Eslam Elkalla

Sumera Khizar
email: sumera.khizar@univ-lyon1.fr

Zouhair Ait-Touchente

Noureddine Lebaz

Marie Hangouët

Guy Raffin

Nadia Zine

Abdelhamid Elaissari

Synthesis and Characterization of Silica-Coated Magnetic Oil-in-Water (O/W) Magnetic Emulsion

Keywords: Oil-in-water emulsion, Organic ferrofluid, Magnetic nanoparticles, Silica shell

A stable emulsion of organic ferrofluid in an aqueous medium, known as oil-in-water (O/W) ferrofluid emulsion, was successfully prepared and characterized. The present study introduces a novel approach to synthesize and emulsify ferrofluid, followed by silica coating of the resulting nanoparticles using sol-gel process. The ferrofluid consisted of magnetic nanoparticles stabilized with oleic acid in octane. Both the ferrofluid and the emulsion exhibited spherical morphology, with average sizes of 20 nm and 200 nm, respectively. Characterization methodologies for instance TEM (transmission electron microscopy), DLS (dynamic light scattering), XRD, FTIR, and magnetic measurements were employed to determine the size distribution, crystallinity, morphology, and chemical composition of the nanoparticles.

Chemical composition analysis of the prepared nanoparticles was performed using thermogravimetric analysis. This research work demonstrates the possibility of encapsulating O/W magnetic emulsion, promising potential for future bioapplications, emphasizing the importance of efficient and stable O/W ferrofluid emulsions.

Introduction:

The research on ferrofluids has significantly improved in last 50 years leading to a substantial extent of scientific knowledge and practical applications of these specific materials. These are artificial materials in which magnetism is present in a liquid state with high magnetic susceptibility [1]. Ferrofluids are acknowledged as nanoscale materials, serving as colloidal suspensions that consist of minute magnetic particles dispersed within aqueous or organic liquids. They exhibit interesting physiochemical characteristics (thermal, optical, magnetic, rheological, apparent density, etc.) under a magnetic stimulus have been at the forefront of research [START_REF] Philip | Magnetic Nanofluids (Ferrofluids): Recent Advances, Applications, Challenges, and Future Directions[END_REF]. The presence of these magnetic properties enables effective control and manipulation of ferrofluids through the application of magnetic fields. Various approaches have been employed to synthesize ferrofluids, including hydrothermal technique, sol-gel pyrolysis, mechanochemical synthesis, flame synthesis, mechanical alloying, thermal decomposition, mechanical milling, coprecipitation, sonochemical method, and one-pot synthesis. [START_REF] Kole | Engineering Applications of Ferrofluids: A Review[END_REF]. Among all, well reported synthetic methods are coprecipitation and thermal decomposition, principally because of their ease and reliability [START_REF] Vuong | Synthesis of High-Magnetization and Monodisperse Fe3O4 Nanoparticles via Thermal Decomposition[END_REF][START_REF] Maity | Synthesis of Iron Oxide Nanoparticles under Oxidizing Environment and Their Stabilization in Aqueous and Non-Aqueous Media[END_REF]. Furthermore, these techniques provide more consistent results, require less equipment, and to best our knowledge are simply more thoroughly documented compared to other methods [START_REF] Paquin | Multi-Phase Semicrystalline Microstructures Drive Exciton Dissociation in Neat Plastic Semiconductors[END_REF]. The applications of magnetic nanoparticles in diverse disciplines are growing day by day. There are countless reported applications of magnetic nanoparticles, counting medical imaging, biosensing, medicinal therapy, magnetic impedance, and magnetic nanoemulsions. Additional applications include energy harvesting and transfer, water treatment, and vibration control [START_REF] Oehlsen | Approaches on Ferrofluid Synthesis and Applications: Current Status and Future Perspectives[END_REF].

Typically, magnetic emulsions have three components: iron oxide nanoparticles, dispersion medium or carrier liquid and a surfactant to prevent their aggregation and to weaken magnetic interactions among them. But depending on the desired magnetic properties and applications, magnetic emulsions can consist of nanoparticles composed of mixed oxides containing cobalt, manganese, barium or nickel. Mostly maghemite (Fe 2 O 3 ) and magnetite (Fe 3 O 4 ) are employed for the synthesis of ferrofluids [START_REF] Wu | Recent Progress on Magnetic Iron Oxide Nanoparticles: Synthesis, Surface Functional Strategies and Biomedical Applications[END_REF]. The stability regarding emulsions not only depends on the affinity between surfactant and carrier liquid, but it also depends on the method of synthesis and size of the nanoparticles. Ferrofluid emulsions have gained significant recognition due to their distinct properties, such as cytocompatibility, affinity to biological systems, nanoscale dimensions, superparamagnetic behavior, and swift magnetic field responsiveness [START_REF] Ait-Touchente | Exploring the Versatility of Microemulsions in Cutaneous Drug Delivery: Opportunities and Challenges[END_REF]. Consequently, they have found diverse applications spanning from the field of biomedicine to bioengineering sectors [START_REF] Zhang | Advanced Materials and Processing for Drug Delivery: The Past and the Future[END_REF]. In the biomedical domain, ferrofluid emulsions find significant utility as carriers for drug delivery, contrast agents, antimicrobial agents, as well as heat mediators in hyperthermia [START_REF] Khizar | Encapsulation of Doxorubicin in Magnetic-Polymer Hybrid Colloidal Particles of Eudragit E100 and Their Hyperthermia and Drug Release Studies[END_REF]. Additionally, their applications extend to biosensors, including magnetic impedance biosensors. The use of magnetic emulsions in biomedical diagnosis is because of superparamagnetic properties of magnetic nanoparticles. Its first application in biomedical diagnosis was in immunoassay. The second use was developed in nucleic acid based diagnostics: specific capture and non-specific capture, purification and concentration of nucleic acid material (DNA and RNA). Another application is related to cell sorting in vivo or in vitro [START_REF] Kritika | Therapeutic Applications of Magnetic Nanoparticles: Recent Advances[END_REF].

One of the challenges of utilizing magnetic nanoparticles is their tendency to aggregate due to their high surface energy and strong magnetic attraction potential [START_REF] Khizar | Magnetic Colloidal Particles in Combinatorial Thin-Film Gradients for Magnetic Resonance Imaging and Hyperthermia[END_REF]. To address this issue, surface modification techniques such as coating with silica shells have been developed. Silica coating provides a protective shell around the magnetic core, which can prevent aggregation and enhance biocompatibility of nanoparticles [START_REF] Fookes | Controlled Fluoride Release for Osteoporosis Treatment Using Orally Administered Chitosan Hydrogels[END_REF]. Stöber Sol-gel method is the simplest route for silica coating that offers a functional interface for attaching biomolecules such as proteins, peptides, and antibodies. This enables precise transportation of magnetic nanoparticles to particular cells or tissues, providing targeted delivery. Silica-coated magnetite nanoparticles (SMNP) recently gained much attraction in biomedical, environmental, construction and nuclear applications. Extensive research has focused on their investigation in treatment applications such as drug delivery, neutron capture therapy (NCT), hyperthermia, imaging applications for instance magnetic resonance imaging (MRI), magnetic particle imaging (MPI) and computed tomography (CT), and separation and detection applications for nucleic acids, cells, and proteins [START_REF] Mokkarat | One-Step Continuous Flow Synthesis of Aminopropyl Silica-Coated Magnetite Nanoparticles[END_REF]. They exhibit excellent properties, high biocompatibility, chemical and thermal stability, and ease of functionalization. Moreover, silica protects magnetic nanoparticles against acid leaching and aggregation as well as increases their physiochemical stability and cytocompatibility [START_REF] Bruckmann | Biological Applications of Silica-Based Nanoparticles[END_REF]. Previous investigations reported that silica-based magnetic nanoparticles also present several biological proprieties, for instance antitumor, anti-protozoal, antibacterial, and stroke treatment [START_REF] Ding | MnFe2O4-Decorated Large-Pore Mesoporous Silica-Coated Upconversion Nanoparticles for near-Infrared Light-Induced and O2 Self-Sufficient Photodynamic Therapy[END_REF]. Liquid capsules can also be formulated using Sol-gel method to contain precise amounts of active ingredients, which can be particularly important for medications with narrow therapeutic windows or where precise dosing is critical [START_REF] Elkalla | Core-Shell Micro/Nanocapsules: From Encapsulation to Applications[END_REF]. This can help to ensure that patients receive the correct dose of medication, which can improve treatment outcomes and reduce the risk of side effects [START_REF] Fookes | Controlled Fluoride Release for Osteoporosis Treatment Using Orally Administered Chitosan Hydrogels[END_REF].

Considering the significance and distinctive characteristics of ferrofluids, the present study aims to prepare oil-in-water (O/W) magnetic emulsion through the emulsification process involving organic ferrofluid in an aqueous medium. The organic ferrofluid was synthesized via coprecipitation method and stabilized in octane. Subsequently, this magnetic fluid was emulsified in an aqueous surfactant solution to achieve a stable emulsion consisting of magnetic colloidal particles. To enhance their stability, the magnetic nanoparticles were subjected to a silica coating using sol-gel process. Various analytical techniques were employed to assess the properties of the prepared ferrofluid, the resulting emulsion, and the silica-coated magnetic nanoparticles [START_REF] Hong | Synthesis of Fe3O4 Nanoparticles without Inert Gas Protection Used as Precursors of Magnetic Fluids[END_REF][START_REF] Elaissari | Magnetic Latex Particles in Nanobiotechnologies for Biomedical Diagnostic Applications: State of the Art[END_REF]. The research work described here is novel and elaborate the encapsulation of SDS stabilized organic ferrofluid by silica shell to form liquid capsules and solid shell. The synthesis of stable emulsion involving combination of organic ferrofluid with SDS is significant from several perspectives. This strategy provides a new, simple, economical, environmentally friendly, efficient, and likely scalable route to encapsulate organic ferrofluid inside silica shell efficiently without evaporation of octane and involvement of the complicated carrier design. The developed silica-coated magnetic particles could be promising efficient diagnostic bioapplications in future.

Materials and Methods

Materials

FeCl 2 .4H 2 O, FeCl 3 .6H 2 O, HCl (35%), NH 3 (25%), oleic acid, ethanol 96%, polyvinylpyrrolidone (PVP), tetraethyl orthosilicate (TEOS), and analytical-grade octane were obtained from Sigma-Aldrich (Germany). Sodium dodecyl sulfate (SDS), an anionic surfactant, was also acquired from Sigma-Aldrich (Germany). Ultra-pure water was employed in the formulation of all solutions. All chemicals and materials were of analytical grades and used as received.

Methods

Aqueous ferrofluid preparation

The coprecipitation technique is commonly employed for synthesizing ferrofluids by reducing metallic elements using various salt conditions [START_REF] Anderson | Preparation and Properties of an Antithyrotropic Substance[END_REF]. To initiate the synthesis, ferrous chloride (3.7 g) and ferric chloride (10.16 g) were dissolved in 80 ml of ultrapure water. Once the iron salts completely dissolved, resulting in a homogeneous orange solution, 80 ml of concentrated ammonia solution (25%) was swiftly introduced into 50 ml thermostated round-bottom four necked flask reactor, equipped with a glass anchor-shaped stirrer, condenser, and nitrogen inlet (same reactor which is used for batch emulsion polymerization reactions) while vigorously stirring at 500 rpm. The immediate formation of a dark precipitate indicated the generation of iron oxide.

2.2.2.

Organic ferrofluid preparation

The aqueous ferrofluid mentioned above was placed in above-mentioned reactor and subjected to gentle stirring. Following one hour, 8 ml of oleic acid was gradually introduced with stirring at a speed of 500 rpm. After a two-hour interval, octane was incrementally added to the solution using a pipette and stirred for duration of 15 minutes. The mixture was left undisturbed overnight. During this time, the iron oxide nanoparticles coated with oleic acid spontaneously migrated from the ammoniac phase to the organic phase, resulting in a distinct separation between a transparent lower phase (aqueous) and an upper phase containing the ferrofluid organic component [START_REF] Bitar | Submicron Silica Shell-Magnetic Core Preparation and Characterization[END_REF][START_REF] Khizar | Magnetic Nanoparticles: From Synthesis to Theranostic Applications[END_REF].

Preparation of magnetic oil-in-water (O/W) emulsion

To obtain the pre-emulsion, the gradual addition of the organic ferrofluid into an aqueous surfactant solution was carried out under moderate stirring conditions. The organic ferrofluid, which had been prepared beforehand, was dispersed within the SDS solution and subjected to intense shearing using an Ultra-Turrax device. Numerous samples were prepared through emulsification of prepared organic ferrofluid with aqueous solution containing SDS as an anionic surfactant. Various concentrations of SDS have been investigated to formulate the stable emulsion ranging from 2 to 16 wt%. The stirring speed (9500 rpm) and time (5 minutes) were kept constant. The emulsion mixture was then subjected to emulsification using an Ultra-Turrax device and subsequently filtered through glass wool.

Synthesis of silica-coated magnetic nanoparticles (Si-MN)

In this study, a sol-gel method was employed to coat magnetic emulsion with silica. Trimethoxysilane (TEOS) was utilized as the silica precursor, while 25% ammonia (NH 3 ) acted as the catalyst. To begin, 10 ml of magnetic emulsion was subjected to a wash with deionized water and then dispersed in a solution containing 3 ml of 10% PVP [START_REF] Bitar | Submicron Silica Shell-Magnetic Core Preparation and Characterization[END_REF]. The resulting solution underwent agitation for duration of three hours. Subsequently, 5 ml of ammonia was introduced and further agitation was carried out for 15 minutes. Following this, different amount of TEOS with 1 ml of ethanol were added to the solution, which was then left under stirring overnight. The aim was to determine the optimal amount of TEOS for achieving the best silica shell coating of magnetic core. The silica-coated particles were subsequently separated using a magnet, washed with deionized water, and filtered through glass wool. The initial dispersion of the organic ferrofluid took place in octane, resulting in the formation of a stable colloidal solution. This dispersion played a crucial role in the subsequent stages of the synthesis process, allowing for a comprehensive investigation of the Si-MN core/shell structure while influenced by octane as the dispersing medium [START_REF] Ahmed | New Oil-in-Water Magnetic Emulsion as Contrast Agent for in Vivo Magnetic Resonance Imaging (MRI)[END_REF].

Characterization techniques

The average hydrodynamic diameter (D h ) and particle size distribution of the organic ferrofluid, emulsion, and silica-coated magnetic nanoparticles were determined using the Malvern Zetasizer (Nano ZS, Malvern Instruments Limited, U.K). The measurements were conducted in a 1 mM NaCl solution at 25 °C. The physical dimensions of the prepared magnetic nanoparticles were examined by employing Transmission Electron Microscopy (TEM, Netherlands) with a Philips CM120 microscope at the "Centre Technologique des Microstructures" (CT) at the University of Lyon (Villeurbanne, France).

Samples for TEM analysis were prepared by dispersing 10 µL of sample in 900 µL of water (aqueous ferrofluid and silicacoated magnetic nanoparticles) and octane (organic ferrofluid), then dropped on grid and dried before analysis. The magnetic properties of the dried magnetic colloids were investigated at room temperature using a vibrating sample magnetometer (Automatic Bench of Magnetic Measurements-ABMM, U.K.) at the Claude Bernard University Lyon-1, Villeurbanne, France. X-ray diffraction (XRD) analysis was performed to determine the crystallinity of the prepared nanoparticles using XRD diffractrometer (Burker D8 advance). Fourier transform infrared (FTIR) analysis was carried out to study and identify chemical functional groups in the synthesized compound with a FTIR spectrometer -Nexus (Thermo-Fisher Nicolet) in the range of 400-4000 cm -1 . Thermogravimetric analysis of the organic ferrofluid and colloids was carried out using the thermogravimetric analyzer (TA Instruments Q50 series) under an inert atmosphere, with the temperature ranging from room temperature to 1000 °C at a heating rate of 10 °C/min.

Results and Discussions

It was found that the most stable emulsion was formulated by using 12 wt% SDS solution. Different samples were prepared by varying the concentration of SDS solution added to organic ferrofluid (solid content of 15 % wt/v) keeping the total volume (12 ml) of magnetic emulsion constant (Table 1). Figure 1 showed different images of oil in water magnetic emulsion prepared by emulsification process. It can be clearly observed from Figure 1, that EK1 is the most stable emulsion since no phases separation is observed. Other samples showed phase separation and sedimentation that revealed their instability. Subsequently, the most stable magnetic emulsion (EK1) was further characterized. The hydrodynamic diameter (D h ) of the ferrofluid nanoparticles was measured to be 25 nm with an average value obtained from measurements carried out three times represented by three different colors (Figure 2a) [START_REF] Nagornyi | Structural Characterization of Concentrated Aqueous Ferrofluids[END_REF]. The findings indicate that the nanoparticles exhibited characteristics indicative of superparamagnetism. Additionally, the polydispersity index (PDI)

was determined and yielded a value of 0.482 (PDI = D w /D n ) that indicates slight polydispersity in size of nanoparticles with a standard deviation (SD) of 13.69 nm. Figure 2b shows the TEM image of ferrofluid nanoparticles. TEM results showed that they have a nearly spherical shape and are uniformly distributed in size. The observed large particle size from DLS compared to TEM analysis can be attributed to the presence of aggregated nanoparticles. This aggregation phenomena can be induced by low colloidal stability of the nanoparticles or attributed to The agglomeration in such nanoparticles indicates the influence of dipolar and magnetic interactions between the particles and has been frequently reported by literature [START_REF] Putra | Darminto Fractal Structures on Fe3O4 Ferrofluid: A Small-Angle Neutron Scattering Study[END_REF][START_REF] Van Silfhout | Colloidal Stability of Aqueous Ferrofluids at 10 T[END_REF]. These results showed that there are both single as well as multidomain magnetic nanoparticles [START_REF] Vékás | Synthesis of Magnetic Nanoparticles and Magnetic Fluids for Biomedical Applications[END_REF]. The average size distribution of an organic ferrofluid showed an average size of 20 nm obtained from three measurements as shown in Figure 3a. This showed that particles possess nanoscale dimensions with PDI of 0.419 and standard deviation (SD) was calculated to be 10.39 nm. TEM image of organic ferrofluid particles (Figure 3b) indicated an average particle size of less than 50 nm. The magnetic particles appeared to be nearly spherical in shape. Results indicated that nanoparticles stabilized in octane showed minimum aggregation that promotes homogeneous distribution [START_REF] Hong | Preparation and Characterization of Fe3O4/Polystyrene Composite Particles via Inverse Emulsion Polymerization[END_REF][START_REF] Beković | Numerical Model for Determining the Magnetic Loss of Magnetic Fluids[END_REF] . Figure 4 gives The size distribution of the most stable emulsion (EK1 in Table 1) is reported in Figure 4. The mean droplet size is around 200 nm. This value was determined by averaging the results of three separate measurements. The PDI value of 0.209 with a standard deviation of 92.28 nm suggests a nearly monodisperse system with excellent colloidal stability as macrospically demonstrated above. The hydrodynamic particle size distribution offers insights into emulsion stability, considering the concentration and type of surfactant used, as well as experimental conditions. However, further studies are required to optimize the emulsion preparation process and ensure consistent hydrodynamic particle size distribution.

Obtaining a TEM image of the magnetic emulsion is challenging due to the presence of octane within the emulsion [START_REF] Montagne | Preparation and Characterization of Narrow Sized (o/w) Magnetic Emulsion[END_REF]. The most colloidally stable magnetic dispersion sample was with attained with 1.25 ml TEOS. The hydrodynamic size distribution analysis of these nanoparticles was performed in water dispersion. These resulting particles comprise magnetic nanoparticles dispersed in octane are coated with a silica shell. The experimental findings displayed a predominant peak average size of 1 μm, as illustrated in Figure 6a. Moreover, Figure 6b exhibits TEM images of the obtained final particles, revealing the presence of a distinct silica shell enveloping the magnetic core. The thickness of the silica shell varied in the range of 9 nm to 19 nm, depending on the quantity of TEOS employed during the coating process. The TEM image distinctly depicts individual Si-MN core/shell nanoparticles, with well-defined core and shell regions [START_REF] Shao | The Shape Effect of Magnetic Mesoporous Silica Nanoparticles on Endocytosis, Biocompatibility and Biodistribution[END_REF]. The core, comprised of magnetic nanoparticles, appears as a dark region, while the silica shell exhibits a lighter color. Additionally, the TEM image captures the presence of the octane solvent, either as a surrounding medium or as residual droplets. This TEM analysis facilitates the observation of the morphology and integrity of the Si-MN core/shell nanoparticles, which significantly influences their performance in various applications. The TEM image of the Si-MN core/shell nanoparticles in the presence of octane serves as a visual confirmation of the successful synthesis of these particles and lays the groundwork for further comprehensive analysis and characterization. Results for silica shell using TEOS are in good agreement with the previously reported literature [START_REF] Chekalil | Multi-Step Synthesis of Core-Shell Magnetic Nanoparticles Bearing Acid-Chelating Functional Moieties[END_REF][START_REF] Rosenholm | Large-Pore Mesoporous Silica-Coated Magnetite Core-Shell Nanocomposites and Their Relevance for Biomedical Applications[END_REF]

Magnetization of aqueous ferrofluid, organic ferrofluid, and O/W magnetic emulsion

The saturation magnetization of the magnetic emulsion, as depicted in Figure 7, was determined by extrapolating the magnetization curve versus (M = f(1/H)) to magnetic field curve strength approaching infinity. Both the aqueous and organic ferrofluids exhibited magnetization values of approximately 80 emu/g and 75 emu/g, respectively, which closely resemble the bulk magnetite value of 92 emu/g [START_REF] Ebadi | Release of a Liver Anticancer Drug, Sorafenib from Its PVA/LDH-and PEG/LDH-Coated Iron Oxide Nanoparticles for Drug Delivery Applications[END_REF]. This similarity suggests a robust magnetic response in both ferrofluids.

Furthermore, the M-H curve in Figure 6 demonstrated a nearly linear increase in magnetization with the applied magnetic field, a characteristic commonly associated with superparamagnetic materials. The superparamagnetic behavior observed in the ferrofluids is attributed to the small size of the iron oxide nanoparticles, which contributes to their exceptional magnetic properties, including remanence [START_REF] Song | A Multifunctional Nanotheranostic for the Intelligent MRI Diagnosis and Synergistic Treatment of Hypoxic Tumor[END_REF]. However, it is worth noting that the magnetization of the organic ferrofluid was slightly lower than that of the aqueous ferrofluid, potentially due to the presence of magnetically inactive oleic acid [START_REF] Matsumura | Stability and Utility of Pyridyl Disulfide Functionality in RAFT and Conventional Radical Polymerizations[END_REF].

Additionally, the magnetization of the magnetic emulsion was determined to be 40 emu/g, which is lower than the magnetization values of both the aqueous and organic ferrofluids. This decrease is attributed to the presence of nonmagnetic components, such as oleic acid and sodium dodecyl sulfate (SDS), within the emulsion. It is important to consider that the magnetization values are influenced by various factors, including particle size, chemical composition morphology, and crystal structure [START_REF] Rahman | Anisotropic Magnetic Microparticles from Ferrofluid Emulsion[END_REF]. Furthermore, the drying process during emulsion preparation can result in particle aggregation, ultimately impacting the magnetic properties of the nanoparticles. Despite the lower magnetization exhibited by the emulsion, it displays promising potential in several biomedical applications, including invitro biomedical diagnosis, sample preparation, drug delivery, magnetic hyperthermia, and magnetic resonance imaging [START_REF] Ahmed | In Vitro MRI of Biodegradable Hybrid (Iron Oxide/Polycaprolactone) Magnetic Nanoparticles Prepared via Modified Double Emulsion Evaporation Mechanism[END_REF].

XRD analysis

The prepared nanoparticles were characterized by XRD for structural determination. The X-ray diffraction patterns of the organic ferrofluid is presented in Figure 8 that contains a series of characteristic peaks at 2 θ= 23.0°, 27.2°, 32.8°, 35.7o, 46.9°, 52.9°, 58.3°, 63.0°, and 78.0° are indexed as the diffractions of (111), ( 220), (311), ( 222), (331), ( 422), (511), (440), and (444) crystalline planes. The analysis of the diffraction pattern showed the formation of a cubic spinel structure in the sample, due to the strongest reflection that proceeds from the (311) plane that is in good accordance with the inverse cubic spinel phase of Fe3O4 (magnetite, Ref-code 01-075-1609). These results confirmed the crystalline structure of obtained magnetite nanoparticles, agreed with the structure of an inverse spinel-type oxide as reported in the literature [START_REF] Tasmia; Shah | Microextraction of Selected Endocrine Disrupting Phenolic Compounds Using Magnetic Chitosan Biopolymer Graphene Oxide Nanocomposite[END_REF][START_REF] Elizondo-Villarreal | Green Synthesis of Magnetic Nanoparticles of Iron Oxide Using Aqueous Extracts of Lemon Peel Waste and Its Application in Anti-Corrosive Coatings[END_REF]. Diffraction patterns of magnetic emulsion showed intense peaks at are 2θ = 6.78°, 20.0°, 21.7°, 22.6°, and 32.2° corresponding to the miller indexes (200), ( 213), (400), and (122), (311) respectively [START_REF] Asab | Synthesis of Silica-Coated Fe3O4 Nanoparticles by Microemulsion Method: Characterization and Evaluation of Antimicrobial Activity[END_REF]. The XRD pattern of magnetic emulsion showed peak at 32.2° that corresponds to magnetite nanoparticles but of very low intensity. The new peaks correspond to SDS characteristics peaks [START_REF] Lun | Enhancing Dispersion of Halloysite Nanotubes via Chemical Modification[END_REF][START_REF] Ibrahim | Surfactant Effects on the Synthesis of Redox Bifunctional V2o5 Photocatalysts[END_REF]. The XRD pattern of silica-coated o/w magnetic particles displayed diffraction peaks at 6.89°, 11.3°, 13.5°, 31.7°, refer to (002), ( 200), (111), and (311) planes, respectively. The additional peak at 6.89° degree corresponds to SiO 2 [START_REF] Islam | Silica Encapsulation of Sonochemically Synthesized Iron Oxide Nanoparticles[END_REF][START_REF] Adhikary | Effects of Carbon Nanotubes on Expanded Glass and Silica Aerogel Based Lightweight Concrete[END_REF]. This peak shows the presence of silica shell encapsulating magnetic o/w emulsion nanodroplets. The reduced intensity of (311) crystalline plane in emulsion and Si-MN is due to random crystal growth in which grains are not oriented correctly to allow diffraction to occur [START_REF] Sjögren-Levin | Separation of XRD Peak Profiles in Single-Phase Metals with Bimodal Grain Structure to Analyze Stress Partitioning[END_REF].

FTIR analysis of magnetic emulsion and silica-coated o/w magnetic emulsion

The FTIR spectra in Figure 9 depict the magnetic emulsion (ME) and silica-coated magnetic particles (Si-MN). Notably, an additional peak at 1220 cm -1 corresponds to the sulfate asymmetric stretch vibration, while two peaks at 1083 and 834 cm -1 relate to the sulfate symmetric stretch vibration [START_REF] Ahmadi | Hybrid Silane Coating Reinforced with Silanized Graphene Oxide Nanosheets with Improved Corrosion Protective Performance[END_REF]. These findings conclusively validate the existence of SDS within the magnetic emulsion samples. Distinctive bands characteristic of SDS molecules in the ME samples are absent in Si-MN.

Instead, Two new peaks emerge at 1092 and 809 cm -1 , indicative of the presence of SiO 2 within the sample [START_REF] Shen | Biofunctional Magnetic Nanoparticles as a General Agent to Immobilize Proteins Contained in Traditional Chinese Medicines[END_REF][START_REF] Foroozan | Effect of Coating Composition on the Anticorrosion Performance of a Silane Sol-Gel Layer on Mild Steel[END_REF]. Please change silica-coated magnetic nanoparticles by silica-coated o/w magnetic emulsion in the figure

Thermogravimetric analysis of magnetic emulsion and silica-coated magnetic nanoparticles

The primary objective of conducting thermogravimetric analysis (TGA) was to assess the thermal behavior and chemical composition of the organic ferrofluid through the process of thermal decomposition. The TGA was carried out under an inert atmosphere, from room temperature to 1000 °C; with a heating rate of 10 °C/min. Figure 10 illustrates the TGA curve of the prepared magnetic emulsion which displayed two-step degradation, since an excess of oleic acid was utilized, resulting in free molecules of oleic acid predominantly orienting towards the water interface. The initial weight loss at 200 °C, is due to the desorption of the physically adsorbed water and the dehydroxylation of hydroxyl groups on the surface of magnetic nanoparticles. The degradation of oleic acid in organic ferrofluid is the cause of second weight loss above 200 °C. [START_REF] Khizar | Magnetic Colloidal Particles in Combinatorial Thin-Film Gradients for Magnetic Resonance Imaging and Hyperthermia[END_REF]. There was no significant thermal variation observed in these investigations. TGA of the magnetic emulsion demonstrated a gradual decrease in mass. Consequently, TGA analysis revealed that the magnetic emulsion comprised approximately 35% iron oxides and 65% oleic acid, which remained bounded to the nanoparticles [START_REF] Sahoo | Aqueous Ferrofluid of Magnetite Nanoparticles: Fluorescence Labeling and Magnetophoretic Control[END_REF].

TGA curve of Si-MN nanoparticles provided insights into the onset and rate of weight loss, revealing the presence of different components within the nanoparticles and their respective thermal decomposition behavior. As shown in Figure 7, the results indicated an overall weight loss of around 80.6% between 25 °C and 1000 °C, primarily attributed to the degradation of organic and volatile components present in the sample. The weight loss observed during TGA analysis could be divided into distinct steps. Initially, there was a weight loss of approximately 5.2% below 300°C associated with the degradation of TEOS and SDS at 120-140°C and 240°C, respectively. The degradation of oleic acid in Si-MN nanoparticles takes place between 250-300 °C [START_REF] Ashraf | Trisaminomethane-Cobalt Complex Supported on Fe3O4 Magnetic Nanoparticles as an Efficient Recoverable Nanocatalyst for Oxidation of Sulfides and C-S Coupling Reactions[END_REF]. PVP degrades at temperature between 400-450 °C [START_REF] Liu | Preparation and Characterization of Thermal-Sensitive Ferrofluids for Drug Delivery Application[END_REF]. Finally, a weight loss above 700°C is due to the phase transition of magnetic nanoparticles (from magnetite to hematite) [START_REF] Gemeay | Chemical Insight into the Adsorption of Reactive Wool Dyes onto Amine-Functionalized Magnetite/Silica Core-Shell from Industrial Wastewaters[END_REF]. Overall, there was a cumulative weight loss of 80%. Based on the mass loss data, the magnetic content within the Si-MN nanoparticles was estimated to be approximately 20%. The results showed that Si-MN nanoparticles are more stable than magnetic emulsion.

A comprehensive understanding of the weight loss pattern and the contribution of different components enables a better comprehension of the chemical composition and thermal characteristics of the sample, thereby facilitating optimization of its synthesis and exploration of potential applications [START_REF] Khizar | Magnetic Colloidal Particles in Combinatorial Thin-Film Gradients for Magnetic Resonance Imaging and Hyperthermia[END_REF][START_REF] Gemeay | Chemical Insight into the Adsorption of Reactive Wool Dyes onto Amine-Functionalized Magnetite/Silica Core-Shell from Industrial Wastewaters[END_REF].

Figure 10. Thermal analysis of the prepared magnetic emulsion and silica-coated magnetic nanoparticles using thermogravimetric analysis (TGA).

Conclusions

The present work is focused on emulsification of organic ferrofluid and afterwards encapsulation of prepared magnetic o/w emulsion in silica shell. The simplest method of coprecipitation was employed to synthesize aqueous and organic ferrofluids. Organic ferrofluid was emulsified by single emulsion to formulate o/w magnetic emulsion consuming SDS as a surfactant. The prepared o/w magnetic emulsion nanoparticles were coated by silica shell by Sol-gel process. The size distribution of an aqueous and organic ferrofluid showed an average size of 25 and 20 nm, respectively. The mean o/w magnetic emulsion droplet size is around 200 nm of magnetic emulsion, determined by dynamic light scattering analysis.

TEM images indicated that the thickness of silica shell around magnetic nanoparticles o/w magnetic submicron droplet varies from 9 nm to 19 nm, depending on the quantity of TEOS employed during synthesis. The saturation magnetization of aqueous and organic ferrofluid was 80 and 75 emu/g, respectively which was higher than its colloidal solution which was found to be 40 emu/g due to surface modification. XRD analysis showed characteristic peaks of magnetite, SDS, and SiO 2 .

FTIR spectra showed characteristic bands of SDS molecules and SiO 2 that showed encapsulation of magnetic nanoparticles inside silica shell. The iron oxide contents were measured by TGA and found to be 35 and 20 % in the magnetic emulsion and silica-coated magnetic nanoparticles. Overall, this investigation establishes a solid foundation for future studies on silica-coated magnetic nanoparticles and their potential applications in the realms of biomedicine and environment.

Figure 1 . 3 . 1 .

 131 Figure 1. Different samples of emulsion prepared by emulsification of organic ferrofluid in SDS aqueous medium. Please change the labeling (should not be by hand) 3.1. Analysis of Aqueous Ferrofluid, Organic Ferrofluid, and Magnetic Emulsion Using Dynamic Light Scattering (DLS) and TEM

Figure 2 .

 2 Figure 2. (a) Dynamic light scattering (DLS) analysis performed three times shown by three different colors and (b) TEM image of highly diluted aqueous ferrofluid.

Figure 3 .

 3 Figure 3. (a) Dynamic light scattering (DLS) analysis performed three times shown by three different colors and (b) TEM image of highly diluted organic ferrofluid (in octane).

Figure 4 .

 4 Figure 4. Dynamic light scattering (DLS) analysis of EK1 performed three times shown by three different colors.

Figure 5 (

 5 Figure5 (a, b, c and d) displays TEM images from various experiments carried out with different amounts of TEOS (50 µL, 100 µL, 150 µL, 200 µL) consecutively, revealing the presence of magnetic particles and silica particles surrounding the magnetic core. However, it is evident from TEM images that clear core-shell morphology has not been achieved[START_REF] Quy | Synthesis of Silica-Coated Magnetic Nanoparticles and Application in the Detection of Pathogenic Viruses[END_REF] and only secondary nucleation (free silica nanoparticles) are produced and no any uniform silica shell is observed.

Figure 5 .

 5 Figure 5. TEM image of Si-MN core/shell nanoparticles. (a) 50 µL, (b) 100 µL, (c) 150 µL, and (d) 200 µL.

Figure 6 .

 6 Figure 6. (a) Dynamic light scattering (DLS) analysis and (b) TEM image of Si-MN core/shell nanoparticles with 1.25 ml TEOS.

Figure 7 .

 7 Figure 7. Magnetization versus magnetic field strength. (what is the unit of magnetization (emu/g) should be added) Ms should be changed by emu/g as used in the text

Figure 8 .

 8 Figure 8. Comparison of XRD patterns of organic ferrofluid, magnetic emulsion, and silica-coated magnetic emulsion. Should be use in the graph above

Figure 9 .

 9 Figure 9. FTIR analysis of o/w magnetic emulsion and silica-coated o/w magnetic emulsion.

Table 1 :

 1 Preparation of magnetic emulsions using different amounts SDS.

	Sample	Organic Ferrofluid	SDS(12 wt%)	Observation
	EK1	2ml	10ml	More stable
	EK2	3ml	9ml	Not stable
	EK3	4ml	8ml	Little
				Sedimentation
	EK4	5ml	7ml	Phase
				separation and
				sedimentation
	EK5	6ml	6ml	Sedimentation

Acknowledgements

Authors acknowledge the support of the ISA for support of present work.

Funding

This project received no external funding.

Data availability

The experimental data used to support the findings of this study are available from the corresponding author upon request.

Authors Contribution

Eslam Elkalla carried out experimental work and drafted a manuscript. Abdelhamid Elaissari designed and coordinated this research as a principal investigator. Sumera Khizar and Zouhair Ait-Touchente assisted in doing experiments and drafting manuscript. Noureddine Lebaz, Marie Hangouet, and Guy Raffin helped out in doing characterization. All other authors read and approved the final manuscript.

Declaration

The authors declare that there are no conflicts of interest regarding the publication of this paper.


Andrés Valenzuela-Sánchez

Benedikt R Schmidt

Claudio Azat

Soledad Delgado

Andrew A Cunningham

Jean-François Lemaître

Jean-Michel Lemaître

Hugo Gaillard

Cayuela

Jean-François Lemaître

Jean-Michel Gaillard

Hugo Cayuela7

Variable rate of ageing within species: insights from Darwin's frogs

Keywords:

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

INTRODUCTION

Actuarial senescence (i.e. the increase in adult mortality risk with increasing age; hereafter 'ageing') is a pervasive process across the animal kingdom [START_REF] Nussey | Senescence in natural populations of animals: widespread evidence and its implications for bio-gerontology[END_REF][START_REF] Jones | Diversity of ageing across the tree of life[END_REF]. Although ageing is widespread in the living world, both the onset and the rate of increase in mortality risk with age (i.e. ageing rate) vary markedly across species [START_REF] Jones | Diversity of ageing across the tree of life[END_REF][START_REF] Colchero | The diversity of population responses to environmental change[END_REF]. Studies on terrestrial vertebrates have shown that variations in ageing patterns across species are commonly associated with body size (i.e. higher ageing rates in smaller species; [START_REF] Ricklefs | Intrinsic ageing-related mortality in birds[END_REF][START_REF] Ricklefs | Life-history connections to rates of ageing in terrestrial vertebrates[END_REF][START_REF] Reinke | Diverse aging rates in ectothermic tetrapods provide insights for the evolution of aging and longevity[END_REF], pace of life (i.e. earlier ageing and steeper ageing rates with faster life histories; [START_REF] Jones | Senescence rates are determined by ranking on the fast-slow life-history continuum[END_REF]Lemaître & Gaillard, 2013;[START_REF] Reinke | Diverse aging rates in ectothermic tetrapods provide insights for the evolution of aging and longevity[END_REF], phylogeny [START_REF] Ricklefs | Intrinsic ageing-related mortality in birds[END_REF][START_REF] Lemaître | Sex differences in adult lifespan and ageing rates of mortality across wild mammals[END_REF] and protective phenotypes (i.e. slower ageing rates in ectothermic tetrapods with physical or chemical protection; [START_REF] Reinke | Diverse aging rates in ectothermic tetrapods provide insights for the evolution of aging and longevity[END_REF].

Although between-species variation in ageing is now well documented, the occurrence of variation in ageing patterns within a given species remains much more debatable (Lemaître et al., 2013). Evidence from humans [START_REF] Vaupel | Biodemography of human ageing[END_REF] and some species of non-human primates [START_REF] Colchero | The long lives of primates and the 'invariant rate of ageing' hypothesis[END_REF] suggests that the rate of ageing can be relatively constant within a species (i.e. the 'invariant rate of ageing hypothesis' sensu [START_REF] Colchero | The long lives of primates and the 'invariant rate of ageing' hypothesis[END_REF]. On the contrary, comparisons of senescence patterns between free-living and captive mammals have shown that, except for species with a very slow pace of life, ageing rates are generally higher in the wild than in captivity (Lemaître et al., 2013;[START_REF] Tidière | Comparative analyses of longevity and senescence reveal variable survival benefits of living in zoos across mammals[END_REF]. Natural selection can also lead to within-species variation in ageing, both in the laboratory and in the wild [START_REF] Stearns | Experimental evolution of ageing, growth, and reproduction in fruitflies[END_REF][START_REF] Reznick | Effect of extrinsic mortality on the evolution of senescence in guppies[END_REF].

Until now, our ability to quantify within-species variation in ageing rates has been limited owing to the scarcity of fine-scale demographic data across multiple wild populations of a given species [START_REF] Colchero | The long lives of primates and the 'invariant rate of ageing' hypothesis[END_REF]. When available, long-term, individual-based capture-recapture studies provide the gold standard for reliable assessment of the variation in ageing rates and to identify the eco-evolutionary drivers of such variation. Here, using a 7-year capturerecapture study, we assess the level of within-species variation in ageing rates of the southern Darwin's frog (Rhinoderma darwinii Duméril and Bibron, 1841), a species endemic to the austral temperate forest of South America. We quantify ageing rates in four disparate Rhinoderma darwinii populations distributed in Chile across a latitudinal gradient of 700 km (38-45°S; Fig. 1A). We also explore associations between ageing rate and pace of life (measured by generation time; [START_REF] Gaillard | Generation time: a reliable metric to measure life-history variation among mammalian populations[END_REF] and environmental factors [i.e. mean environmental temperature, environmental temperature seasonality and presence of the virulent pathogen Batrachochytrium dendrobatidis (Bd)]. These variables are known to influence among-and within-species variation in ageing rates in the wild. A faster pace of life has been associated with a steeper ageing rate at the interspecific [START_REF] Jones | Senescence rates are determined by ranking on the fast-slow life-history continuum[END_REF] and intraspecific (Cayuela et al., 2020) levels. Cayuela et al. (2021) found that the ageing rate was positively associated with mean annual temperature in four amphibian species from North America and Europe. Temperature seasonality explains among-population variation in body size in Rhinoderma darwinii (i.e. a larger body size in more seasonal areas; [START_REF] Valenzuela-Sánchez | Geographic body size variation in ectotherms: effects of seasonality on an anuran from the southern temperate forest[END_REF], and body size is positively associated with ageing rate at the interspecific scale [START_REF] Jones | Senescence rates are determined by ranking on the fast-slow life-history continuum[END_REF]Lemaître & Gaillard, 2013). Also, there is evidence that infection with Mycobacterium bovis can accelerate ageing in badgers [START_REF] Mcdonald | Mortality trajectory analysis reveals the drivers of sex-specific epidemiology in natural wildlifedisease interactions[END_REF][START_REF] Hudson | Analysis of lifetime mortality trajectories in wildlife disease research: BaSTA and beyond[END_REF]. We end by discussing why among-population variation in ageing rates might occur, and the available empirical evidence of withinspecies variation in ageing rates.

MATERIAL AND METHODS

STUDY DESIGN

From 2014 to 2020, we carried out a capture-recapture study of four Rhinoderma darwinii populations in Chile (Fig. 1A): Monumento Natural Contulmo (CON); Reserva Biológica Huilo Huilo (HUI); Parque Tantauco (TAN); and Reserva Natural Melimoyu (MER). The populations are within native old-growth forests sharing similar characteristics, such as the absence of anthropogenic stressors besides the emerging chytrid fungus, Bd [START_REF] Valenzuela-Sánchez | Interpopulation differences in male reproductive effort drive the population dynamics of a host exposed to an emerging fungal pathogen[END_REF]. The elevation is ~500 m a.s.l. at CON and HUI and ~10 m a.s.l. at TAN and MER. Each year, we carried out surveys during one period in early summer at the peak of the reproductive season (January-February; see [START_REF] Valenzuela-Sánchez | Home range and social analyses in a mouth brooding frog: testing the coexistence of paternal care and male territoriality[END_REF]. Briefly, all captured frogs were measured [snout-to-vent length (SVL)] and photographed for individual recognition (for further details, see [START_REF] Valenzuela-Sánchez | Home range and social analyses in a mouth brooding frog: testing the coexistence of paternal care and male territoriality[END_REF], 2017, 2022).

ASSESSMENT OF AGEING PATTERNS

We used the Bayesian survival trajectory analysis (BASTA) package in R [START_REF] Colchero | BaSTA: an R package for Bayesian estimation of age-specific survival from incomplete mark-recapture/recovery data with covariates[END_REF] to assess interpopulation variation in trajectories of age-specific mortality during adulthood. This model allows the estimation of apparent mortality rate at a given age, while accounting for imperfect detection, left-truncated (i.e. unknown birth date) and right-censored (i.e. unknown death date), from capture-recapture data. Birth dates were estimated for a variable proportion of adult frogs that were first captured as juveniles (Supporting Information, Table S1), and death dates were unknown for all adults (and estimated as a latent variable using BASTA). Specifically, the year of birth of each adult frog that was first captured as a juvenile was derived using the population-specific SVL of recently metamorphosed frogs (i.e. size at birth; CON = 9.30 mm, HUI = 10.12 mm, TAN = 8.19 mm and MER = 8.88 mm) and regression coefficients (slope and intercept) of a population-specific growth model describing the change in SVL in juveniles from year t to year t + 1. Given that we previously detected time variation in recapture probability across primary capture periods [START_REF] Valenzuela-Sánchez | Cryptic disease-induced mortality may cause host extinction in an apparently stable host-parasite system[END_REF], 2022), we allowed this parameter to vary among primary capture periods. In BASTA, we fitted a Siler model on age-specific mortality data [START_REF] Siler | A competing-risk model for animal mortality[END_REF] to obtain comparable metrics for each population (for a justification of the use of this model, see [START_REF] Lemaître | Sex differences in adult lifespan and ageing rates of mortality across wild mammals[END_REF]. The Siler model is given by:

µ (x) = a 0 exp (-a 1 x) + c + b 0 exp (b 1 x)
where a 0 , a 1 , b 0 , b 1 and c are the parameters of the mortality function, x the age in years, and µ(x) the age-specific rate of mortality. The exponential function with a parameters describes the changes in mortality in the early adult stage, whereas c gives the lower limit of mortality during the adult stage. The exponential function with b parameters corresponds to the increase in mortality during the senescent stage. The parameter b 1 measures the exponential increase in mortality rate with increasing age during the senescent stage and corresponds to the ageing rate in vertebrates [START_REF] Lemaître | Sex differences in adult lifespan and ageing rates of mortality across wild mammals[END_REF]. We ran four Markov chain Monte Carlo chains with 40 000 iterations, a burn-in of 10 000 and no thinning. Chain convergence was evaluated using the potential scale reduction factor [START_REF] Colchero | BaSTA: an R package for Bayesian estimation of age-specific survival from incomplete mark-recapture/recovery data with covariates[END_REF].

We conducted a pairwise comparison to evaluate differences in ageing rate between populations. For each pair of populations, we evaluated the proportion of the posterior distributions of b 1 that did not overlap using the R package 'overlapping' [START_REF] Pastore | Overlapping: a R package for estimating overlapping in empirical distributions[END_REF]. This value can range from zero to one, where value of zero indicates perfect overlap between the two distributions and a value of one indicates the total absence of overlap. Therefore, this value can be interpreted as the probability that the ageing rate differs between a given pair of populations. Finally, we conducted an exploratory visual analysis to identify factors that might explain amongpopulation variation in ageing rate. We plotted ageing rate estimated for each of the four studied populations against population-specific generation time, mean annual temperature (extracted from the study by [START_REF] Valenzuela-Sánchez | Geographic body size variation in ectotherms: effects of seasonality on an anuran from the southern temperate forest[END_REF], temperature seasonality (extracted from the study by [START_REF] Valenzuela-Sánchez | Geographic body size variation in ectotherms: effects of seasonality on an anuran from the southern temperate forest[END_REF] and the presence of Bd infection (extracted from the study by Valenzuela-Sánchez et al., 2022) in these populations. Generation time was calculated as the inverse of the elasticity of the population growth rate to changes in fecundity [START_REF] Bienvenu | A new approach to the generation time in matrix population models[END_REF]. This parameter was calculated using the R package POPBIO v.2.7 [START_REF] Stubben | Estimating and analyzing demographic models using the popbio package in R[END_REF] on a prebreeding census, agestructured matrix population model parameterized with population-specific vital rates (for further details, see [START_REF] Valenzuela-Sánchez | Interpopulation differences in male reproductive effort drive the population dynamics of a host exposed to an emerging fungal pathogen[END_REF].

RESULTS

The total number of captures, number of adults found, and the percentage of adults with known birth date are presented in the Supporting Information (Table S1).

We found two different patterns of ageing, which did not follow a latitudinal cline (Fig. 1B,C). Ageing rates were higher (more than a twofold difference) in CON [b 1 = 1.010, 95% Bayesian credible interval (CRI) = 0.654-1.366] and TAN (b 1 = 0.998, 95% CRI = 0.793-1.207) than in HUI (b 1 = 0.423, 95% CRI = 0.253-0.649) or MER (b 1 = 0.481, 95% CRI = 0.097-0.820). The pairwise analysis of overlap between the posterior distributions of ageing rates provided strong evidence of a difference between populations with fast and slow ageing (i.e. the probability that b 1 was different between a pair of populations with high vs. low ageing rate was consistently > 0.9; Fig. 1C).

A negative association occurred between ageing rate and generation time: the two populations with higher ageing rates had a shorter generation time (i.e. 2.7 and 3.2 years in CON and TAN, respectively) than the two population with the lower ageing rates (4.8 and 3.7 years in HUI and MER, respectively; Fig. 1D). None of the environmental factors we analysed accounted for population differences in ageing rate (Fig. 1D), although small sample size might have prevented a proper quantitative assessment of these putative associations.

DISCUSSION

Our findings in Rhinoderma darwinii provide clear evidence of large among-population variation in ageing rates within this species (Fig. 1).

Among-population variations in ageing rate are expected to occur by at least two intertwined mechanisms. First, as ageing rate increases in organisms with fast life-history strategies [START_REF] Jones | Senescence rates are determined by ranking on the fast-slow life-history continuum[END_REF], population-specific variation in biological times, such as generation time, are expected to be associated with variation in ageing rate. Our results support the existence of a positive association between ageing rate and pace of life among populations of Rhinoderma darwinii: the two populations with the highest ageing rates were the two with the shortest generation times (Fig. 1D). These results are similar to those reported for the amphibian Bombina variegata (Cayuela et al., 2020). Second, as ageing rate increases with increasing mean mortality rate during adulthood [i.e. Williams' (1957) second prediction of ageing evolution, reviewed by [START_REF] Gaillard | The Williams' legacy: a critical reappraisal of his nine predictions about the evolution of senescence[END_REF], population-specific patterns of environmentally driven adult mortality are expected to cause variation in ageing rate. This could explain the acceleration in ageing rate observed in badgers infected with M. bovis [START_REF] Mcdonald | Mortality trajectory analysis reveals the drivers of sex-specific epidemiology in natural wildlifedisease interactions[END_REF][START_REF] Hudson | Analysis of lifetime mortality trajectories in wildlife disease research: BaSTA and beyond[END_REF]. Owing to the lethal effect of Bd infection, adults from CON have the highest mortality rate among the four studied Rhinoderma darwinii populations (see [START_REF] Valenzuela-Sánchez | Cryptic disease-induced mortality may cause host extinction in an apparently stable host-parasite system[END_REF], 2022). In agreement with William's second prediction, this population also exhibited the highest ageing rate. The HUI and TAN populations, however, had dissimilar ageing patterns despite having similar mean mortality rates (Supporting Information, Fig. S1).

Among-population variation in ageing rates in amphibians and other organisms has been attributed to local environmental conditions such as environmental temperature [START_REF] Ganetzky | On the relationship between senescence and age-related changes in two wild-type strains of Drosophila melanogaster[END_REF][START_REF] Valenzano | Temperature affects longevity and agerelated locomotor and cognitive decay in the short-lived fish Nothobranchius furzeri[END_REF][START_REF] Lee | Regulation of the longevity response to temperature by thermosensory neurons in Caenorhabditis elegans[END_REF]Cayuela et al., 2020Cayuela et al., , 2021)). For example, Cayuela et al. (2021) found that the among-population variation in ageing rate was positively associated with mean annual temperature across four amphibian species. We did not find any clear association between ageing rate and environmental temperature in our study system. A link between ageing rate and environmental temperature could be caused by several behavioural, physiological and genetic mechanisms. For instance, Cayuela et al. (2021) argued that metabolic depression during hibernation Downloaded from https://academic.oup.com/biolinnean/advance-article/doi/10.1093/biolinnean/blac119/6865150 by OUP site access, andresvalenzuela.zoo on 03 December 2022 could slow down the ageing process, leading to the slow ageing rate they observed in populations of Rana luteiventris, Anaxyrus boreas and Rana temporaria inhabiting cold areas. In Rhinoderma darwinii, the duration of hibernation is most likely to be driven by temperature seasonality, because more seasonal areas exhibit colder and longer winters [START_REF] Valenzuela-Sánchez | Geographic body size variation in ectotherms: effects of seasonality on an anuran from the southern temperate forest[END_REF]. In addition, given that seasonality is positively associated with body size in Rhinoderma darwinii [START_REF] Valenzuela-Sánchez | Geographic body size variation in ectotherms: effects of seasonality on an anuran from the southern temperate forest[END_REF], allometric constraints on ageing rate [START_REF] Ricklefs | Intrinsic ageing-related mortality in birds[END_REF][START_REF] Ricklefs | Life-history connections to rates of ageing in terrestrial vertebrates[END_REF] could lead to faster ageing in populations within less seasonal areas. In our study, TAN and HUI populations inhabit, respectively, the least and most seasonal areas known for populations of this species [START_REF] Valenzuela-Sánchez | Geographic body size variation in ectotherms: effects of seasonality on an anuran from the southern temperate forest[END_REF]. Among these populations, the observed pattern of ageing is as expected, with TAN exhibiting a faster ageing than HUI (Fig. 1D). Individuals from CON and MER, however, experience a moderate and similar seasonality but contrasting ageing patterns (Fig. 1D). Altogether, our results from this small set of populations suggest that ageing patterns in Rhinoderma darwinii are unlikely to be driven by a single major structuring factor. Instead, the ageing rate might be determined by the interactions of multiple factors, such as seasonality and Bd infection.

Among-population variation in ageing rate is now available for a range of amphibian species (Fig. 2). These studies show that ample variation in ageing rates can exist within species in this taxonomic group. For example, the magnitude of variation in ageing rates across populations of Rana luteiventris and A. boreas was larger than the overall variation estimated across 101 mammalian species [START_REF] Lemaître | Sex differences in adult lifespan and ageing rates of mortality across wild mammals[END_REF]Cayuela et al., 2021). These findings, along with those of the present study, highlight amphibians as emerging models to study the patterns and mechanisms of intraspecific variation in ageing rates in natural populations. We encourage researchers to take advantage of the increasing number of long-term, individually based studies in amphibians and other poorly studied taxonomic groups in terms of ageing The range of minimum and maximum values (lines), the median (dots; only for species with more than two studied populations) and the number of populations studied displayed. In the inset, we show the geographical location of the studied populations. Data were obtained as follows: Anaxyrus (Cayuela et al., 2021), Bombina variegata (Cayuela et al., 2020), Bufo bufo (Cayuela et al., 2021), Rana luteiventris (Cayuela et al., 2021), Rana temporaria (Cayuela et al., 2021), Rhinoderma darwinii (this study) and Salamandra salamandra et al., 2019).

patterns to conduct comparative analyses to explore within-species variation in ageing. Such analyses are likely to provide important insights into the forces driving the evolution, maintenance and variation of ageing across natural populations.

Figure 1 .

 1 Figure 1. Actuarial senescence patterns in adults from four southern Darwin's frog (Rhinoderma darwinii) populations located in Southern Chile. A, study area showing the studied populations: 1 = Contulmo (CON); 2 = Reserva Biológica Huilo Huilo (HUI); 3 = Parque Tantauco (TAN); and 4 = Reserva Elemental Melimoyu (MER). B, age-specific mortality rate for each population. Note that this is a hazard rate, not a probability (i.e. it is not one minus survival). The shaded areas represent the Bayesian 95% credible intervals. C, density plots for the posterior distributions of the ageing rate parameter, b 1 , from the Siler model. This parameter represents the magnitude of the exponential increase in mortality rate with age during the ageing stage. D, relationship between ageing rate and generation time (extracted from the study by Valenzuela-Sánchez et al., 2022), mean annual temperature (extracted from the study by Valenzuela-Sánchez et al., 2015), temperature seasonality (extracted from the study by Valenzuela-Sánchez et al., 2015) and presence of Batrachochytrium dendrobatidis (Bd) infection (extracted from the study by Valenzuela-Sánchez et al., 2022) in each population.

Figure

  Figure Among-population variation in ageing rate (i.e. b 1 from Siler model) in amphibians. The range of minimum and maximum values (lines), the median (dots; only for species with more than two studied populations) and the number of populations studied displayed. In the inset, we show the geographical location of the studied populations. Data were obtained as follows: Anaxyrus(Cayuela et al., 2021), Bombina variegata(Cayuela et al., 2020), Bufo bufo(Cayuela et al., 2021), Rana luteiventris(Cayuela et al., 2021), Rana temporaria(Cayuela et al., 2021), Rhinoderma darwinii (this study) and Salamandra salamandra et al., 2019).

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© 2022 The Linnean Society of London, Biological Journal of the Linnean Society, 2022, XX, 1-7

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ACKNOWLEDGEMENTS

This study is part of an ongoing long-term monitoring programme focused on this threatened species led by the Chilean non-profit organization ONG Ranita de Darwin (www.ranitadedarwin.org/monitoreo). This project has been funded by Zoo Leipzig, Weeden Foundation, The National Geographic Society, Rufford Foundation, Mohamed Bin Zayed Species Conservation Fund, Wild Lama, Froens, Fundación Huilo Huilo, Fundación MERI and Parque Tantauco. We thank the many volunteers who have kindly participated during this project. A.V.-S. and C.A. were supported by Fondo Nacional de Desarrollo Científico y Tecnológico (FONDECYT) (grant numbers 3180107 and 1211587, respectively). This research project was approved by the Animal Welfare Committee at the Universidad Andrés Bello, Chile (number 13/2015) and by the Zoological Society of London's Ethics Committee (WLE709) and was conducted in accordance with Chilean law under permit numbers 5666/2013, 230/2015, 212/2016, 1695/2018 and 6618/2019 of the Servicio Agrícola y Ganadero de Chile and numbers 026/2013, 11/2015 IX and 10/2018 IX of the Corporación Nacional Forestal de Chile. The authors declare no conflicts of interest.

DATA AVAILABILITY

Data are available at https://doi.org/10.5281/ zenodo.7072271.

SUPPORTING INFORMATION

Additional Supporting Information may be found in the online version of this article at the publisher's web-site: Table S1. Details of the 7-year capture-recapture study (2014)(2015)(2016)(2017)(2018)(2019)(2020) of four populations of the southern Darwin's frog (Rhinoderma darwinii) in southern Chile.