. EF1α was used as a reliable normalization gene as no significant difference (p < 0.05) of Ct values was observed between the different samples compared. Coefficient of variation of EF1α was less than 5% for all the tissue samples and experimental conditions. Thus, the relative level of expression of the target gene was calculated by using the following formula: N = 2 (Ct Cragi-EF1α -Ct target cDNA) . The PCR amplification efficiency (E = 10 (-1/slope) ) for each primer pair was determined by linear regression analysis of a dilution series to ensure that E ranged from 1.98 to 2.02.

Statistical analysis

Gene expression levels between different tissues and between samples at different reproduction stages were compared using one-way ANOVA followed by a Tukey post hoc test. Expression levels between animals in different feeding or salinity conditions were compared using an unpaired Student's t test. Significance was set at p < 0.05.

3 Results.

3.1

Molecular processing of Cragi-ELH precursor.

The sequence of the precursor encoding C. gigas ELH (Cragi-ELH) described previously [START_REF] Stewart | Neuropeptides encoded by the genomes of the Akoya pearl oyster Pinctata fucata and Pacific oyster Crassostrea gigas: a bioinformatic and peptidomic survey[END_REF] corresponding to the full-length sequence could be detected. In contrast, despite the presence of a monobasic putative processing site, no Cragi-ELH2 processed peptides could be detected.

Structure determination of Cragi-ELH peptides by NMR

The NMR analysis of ELH1a, as the longest processed peptide from predicted ELH1 sequence and ELH2 in presence of deuterated DPC was performed using the sequential assignment strategy [START_REF] Wüthrich | NMR of Proteins and Nucleic Acids[END_REF]. As presented in supplementary tables 1 and 2, the complete chemical shift assignment of ELH1a was successfully performed whereas severe overlapping prevented the full assignment of ELH2. The three-dimensional structure of peptide ELH1a in DPC micelles was solved. The superposition of the 18 lowest energy conformers, fitted on segment 4-17 backbone atoms is presented in Fig. 2B. Summary of the refinement statistics are given (Suppl. table 3). An alpha helix is clearly defined between the residues 4 to 17. Incomplete assignment of ELH2 prevents the structure determination, however the NOE connectivity pattern is also characteristic of a helical structuration of the ELH2 peptide in DPC micelles with numerous inter residue i -i+3 connectivities between the residues 4-19 (Suppl. Fig. 2).

Characterization of Cragi-ELHR

A BLAST search using the Drosophila DH44 Receptor 1 (DH44R1: AAF58250.1) sequence in the Gigaton database [START_REF] Riviere | GigaTON: an extensive publicly searchable database providing a new reference transcriptome in the pacific oyster Crassostrea gigas[END_REF] allowed the identification of a full-length transcript (CHOYP_CRHR.1.2) encoding a seven transmembrane domain protein typical of a GPCR (Suppl. Fig. 3). Alignment of the oyster receptor named Egg-laying Hormone Receptor (Cragi-ELHR) with vertebrate and protostome GPCRs highlights its high degree of homology with functionally characterized insect DH44Rs, lophotrochozoan putative DH44/CRHRs and vertebrate CRHRs (Suppl. Table 4 and Suppl. Fig. 4). A phylogenetic analysis showed that all lophotrochozoan receptors form a single clade and were phylogenetically related to vertebrate CRHRs and Ecdysozoan DH44Rs (Fig. 3). Transiently transfected HEK293T cells expressing Cragi-ELHR were challenged with Cragi-ELH1 and Cragi-ELH2 as well as with all their predicted or characterized processed peptides (Cragi-ELH1a, Cragi-ELH1b, Cragi-ELH1c, Cragi-ELH123-40, Cragi-ELH2a and Cragi-ELH2b) at a single concentration of 10 -5 M. Both

Cragi-ELH1 and Cragi-ELH2 activated Cragi-ELHR resulting in the activation of the adenylylmediated cAMP transduction pathway. Only N-terminal peptides of both Cragi-ELH1 (Cragi-ELH1a) and Cragi-ELH2 (Cragi-ELH2a) were efficient in activating Cragi-ELHR though Cterminal and central peptides were not (Fig. 4A). Thus, a dose-dependent activation of Cragi-ELHR was investigated using the active peptides. Both Cragi-ELH1 and Cragi-ELH2 elicited a gradual response with an EC50 of respectively 26.2 ± 3.2 nM and 81.3 ± 8.9 nM whilst ELH1a and ELH2a showed an EC50 of 2.88 ± 0.76µM and of 3.11 ± 1.29 µM respectively (Fig. 4B).

Only micromolar concentrations of ELH1 and ELH2 triggered the phospholipase Cβ-mediated calcium transduction pathway (data not shown)

3.4

Gene expression of Cragi-ELHR and Cragi-ELH.

The expression of Cragi-ELHR and Cragi-ELH precursor transcripts was investigated by RT-qPCR in several adult tissues, in VG and gonads during reproduction and in VG of animals exposed to different stresses (Supplementary table 5). Both Cragi-ELHR and Cragi-ELH precursor genes were majorly expressed in the VG and at very much lower levels in most adult tissues (Fig. 5A andB). During a cycle of reproduction, no significant change in expression of Cragi-ELHR was observed both in male and female VG or gonads (Fig. 5C andD). In contrast, a significantly higher expression of Cragi-ELH precursor gene was noticed only in VG at stage 3 in both males and females (Fig. 5C). Given the implication of vertebrate CRH in mediating stress, two stress conditions were applied to oysters. The first stress was to starve animals for a period of four weeks. In these conditions, no significant change of expression of Cragi-ELH or Cragi-ELHR transcripts was observed in the VG of starved animals as compared to VG of fed animals (Fig. 6A andB). The second stress was to expose oysters to brackish water either suddenly (acute stress) or by progressively changing the salinity over a period of three days (mild stress). Only Cragi-ELHR gene expression was found to be significantly stimulated in mild stress conditions in the VG whilst no change was found for Cragi-ELH transcripts for both stresses in the VG (Fig. 6C andD).

Discussion

The present work describes the functional characterization of a lophotrochozoan G proteincoupled receptor (GPCR) phylogenetically related to insect DH44Rs and vertebrate CRH family member receptors. Our phylogenetic analysis is congruent with previous studies [START_REF] Cardoso | New insights into the evolution of vertebrate CRH (corticotropin-releasing hormone) and invertebrate DH44 (diuretic hormone 44) receptors in metazoans[END_REF] and suggests that mollusk and annelid receptors are more similar to chordate CRHRs than to insect DH44Rs. Interestingly, the receptors obviously cluster according to the animal phylogeny, whereas among the ligands, gastropod ELHs segregate apart and form an independent and specific clade beside the group gathering the remaining mollusk and lophotrochozoan ELH/DH44 sequences [START_REF] De Oliveira | Extensive conservation of the proneuropeptide and peptide prohormone complement in mollusks[END_REF]. This suggests a specific evolution of gastropod ligands, potentially in association with their specific role in the control of reproductive behavioral processes [START_REF] Scheller | A family of genes that codes for ELH, a neuropeptide eliciting a stereotyped pattern of behavior in Aplysia[END_REF][START_REF] Vreugdenhil | Isolation, characterization, and evolutionary aspects of a cDNA clone encoding multiple neuropeptides involved in the stereotyped egg-laying behavior of the freshwater snail Lymnaea stagnalis[END_REF]. Among mollusks, cephalopods also display a complex pattern of reproductive behaviors (von [START_REF] Boletzky | Sepia officinalis[END_REF]) involving a variety of neuropeptides [START_REF] Henry | Peptidergic control of egg-laying in the cephalopod Sepia officinalis: involvement of FMRFamide and FMRFamide-related peptides[END_REF][START_REF] Zatylny-Gaudin | Neuropeptidome of the cephalopod Sepia officinalis: identification, tissue mapping, and expression pattern of neuropeptides and neurohormones during egg laying[END_REF][START_REF] Zatylny | The SepOvotropin: a new ovarian peptide regulating oocyte transport in Sepia officinalis[END_REF]. However, no ELH-related peptide was identified from the neurotranscriptome and the neuropeptidome of spawning cuttlefish (Sepia officinalis) [START_REF] Zatylny-Gaudin | Neuropeptidome of the cephalopod Sepia officinalis: identification, tissue mapping, and expression pattern of neuropeptides and neurohormones during egg laying[END_REF]. It has been suggested that the ELH-encoding gene may have been lost in cephalopods [START_REF] De Oliveira | Extensive conservation of the proneuropeptide and peptide prohormone complement in mollusks[END_REF]. The present study clearly shows the existence of an ELHR in the cephalopod Octopus bimaculoides, noticeably phylogenetically distant from the other lophotrochozoan receptors. This possibly indicates that it binds peptide ligands divergent from their lophotrochozoan counterparts.

In the same way as other bivalve precursors [START_REF] Stewart | Neuropeptides encoded by the genomes of the Akoya pearl oyster Pinctata fucata and Pacific oyster Crassostrea gigas: a bioinformatic and peptidomic survey[END_REF][START_REF] Zhang | Identification and characterization of neuropeptides by transcriptome and proteome analyses in a bivalve mollusc Patinopecten yessoensis[END_REF], the Cragi-ELH precursor potentially generates two homologous ELHs (Cragi-ELH1 and Cragi-ELH2) of 40 and 38 amino acids, respectively [START_REF] Stewart | Neuropeptides encoded by the genomes of the Akoya pearl oyster Pinctata fucata and Pacific oyster Crassostrea gigas: a bioinformatic and peptidomic survey[END_REF]. Both peptides specifically activate

Cragi-ELHR at nanomolar concentrations, as observed for insect DH44Rs and vertebrate

CRHRs and their respective ligands [START_REF] Chen | Expression cloning of a human corticotropin-releasing-factor receptor[END_REF][START_REF] Hector | Functional differences between two CRF-related diuretic hormone receptors in Drosophila[END_REF][START_REF] Johnson | A novel diuretic hormone receptor in Drosophila: evidence for conservation of CGRP signaling[END_REF][START_REF] Liaw | Cloning and characterization of the human corticotropin-releasing factor-2 receptor complementary deoxyribonucleic acid[END_REF]. Cragi-ELHR increased cAMP levels upon activation, as most receptors of this family do [START_REF] Chen | Expression cloning of a human corticotropin-releasing-factor receptor[END_REF][START_REF] Hector | Functional differences between two CRF-related diuretic hormone receptors in Drosophila[END_REF][START_REF] Johnson | A novel diuretic hormone receptor in Drosophila: evidence for conservation of CGRP signaling[END_REF][START_REF] Liaw | Cloning and characterization of the human corticotropin-releasing factor-2 receptor complementary deoxyribonucleic acid[END_REF], but which are otherwise promiscuous as to the G type they activate [START_REF] Grammatopoulos | Insights into mechanisms of corticotropin-releasing hormone receptor signal transduction[END_REF].

In our experimental conditions, only micromolar concentrations of Cragi-ELH1 and Cragi-ELH2 increased intracellular calcium, as found for DH44R1 in Drosophila [START_REF] Johnson | Drosophila CG8422 encodes a functional diuretic hormone receptor[END_REF]. Structure-activity relationships were examined, and demonstrated that like vertebrate CRHs [START_REF] Deussing | The Corticotropin-Releasing Factor Family: Physiology of the Stress Response[END_REF], synthetic N-terminal fragments of Cragi-ELH1 and Cragi-ELH2 were efficient to activate Cragi-ELHR, though with less potency than their respective full-length forms. Both the N-and C-terminal moieties of Cragi-ELHs display very high conservation with other CRH/DH44/ELH peptides. This suggests that, as in vertebrate CRHs, the N-terminal part of the ligand is crucial for receptor binding and activation, although the Cterminal part would be responsible for the affinity and specificity of the ligand [START_REF] Deussing | The Corticotropin-Releasing Factor Family: Physiology of the Stress Response[END_REF]. Although the conformation of oyster ELHs had been predicted [START_REF] Stewart | Neuropeptides encoded by the genomes of the Akoya pearl oyster Pinctata fucata and Pacific oyster Crassostrea gigas: a bioinformatic and peptidomic survey[END_REF], their high-resolution structure was determined by NMR in this study. From a structural point of view, there was no defined structure of the ELH1a and ELH2 peptides in water, as often encountered with small peptides. However, an extended alpha helix was found in a micellar medium after molecular modeling using NMR constraints. Also observed in human and rat CRHs [START_REF] Spyroulias | Monitoring the structural consequences of Phe12 → D-Phe and Leu15 → Aib substitution in human/rat corticotropin releasing hormone: Implications for design of CRH antagonists[END_REF] and more generally in Class B GPCR ligands [START_REF] Neumann | Class-B GPCR activation: is ligand helix-capping the key?[END_REF], such a structuring of Cragi-ELH1a and Cragi-ELH2 in close proximity to the membrane is consistent with the idea that peptides can adopt a helical structure in the vicinity of their membrane receptors.

It was somewhat intriguing to observe that Cragi-ELH1 was much more potent than Cragi-ELH2 to activate Cragi-ELHR. Yet, unlike Cragi-ELH2, Cragi-ELH1 does not seem to exist as a molecular entity in oyster: it is processed into three unrelated peptides (ELH1a, ELH1b and ELH1c). We cannot exclude the possibility that Cragi-ELH1 was not detected by MS due to an inappropriate extraction procedure or poor ionization of the peptide. However, we think this is unlikely because Cragi-ELH1 cleavage peptides have been easily identified by MS, which means that Cragi-ELH1 occurs in rather small amounts if it does exist. As a result, Cragi-ELH2 appears as the main activator of the ELH signaling system in C. gigas given the poor potency of ELH1a in activating Cragi-ELHR. Potency differences between Cragi-ELH2 and Cragi-ELH1a may reflect respective roles as a neurohormone and a neurotransmitter. Nevertheless, apart from Cragi-ELH1a, the other two neuropeptides generated by the processing of Cragi-ELH1 constitute potential biologically active factors regulating target tissues via distinct receptors. The processing of Cragi-ELH1 is somewhat reminiscent of that of Aplysia californica ELH and Lymnaea stagnalis CDCH precursors: multiple short peptides (BCPs,  CDCPs) are generated that play a role as neurotransmitters [START_REF] Scheller | A single gene encodes multiple neuropeptides mediating a stereotyped behavior[END_REF] and control the activity of various target neurons implied in the reproductive behavior of L. stagnalis [START_REF] Koene | Neuro-endocrine control of reproduction in hermaphroditic freshwater snails: mechanisms and evolution[END_REF]. Out of the three neuropeptides generated from the processing of Cragi-ELH1, the C-terminal tetrapeptide LFRYamide (Cragi-ELH1c) exhibited a sequence identical to the C-terminal sequence of Cragi-LXRYamide (Réalis-Doyelle et al., 2021), and its structure clearly resembled that of the LFRFamide family of neuropeptides identified in C. gigas [START_REF] Bigot | Functional characterization of a short neuropeptide F-related receptor in a lophotrochozoan, the mollusk Crassostrea gigas[END_REF]. In agreement with a previous work [START_REF] Réalis-Doyelle | Transcriptome profiling of the pacific oyster Crassostrea gigas visceral ganglia over a reproduction cycle identifies novel regulatory peptides[END_REF], the present study shows that Cragi-ELH gene expression peaks in the VG of reproductively mature male and female oysters. Interestingly, the expression of the Cragi-LXRYamide-encoding gene increased in parallel to that of the Cragi-ELH-encoding gene in the VGs of reproductively mature oysters [START_REF] Réalis-Doyelle | Transcriptome profiling of the pacific oyster Crassostrea gigas visceral ganglia over a reproduction cycle identifies novel regulatory peptides[END_REF]. In addition, LFRFamide signaling regulates reproduction and feeding processes in mollusks [START_REF] Bigot | Functional characterization of a short neuropeptide F-related receptor in a lophotrochozoan, the mollusk Crassostrea gigas[END_REF][START_REF] Hoek | LFRFamides: a novel family of parasitation-induced -RFamide neuropeptides that inhibit the activity of neuroendocrine cells in Lymnaea stagnalis[END_REF]. Thus, although encoded by different genes, these closely structurally related neuropeptides may synergistically activate the same receptor and contribute to regulate reproduction-and feeding-associated functions in oysters. The significant increased expression of Cragi-ELH in the VGs of mature oysters suggests a role in the regulation of reproduction. However, considering the low and stable expression of Cragi-ELHR in oyster gonads during a reproductive cycle, it is not clear whether Cragi-ELH signaling is directly involved in spawning. The highest levels of expression of Cragi-ELHR and Cragi-ELH in the VGs compared to other adult tissues rather suggest a reproduction-associated neuroregulatory role in the central nervous system. Cragi-ELH signaling is likely not involved in the regulation of feeding, as no significant change was recorded in the expression of ELH receptor-and ligand-encoding genes during a nutritional stress. This is in contrast with the reported role of DH44 in inducing satiety in the desert locust Schistocerca gregaria [START_REF] Van Wielendaele | CRF-like diuretic hormone negatively affects both feeding and reproduction in the desert locust, Schistocerca gregaria[END_REF]. Given the major role of CRH and DH44 in mediating the stress response and the regulation of osmolarity, it was rational to investigate the possible role of Cragi-ELH signaling during a hypoosmotic stressa common environmental pressure experienced by oysters in estuaries [START_REF] Guo | Immune and stress responses in oysters with insights on adaptation[END_REF]. Cragi-ELH tended to increase in the VG following a mild or acute hypoosmotic stress, but these changes CT1b/CTR expression decreased, whereas CT2/CTR2 expression increased [START_REF] Schwartz | Characterization of an evolutionarily conserved calcitonin signalling system in a lophotrochozoan, the Pacific oyster (Crassostrea gigas)[END_REF].

Conclusion

An ELH signaling system was characterized for the first time in a lophotrochozoan species.

This signaling system is evolutionarily related to the vertebrate CRH and arthropod DH44 signaling systems. The involvement of ELH signaling in the regulation of reproduction-related functions in oysters is very likely, while its involvement in mediating the stress response remains doubtful.

  The reaction was stopped by incubation at 70 °C for 10 min. The GoTaq® qPCR Master Mix (Promega) was used for real time monitoring of amplification (5 ng of cDNA template, 40 cycles: 95 °C/15s, 60 °C/15 s) with transcript specific primers for Cragi-ELH QF (5'-GTTCCGATACGGCAAAAGAA -3') and QR (5'-AATCAAGGGCGTTTGATCTG -3') and for Cragi-ELHR QF2 (5'-GGTCCCAATCGTTGCAAATCAA-3') and QR2 (5' -CCCTCAACAAGCATCCAGAAGA-3') as forward (QF) and reverse (QR) primers respectively. Accurate amplification of the target amplicon was checked by performing a melting curve. A parallel amplification of C. gigas Elongation Factor1α (EF1α) QS-Cg-EF1α

  Fig.1, iProX database IPX0007327001). A peptide corresponding to the predicted full length

Figure 1 :

 1 Figure 1: Multiple sequence alignment of Cragi-ELH and ELH-related family

Figure 2 :Figure 3 :

 23 Figure revisionClick here to access/download;Figure;Revision Figures Cragi ELHR.docx

Figure 4 :

 4 Figure 4: Activation modalities of Cragi-ELHR by Cragi-ELH peptides. A: Activation of

Figure 5 :

 5 Figure 5: Expression of mRNAs encoding Cragi-ELHR and Cragi-ELH precursor in adult

Figure 6 :

 6 Figure 6: Expression of mRNAs Cragi-ELH precursor and Cragi-ELHR in VG of

  were not statistically significant, whereas Cragi-ELHR increased significantly in mild stress conditions. Complementary experiments should be carried out on animals exposed to other stress conditions or other stressors (i.e., temperature or air exposure), but our results suggest that Cragi-ELH signaling does not represent a key neuroendocrine pathway of the stress response or osmotic regulation in C. gigas. In contrast, decreased expression of the CCAP signaling components has been evidenced in the gills of oyster[START_REF] Realis-Doyelle | Molecular and physiological characterization of a crustacean cardioactive signaling system in a lophotrochozoanthe pacific oyster (Crassostrea gigas): a role in reproduction and salinity acclimation[END_REF] in similar mild hyposaline conditions. Conversely, the two distinct calcitonin (CT) signaling systems of C. gigas showed opposite responses in lower saline concentration conditions:

Fundings:

This work was supported by the ANR project NEMO (Agence Nationale de la Recherche 14CE02 0020) and by the Council of the Normandy Region (RIN ECUME: 18E01643-18P02383) to P. Favrel. Credit authorship contribution statement: PF, JL and AB designed the study; MPD, JP, JS, BB, LM, GR, JL and AB performed the experiments and analyzed, PF wrote the paper. All authors reviewed and edited the manuscript.

Competing interests:

The authors declare no competing or financial interests

Supplementary Material revision

Click here to access/download Supplementary Material Revision Suppl data Cragi ELH signaling.pdf


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