Pelage Latifa
email: latifa.pelage3@gmail.com

Júlio Gonzalez

Guazzelli

Leloch François

Ferreira Valdimere

Munaron Jean-Marie

Lucena-Frédou Flávia

Frédou Thierry

Latifa Pelage

Importance of estuary morphology for ecological connectivity with their adjacent coast: A case study in Brazilian tropical estuaries

Coastal habitats are interlinked by ecological connectivity, defined as the exchange of organic matter or organisms between habitats. The degree of this connectivity will depend in particular on the geometric properties of the landscape. The increasing anthropogenic alterations in estuaries therefore raises the need to understand how their morphological characteristics influence fluxes between habitats. We used stable isotopes to investigate the variability of ecological connectivity between three estuaries and their adjacent coastal areas by tracking the origin of the organic matter (estuarine vs coastal) underlying the diet of the migrant species Eucinostomus argenteus. The chosen estuaries were geomorphologically distinct, exhibiting, in particular, differences in their degree of connection to the sea, corresponding to the morphological features (shape, mouth width) controlling key physico-chemical variables in this habitat (e.g. salinity). The sampling of the basal food sources contributing to the food web was performed in the three estuaries and in their adjacent coasts. The variability in stable isotope ratios between estuaries was examined for both fish and sources of organic matter. In the three estuaries, Bayesian models were applied for each season (dry and rainy) to quantify the relative contribution of sources from estuarine and coastal environments supporting the diet of the silver mojarra. The share of coastal organic matter increased with the degree of sea connection, indicating that the properties of the seascape can regulate the intensity of interactions between ecosystems. Variations in ecological connectivity are likely to affect the functioning of ecosystems as they influence trophic pathways and energy flows between adjacent habitats. Morphological modifications could thus significantly disturb ecosystems by altering the structure of food web, thereby affecting certain ecosystem services such as the availability of living marine resources.

Introduction

The emergence of seascape ecology brought the need to consider coastal habitats as a single continuum rather than as separate biomes [START_REF] Green | Habitat connectivity and spatial complexity differentially affect mangrove and salt marsh fish assemblages[END_REF][START_REF] Berkström | Ecological connectivity and niche differentiation between two closely related fish species in the mangrove-seagrass-coral reef continuum[END_REF]. This conceptual change aimed at a more integrated management of the coastal zone [START_REF] Beger | Conservation planning for connectivity across marine, freshwater, and terrestrial realms[END_REF]. In particular, addressing the connectivity between habitats is essential for fisheries management because coastal habitats provide complementary ecological functions that are critical for marine living resources [START_REF] Sheaves | Consequences of ecological connectivity: the coastal ecosystem mosaic[END_REF]. Coastal habitats should therefore not be considered as isolated patches in which different sub-populations occur, since a comprehensive view reflects population dynamics more accurately [START_REF] Burgess | Beyond connectivity: how empirical methods can quantify population persistence to improve marine protected-area design[END_REF].

Ecosystem interactions can be primarily subdivided into biological, chemical and physical interactions [START_REF] Ogden | Ecosystem interactions in the tropical coastal seascape[END_REF]. The connectivity degree between coastal habitats will depend in particular on the geometric properties of the landscape (Olds et al., 2017). More specifically, landscape characteristics affect ecological connectivity which consists of the Interactions between ecosystems through the movement of organisms and the exchange of nutrients and organic matter that are involved in ecological processes within these systems. [START_REF] Nagelkerken | Ecological connectivity among tropical coastal ecosystems[END_REF]. Previous work demonstrated that variability in the geometric characteristics of the seascape, such as the distance between habitats, can affect the intensity of flows between habitats [START_REF] Mumby | Mangroves enhance the biomass of coral reef fish communities in the Caribbean[END_REF][START_REF] Berkström | Ecological connectivity and niche differentiation between two closely related fish species in the mangrove-seagrass-coral reef continuum[END_REF].

Estuaries include a wide range of different transitional water bodies with distinct landscape characteristics [START_REF] Flemming | 02 Geology, Morphology, and Sedimentology of Estuaries and Coasts[END_REF]. Thereby, the classification of estuaries requires the consideration of physico-chemical variables such as hydrodynamics and bathymetry, as they determine not only the dynamics and structure of the sediments, but also the nature of the primary producers [START_REF] Elliott | The Need for Definitions in Understanding Estuaries[END_REF][START_REF] Whitfield | Ecosystem and Biotic Classifications of Estuaries and Coasts[END_REF]. Consequently, the morphology of estuaries and, in particular, the set of geometrical variables controlling the degree of sea water dilution in the estuary (like depth and mouth width), could have an influence on ecological connectivity. This morphological variability, which can be viewed as their degree of connection to the sea, is likely to enhance or prevent flows with adjacent coastal habitats. In addition, estuarine morphologies suffer from increasing anthropic alterations, such as the construction of polders, harbours or dykes which can drastically change the sedimentary dynamics and the composition of estuarine J o u r n a l P r e -p r o o f biological communities [START_REF] Wetzel | The ecological quality status of the Elbe estuary. A comparative approach on different benthic biotic indices applied to a highly modified estuary[END_REF][START_REF] Du | Recent human impacts on the morphological evolution of the Yangtze River delta foreland: A review and new perspectives[END_REF][START_REF] Lechêne | Nekton communities of tidally restored marshes: A whole-estuary approach[END_REF]. It is thus relevant to investigate how estuarine morphological characteristics influence interactions between habitats. The implications of this variability in terms of ecosystem functioning need to be taken into account before deciding to modify seascape features or implement restoration measures through eco-engineering [START_REF] Elliott | Ecoengineering with Ecohydrology: Successes and failures in estuarine restoration[END_REF].

Understanding ecological connectivity involves studying energy pathways in food webs and their intrinsic trophic relationships in order to assess community structure and functional role of species living in the ecosystems [START_REF] Pasquaud | Determination of fish trophic levels in an estuarine system[END_REF][START_REF] França | Assessing food web dynamics and relative importance of organic matter sources for fish species in two Portuguese estuaries: A stable isotope approach[END_REF]. Hence, estuarine food webs can be described by characterizing trophic relationships, sources of organic matter and energy flows between system components [START_REF] Pasquaud | A preliminary investigation of the fish food web in the Gironde estuary, France, using dietary and stable isotope analyses[END_REF]. In these complex and dynamic ecosystems, food webs can be supported by the production of various local primary producers, as well as the transport of organic matter from adjacent coastal and riverine areas [START_REF] Choy | Quantifying the trophic base for benthic secondary production in the Nakdong River estuary of Korea using stable C and N isotopes[END_REF][START_REF] Selleslagh | Feeding habitats, connectivity and origin of organic matter supporting fish populations in an estuary with a reduced intertidal area assessed by stable isotope analysis[END_REF]. However, identifying the origin of the organic matter at the base of estuarine fish food web can be difficult, in particular because conventional methods like gut content analysis provide only a snapshot of the diet at a given time [START_REF] Pasquaud | A preliminary investigation of the fish food web in the Gironde estuary, France, using dietary and stable isotope analyses[END_REF][START_REF] Pasquaud | Determination of fish trophic levels in an estuarine system[END_REF][START_REF] França | Assessing food web dynamics and relative importance of organic matter sources for fish species in two Portuguese estuaries: A stable isotope approach[END_REF]. Moreover, gut analyses do not enable a comprehensive food web analysis to reliably track the source of the organic matter underlying the diet of high trophic level consumers that do not feed directly on primary producers.

Stable isotope methods produce estimates of trophic position that can both capture complex trophic interactions and track energy flows between habitats [START_REF] Carvalho | The trophic structure of fish communities from streams in the Brazilian Cerrado under different land uses: an approach using stable isotopes[END_REF][START_REF] Whitney | Using Stable Isotopes to Assess the Contribution of Terrestrial and Riverine Organic Matter to Diets of Nearshore Marine Consumers in a Glacially Influenced Estuary[END_REF][START_REF] Gonzalez | Trophic resource partitioning of two snook fish species (Centropomidae) in tropical estuaries in Brazil as evidenced by stable isotope analysis[END_REF]. Since a consumer's stable isotope ratios reflect the values of its food sources, trophic position assessment is possible if the differences in isotopic composition between an animal and its prey, i.e. trophic level enrichment, are known [START_REF] J O U R N A L P R E -P R O O F Caut | Variation in discrimination factors (Δ15N and Δ13C): the effect of diet isotopic values and applications for diet reconstruction[END_REF]. Indeed stable isotopes of nitrogen (δ 15 N) and carbon (δ 13 C) have been successfully used to study ecosystem functions and food webs [START_REF] Le | Importance of seagrassmangrove continuum as feeding grounds for juvenile pink ear emperor Lethrinus lentjan in Setiu Lagoon, Malaysia: Stable isotope approach[END_REF]. δ 15 N can constitute a proxy for the trophic position of an organism since it increases considerably with the trophic level [START_REF] Post | Using stable isotopes to estimate trophic position: models, methods, and assumptions[END_REF][START_REF] J O U R N A L P R E -P R O O F Caut | Variation in discrimination factors (Δ15N and Δ13C): the effect of diet isotopic values and applications for diet reconstruction[END_REF]. On the other hand, δ 13 C differs substantially between primary producers [START_REF] Post | Using stable isotopes to estimate trophic position: models, methods, and assumptions[END_REF]Herzka, 2005), providing an overview of the origin of organic matter [START_REF] Fry | Stable isotope ecology[END_REF] .

J o u r n a l P r e -p r o o f

In estuaries, the origin of organic matter has already been studied with stable isotopes and a high contribution of marine basal sources has been highlighted in some studies [START_REF] Pasquaud | A preliminary investigation of the fish food web in the Gironde estuary, France, using dietary and stable isotope analyses[END_REF][START_REF] Selleslagh | Feeding habitats, connectivity and origin of organic matter supporting fish populations in an estuary with a reduced intertidal area assessed by stable isotope analysis[END_REF]. Nevertheless, other studies conducted in estuaries have shown that in situ primary production can outweigh other food sources and contribute significantly to fish growth [START_REF] Lobry | Diversity and stability of an estuarine trophic network[END_REF]. Therefore, knowledge on the extent of ecological connectivity between estuaries and their adjacent coastal areas is essential for designing the scale of conservation measures, particularly in the case of migratory species using multiple habitats [START_REF] Vasconcelos | Nursery use patterns of commercially important marine fish species in estuarine systems along the Portuguese coast[END_REF][START_REF] Reis-Santos | Reconciling differences in natural tags to infer demographic and genetic connectivity in marine fish populations[END_REF].

Migratory species constitute a good proxy to study ecological connectivity between two habitats since their movements represent linkages across the seascape [START_REF] Selleslagh | Feeding habitats, connectivity and origin of organic matter supporting fish populations in an estuary with a reduced intertidal area assessed by stable isotope analysis[END_REF]. In this study, we chose Eucinostomus argenteus Baird & Girard (1855) to investigate the intensity of the flows between habitats.

The silver mojarra is a member of the Gerreidae family comprising species with complex life strategies that are important for artisanal fisheries in north-eastern Brazil [START_REF] Pinto | Ethnotaxonomical considerations and usage of ichthyofauna in a fishing community in Ceará State, Northeast Brazil[END_REF]. E. argenteus is one of the main Gerreidae in the region and can be classified as an estuarine-dependent species, as its juveniles are found in great abundance in the estuarine habitats (Potter et al., 2013) where they reside since their reduced size at this ontogenetic stage (less than 13.5 cm) [START_REF] Bouchon-Navaro | Weight-length relationships for 50 fish species collected in seagrass beds of the Lesser Antilles[END_REF] does not allow them to make large migrations [START_REF] Franco | Patterns of spatial distribution of five species of mojarras (Actinopterygii: Gerreidae) in a small tropical estuary in south-eastern Brazil[END_REF]. In those habitats, they were classified as second-order consumers, feeding opportunistically as well on microcrustaceans (amphipods, copepods, tanaidaceous, ostracods) as on detritus with variations in the proportion and frequency of different items according to their ontogeny and food availability (Chi-Espínola et al., 2018). Although adults of these species are more abundant in adjacent coastal areas [START_REF] Ramos | Seasonal and spatial ontogenetic movements of Gerreidae in a Brazilian tropical estuarine ecocline and its application for nursery habitat conservation[END_REF], they use estuaries as a feeding ground, predating mostly on Bivalvia siphons and polychaetes [START_REF] Vasconcellos | Do closely related species share of feeding niche along growth? Diets of three sympatric species of the mojarras (Actinopterygii: Gerreidae) in a tropical bay in southeastern Brazil[END_REF] or as a reproductive ground [START_REF] Chaves | Use of mangrove habitat for reproductive activity by the fish assemblage in the Guaratuba Bay, Brazil[END_REF]. The degree of ecological connectivity between estuaries and adjacent coast would be reflected by the dominant origin of the carbon sources (coastal vs estuarine origin) on which the species diet is based.

The objective of this study is to understand the variability in ecological connectivity between estuarine and coastal habitats. We are thus investigating the flows between three morphologically distinct estuaries and their adjacent coastal areas by tracking the origin of the organic matter (coastal or estuarine) underlying the J o u r n a l P r e -p r o o f diet of E. argenteus. Our hypothesis is that geomorphological disparities between estuaries influence the origin of the organic matter that supports the E. argenteus regime, based on the assumption that flows are enhanced in the estuaries most connected to the sea. The area under study encompasses estuarine areas and shallow coastal waters of the continental shelf of the state of Pernambuco, Brazil (Figure 1). This narrow continental shelf (35 km on average),is characterized by shallow and warm waters (typically 26-29°C), soft slope, shelf break between 50 and 60 m, high salinity (>36) and a sedimentary cover composed of carbonatic and terrigenous sediments [START_REF] Manso | Morfologia e sedimentologia da plataforma continental interna entre as Praias Porto de Galinhas e Campos-Litoral Sul de Pernambuco, Brasil[END_REF] J o u r n a l P r e -p r o o f [START_REF] Assunção | 3D characterisation of the thermohaline structure in the southwestern tropical Atlantic derived from functional data analysis of in situ profiles[END_REF]. Three estuaries, the Santa Cruz Channel, Suape and Sirinhaem, were chosen because of their diverse morphological characteristics, which differ in particular in the geometric variables controlling the degree of dilution of the sea water in the estuary (such as the depth and width of the mouth) (Table 1, Figure 1). This different degree of connection to the sea is manifested notably by a difference in salinity (Table 1). , 2011). These two mouths are 0.7 km and 1.3 km wide, respectively, providing it with a high degree of connection with the open sea, resulting in high salinity (Table 1). In addition, the city of Itapissuma is the main fishing centre of Pernambuco, and also one of the most touristic destinations of the region. [START_REF] Quinamo | Pesca artesanal e meio ambiente em áreas de manguezais no complexo estuarino-costeiro de Itamaracá, Pernambuco: o caso de Itapissuma[END_REF]. Aquaculture, artisanal fishing, industry and agriculture are all important anthropogenic activities in the area [START_REF] Moura | A multi-temporal remote sensing and gis based inventory of the mangroves at itamaracá estuarine system, Northeastern Brazil[END_REF].

Material and Methods

Study Area

The Suape estuary, situated in the south coast, is a coastal lagoon with a flattened geomorphological shape [START_REF] Santos | Complexo industrial de Suape: estudo hidrogeológico preliminar. Simpósio de Geologia do Nordeste[END_REF]. The geomorphological and hydrodynamic conditions were altered by the construction of the port in 1979 [START_REF] Muniz | Hydrological impact of the port complex of Suape on the Ipojuca River (Pernambuco-Brazil)[END_REF]. The generated landfills blocked the flow of two of the four rivers that previously drained into the Suape estuary. Suape is now formed by two main rivers that flow into a large brackish lagoon and has a restricted connection to the ocean.

Also located in the south littoral of Pernambuco, Sirinhaem is the smallest estuary among the three and presents the narrowest connection to the sea [START_REF] Cprh | Diagnóstico socioambiental do litoral norte de Pernambuco[END_REF]. This enclosed lagoon exhibits low salinity and low depth (Table 1). Artisanal fishing, agroindustry and trade are important socio-economic activities in the zone [START_REF] Valença | Prospective scenarios of the environmental management of the tourist destination of Porto de Galinhas based on the enlargement of the industrial and portuary complex of Suape, Pernambuco[END_REF].

Sampling

Fish and baseline

E. argenteus was captured in the three estuaries above described, in shallow areas (Figure 1). The specimens were collected in 2015 from March to August and from September to January, which represent the local rainy and dry seasons, respectively [START_REF] Medeiros | The Itamaracá estuarine ecosystem, Brazil, in: Coastal Marine Ecosystems of Latin America[END_REF] with the help of local fishermen using artisanal fishing gears. All gears characteristics are detailed in the Supplementary Table 1. After identification, all fish were measured to the nearest millimetre (Total length) and weighed to the nearest gram. In addition, a long-lived primary consumer (mangrove oyster Crassostrea rhizophorae) was collected to represent the baseline for estimating the trophic position of E. argenteus.

Organic matter sources

Organic matter sources were sampled in the estuaries, to provide a background of stable isotope ratios at the base of E. argenteus diet. Since mojarras are characterized by their highly protrusible mouth which they use to feed on items they remove from the sediment [START_REF] Chen | Relationships among four genera of mojarras (Teleostei: Perciformes: Gerreidae) from the western Atlantic and their tentative placement among percomorph fishes[END_REF], sources associated to the benthic J o u r n a l P r e -p r o o f system were selected. Thus, macroalgae (Sargassum sp. and Ulva sp.), microphytobenthos, rotten mangrove leaves and particulate organic matter in the sediment (SOM) were collected along with seagrass when present in the area.

The SOM was also collected from the adjacent coast (shallow coastal waters) of the Santa Cruz Channel and Sirinhaem to measure the influence of coastal sources. However, as an industrial port is settled in the coast in front of the Suape estuary, the sampling of the coastal SOM could not be realized due to the restricted access to the area. We thus chose to use the coastal SOM isotopic ratios of Sirinhaem as a proxy for Suape considering the vicinity of the two areas.

The rotten mangrove leaves, seagrass and macroalgae were picked up manually during low tide. The SOM was collected from the first 2 mm layer of sediment while microphytobenthos (i.e. benthonic microalgae)

was sampled from the sediment surface at low tide and extracted in the laboratory.

Isotope analysis

For stable isotopic analysis, a fragment of dorsal white muscle from each fish was extracted and cleaned with distilled water to remove any remaining scales or bones. The isotope analysis of the basal sources was performed on the whole collected sample. The material was dried in an oven at 60°C for 48 hours. Then, dried samples were grinded into a homogeneous fine powder with a mortar and pestle. The SOM samples were divided into two subsamples. One was acidified to remove inorganic carbon before the δ 13 C analysis and the other was used for δ 15 N analysis [START_REF] Pinnegar | Differential fractionation of δ13C and δ15N among fish tissues: implications for the study of trophic interactions[END_REF]. Analysis of the carbon and nitrogen isotope ratios were performed at the Pôle de Spectrométrie Océan (PSO -IUEM, Plouzané, France) with an elemental analyzer (Thermo Flash 2000, interface Thermo ConFio IV) interfaced to a mass spectrometer (Thermo Delta V+).

The isotope ratio for carbon (δ 13 C) and nitrogen (δ 15 N) were calculated from the relation between the sample isotopic value and a known standard:

δ 13 C or δ 15 N = [(Rsample / Rstandard)-1] x 1000.
In this equation, R represents the ratio 13 C/ 12 C or 15 N/ 14 N. The R standard is relative to international standards, the Air and V-PDB (Vienna PeeDee Belemnite) for nitrogen and carbon, respectively. The J o u r n a l P r e -p r o o f precision of the analysis was monitored through a known standard (Thermo -Acetanilide) every six samples and defined as ± 0.11‰ (standard error) and ±0.07‰ for carbon and nitrogen, respectively.

Statistical Analyses

All the statistical analyses were performed with the software R version 3.5.2 (R Core Team, 2019). Mann

Whitney tests were carried out to determine if there were any differences in δ 13 C and δ 15 N between the estuarine and coastal SOM. Spearman ranking tests correlation tests were performed to examine the relationship between fish δ 13 C or δ 15 N and total fish length in each estuary separately and for all estuaries together. Mann Whitney tests were applied to check whether the δ 13 C and δ 15 N of the fish varied seasonally in each estuary.

The trophic positioning of Eucinostomus argenteus was determined using the tRophicPosition package ("tRophicPosition: Bayesian Trophic Position Calculation with Stable Isotopes" [START_REF] J O U R N A L P R E -P R O O F Quezada-Romegialli | tRophicPosition, an R package for the Bayesian estimation of trophic position from consumer stable isotope ratios[END_REF].The trophic position estimate is influenced by the baseline's representativeness of the target consumer food chain [START_REF] Post | Using stable isotopes to estimate trophic position: models, methods, and assumptions[END_REF]. In view of the zoobenthivorous diet of the species studied, we proposed an estimate based on a long-lived filter feeder as a reference base (C. rhizophorae).

Kruskal-Wallis tests together with Dunn's post hoc tests were used to see if there were differences of δ 13 C and δ 15 N for the fish and the baseline (C. rhizophorae) among the different estuaries.

Moreover, the Standard Elliptical Area (SEA) method was applied to compare the isotopic composition of the fish caught in each estuary. The SEA encompasses about 40% of the data (mean and standard deviation of covariables δ 13 C and δ 15 N) and provides an appropriate method for comparing the isotope niche (variability in isotope ratios) of groups belonging to several communities by eliminating extreme values that would bias the analysis if only the amplitude was considered [START_REF] Jackson | Comparing isotopic niche widths among and within communities: SIBER-Stable Isotope Bayesian Ellipses in R[END_REF]. The SEAc (corrected standard ellipse area) was calculated to overcome the underestimation for small sample sizes [START_REF] Jackson | Comparing isotopic niche widths among and within communities: SIBER-Stable Isotope Bayesian Ellipses in R[END_REF]). The SEAc was estimated for each estuary and season. These analyses were performed with the Stable isotope Bayesian analysis in R (SIBER) package [START_REF] Jackson | Comparing isotopic niche widths among and within communities: SIBER-Stable Isotope Bayesian Ellipses in R[END_REF].

Bayesian stable isotope mixing models, using the MixSiar package [START_REF] Stock | MixSIAR[END_REF] were built to estimate the relative contributions of basal sources to E. argenteus diet in each estuary. MixSiar allows the inclusion of covariates to better understand the variance among both consumer and source tracer values J o u r n a l P r e -p r o o f [START_REF] Stock | Analyzing mixing systems using a new generation of Bayesian tracer mixing models[END_REF]. As estuarine environments are highly dynamic, the factor season was added for both the fish and the sources. The use of separate models for each season would have increased the residual error [START_REF] Stock | Analyzing mixing systems using a new generation of Bayesian tracer mixing models[END_REF]. The input data were ratios of carbon and nitrogen from fish along with the respective mean and standard deviations of sources for a given estuary and season. When sources were not significantly different between seasons, the annual average for both the rainy and dry seasons was used.

Mixing models are highly influenced by the selection of a proper trophic enrichment factor (TEF) [START_REF] Parnell | Source partitioning using stable isotopes: coping with too much variation[END_REF]. The TEF consists in the isotopic fractionation from source to consumer [START_REF] Post | Using stable isotopes to estimate trophic position: models, methods, and assumptions[END_REF]. Since this TEF varies with both the diet and the habitat (Bunn, Leigh, and Jardine 2013), we chose a TEF proper to apply between estuarine zoobenthivorous fish (which is a second-degree consumer) and its basal sources of organic matter (primary producers): 2.0 ± 0.6 ‰ and 5.6 ± 1.5 ‰ for δ 13 C and δ 15 N, respectively (see Kostecki et al., 2012;[START_REF] Selleslagh | Feeding habitats, connectivity and origin of organic matter supporting fish populations in an estuary with a reduced intertidal area assessed by stable isotope analysis[END_REF]. As the δ 15 N of fish in each estuary were not significantly different between seasons (Supplementary Table 2), the same TEF was applied for both seasons assuming that E. argenteus fed at the same trophic levels during the dry and rainy seasons. The signatures of the sources after application of TEF were represented on the same two-dimensional space as the isotope ratios of the consumers in order to verify a geometric prerequisite for the execution of Mixsiar. The mixing models were only run when the consumer isotope ratios were within the mixing polygon bounded by the base source signatures representing the trophic limits of the system [START_REF] Phillips | Source partitioning using stable isotopes: coping with too many sources[END_REF].

Results

Fish isotopic variability and trophic position

A total of 46 samples of E. argenteus, 25 C. rhizophorae samples and 112 of basal food sources had their δ 13 C and δ 15 N isotope values analysed (Supplementary Table 2 and3). Estuarine fish size ranged from 3.6 cm (in Suape) to 16.6 cm (in Sirinhaem) (Figure 2). The size of the fish was not correlated to their δ 15 N nor their δ 13 C values when considering all estuaries together (Supplementary Table 4). However, fish size in the Santa Cruz Channel was positively correlated with their δ 15 N values and the fish size in Suape were negatively correlated with their δ 13 C (Supplementary Table 4, Figure 2). Seasonal variations in terms of δ 15 N were only observed for fish captured in the Santa Cruz Channel (Supplementary Table 5). Seasonal differences in δ 13 C were detected only for Sirinhaem (Supplementary Table 5, Figure 2, 3). In addition, Sirinhaem was the only estuary where the ellipses of both seasons did not overlap. However, the overlap between the rainy and dry ellipses was small for Suape and the Santa Cruz Channel.

J o u r n a l P r e -p r o o f

Sirinhaem ellipse during the rainy season completely overlaps the Suape ellipse for the rainy season and overlapped an important part of Suape ellipse for the dry season. Sirinhaem fish collected during the rainy season had the most depleted δ 13 C, followed by the Suape fish in both seasons (Figure 3) while the δ 13 C of Sirinhaem (Dunn's test, p-value < 0.05).Likewise, the C. rhizophorae δ 15 N did not exhibit differences among estuaries (Kruskal-Wallis chi-squared = 5.6029, df = 2, p-value > 0.05) but their δ 13 C were different (Kruskal-Wallis chi-squared = 15.382, df = 2, p-value > 0.05). The trophic positions of E. argenteus were similar among the estuaries: 2.78 ± 0.17 in Sirinhaem, 2.75 ± 0.14 in Suape and 2.66 ± 0.11 in the Santa Cruz Channel.

Sources contributions to E. argenteus diet

J o u r n a l P r e -p r o o f

J o u r n a l P r e -p r o o f

In all three estuaries, several basal sources exhibited overlapping isotopic signatures (Figure 4). Indeed, the macroalgae ratios were close to those of the SOM in the Santa Cruz Channel while in Suape, depending on the season, both the microphytoplankton and mangrove leaves signatures were similar to the isotopic ratios of the estuarine SOM. Likewise, in Sirinhaem, the macroalgae overlapped the coastal SOM during the dry season. Most of the Sirinhaem fish ratios during the dry season were close to those of the macroalgae and the coastal SOM but were distant from the other sources. During the dry season, most of the Sirinhaem E.

argenteus points displayed on the dual plot were outside the mixing polygon delimited by the basal sources.

This pattern suggests that the selected sources were not appropriate to adequately describe the organic matter that supports the diet of E. argenteus. Thus, the MixSiar model was run only for the rainy season in this estuary.

J o u r n a l P r e -p r o o f Overall, the relative contributions of the basal sources were different between estuaries (Figure 5, Supplementary Table 6). The proportion of coastal source compared to estuarine sources increased with the sea connection (Figure 5, 6 and Supplementary Table 6). The relative contribution of the coastal source is high in Santa Cruz Channel, lower in Suape and minimal in Sirinhaem (Figure 5,6).

J o u r n a l P r e -p r o o f

The contribution of the coastal SOM in the Santa Cruz Channel was the largest in both seasons, accounting for slightly more than 50% of the diet of E. argenteus. During the dry season, the seagrass had an important contribution during the dry season (23 %) whereas it contributed less during the rainy season (13%). The microphytobenthos contribution was also relevant in both dry and rainy season (around 12 %). The other sources contributions are low (less than 10%).

The coastal SOM had also the highest contribution during both seasons in Suape attaining the value of 32%

during the dry season and 29% during the rainy season, but its relative participation was less substantial than in the Santa Cruz Channel. The other sources with relevant contribution in that area were the macroalgae during the dry season (25 %), and the SOM in the rainy season (20 %). The contributions of the other sources were similar, fluctuating between 10 and 20 %.

On the other hand, in Sirinhaem, the Coastal SOM exhibited the lowest contribution during the rainy season. No sources contribution was higher than 30 %. The two sources with relevant contribution were the estuarine SOM (27%) and the mangrove leaves (24%). The rest of the source contributions varied between 10 and 20%. Yet, the amplitude of the curves expressed a high variability among individuals.

Landscape properties and ecological connectivity

Our study showed that E. argenteus diet varied between the three estuaries studied and that the contribution of coastal resources increased with the connection to the sea. This connection with the sea, embodied by the morphological features (shape, mouth width) controlling key physico-chemical variables in this habitat (depth, salinity), is thus a suitable indicator of the connectivity between estuarine and coastal habitat. Thereby, ecological connectivity was promoted by the high sea connection in the Santa Cruz

Channel. The fish of this estuary had high δ 13 C values and the coastal SOM was therefore the source that contributed most to the diet.

The coastal SOM was also important in Suape but to a lesser extent since the geomorphological alterations, induced by the construction of the port, resulted in a restricted land-sea connection, isolating the two environments. The landfills caused by the dredging have led to a reduction in the river regime and hence to sedimentation, as well as to a reduction in water exchange and depth [START_REF] Muniz | Hydrological impact of the port complex of Suape on the Ipojuca River (Pernambuco-Brazil)[END_REF]. In Sirinhaem, estuarine sources were predominant, showing that ecological connectivity is reduced in this enclosed lagoon during the rainy season.

Tracing the origin of basal sources of organic matter was important to illustrate the complexity of trophic pathways between coasts and estuaries. In addition, E. argenteus was an appropriate indicator to provide information on the ecological connectivity between these two systems, since the adults (>13 cm) that inhabit preferentially coastal areas, enter the estuaries to feed [START_REF] Ramos | Seasonal and spatial ontogenetic movements of Gerreidae in a Brazilian tropical estuarine ecocline and its application for nursery habitat conservation[END_REF]. Thus, no correlation was found between the δ 13 C and the sizes of the fish in Sirinhaem or in the Santa Cruz Channel while the larger individuals in Suape presented depleted δ 13 C values, characteristics of estuarine sources [START_REF] Moens | Tracing organic matter sources of estuarine tidal flat nematodes with stable carbon isotopes[END_REF]. This corroborates the fact that adults of this species use estuaries as a feeding area, as is the case for many species of the Gerreidae family [START_REF] Ramos | Ingestion of nylon threads by Gerreidae while using a tropical estuary as foraging grounds[END_REF].

Though the movements performed by adult fish might exacerbated the connectivity patterns, local variations in marine intrusion are likely to explain the gradient of connectivity seen in the three estuaries.

Organisms that have limited mobility can be influenced by this connectivity as estuarine food webs can be supported by coastal organic matter [START_REF] Selleslagh | Feeding habitats, connectivity and origin of organic matter supporting fish populations in an estuary with a reduced intertidal area assessed by stable isotope analysis[END_REF]. Methods using only stable isotopes cannot J o u r n a l P r e -p r o o f separate the effect of fish movement from the effect of marine intrusion. A tagging study with artificial loggings would have provided information on the frequency of foraging movements (Olds et al., 2017) and would have helped to distinguish these two components of ecological connectivity. Yet, the influence of ontogenetic migrations on the results seems to be reduced since fish sizes were not correlated to their respective δ 13 C and δ 15 N values when the three estuaries were grouped. Thus, differences in diet observed among estuaries cannot be attributed to differences in fish size. Furthermore, although fish sizes differed from one estuary to another, trophic positions were similar among estuaries. However, the low trophic position values found in this study (about 2.7) could be due to the tendency of trophic position underestimation with isotopic methods [START_REF] Hussey | Rescaling the trophic structure of marine food webs[END_REF] . Indeed, studies in the region of study did find a trophic position a little higher (i.e. 2.95) [START_REF] Lira | Trophic flow structure of a neotropical estuary in northeastern Brazil and the comparison of ecosystem model indicators of estuaries[END_REF]) and diet studies in estuaries classified E.

argenteus as a second degree consumer (Chi-Espínola et al., 2018;[START_REF] Vasconcellos | Do closely related species share of feeding niche along growth? Diets of three sympatric species of the mojarras (Actinopterygii: Gerreidae) in a tropical bay in southeastern Brazil[END_REF].

Coastal and estuarine sources have been successfully distinguished using Bayesian mixing models. This analysis, despite the need for careful parameterisation, remain the best method for quantifying the relative contributions of sources in a diet [START_REF] Parnell | Source partitioning using stable isotopes: coping with too much variation[END_REF]. They offered a useful tool for identifying differences in flows between habitats based on landscape properties. Nevertheless, the trophic enrichment factor, which strongly affects source contributions, should be specifically measured in the laboratory (Herzka, 2005). Yet, since the TEF chosen was consistent with those measured in the laboratory for zoobenthivorous fish (Kostecki et al., 2012), our results should appropriately reflect the relative contribution of coastal and estuarine sources.

Another weakness of mixing models is their lack of accuracy in an environment where several sources present overlapping isotope ratios, which usually occurs in estuaries [START_REF] Phillips | Best practices for use of stable isotope mixing models in food-web studies[END_REF]. Nonetheless, as our objective was to relate the degree of connection of the sea to the contribution from coastal sources, uncertainties in determining the participation of low contribution overlapping estuarine sources were not an issue. The fact that the coastal SOM was not available in Suape is not likely to bias the model, since

Suape and Sirinhaem are part of the same coastal region with homogeneous habitats [START_REF] Assis | Carta de caracterização dos agregados marinhos da plataforma continental rasa do estado de Pernambuco[END_REF].

Furthermore, the anthropogenic pressures locally occurring in Suape are not expected to significantly alter the isotope ratios of its coastal area, since Suape is a small estuary.

J o u r n a l P r e -p r o o f

Contributions of basal sources were also influenced by seasonality. For example, marked seasonal variations in isotope ratios of E. argenteus occurred in Sirinhaem, the smallest estuary, due to the fact that in small tropical estuaries, where freshwater inputs are intermittent, seasonal nutrient inputs from terrestrial systems are favoured [START_REF] Abrantes | Habitat-specific food webs and trophic interactions supporting coastal-dependent fishery species: an Australian case study[END_REF]. Indeed, Sirinhaem location in the south, where rainfall is higher than in the north of the state [START_REF] Cprh | Diagnóstico socioambiental do litoral norte de Pernambuco[END_REF], allows for a large difference in freshwater supply between seasons. Differences in land use can also affect the seasonal intensity of inputs, but as Sirinhaem is the least anthropized estuary [START_REF] Pelage | Coastal land use in Northeast Brazil: mangrove coverage evolution over three decades[END_REF] , environmental fluctuations seems to be the main driver of seasonal patterns in this region. Hence, during the dry season, we could not run the model for Sirinhaem because consumers were not within the trophic polygon of sources suggesting the existence of unknown sources probably derived from the sea due to their enriched δ 13 C ratios. This implies that the low connection to the sea, inherent in the morphological characteristics of this enclosed lagoon, was not the factor that influenced the ecological connectivity during this season. Therein, high marine intrusion, fostered by high evaporation during the dry season and reduced river flow [START_REF] Potter | The concept of an estuary: a definition that incorporates systems which can become closed to the ocean and hypersaline[END_REF], may have caused a diet shift. During the rainy season, freshwater discharges reduce salinity, which is propitious for the development of polychaetes, while during the dry season, evaporation increases salinity, promoting the presence of marine benthic larvae (Chi-Espínola et al., 2018). This shift in marine invertebrates community could thus explain the higher δ 13 C values observed in Sirinhaem during the dry season.

Despite the lack of significant differences in δ 13 C values between seasons in Suape and in the Santa Cruz Channel, the seasonal shift observed in the models and with the ellipses may also be attributed to a difference in fish size within each estuary. Especially the differences in size between seasons could reflect the reproduction period of E. argenteus in the study area, which takes place at the beginning of the dry season and resulted the capture of smaller fish. Ontogenetic shifts were already observed in estuaries and hypersaline lagoons, where smaller specimens fed on zooplankton and larger individuals preyed on polychaetes [START_REF] Silva | Distribution and feeding ecology of three juvenile mojarras in a hypersaline tropical estuary in Northeastern Brazil[END_REF]Chi-Espínola et al., 2018).

Implication for ecosystem functioning

Greater variability in fish isotope ratios was measured at Sirinhaem during the rainy season, translated by a large ellipse area and a high variability in sources contributions. The size range of Sirinhaem fish during that J o u r n a l P r e -p r o o f season was narrow so its variability cannot be attributed to ontogenetic shifts. This may be due to resource partitioning to avoid interspecific competition in this closed estuary thanks to the plasticity of E. argenteus diet. Diet plasticity is generally associated with the quantification of the extent of the trophic niche, i.e. the set of resources that can be exploited by a population to maintain acceptable growth parameters [START_REF] Polis | Age structure component of niche width and intraspecific resource partitioning: can age groups function as ecological species?[END_REF]. The ability to occupy wider trophic niches, thus feeding on a wide range of different items, ensures greater adaptability [START_REF] Pörtner | Niche dimensions in fishes: an integrative view[END_REF]. The opportunistic behaviour of the silver mojarra was already registered (Chi-Espínola et al., 2018) and could promote species adaptation in closed lagoons where coastal foraging opportunities are limited. In tropical estuaries, spatial differences in the diet of E. argenteus have already been reported [START_REF] Guedes | Partitioning of the feeding niche along spatial, seasonal and size dimensions by the fish community in a tropical B ay in S outheastern B razil[END_REF]. This habit could mitigate the adverse effects caused by natural or anthropic isolation between estuarine and coastal environments. However, more detailed health studies need to be conducted to assess the actual status of the fish population since variation in food web carbon sources, may cause differences in growth and condition [START_REF] Isnard | Getting a Good Start in Life? A Comparative Analysis of the Quality of Lagoons as Juvenile Habitats for the Gilthead Seabream Sparus aurata in the Gulf of Lions[END_REF]. Hence, variability in ecological connectivity and thus in diet could lead to variation in growth parameters.

Estuaries are suffering from various anthropic geomorphological modifications (Wetzel et al., 2012Du et al., 2016;[START_REF] Lechêne | Nekton communities of tidally restored marshes: A whole-estuary approach[END_REF] which could alter the flows with adjacent coastal areas. Variations in ecological connectivity are likely to affect the functioning of ecosystems as they influence trophic pathways and energy flows between adjacent habitats. Besides, this study shows that ecological connectivity extent evolves with sea connection degree which controls essential environmental variables such as salinity and depth. These variables are key structuring factors for the distribution and abundance of species. Indeed, E. argenteus is a saltwater species, and in general Gerreidae abundance is usually positively correlated with salinity [START_REF] Corrêa | Spatial and temporal distribution patterns of the silver mojarra Eucinostomus argenteus (Perciformes: Gerreidae) in a tropical semi-enclosed bay[END_REF]. In the Santa Cruz Channel, where sea connection is strong, E. argenteus was the second most abundant species, while in the other two estuaries, Suape and Sirinhaem, the silver mojarra was not among the ten most abundant species [START_REF] Merigot | Fish assemblages in tropical estuaries of northeast Brazil: A multicomponent diversity approach[END_REF].

Indeed, the availability of the prey is controlled by environmental factors [START_REF] Arula | Diet composition and feeding activity of larval spring-spawning herring: importance of environmental variability[END_REF][START_REF] Martino | Recruitment of striped bass in Chesapeake Bay: spatial and temporal environmental variability and availability of zooplankton prey[END_REF]) and a narrow sea connection degree does influence estuarine resources access for saltwater species and coastal resources for estuarine resident. Reduced access to food resources resulting from habitat fragmentation is likely to exacerbate competition patterns [START_REF] Hasegawa | Displacement of native white-spotted charr Salvelinus leucomaenis by nonnative brown trout Salmo trutta after resolution of habitat fragmentation by a migration barrier[END_REF]. In the case of a change J o u r n a l P r e -p r o o f in the sea connection such as during the construction of the port, coastal preys were less available for E.

argenteus juveniles but the diet plasticity of this species may have allowed it to switch to a more estuarine diet, which may have increased competition with estuarine resident species.

In addition, marine migratory species whose juveniles use estuaries as nurseries and whose adults extensively use estuaries as feeding or reproductive grounds (e.g. Mugilidae, Gerreidae) represent an important yield for small-scale estuarine and coastal fisheries in tropical areas (Salas, 2011). Modification in ecological connectivity through alterations in estuarine morphology is susceptible to have adverse effects on these species and could have socioeconomical repercussion since they constitute essential source of proteins for local populations. In addition, they represent key prey for piscivorous fish, therefore a change in their abundance due to habitat degradation would have drastic repercussions on the ecosystem.

Conclusion

Differences in the degree of connection to the sea in estuaries influenced the origin of the organic matter supporting the diet of E. argenteus, chosen as an indicator of ecological connectivity with adjacent coastal habitats. Consequently, the geomorphological disturbances caused by the increasing human modification of estuaries are likely to seriously affect the functioning of these habitats, by altering the energy flows that support their food networks. The influence of seascape properties on biological processes is complex and assessing their effects on ecosystem services such as fisheries yields is a difficult but necessary task in a context of global change and dwindling resources. Preliminary multidisciplinary studies should be carried out before implementing any changes to the seascape that alter the connectivity between habitats, especially in the case of habitats of significant importance for migratory species. In addition, understanding the extent to which the restoration of geometric properties through eco-engineering allows for ecosystem resilience in terms of connectivity would be of interest.

Acknowledgements

This

Figure 1 :

 1 Figure 1: Map of the area under study showing the three estuaries (Santa Cruz Channel, Suape and Sirinhaém) and their adjacent coast where sampling was performed in the state of Pernambuco, Brazil. The widths of the mouths are indicated in kilometers for each estuary to show their geomorphological disparities, providing them with different degrees of connection with the sea.

  , 2011) † Data obtained with the soRware ArcGis 10.1 based on satellite images from LANDSAT 7. ‡ Personal data collected during surveys in 2015. Type: geomorphological classification on the type of estuary according to Pritchard's classification. Estuary: estuary's area as the sum of mangrove and watershed areas. Salinity: Salinity at mid-depth of the water column. Pluviometry: Annual mean between 2014-2018 measured in stations near sampling points (APAC, 2020). Activity: existent anthropogenic activity with potential impact on the estuary.The Santa Cruz Channel is located on the north coast and forms the largest estuarine complex in Pernambuco. It displays a U-shaped surface area of 22 km and has two accesses to the Atlantic Ocean (Silva + connected -connected J o u r n a l P r e -p r o o f et al.

Figure 2 :

 2 Figure 2: Biplot showing A) the δ 13 C and B) the δ 15 N of fish according to their total length in in each estuary (Santa Cruz Channel, Sirinhaem and Suape) during both seasons (dry and rainy).

  J o u r n a l P r e -p r o o f the Santa Cruz Channel fish caught in both seasons exhibited more enriched δ 13 C values, comparable to those of the coastal SOM. Fish caught in Sirinhaem during the dry season had the highest values of δ 13 C.The most elevated values of SEAc were observed for Sirinhaem during the rainy season (10.1‰²). All the other estuaries /seasons values exhibited low SEAc (between 1.9 and 3.7‰²).

Figure 3 :

 3 Figure 3: Fish standard ellipses of the three estuaries during both dry (solid line) and rainy (dashed line) seasons. The black points represent the fish sampled in coastal areas which are plotted to highlight the differences in the degree of connection to the sea among the three estuaries (Santa Cruz Channel, Suape and Sirinhaem). The δ13C values of the SOM collected in estuaries (-25 ± 1.2 ‰) and at the coast (-17 ±1.6 ‰ ) are shown in the graph to give an indication of the δ13C values at the base of each environment.

Figure 4 :

 4 Figure 4: Dual plots of the sources (represented by the squares and whiskers showing mean values ±SD of the δ 13 C and δ 15 N) and the consumers (represented by the points) after applying the trophic enrichment factor to the basal sources in the three estuaries (Santa Cruz Channel, Suape and Sirinhaem). SOM: Sedimentary organic matter; mangrove: mangrove leaves; MPB: microphytobenthos.

Figure 5 :

 5 Figure 5: Density plots representing seasonal proportion of the basal sources in the diet of E. argenteus at the Santa Cruz Channel, Suape and Sirinhaem. SOM: Sedimentary organic matter; mangrove: mangrove leaves; MPB: microphytobenthos.

Figure 6 :

 6 Figure 6: Conceptual model summarizing the relative contribution of estuarine and coastal organic matter supporting the diet of E. argenteus in the three estuaries studied. In Sirinhaem, the relative contributions of organic matter correspond only to the rainy season.

Table 1 :

 1 Summary of morphological characteristics and anthropogenic activities of the three estuaries along the coast of Pernambuco, Brazil.

			Estuary
	Characteristics	Santa Cruz	Suape	Sirinhaem
	Type	Ria	Coastal lagoon	Coastal plain
	Estuary (km²) †	73.5	29.7	18.7
	Vegetated area (km²) †	48.0	23.1	17.0
	Watershed area (km²) †	25.5	6.6	1.7
	Mean depth (m)	7.5 (North) /3.0 (South)	3.1	2.6
	Mouth width (km) †	1.3/0.7	0.6/0.2	0.2
	Salinity (mean± SD) ‡	31.2 ± 2.9	17.8± 2.4	9.6 ± 3.7
	Pluviometry (mm; mean± SD)	1517 ±122	1869±367	2053±699
				Industrial
		Aquaculture,	Industrial harbour,	domestic
	Activity	industrial and	industrial and	and
		domestic waste	agriculture waste	agriculture
				waste
		(Medeiros &
	Reference	Kjerfve, 1993; Guimarães et al., 2010; Medeiros et al., 2001; Silva et	(CPRH, 2001; Borges, 2011; Silva et al., 2011)	(CPRH, 2001; Silva et al.
		al., 2011)

  study was funded in part by the INCT (National Institute of Science and Technology, CNPq n° 610013/2011-4) in Tropical Marine Environments -INCT-AmbTropic, the CNPq (National Council for Scientific and Technological Development, CNPq n° 479845/2013-1, n° 407125/2013-2 and n°

		J o u r n a l P r e -p r o o f
		Environment Area	DRY n δ 13 C	δ 15 N	RAINY n δ 13 C	δ 15 N
			SCC	6	-23.8±1.4 4.5±1.0	6	-24.0±1.0 4.2±0.7
		Estuary	SUAPE 6	-25.3±0.5 4.2±0.9	6	-26.0±0.4 2.7±0.6
	SOM		SIR	6	-25.9±0.7 4.7±0.5	6	-25.0±1.0 4.0±0.3
		Coast	SCC Sir	6 6	-21.2±1.2 5.5±1.2 -16.8±0.4 3.9±0.6	6 6	-20.6±1.1 5.8±1.1 15.9±0.1 3.3±0.2
	Algae (Ulva sp)	Estuary	SCC	3	-23.4±0.4 8.9±0.2	3	-23.0±0.4	6.3±0.2
	Algae Sargassum sp.	Estuary	SUAPE 3 SIR	-18.2±0.3 8.9±0.1 --	3 3	-16.5±2.3 7.0±0.2 -16.8±1.3 5.4±0.4
	Microphyto benthos	Estuary	SCC SUAPE 1 3 SIR 4	-20.3±0.2 3.9±0.4 -26.4 4.7 -24.8±0.7 4.2±0.5	2 -3	-19.6±2.8 2.7±0.4 -22.0±1.1 5.9±0.2
	Seagrass	Estuary	SCC	3	-27.6±0.6 2.4±0.5	3	-28.0±0.3 1.4±0.4

J o u r n a l P r e -p r o o f

Source

Declaration of interests

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

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


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X-ray imaging with Micromegas detectors with optical readout

Introduction 1 2 Beam test at the SOLEIL synchrotron 1 2.1 Experimental setup 2 2.

A

: In the last years, optical readout of Micromegas gaseous detectors has been achieved by implementing a Micromegas detector on a glass anode coupled to a CMOS camera. Effective X-ray radiography was demonstrated using integrated imaging approach. High granularity values have been reached for low-energy X-rays from radioactive sources and X-ray generators.

Detector characterization with X-ray radiography has led to two applications: neutron imaging for non-destructive examination of highly gamma-ray emitting objects and beta imaging for the single cell activity tagging in the field of oncology drug studies.

First measurements investigating the achievable spatial resolution of the glass Micromegas detector at the SOLEIL synchrotron facility with a high-intensity and flat irradiation field will be shown in this article.

K

: Micromegas, Optical detector readout concepts, Gaseous detectors, Synchrotron A X P : xxxx.xxxxx

Introduction

The optical readout of gaseous detectors is an emerging technology being already used in many different fields. Optical Time Projection Chambers (OTPC) are able to reconstruct 3D tracks in dark matter experiments [1] and could also be used in the medical field for proton therapy beam monitoring [2]. Gaseous Electron Multipliers (GEM) detectors coupled with optical readout have also shown good performances regarding the gain and light yield for many different applications [3].

Recent studies have shown that the Micromegas detector [4,5] is well suited for imaging when it is implemented on a transparent anode and coupled with a CMOS camera. This detector obtained high spatial resolution under X-ray and neutron irradiation and was able to detect very low activity 𝛽-ray emitting samples [6,7].

One major strength of optical readout is the ability to integrate the light scintillated by the gas for real-time imaging. The device gives images with high contrast and granularity thanks to the Micromegas detector high gain and micromesh structure coupled with a CMOS camera of several mega pixels resolution.

A beam test at the SOLEIL synchrotron facility [8,9] took place in November 2022 for characterization of the glass Micromegas detector. This test aimed to measure the spatial resolution and scintillation light response of the detector. This article describes the measurement of the Point Spread Function (PSF) of the detector for different configurations.

Beam test at the SOLEIL synchrotron

For a precise characterization of the optical readout Micromegas detector, it is essential to measure the impulse response of the detector. This is given by the PSF which describes the response of the detector to a point source. The SOLEIL synchrotron is a first-class solution, providing a very thin parallel beam of hard X-rays at very high flux. This test studies the origins of the blurring of the final image, namely the diffusion, the optical aberrations and the light reflection inside the detector. In this section, preliminary results from a test at the SOLEIL synchrotron are presented.

Experimental setup

This experiment was conducted at the SOLEIL Metrologie beam line [8,9], the main characteristics of which are described in this paragraph. The beam size is set at 5 × 5 µm . We assume that 5 µm is at a scale far lower than the intrinsic spatial resolution of the detector and the beam can be approximated to a point source. The photons are extracted from the storage ring of the synchrotron more than 20 m away from the detector and are expected to have parallel trajectories. The energy of the beam was set at 6 keV and the measured beam flux at the detector was 3.69 × 10 5 photons/s. The beam size was measured by a Basler camera [10]. The camera, the power supply, the table position and the beam energy were controlled remotely from the control room.

The properties of the detector are depicted in details in [6] while the main features are presented here. The drift gap was set at 3 mm for this first measurement while a wide range of operating voltages was scanned. The Micromegas detector was built on a 5 mm quartz coated with 150 nm of Indium Thin Oxide (ITO). A standard mesh (18 µm wire diameter, 45 µm opening between wires) is attached to the crystal by the bulk process [11] with pillars of 500 µm diameter and 6 mm pitch. The gas mixture is composed of 90% of Argon and 10% of Carbon tetrafluoride (CF 4 ) which scintillation spectrum contains a wide emission band around 630 nm [3]. The camera is an ORCA-Quest qCMOS imaging sensor from Hamamatsu [12], sensitive to single photons. The camera was coupled with a fast lens from Schneider group [13] of large aperture (f/0.95) for low light imaging applications. The detector and the camera are fixed on one solid plate to ensure the optical alignment and interconnected by a "light-tight" black cover made by 3D printing. The mesh was grounded while the cathode and the anode were at a negative and positive voltage respectively. The drift field (E drift ) is defined between the cathode and the mesh and the amplification field (E amp ) between the mesh and the anode. For every measurements, 100 frames of 1 s of beam signal were acquired.

Analysis and results

Here we describe the image treatment processes and the different operations involved in the analysis. The background noise coming from the dark current and the readout noise of the camera constitutes the pedestal of the image. The pixels intensity of dark images correspond to 0 photons and varies from one pixel to another. For this reason, the mean value over 100 frames of every pixel i intensity (𝜇 𝑖 ) is computed and subsequently suppressed from the beam signal. Hence, the pedestal and most of the hot pixels are removed. In the follow-up analysis, the beam signal is characterized by the two 1D shapes instead of one 2D shape (Figure 1 left) by integrating the image over the horizontal and vertical axes (Figure 1 right). No intensity threshold is applied because the light spreading effects of low light intensity could be removed.

Figure 1 (right) represents the beam intensity profile projected along the horizontal and the vertical axes. Two main contributions to the light spreading have to be considered: the electron diffusion in the gas and the optical effects. The diffusion of charges by multiple collisions in the gas follows a Gaussian distribution with a standard deviation (STD) given by 𝜎 𝑥 = √ 2𝐷𝑡 after a time 𝑡, drifting through a distance 𝑥. The diffusion coefficient 𝐷 is a function of the charge velocity and strongly depends on the drift field. Thus, the diffusion increases as a function of √ 𝑥 and is given in units of 𝜇𝑚/ √ 𝑐𝑚. When a hard X-ray penetrates the gas volume, it can interact at any position in the drift gap, given a certain probability 𝜖 𝑥 (absorption coefficient). Any photon interacting at a specific distance from the mesh implies a diffusion process of unique 𝜎 𝑥 that differs from other photons of a different position of interaction. Therefore, the contribution of the diffusion in Figure 1 (green cone) is defined by a sum of an infinite number of Gaussian curves with different 𝜎 𝑥 values. All the Gaussian distributions have the same mean value (𝜇 𝐷 ) because the X-ray beam is meant to be parallel to the drift field lines. Hence, the diffusion is modeled as one single Gaussian distribution of STD value 𝜎 𝐷 described in (2.1) where 𝜖 𝑥 is the X-ray absorption coefficient at a distance 𝑥 from the cathode and 𝑑 is the drift gap thickness. Nevertheless, optical effects such as optical aberrations or light reflections in the detector produce an additional contribution in the beam signal shape. Unlike optical TPCs where low luminosity events are captured, this detector aims to integrate high light intensity exposing the camera to third order aberrations. An important spreading pattern is brought from the optical aberration called Coma [14]. Positive Coma causes a "comet-like" blur directed away from the optic axis which grows quadratically with the distance of the source from the optic axis. The lens has a large aperture ( 𝑓 /0.95) allowing photons that are far away from the optical axis to interact with the lens, increasing the aberration. As shown in Figure 1 (orange curve), if the X-ray beam does not hit the detector at the exact center in alignment with the camera, the beam signal shape becomes asymmetric. In other words, the Gaussian distribution associated to the optical effects of mean value 𝜇 𝑂 is off-centered with the diffusion contribution. Furthermore, the inox micro-mesh being shinny, it produces reflections and adds some extra spread of the light. For the same reason, the cathode is made black to diminish reflections. In addition, the two 5 mm-thick quartz windows that light crosses before reaching the CMOS camera are sources of light reflection and refraction.

In order to calculate the uncertainty on the measurement of the beam size, the standard deviation of the sum of all the contributions is computed. The STD of N added up Gaussian shapes is given by (2.2). Since 𝜇 𝑂 ≠ 𝜇 𝐷 , the STD for N=2 (2.3) contains an additional term involving the shift (𝜇 𝑂 -𝜇 𝐷 ) 2 between the distributions. The STD and amplitude of the optical effects distribution are respectively 𝜎 𝑂 and 𝑝 𝑂 .

𝜎 2 𝐷 = ∫ 𝑑 0 𝜖 𝑥 𝜎 2 𝑥 𝑑𝑥 (2.1) 𝜎 2 𝐹 𝑖𝑛𝑎𝑙 = 𝑁 ∑ 𝑖=1 𝑝 2 𝑖 𝜎 2 𝑖 + 𝑁 -1 ∑ 𝑖=1 𝑁 ∑ 𝑗=𝑖+1 𝑝 𝑖 𝑝 𝑗 (𝜎 2 𝑖 + 𝜎 2 𝑗 + (𝜇 𝑖 -𝜇 𝑗 ) 2 ) (2.2) 𝜎 2 𝐹 𝑖𝑛𝑎𝑙 = (1 -𝑝 𝑂 )𝜎 2 𝐷 + 𝑝 𝑂 𝜎 2 𝑂 + 𝑝 𝑂 (1 -𝑝 𝑂 ) (𝜎 2 𝐷 + 𝜎 2 𝑂 + (𝜇 𝐷 -𝜇 𝑂 ) 2 ) (2.
3)

The electric fields in the Micromegas detector are key parameters of the diffusion and amplification and directly affect the final signal standard deviation. Measurements were realized with five different amplification fields and six different drift fields in order to study the STD dependency on these fields. The minimum value of the STD was measured at E drift = 330 V/cm (Figure 2, left) which corresponds to the minimum of the simulated diffusion coefficient (Figure 2, right). At low amplification field (Figure 2, left plot, red curve), the amount of light is small and the Signal to Noise Ratio is dominated by the readout noise, increasing the STD value. At high amplification field (Figure 2, left plot, purple curve), the Micromegas detector enters the non-linear region and suffers from instabilities, increasing the light spreading.

Conclusion

A test run at the SOLEIL Metrologie beam line was performed for measuring of the Point Spread Function (PSF) of the optical Micromegas detector. The PSF of the device was computed at different electric field values allowing us to establish the dependency of the resolution on the diffusion and the amplification of the detector. Various sources of the light spreading have been identified as well as the effect of optical aberrations and the lens settings and will be studied further. Blackening of the mesh will be implemented in future set-ups for reflection investigation. The full analysis of the data is in progress and will allow us to exctract the response of the detector for different beam energies and sizes, detector positions and drift gaps. This will open the way to deconvolution of 2D images for high resolution imaging.

Figure 1 :

 1 Figure 1: 2D intensity image (left) and 1D projections on horizontal and vertical axes (center, right) for E amp = 37 𝑘𝑉/𝑐𝑚 and E drift = 330 𝑉/𝑐𝑚.

Figure 2 :

 2 Figure 2: STD of the beam signal as a function of the amplification and drift fields (left). Simulated diffusion coefficient for a gas mixture with 10% of CF4 (right).

Acknowledgments

The authors acknowledge the financial support of the Cross-Disciplinary Program on Instrumentation and Detection (PTC-ID) of the French Alternative Energies and Atomic Energy Commission (CEA), of the «P2IO LabEx (ANR-10-LABX-0038)» in the framework "Investissements d'Avenir" (ANR-11-IDEX-0003-01) managed by the Agence Nationale de la Recherche (ANR), France and of the P2I Department of Paris-Saclay University. We acknowledge SOLEIL for provision of synchrotron radiation facilities (proposal number 99220033) and we would like to thank Pascal Mercere and Paulo Da Silva for assistance in using beamline METROLOGIE. The authors would like to thank Óscar Pérez and Luis Obis for their contribution to the data taking shifts during the beam test.

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