Christian Grenz
email: christian.grenz@mio.osupytheas.fr

Renaud Fichez

Carlos Álvarez Silva

Laura Calva Benítez

Pascal Conan

Adolfo Contreras

Ruiz Ruiz

Lionel Denis

Silvia Díaz Ruiz

Pascal Douillet

Margarita E Gallegos Martinez

Ruiz Esparza

Silvia Díaz Ruiz

Jean-François Ghiglione

Francisco José Gutiérrez Mendieta

Montserrat Origel-Moreno

Antonio Zoilo Marquez

Alain Muñoz Caravaca

Mireille Pujo-Pay

Rocío Torres

Jorge Zavala-Hidalgo

Benthic ecology of tropical coastal lagoons: Environmental changes over the last decades in the Términos Lagoon, Mexico

Keywords: Multidisciplinary Study, Ecosystem, Estuaries, Coastal lagoon, Terminos Lagoon, Mexico

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

Introduction

In recent years, global concern has arisen concerning the risks of decline in Marine ecosystem health and the associated functions and services they provide. This has brought about a number of innovative studies and much path-breaking research and has led to the development of guidance and policies worldwide. Most of this information underlines the need to observe and manage the state of marine ecosystems as a whole [START_REF] Tett | Framework for understanding marine ecosystem health[END_REF], Grand Challenge 4 in Borja, 2014). Moreover, scientific literature about marine ecosystems has increased exponentially during the last 15 years [START_REF] Borja | Grand challenges in marine ecosystems ecology[END_REF]. Coastal ecosystems are likely to have been altered substantially by human activities and probably also by climate drivers of change (climate change related key drivers) [START_REF] Parry | Climate Change 2007: Impacts, Adaptation and Vulnerability. Contribution of Working Group II to the Fourth Assessment Report of the Intergovernmental Panel on Climate Change[END_REF]. As stated by [START_REF] Halpern | A Global Map of Human Impact on Marine Ecosystems[END_REF] no marine system is unaffected by human influence and a large fraction (41%) is strongly affected by multiple drivers.

Marine ecosystems are highly diverse given their biogeographical characteristics on a global scale. Their morphology controls internal hydrodynamics and exchanges with the adjacent sea, regulating in this way the level of anthropogenic pressures and sensitivity to eutrophication [START_REF] Kennish | Environmental threats and environmental future of estuaries[END_REF]. Semi-enclosed coastal systems (SECS), which usually include open, leaky and choked lagoons and transitional waters [START_REF] Eu | Directive 2000/60/EC of the European Parliament and of the Council establishing a framework for Community action in the field of water policy[END_REF], are sentinel systems and hotspots of coastal vulnerability at a global scale [START_REF] Newton | Hotspots of coastal vulnerability: A DPSIR analysis to find societal pathways and responses[END_REF]Newton et al., 2014 and references therein). Because of the physical connection between the benthic layer and the whole water column, the geochemical and biological dynamics extensively depend on benthic-pelagic interactions.

Terminos Lagoon in the southern Gulf of Mexico is one of the largest lagoons situated in the intertropical zone.

Considering the extension and shallowness of Terminos Lagoon as well as its potential sensitivity to environmental change, the objective of this paper is to review and combine existing information on what has been identified as a model tropical coastal lagoon in the Gulf of Mexico [START_REF] García-Ríos | Towards a coastal condition assessment and monitoring of the Gulf of Mexico Large Marine Ecosystem (GoM LME): Terminos Lagoon pilot site[END_REF] where seasonal signals are distinct from those in temperate regions. The following sections present an overview of the general features of Terminos Lagoon, as well as hydrodynamic characteristics, and benthic habitats with special attention to trophic status and benthic communities as they strongly contribute, together with external loadings, to the biogeochemistry of the water column. In the context of expected Global Change in the Centro America region, the present review also presents a number of specific drivers of change and pressures including land-use, watershed management, river inputs, coastal erosion processes and contaminant levels, to assess what major changes can be expected and what effect they might have on the current environmental status, finally issuing recommendations for a long term environmental survey strategy.

General features

Terminos Lagoon borders the southern Gulf of Mexico in Campeche and is, by area and volume, Mexico's second largest estuarine system. The lagoon borders two geologic provinces: to the east the Yucatan Peninsula (low rainfall, calcareous soils, and very low surface drainage) and to the west and south the lowlands of Tabasco and the highlands of Chiapas and Guatemala, an area of high rainfall and fluvial soils (Fig. 1). Three main rivers discharge directly into the lagoon: the Candelaria, the Chumpan, and the Palizada (a tributary of the Grijalva-Usumacinta) with a catchment area totaling 49 700 km 2 .

The average freshwater flow rate is approximately 12.5 10 9 m 3 yr -1 , with the Palizada River catchment area on the western coast of the lagoon accounting for most of the fresh water inputs. Tidal regime is mixed, mostly diurnal with a mean range of 0.3 m [START_REF] Contreras Ruiz Esparza | Tidal dynamics of the Terminos Lagoon, Mexico: observations and 3D numerical modelling[END_REF].

The continental shelf (Campeche Sound) is one of the most important fishery areas in the western central Atlantic region.

Campeche Sound (Tabasco/Campeche) contributes 34% of the total Mexican fishery yield in the Gulf and Caribbean coasts, including penaeid shrimps, mollusks, demersal and pelagic fishes [START_REF] Conapesca | Anuario estadístico de Pesca[END_REF].

Parallel to fisheries, crude oil extraction in the Gulf of Mexico represents a large economic activity with, Petroleros Mexicanos (PEMEX), a major non-OPEC oil producer (top 10) operating here since 1938. PEMEX is a state-owned company extracting around 2 million barrels per day in Bahia de Campeche (Cantarell oil field). More significantly, revenues from the oil industry (including taxes and direct payments from PEMEX) accounted for about 32% of total Mexican government revenues in 2013. This activity has generated the urban development of Ciudad del Carmen, the largest town (located on the western tip of the island) and in 1994, it contributed to a great extent to the declaration of Terminos Lagoon as a Protected Area of Flora and Fauna (APFFLT). Ten years later the lagoon was designated as a wetland of international importance (RAMSAR) and today represents the largest such zone in Mexico, at 705,016 hectares (www.ramsar.org). Unfortunately, PEMEX's activity continues to grow within the protected area, potentially threatening the whole ecosystem through accidental oil spills and pipeline leaks (e.g. Ixtoc-1 in 1979Ixtoc-1 in -1980)).

The vast wetlands surrounding Terminos Lagoon provide ideal conditions for the migration and breeding of numerous species such as sea bass, shellfish, shrimp, and manatees, amongst others. This area also harbors the highest concentration of dolphins in the Gulf. This protected area is also one of the most important bird wintering areas in the Gulf of Mexico and its level of marine and terrestrial flora and fauna diversity is considered as very high (Yañez-Arancibia et. al,. 1988). The coastal fringe is occupied by vast areas of mangroves and the lagoon comprises various habitats including important oyster and sea grass beds [START_REF] Moore | The distribution and productivity of seagrasses in the Terminos lagoon[END_REF].

The area presents two climatic seasons [START_REF] Yáñez-Arancibia | Environmental Behavior of Terminos Lagoon Ecological System, Campeche, Mexico[END_REF]: the rainy season (Jun-Sep) and the dry period which splits into a period with dominant north winds and winter storms (Nov-Mar) and a second with lower wind forcing (Feb-May). The average evaporation is 1512 mm a -1 and the average rainfall is 1805 mm a -1 (Yáñez-Arancibia & Day, 1988). Nevertheless, a strong variability in rainfall and subsequent freshwater discharge to the lagoon has recently been linked to climate variability and to the ENSO regime [START_REF] Fichez | Global climate change and local watershed management as potential drivers of salinity variation in a tropical coastal lagoon (Laguna de Términos, México)[END_REF].

Terminos Lagoon stretches over more than 2000 km 2 (Fig. 1). The lagoon is connected to the sea through two inlets:

'Carmen Inlet' on the western side (4 km width) and 'Puerto Real Inlet' on the eastern side (3.3 km width). These are separated by a carbonate-rich barrier (Carmen Island, 30 km long and 2.5 km wide). Terminos Lagoon is shallow with an average depth of 2.4 m, with the exception of the tidal flats and the deep channel on the eastern part of each inlet.

The eastern entrance is influenced by transparent marine waters which form an interior delta. In the western inlet, the suspended terrigenous materials of the Palizada River estuary, generate high turbidity and contribute to the formation of an exterior delta oriented towards the west in the Gulf of Mexico. Waters are mostly oligo-to mesohaline, with a tendency to euhaline in some parts of the system; the east inlet is characterized by marine waters whereas the west inlet is mesohaline to oligohaline near the river mouths [START_REF] Bach | Resumen del Perfil de Primer Nivel del Sitio Laguna de Términos y su Cuenca[END_REF]. Predominant sediments are silt and clay, with calcareous sands (shells and shell fragments) present in the eastern part and close to the barrier island only. The sediments of the western part are silty-clay [START_REF] Yáñez-Arancibia | Environmental Behavior of Terminos Lagoon Ecological System, Campeche, Mexico[END_REF].

A study by Yañez-Correa (1963) compared the sediment structures of both inlets, and showed that sediment accumulation outside Carmen Inlet in the west contributed to the formation of a small alluvial delta outside the lagoon, due to the predominant seaward direction of the currents. Conversely, in the east at Puerto Real Inlet, the existence inside the lagoon of a submarine alluvial delta composed of coarse sediment fraction of marine origin reveals an inward component of the currents; one of the main sources of sediments is thus offshore derived waters entering the lagoon.

The few published studies dealing with hydrodynamic modeling in Terminos Lagoon [START_REF] Kjerfve | Modeling of circulation and dispersion in Términos Lagoon[END_REF][START_REF] Jensen | Remote sensing and numerical modeling of suspended sediment in Laguna de Terminos, Campeche, Mexico[END_REF][START_REF] Contreras Ruiz Esparza | Tidal dynamics of the Terminos Lagoon, Mexico: observations and 3D numerical modelling[END_REF] converge to describe a general circulation pattern from east to west, with a net westward transport of the water masses entering the lagoon through Puerto Real Inlet and exiting through Carmen Inlet during the dry season, and a dominant net export of water through both inlets during the wet period. Tidal effects combined with freshwater inputs tend to bend the southwestward current into a cyclonic circulation inside the lagoonwith a vortex core located in the northeast part -while predominant winds from the east (67 % from 45° to 135°) tend to drive the river plumes toward Carmen Inlet [START_REF] Contreras Ruiz Esparza | Tidal dynamics of the Terminos Lagoon, Mexico: observations and 3D numerical modelling[END_REF]. Based on tidal current measurements in and around the inlets, [START_REF] David | Tides and Currents in a Two-Inlet Coastal Lagoon: Laguna de Términos, México[END_REF] estimated that fifty percent of the lagoon water volume was renewed in 9 days. However, long term trends of residence times of the water masses for the whole lagoon remain difficult to estimate, considering that water replacement in Terminos Lagoon does not behave linearly but decays exponentially (Yanez-Arancibia & Day. J. 2005). The recent development of a 3-D hydrodynamic modeling approach of Terminos Lagoon accounted for tidal forcing only [START_REF] Contreras Ruiz Esparza | Tidal dynamics of the Terminos Lagoon, Mexico: observations and 3D numerical modelling[END_REF] and thus prevented the computing of water mixing and renewal indicators [START_REF] Umgiesser | Comparative hydrodynamics of 10 Mediterranean lagoons by means of numerical modeling[END_REF]. However, a relatively simple water budget could be established using the net freshwater inputs (river + groundwater + net precipitation) previously estimated at 12.5 10 9 m 3 yr 1 [START_REF] Fichez | Global climate change and local watershed management as potential drivers of salinity variation in a tropical coastal lagoon (Laguna de Términos, México)[END_REF]) and the water volume of 4.65 10 9 m 3 (Contreras Ruiz Esparza et al, 2014), yielding an average flushing time (Tf, [START_REF] Monsen | A comment on the use of flushing time, residence time, and age as transport time scales[END_REF] of 135 days, a value within the 1 to 5 months range previously reported by [START_REF] Robadue | Characterization of the region of the Terminos Lagoon: Campeche, Mexico. PlusDraft 1 Site Profile[END_REF] for different seasonal conditions.

Water column biogeochemistry

Terminos Lagoon is a "choked" lagoon subject to significant fresh water inputs, so salinity is strongly variable at the spatial as well as temporal scale. Recent results from a survey (currently October 2008 to September 2010, 10 samplings over a network of 34 stations) yielded an average salinity of 25.7 at 1 m depth (standard deviation = 8.1) with values ranging from 3 to 37 [START_REF] Fichez | Global climate change and local watershed management as potential drivers of salinity variation in a tropical coastal lagoon (Laguna de Términos, México)[END_REF]. Such high variability may be related to the high variability in precipitation and subsequent river discharge, sea level and wind patterns, as documented in [START_REF] Fuentes-Yaco | Environmental forcing in a tropical estuarine ecosystem: the Palizada River in the southern Gulf of Mexico[END_REF].

Nevertheless, the distribution of salinity in Terminos Lagoon for January 2010 (Fig. 2) may be considered as a good example of the general pattern of spatial variability.

Lower salinities are generally measured in the vicinity of Palizada River, with Candelaria and Chumpan Rivers contributing to a lesser extent to freshwater inputs to the lagoon. Highest salinities in the range of 33 to 36, similar to those of adjacent marine waters, are measured close to Puerto Real Inlet and, according to the general hydrodynamic pattern, the more marine water bodies tend to travel to the southwest along the coast of Carmen Island. Salinity varies considerably within the lagoon as a function of the imbalance between those two sources of input.

Quite surprisingly, the trophic status (dissolved inorganic and organic material and particulate organic material) of Terminos Lagoon has been poorly documented. Silicate concentrations generally correlate to salinity as freshwater transports huge quantities of silica originating from soil leaching. In the Gulf of Mexico, marine offshore waters have silicate concentration of roughly 2 µM whereas coastal waters are generally in the range of 20-40 µM. Concentrations in the lagoon are around 70 µM on average with strong temporal and spatial variability, as for salinity.

Inorganic nitrogen concentrations generally come within the 0 to 4 µM range with some very occasional peaks close to or above 10 µM in the vicinity of the Palizada River. Concentrations in the middle of Terminos Lagoon are -in most cases -significantly below 1 µM. Nitrates + nitrites concentrations are higher during the wet season than during the dry season.

Phosphate (orthophosphate) concentrations come within the 0 to 1.00 µM range with an average value of roughly 0.13 µM. It is clear from the recent study by [START_REF] Conan | Combining biogeochemical characterisation and microbial biomass and activities to assess pelagic cycling in a large and shallow tropical lagoon (Terminos Lagoon, Mexico)[END_REF] that phosphate distribution in the lagoon is for the most part (spatially and temporally) disconnected from nitrogen. During their study, the authors identified the Palizada River and Puerto Real Inlet as the two main sources of NO3 and NH4, whereas PO4 originated from Candelaria and Chumpan R. inputs, and primarily from the mineralization of organic phosphorus (PP and DOP) by bacterial activity. This impacted the stoichiometry of particulate organic matter (N:P ratio) throughout the lagoon. When averaged over the whole lagoon, dissolved inorganic N:P ratio ranges from 3:1 to 18:1 with an average value of 9:1, thus suggesting that nitrogen is the most probable element to limit pelagic primary production. However, ratios well over the 16:1 Redfield Ratio and as high as 353:1 were occasionally measured (mostly close to the Palizada River mouth), demonstrating that such an assumption must not be applied uniformly to the whole lagoon system.

On average, concentrations in dissolved organic nitrogen and phosphorus are around 15 and 0.6 µM, respectively. Some strong spatial variation can be observed with occasional high concentrations in the north-east part of the lagoon, possibly due to export of organic compounds from the mangrove. On average, concentrations in particulate organic carbon, nitrogen and phosphorus are around 70, 10 and 0.5 µM, respectively.

Chlorophyll a as an indicator of phytoplankton biomass and potential eutrophication process has been more extensively documented. Average concentration in the lagoon, as reported by various authors, ranged broadly between 1 and 10 mg m -3 , with the lower values being measured as a whole in Puerto Real and the highest close to the Palizada estuary.

Chlorophyll a versus total chloropigment ratio was recorded as being recurrently close to 0.75, demonstrating a relatively healthy population of pelagic primary producers. There is virtually no assessment of primary production barring two publications, which report a gross primary production rate of 478 g C m -2 a -1 at one station of the lagoon [START_REF] Rivera-Monroy | Seasonal coupling of a tropical mangrove forest and an estuarine water column: enhancement of aquatic primary productivity[END_REF] and variations from 20 to 300 gC m -2 a -1 [START_REF] Day | Primary production in the Laguna de Terminos, a tropical estuary in the Southern Gulf of Mexico. Coastal Lagoons[END_REF], suggesting a potential shift from oligotrophic to eutrophic conditions.

A recent study on biogeochemical cycling in Terminos Lagoon has shown that the water column behaves globally as a sink, and in particular as a "nitrogen assimilator" due to the elevated production of particulate and dissolved organic matter and the weak exportation of autochthonous matter to the Gulf of Mexico [START_REF] Conan | Combining biogeochemical characterisation and microbial biomass and activities to assess pelagic cycling in a large and shallow tropical lagoon (Terminos Lagoon, Mexico)[END_REF]. The same study has revealed that "bottom-up" control largely accounts for the observed variability in phytoplankton productivity.

Phosphates delivered by Palizada River inputs and strong on site bacterial mineralization of organic matter result in maximal phytoplankton production in the western part of the lagoon.

A nutrient and carbon budget calculated by [START_REF] Reyes | Comparison of carbon, nitrogen and phosphorus fluxes in Mexican coastal lagoons[END_REF] and [START_REF] David | Laguna de Términos, Campeche. Mexican and Central American coastal lagoon systems: Carbon, Nitrogen and Phosphorus fluxes[END_REF] showed that Terminos Lagoon is slightly autotrophic (carbon sink) on a yearly basis with a net production rate of +0.2 mol C m -2 yr -1 , even though the authors themselves underlined the fact that such results should be taken with great caution due to the evident gaps in available datasets.

Seagrass

Seagrass meadows are known to play a number of important ecological roles in estuarine and shallow-water coastal ecosystems: they enhance primary production and nutrient cycling, stabilize sediments, elevate biodiversity, and provide nursery and feeding grounds for a range of invertebrates and fish. [START_REF] Orth | Longterm trends in submersed aquatic vegetation (SAV) in Chesapeake Bay, USA, related to water quality[END_REF][START_REF] Cullen-Unsworth | Seagrass meadows, Ecosystem Services and Sustainability[END_REF]. The highest productivity of seagrasses is during the dry season as a result of higher water clarity, according to [START_REF] Moore | The distribution and productivity of seagrasses in the Terminos lagoon[END_REF]. In Terminos Lagoon, the distribution of the seagrass communities clearly reflects the conditions of water movement, clarity and water salinity. Seagrasses develop denser meadows along the lagoon shoreline of Carmen Island and in particular in the delta of Puerto Real where high water transparency and salinities, and high percentage of calcium carbonate in the sediments, have been measured [START_REF] Calva | Carbono orgánico y características texturales de sedimentos en áreas del pasto marino Thalassia testudinum en ecosistemas costeros del Sureste del Golfo de México[END_REF].

The communities are dominated by the turtle grass Thalassia testudinum and in lower density by Halodule wrigtii (Raz-Guzmán & Barba, 2000). H. wrigtii grows in the shallowest areas while T. testudinum extends down to depths of 3 m [START_REF] Ortega | Observaciones del fitobentos de la Laguna de Términos, Campeche, México[END_REF]. Total primary production of T. testudinum in the Lagoon is estimated at 260 T a -1 [START_REF] Moore | The distribution and productivity of seagrasses in the Terminos lagoon[END_REF]. Epifauna associated to the seagrass meadows is abundant and includes epibenthic amphipods and their predators, i.e. crustacean species of Hippolyte and Pink shrimp [START_REF] Negreiros-Fransozo | The species of Hippolyte leach (Crustacea, Caridea, Hippolytidae) from Terminos Lagoon, southwestern Gulf of Mexico[END_REF][START_REF] Corona | Epibenthic amphipod abundance and predation efficiency of the pink shrimp Farfantepenaeus duorarum (Burkenroad, 1939) in habitats with different physical complexity in a tropical estuarine system[END_REF].

Mangroves

Mangrove forests play an important role in the functioning of tropical coastal systems. A classification of mangrove forest based on the importance of hydrodynamic exchange describes different categories that are present in Terminos Lagoon: (1) riverine tidal forests that receive high inputs of freshwater, (2) fringe forests which are flushed regularly by tide, (3) basin forests which are less regularly flushed, and (4) scrub forests which have very slow flushing and low nutrient levels [START_REF] Lugo | The ecology of mangroves[END_REF]. Terminos Lagoon is bordered almost completely by extensive mangrove swamps composed by three dominant species: Rhizophora mangle L. (red mangrove), Avicenia germinans L. (black mangrove) and Laguncularia racemosa Gaertn.f. (white mangrove) (Day et al., 1987). These authors measured higher productivity near the river mouths, probably caused by a number of factors which include lower salinity, higher nutrient inputs and a lower hydrogen sulfide level due to lower sulfate concentrations in freshwater. Since stresses are lower, productivity is higher, resulting in more available energy for building biomass. The productivity and the extent of mangrove forests in the area are directly related to high fisheries productivity (Yanez-Arancibia & Day, 1982).

Benthic fauna

Besides the importance of benthic diversity that has been underlined in numerous studies (Yáñez-Arancibia & Day, 1988) benthos in Terminos Lagoon represents a major trophic resource, and plays an important role in the biogeochemical budget of such a shallow system. [START_REF] Ayala-Castañares | Sistemática y distribución de los foraminíferos de la Laguna de Términos, Campeche, México[END_REF] and Phleger and Ayala-Castañares (1971) described the taxonomy and the distribution of foraminifers in Terminos Lagoon. They reported densities in the range of 250-900 ind.10 cm -2 , which suggests relatively high rates of organic matter production, probably consecutive to high river runoffs. Their distribution reflects the circulation in the lagoon, with open-gulf foraminifers close to Puerto Real Inlet and along the southeast side of Carmen Island, while lagoon foraminifers occur close to Carmen Inlet. A mixed fluvial assemblage was also observed in and near the river mouths.

Marrón-Aguilar (1975) described the systematic and abundances of polychaetes throughout Terminos Lagoon, while [START_REF] Reveles | Contribución al estudio de los anélidos poliquetos asociados a praderas de Thalassia testudinum en la porción este-sur de la Laguna de Términos[END_REF], focused on polychaete species associated with seagrass beds of Thallasia testudinum, and Hernández and Solís-Weiss (1991) examined those found in the mangrove Rhizophora mangle. In the seagrass beds and mangroves, polychaetes dominated in both abundance and species diversity. Their distribution is closely linked to the salinity gradient, turbidity and sediment types, and three assemblages may be identified: a first group, localized in the eastern part of the lagoon, is mainly characterized by families Spionidae and Cirratulidae. The second group is located in the central part and the south of the lagoon, with families Cirratulidae and Lumbrinereidae, while the third group located close to Carmen Island is characterized by families Capitellidae and Nereidae.

Mollusks have received extensive attention due to the exploitation of two bivalve species. [START_REF] García-Cubas | Caracteristicas ecológicas de los moluscos de la Laguna de Términos[END_REF] published a review of all the mollusks encountered in this lagoon, and several studies have been carried out on the exploited bivalves Rangia cuneata and Crassostrea virginica, mainly concerning their distribution [START_REF] Ruiz | Estudio ecológico preliminar de las almejas comerciales del sistema lagunar de Términos, Campeche, Rangia cuneata (Gray, 1831)[END_REF], the physiology of their reproduction (Rogers-Nieto and García-Cubas, 1981) as well as the behaviour of larval stages [START_REF] Chávez | Desarrollo larvario de Crassostrea virginica (Gmelin, 1792) y Rangia cuneata (Gray, 1831) (Mollusca: Bívalvia), procedentes del área Atasta-Pom de la Laguna de Términos, Campeche, México[END_REF].

Based on previous works, García-Cubas (1988) identified four typical subsystems in Terminos Lagoon: a-Interior lagoon (western part of the area, but with limited connections to Terminos Lagoon -salinity lower than 10 in summer periods) associated with rivers and characterized by 3 bivalve species of commercial interest (Rangia flexuosa, R. Additional extensive studies have been conducted on fish distributions in the lagoon. Most fish have a direct trophic link with benthic macrofauna (Yáñez-Arancibia and Day, 1988), even if only 10% of the species are permanent residents in the lagoon (45% use the lagoon as a nursery, and 45% are occasional visitors). While half of the fishes are primarily carnivorous, a quarter are higher carnivorous and the last quarter is constituted of herbivorous, detritivorous or omnivorous fishes. A maximal juvenile flow into the lagoon was recorded in September-November, mainly via Puerto Real inlet.

As described above, the diversity of benthic communities is strongly structured by the balance between marine and riverine inputs and this structuration strongly influences the benthic metabolism and its coupling with the biogeochemistry of the water column.

Benthic metabolism

Dissolved and particulate exchanges between sediments and the water column remain poorly documented, and most budgets are based on the assumption that the benthic boundary layer is in balance (Gomez- [START_REF] Reyes | Comparison of carbon, nitrogen and phosphorus fluxes in Mexican coastal lagoons[END_REF][START_REF] David | Laguna de Términos, Campeche. Mexican and Central American coastal lagoon systems: Carbon, Nitrogen and Phosphorus fluxes[END_REF]. Studies of sediment-water exchanges of nutrients remain poorly documented, except in the mangrove forest [START_REF] Rivera-Monroy | Flux of nitrogen and sediment in a fringe mangrove forest in Terminos lagoon, Mexico[END_REF][START_REF] Day | A 7 year record of above-ground net primary production in a southeastern Mexican mangrove forest[END_REF] or seagrass beds of Thalassia testudinum.

In a fringe mangrove, sediment and nitrogen exchanges at the sediment-water interface have been estimated using a 12 m-long flume extended through a fringe forest from a tidal creek to a basin forest. [START_REF] Rivera-Monroy | Flux of nitrogen and sediment in a fringe mangrove forest in Terminos lagoon, Mexico[END_REF] demonstrated that the tidal creek was the main source of ammonium (0.53 g m -2 a -1 ) and nitrate and nitrite (0.08 g m -2 a - 1 ), while the basin forest was the principal source of total suspended sediments (210 g m -2 a -1 ). On the contrary, net export of particulate nitrogen occurred from the fringe forest to the tidal creek (0.52 g m -2 a -1 ), while less particulate nitrogen was exported to the basin forest (0.06 g m -2 a -1 ). They also demonstrated that the exchanges were highly variable with seasonal weather forcing, as salinity (hence nutrient concentrations) in the creek was influenced by inputs from rainfall and river discharge to the lagoon. Moreover, as the Carbon to Nitrogen ratio of particulate matter exported during ebb tides was generally higher than particulate matter imported into the forest during flooding, they suggested that there is a greater nitrogen loss during ebb tide caused by the export of nitrogen deficient detritus from fringe and basin mangroves.

Several works describing nutrient dynamics have been published on Thalassia testudinum seagrass beds located off the inner littoral of Carmen Island [START_REF] Stevenson | Sources of new nitrogen in a tropical seagrass system, Terminos Lagoon, with special reference to N-fixation[END_REF][START_REF] Hopkinson | Nitrogen pools and turnover times in a tropical seagrass system, Terminos Lagoon[END_REF][START_REF] Kemp | Ammonium regeneration in the sediments of a tropical seagrass beds (Thalassia testudinum) community, Terminos Lagoon[END_REF]. Rates of NH4 regeneration in sediments of T. testudinum beds were ten times higher in surficial sediments (0 to 2 cm) than at depth (18 to 20 cm). Turnover-time for ammonium pools in surface sediments were about 1 day. Both anaerobic decomposition and denitrification are important biogeochemical processes in Terminos Lagoon seagrass beds and rates of ammonium regeneration were sufficient to supply >70% of the nitrogen required for seagrass growth in this system [START_REF] Kemp | Ammonium regeneration in the sediments of a tropical seagrass beds (Thalassia testudinum) community, Terminos Lagoon[END_REF]. Nitrogen uptake measured in intact cores showed low rates ranging from 0.8 µmolN m -2 d -1 in February ('Nortes') to 50 µmolN m -2 d -1 in August (rainy season). Separate nitrogen uptake by leaf, root, rhizome, and sediment components measured in small serum bottles suggest that N fixation provides 10 to 40% of nitrogen demand of the seagrasses [START_REF] Stevenson | Sources of new nitrogen in a tropical seagrass system, Terminos Lagoon, with special reference to N-fixation[END_REF]. The highest uptake rates occurred just prior to the Thalassia testudinum production peak in February [START_REF] Rojas-Galaviz | Estuarine primary producers: the Terminos Lagoon case study[END_REF]. Measurements of stocks of organic and inorganic nitrogen in sediment, water and the biota indicates that biotic stocks of 13220 µmol m -2 dominated abiotic stocks of 19 µmol m -2 of nitrogen in the Thalassia system, with less than 0.2% of the nitrogen being in the inorganic form [START_REF] Hopkinson | Nitrogen pools and turnover times in a tropical seagrass system, Terminos Lagoon[END_REF]. A large percentage of the total organic nitrogen pool (94%) is contained in dead material (746 versus 12610 µmol m -2 , living and dead material, respectively) and 97% of the organic nitrogen is located in the sediments, as opposed to the water column. Approximately 75% of the inorganic nitrogen is in the sediments, as opposed to the water column [START_REF] Hopkinson | Nitrogen pools and turnover times in a tropical seagrass system, Terminos Lagoon[END_REF], while inorganic nitrogen uptake requirements are 7.5, 2.5, and 4.0 mmol N m -2 d -1 for phytoplankton, epiphytes and T. testudinum, respectively. The nitrogen turnover times ranged from less than 1 day for inorganic nitrogen in the water column to over 3000 days for sedimentary organic nitrogen [START_REF] Hopkinson | Nitrogen pools and turnover times in a tropical seagrass system, Terminos Lagoon[END_REF].

Drivers of environmental change

Terminos Lagoon and its surrounding wetlands are oppositely impacted by their conservation status and by human development pressure. Urbanization, wastewater discharge, industrialization, alteration of the hydrologic regime, agricultural and cattle production, petroleum extraction and fishing have been exhaustively addressed as the most important deleterious issues [START_REF] Robadue | Characterization of the region of the Terminos Lagoon: Campeche, Mexico. PlusDraft 1 Site Profile[END_REF]. Land cover is one of the most systemic indicators of environmental change in the protected area surrounding Terminos Lagoon. Between 1974 and 2001, 31 % of the area was detrimentally affected, resulting mainly from the replacement of tropical forest and mangroves by grassland and urban areas [START_REF] Soto-Galera | Spatial and temporal land cover changes in Terminos Lagoon Reserve, Mexico[END_REF].

Between 1986 and 2001 however, the protected status of Terminos Lagoon prompted some positive action in terms of environmental restoration and protection, allowing to reduce losses in natural habitats by half and demonstrating the potential of efficient remediation strategies. A mangrove restoration experiment headed by the Gulf of Mexico Large Marine Ecosystem Based Assessment and Management Project (GEF LME-GoM) identified increasing salinity in the porewater of mangrove soils as responsible for the most long term deleterious effects and defined remediation based on hydrological restoration and local community involvement which yielded an inversion in the recessing mangrove dynamics (Zaldivar-Jiménez et al., 2017).

Currently, the fluvial-lagoon systems of Terminos Lagoon are disturbed. According to [START_REF] Herrera | Análisis de la calidad ambiental usando indicadores hidrobiológicos y modelo hidrodinámico actualizado de Laguna de Términos[END_REF] and [START_REF] Epomex | Ecología del Paisaje y Diagnóstico Ambiental del ANP Laguna de Términos[END_REF], the zones having registered the most change are the river-mouths of the three main rivers (the Candelaria, the Chumpan, and the Palizada), due to excessive inputs of terrigenous materials and growth of oyster reefs.

According to [START_REF] Márquez-García | Características sedimentológicas del Sur del Golfo de México, Tesis de Doctorado[END_REF], about 30% of the lagoon is in the process of sediment deposition meaning that the lagoon is facing a major sediment accumulation problem. The formation of an internal delta at the mouth of Puerto Real involves the retention of sandy sediments in Terminos Lagoon due to the decrease in current velocity within the system. Conversely, the processes of erosion-deposition in the lagoon generate changes in the depth of the system and erosive morphology of some parts of the coastal area. This can lead to various problems ranging from the siltation or death of seagrasses to constraints in small boat navigation. Approximately 42 m of beach have been lost in the eastern side of Puerto Real Inlet, a process aggravated by poorly engineered breakwaters that partially disrupt littoral transport (Márquez-García et al., 2013). These changes primarily in the east part of the lagoon have likely transformed the biogeochemical characteristics of the sediments, thus impacting the distribution of benthic species and lagoon biodiversity. Significant changes in fish population have been reported since 1980, including alteration in fish taxonomic and functional diversity combined, with decreases of 41 % and 58 % in fish abundance and biomass, respectively (Ramos Miranda et al., 2005;[START_REF] Villéger | Contrasting changes in taxonomic vs. functional diversity of tropical fish communities after habitat degradation[END_REF][START_REF] Sirot | Combinations of biological attributes predict temporal dynamics of fish species in response to environmental changes[END_REF][START_REF] Abascal-Monroy | Functional and Structural Food Web Comparison of Terminos Lagoon, Mexico in Three Periods[END_REF][START_REF] Sirot | Using otolith organic matter to detect diet shifts in Bardiella chrysoura, during a period of environmental changes[END_REF]. Such changes in biota have been tentatively linked to a loss of favorable habitats and a sustained increase in salinity due to decreasing river inputs to the lagoon [START_REF] Sosa-Lopez | Fish species richness decreases with salinity in tropical coastal lagoons[END_REF]. However, such a sustained trend in salinity increase is supported neither by long term surveys of rainfall and river discharge, nor by modeling outputs that foresee stability in rainfall during the first half of the 21st century. The observed differences have been related to strong inter-annual variability in runoff, potentially related to ENSO conditions [START_REF] Fichez | Global climate change and local watershed management as potential drivers of salinity variation in a tropical coastal lagoon (Laguna de Términos, México)[END_REF]. It is true, however, that the very same climate change modeling approaches foresee reductions of rainfall by 5 to 30 % in the region during the second half of the 21st century [START_REF] Biasutti | Projected changes in the physical climate of the Gulf Coast and Caribbean[END_REF][START_REF] Hidalgo | Hydrological climate change projections for Central America[END_REF], corresponding to an overall reduction in runoff of up to 80 percent in the central Yucatan Peninsula and Guatemalan highlands, and of 20 percent in the Veracruz, Tabascan and Campeche lowlands [START_REF] Imbach | Modeling potential equilibrium states of vegetation and terrestrial water cycle of Mesoamerica under climate change scenarios[END_REF][START_REF] Kemp | Can Continental Shelf River Plumes in the Northern and Southern Gulf of Mexico Promote Ecological Resilience in a Time of Climate Change?[END_REF]. Such alteration in hydrologic regime will dovetail with sea level rise, leading to flooding of the most coastal lowlands including Terminos Lagoon, whose surrounding wetlands have been identified as some of the most critical areas in the region in terms of submersion risk and flooding (Ramos [START_REF] Ramos-Reyes | Indicadores geomorfológicos para evaluar la vulnerabilidad por inundación ante el ascenso del nivel del mar debido al cambio climático en la costa de Tabasco y Campeche, México[END_REF].

Considering pollutants, the highest dissolved Total Polycyclic Aliphatic Hydrocarbons (PAH) concentrations were reported from the Carmen Inlet, whereas water masses close to the Palizada River estuary supported an abundant bacterial community of PAH degraders [START_REF] Conan | Combining biogeochemical characterisation and microbial biomass and activities to assess pelagic cycling in a large and shallow tropical lagoon (Terminos Lagoon, Mexico)[END_REF]. PAH concentrations in oyster tissues were reported to range from 2.5 to 42.5 µg g -1 (Norena- [START_REF] Norena-Barroso | Polynuclear aromatic hydrocarbons in American oysters Crassostrea virginica from the Terminos Lagoon, Campeche, Mexico[END_REF]. The predominance of low and medium molecular weight alkylated compounds over their parent compounds indicated the petrogenic source of these PAHs, hence pointing to offshore oil activities as a major source of PAHs inputs. A study in the western part of Terminos Lagoon showed that PAH concentrations in fish flesh exceeded the maximum values recommended by international regulations (greater than 40.0 μg g -1 for the epibenthic Petenia splendida) [START_REF] Orozco-Barajas | Hidrocarburos Aromáticos Policíclicos (hap) y peces del sistema Pom-Atasta[END_REF].

Chlorpyrifos was detected in the water at concentrations up to 72 pg l -1 , and amongst organochlorine compounds, PolyChloroBiphenyl (PCB) averaged 1177 pg l -1 and DDT 279 pg l -1 , respectively (Carvalho et al., 2009a). Residues of chlorinated compounds were present in both the sediments and the biota, with DDT averaging 190 pg g -1 and 5876 pg g -1 in sediments and oysters, respectively. Concentrations of residues did not reach alarming levels and were in fact lower than those reported for other coastal lagoons of the region (Carvalho et al., 2009b). The Palizada River is by far the major contributor (85 % to 99 % depending on the considered metal) of riverborne metal inputs to the lagoon [START_REF] Páez-Osuna | Trace metals in the fluvial system of Terminos lagoon, Mexico[END_REF]. Reported concentrations of total metals in lagoon water were higher than in pristine environments [START_REF] Vazquez | Heavy metals in a coastal lagoon of the Gulf of Mexico[END_REF] but not very different than in other coastal waters. The authors related the highest metal concentrations in the lagoon to anthropogenic inputs from rivers as well as to more diffuse atmospheric input from the nearby petroleum industry, but no direct evidence was provided.

Comparisons between metal concentrations in the oyster Crassostrea virginica with previous data [START_REF] Vazquez | Trace metals in the oyster Crassostrea virginica of the Terminos lagoon, Campeche, Mexico[END_REF] showed that whereas zinc concentrations remained virtually unchanged since the mid-1970s, the levels of cadmium decreased and concentrations of copper and lead increased significantly. In a recent paper dealing with the study of the effect of various contaminants on the oyster C. virginica [START_REF] Gold-Bouchot | Hydrocarbon Concentrations in the American Oyster (Crassostrea virginica) in Laguna de Terminos, Campeche, Mexico[END_REF] the authors concluded that the pollution level in Terminos Lagoon could be considered as moderate.

Long-term effects of pesticides and heavy metals were studied in aquatic mammals inhabiting Terminos Lagoon. Heavy metals and pesticides in bottlenose dolphins (Tursiops truncatus) were present in skin and blubber biopsy samples, albeit in lower concentrations than those recorded in other studies [START_REF] Delgado-Estrella | Effects of pollutant discharges on the aquatic mammal populations of Terminos Lagoon, Campeche, Mexico[END_REF].

Conclusions and recommendations

The great variability and heterogeneity in the numerous variables that control and characterize transitional water systems greatly hinders the potential for inter-comparison between very diverse sites, because tropical coastal lagoon systems are driven, for example, by very specific factors when compared to temperate ones (Ortiz-Lozano et al,, 2005;Camacho-Ibar & Rivera-Monroy, 2014). While most of the coastal lagoons in the world are more or less exposed to the same set of detrimental factors arising from human development and sea level rise, global climate change impacts can on the other hand be strongly geographically dependent, even at the regional scale. This variability is particularly strong in the Gulf of Mexico and Central America region, and joint simulations foresee an increase in the mean annual discharge of the Mississippi-Atchafalaya River by 11 to 63 percent, compared to a severe decrease by up to 80 percent for the Usumacinta-Grijalva River [START_REF] Kemp | Can Continental Shelf River Plumes in the Northern and Southern Gulf of Mexico Promote Ecological Resilience in a Time of Climate Change?[END_REF]. Such a contrasting forecast regarding the two major watersheds of the Gulf of Mexico is of major consequence to their respective downstream coastal systems and prefigures a drastic environmental shift in Terminos Lagoon.

Considering the sensitivity of the biota to the balance between riverine and marine influences, salinity is obviously an essential indicator of the main alteration process that will impact the Terminos Lagoon system on a long term and global scale. In the absence of a structured environmental monitoring plan focusing on the hydrology of Terminos Lagoon in particular, urgent action should be taken to gather information on the ongoing trends in environmental change. Indeed, the previously exposed arguments demonstrate that salinity is the most obvious basic indicator of environmental change to be continuously measured in various representative parts of the lagoon.

Larger scale studies are required to more precisely capture the dimensions and nature of the changes produced and their effects on the structure and function of the landscape. Additionally, deeper examination of the history of land use should be conducted to better interpret elements concerning the causes of changes and modifications occurring in the area.

Regional trends over the past 30 years have replaced forests with pastures, and there is a call today for more efficient and proactive conservation policies through paid environmental services.

Regarding pollutants, environmental threats seem to be primarily localized around urban areas and river mouths, or restricted to specific ecosystem components, especially in the highest trophic levels. Furthermore, studies on emerging pollutants are entirely absent in Terminos Lagoon and are urgently needed.

Finally, even if several compartments of the benthic ecosystem have been studied, some topics remain to be assessed, such as sediment-water exchanges of nutrients and benthic carbon mineralization in the lagoon, as well as the processes related to microphytobenthic productivity. Such information may be of prime importance in understanding the functioning of Terminos Lagoon and for calculating accurate budgets in order to preserve this protected but threatened ecosystem.

Fig. 1 .

 1 Fig. 1. Bathymetric map of the study area with major rivers and connections to the Gulf of Mexico.

Fig. 2 .

 2 Fig. 2. Spatial variability in salinity measured in Terminos Lagoon in 2010 during the dry (left) and rainy (right) seasons (modified from Fichez et al., 2017).

  cuneata and Polymesoda caroliniana); b-lagoons joined to the southwest part of Terminos Lagoon where naturally occurring reefs of Crassostrea virginica and their typically associated community are found (salinity in the range 0-15); c-The main lagoon central basin, where eight gastropod species and nine bivalve species constitute the mollusk community and are submitted to a wide range of salinity throughout the year (10-36);d-An area of strong marine influence located close to Carmen Island (salinity ranging from 28 to 38), where a broad community of eight gastropods and nine bivalves dominates, mainly in association with Thalassia testudinum beds.

Acknowledgements

This study was financially supported by the Institut de Recherche pour le Développement (IRD), the Centre National de la Recherche Scientifique (CNRS), the Université de Lille, the Universidad Autónoma Metropolitana-Iztapalapa (UAM-I) and the French National Program Ecosphère Continentale et Côtière -Dynamique et Réactivité des Interfaces Littorales (EC2CO-DRIL). Acknowledgements are also due to Instituto de Ciencias del Mar y Limnologia, Universidad Nacional Autónoma de México (ICMyL-UNAM) for providing access to their field station in Ciudad del Carmen. French Bi National collaboration was federated by ECOS-Nord (ECOS/ANUIES/SEP/CONACyT contract M12-U01) and DANONE-BONAFONT S.A. de C.V.


Fuwei Pi

Pierre Dillard

Laurent Limozin

Anne Charrier

Kheya Sengupta
email: sengputa@cinam.univ-mrs.fr

A Versatile Approach to Fabrication of Protein Nano-dot Arrays on Soft Substrates

Keywords: Nano-bio-functionalization, Particle Lithography, Adhesion of T-lymphocytes, Soft substrate, Surface sensitive imaging

We present a cost effective simple colloidal bead self-assembly based bench-top protocol for fabricating protein nano-dot arrays on hard and soft substrates. An improved protocol for creating evaporation masks by self-assembly of micro-beads is developed. The masks are subsequently used to create regularly spaced protein dots of variable size, down to about 80 nm, and covering macroscopic areas on glass. The various steps of the fabrication process are characterized by atomic force and epi-fluorescence microscopy. The glass supports can be either used as is for advanced optical microscopic studies (including surface sensitive techniques like total internal reflection microscopy and reflection interference microscopy) of living cells, or can be used as template for further functionalization of soft substrates by contact printing. Glass supported polydimethylsiloxane (PDMS) thin layers, with stiffness ranging from 30 KPa to 3 MPa were functionalized with fluorescent proteins and imaged with an optical microscope.

In the context of nano-biotechnology a lot of advances have been made in production of biocompatible nano-particles or surfaces exhibiting nano-topography, but techniques for biochemical patterning of surfaces with nano-dots of bioactive molecules remain restricted. One of the most flexible methods in terms of choice of molecules and surfaces is dip pen nanolithography, 11 but even with massive parallelization, 12,13 due to the requirement of large coverage for cell biology studies, this remains too slow for regular use in a biology laboratory. Techniques based on contact printing, have been limited mainly by the material properties of the stamp that limit the smallest feature size. 14 Two alternative approaches -one based on colloidal gold precipitated inside block co-polymer micelles and the other on nano-imprint lithography followed by etching and gold-paladium evaporation, have been successfully used in cell adhesion studies. 4,5,10,15,16 However, both these approaches rely on selective functionalization of pre-formed gold nano-dots and are therefore limited by the bio-chemistry and optical properties of gold. Of these approaches, only the block co-polymer micelle approach has been successfully adapted to soft substrates, 17,18 but again, they rely on the use of gold nano-clusters.

A different promising approach for nano-patterning is the use of self-assembled mask for vapor deposition of organo-aminosilane molecules that are subsequently functionalized with the desired protein. The amino-silane nano-dots offer virtually no interaction with light, at the same time permitting a large range of strategies for subsequent bio-functionalization. The evaporation mask can be made via phase separation of polymers, 19 from ordered anodic alumina membranes, 20 or via particle lithography 21,22 (see Ref. 23 for a detailed review). Of these, particle lithography has seen considerable development in the past few years, including improvements in ordering, 24 in functionalization, 25 use of binary mixture, 26 independent control of dot size and spacing 27 etc.

The two main concerns still remaining are the issue of controlling the ordering over large surfaces to facilitate applications in biology and the nano-dot size, which ideally should be controlled down to single molecule but to this day remains around 200 nm on hard inorganic substrate. Particle lithography has so far never been used for functionalization of soft or flexible substrates.

Here we present a versatile and facile technique for large-scale coverage of glass or a soft polymer with ordered protein nano-dots. A novel protocol for producing an evaporation mask by colloidal bead self-assembly is developed. The mask, assembled on glass, serves to successively deposit (a) molecules of organo-amino-silane in the form of a cushion with regularly spaced holes, (b) reactive polyetheleneglycol (PEG) molecules that bind to the amine group, and (c) the protein of interest that binds wherever PEG is not present. On removal of the mask, the slides can be used as is, for example for cell adhesion studies as reported here, or they can be used as "masters" for functionalization of soft substrates. For this, the nano-dots of protein are transferred by contact printing to the target soft substrates comprising glass supported planar Polydimethylsiloxane (PDMS) cushions. The stiffness of the PDMS layer is varied by varying the base to cross-linker concentration in the range of 30 KPa to 3 MPa. Fig. 1(a) illustrates the procedure for obtaining large area coverage of a monolayer of colloidal microspheres. In brief, a controlled volume of polystyrene or silica microsphere suspension of a controlled concentration is allowed to spread under gravity on a slightly inclined glass cover-slip, which is pre-cleaned to render it highly hydrophilic. Two simple improvements facilitate uniform coverage: first, the inclination angle is slowly decreased and second, a backflow of the drained bead suspension is effectuated so that the defects in the monolayer formed during the first spreading step are filled over. Using such improved strategy, large scale patterns (up to about 24  24 mm -corresponding to full coverage of the coverslide) of microspheres could be easily obtained without special control of temperature, humidity or other environmental conditions. The important factors that control the quality of the formed monolayers in terms of uniformity and the area covered are: hydrophilicity of the glass surface, the concentration and volume of microsphere suspension. The microsphere size was varied from 500 nm to 6  m. The overall strategy is the same for both polystyrene and silica microspheres and for the different sizes. The main parameters to be optimized are the concentration and volume used (See Supporting Information for details).

The colloidal bead monolayer was used as a mask to deposit organo-amino-silane molecules on a glass coverslip from the vapor phase. We could successfully graft APTES ((3-Aminopropyl)triethoxysilane) and OTS (Octadecyltrichlorosilane), the former being more suitable for subsequent functionalization. The success of the silanization process was tested by measuring the contact angle of a droplet of water to verify that the coverslip was rendered hydrophobic.

To characterize the grafting of APTES at the molecular scale, we imaged the surface of the coverslip, after removal of the colloidal bead mask, with atomic force microscopy (AFM). For further discussion here we describe the patterns fabricated with polystyrene microspheres with 1  m diameter as representative. We have verified that the results are similar for other bead sizes and for silica beads. Fig. 2a shows AFM image after the APTES deposition stage. An array of circles is seen. The spacing of 980  10 nm corresponds well to the diameter of the beads used (Fig. 2b; see also Table 1a for all statistics). The inner radius of the circles is 120  20 nm. The height profile across a single circle demonstrates that the inside of the circle either remains bare or is very sparsely grafted with APTES (Fig. 2c andd). The height difference of 1.5  0.2 nm (statistics from >10 motifs) between the inside and the outside corresponds well to the expected size of an APTES molecule. We conclude that the APTES molecules are grafted on the glass surface in the form of a monolayer, but in the confined space between the bead and the substrate they form multilayers.

At this stage, the substrate is patterned with patches of bare glass surrounded by a sea of APTES molecules. To proceed further with the functionalization, PEG chains were grafted on to APTES by submerging the silanized glass coverslip into an aqueous solution of NHS-PEG ester (Fig. 1b). An alternative strategy was to graft PEG-COOH via peptide bonds in an alkaline medium overnight (see Supporting Information for details). While the first strategy is quick and facile, the second offers more flexibility in terms of availability of different kinds of PEG molecules. In both cases, the quality of the pattern is better if the colloidal bead mask is removed after, rather than before, grafting the PEG molecules. The success of the PEG binding step could be easily verified by measuring the contact angle of a droplet of water, showing that the slides become hydrophilic again after this step. At this stage, the glass coverslip is patterned with submicron holes of bare glass in a sea of PEG. In the subsequent step, the bare glass holes are filled with a desired protein, with the PEG molecules acting as repellers to prevent protein deposition elsewhere. Different chain lengths of PEG were tested and the one most suitable for the subsequent protein binding step, namely 36 units, was retained.

The coverslips with patterned PEG were functionalisatized with the protein of interest by incubating in a suitable solution of the protein molecule for a controlled amount of time (Fig. 1c).

The protein molecules are expected to adsorb only on the patches of bare glass corresponding to the contact sites of microspheres in the original mask, thus forming a regular protein island array corresponding to the original pattern of the microspheres. Typically, fluorescent bovine serum albumin (BSA) was used for establishing the protocol. The robustness of the protocol was then tested using BSA-biotin which was subsequently functionalized with the avidin analogue fluorescent neutravidin.

The fluorescent protein islands were imaged with epi-fluorescence (Fig. 3) or atomic force (Fig. 4) microscopy. Fig. 3 shows a few typical examples of the patterns formed with different parameters for the bead-mask and different choice of proteins. The pitch corresponds to the diameter of the beads used to fabricate the mask. Comparison of AFM and optical imaging data for bead sizes of 500 nm, 1  m or 2  m diameter (Table 1a), shows that for both the silica and the polystyrene beads, the size of the protein nano-dots measured in epi-fluorescence from the full width at half maximum of the intensity trace, is larger than the size measured in AFM. This is probably because the optical images are resolution limited. Fig. 3g and 3h are of particular interest -they illustrate the possibility of changing the patch size without changing the inter-patch distance 28 . To achieve this, the colloidal bead mask, prepared from 6  m PS beads, is gently heated after its formation and just prior to APTES deposition. This melts the polymer and increases the foot-print of the bead on the glass surface thus giving rise to an expanded patch size.

AFM image of BSA protein patches (fabricated with 1  m diameter microspheres), is presented in Fig. 4a. This can be compared with Fig. 3a where epi-fluorescence images (fluo) of the same substrate is presented. The measured pitch of 1000  30 nm (fluo) and 960  65 nm (AFM) correspond closely to the pitch of APTES circle arrays imaged in Fig. 2, as well as to the diameter of the colloidal beads used to fabricate the mask. The patch size measured in fluorescence images (390 ± 20 nm) is larger than the true size (115 ± 20 nm) measured from AFM. The concentration of the solution from which the protein is deposited regulates the amount of protein in the patches, as measured from the height of the protein patch with respect to the surrounding layer of PEG (data not shown). When deposited from 10  g/ml BSA/PBS solution, the height difference is about 3 nm (Fig. 4d). Comparing Fig. 4 In all the cases tested, the pitch of the final protein nano-dot array equals the diameter of the bead type used to fabricate the mask. The nano-dot size however is only weakly dependent on the diameter of the beads used in the mask (Table 1a). The smallest size obtained is for 500 nm diameter beads and is 80 nm as measured by AFM.

These protein patterned glass slides were used as support for pilot experiments on Tlymphocyte adhesion. For this, BSA-biotin was patterned as described and was subsequently functionalized with fluorescent streptavidin (visualized in Fig. 5a), which in turn was further functionalized with mono-biotinylated anti-CD3 (OKT3). T-lymphocytes (cell line Jurkat) were allowed to interact with these substrates. It is expected that the sea of PEG provides a passive background with activating anti-CD3 islands at regular intervals. The live cells were monitored with RICM 29 that the cells adhere not only on the functionalized patches but also on the PEG. This is consistent with control experiments on PEG alone. As expected, the TCR forms micro-clusters on the cell surface [START_REF] Kaizuka | Proc. Natl. Acad. Sci[END_REF] but unexpectedly, the TCR microclusters are distributed randomly, as also seen in control experiments on uniform anti-CD3, rather than co-localizing with the anti-CD3 patches. These pilot experiments confirm that the current organo-amino-silane based functionalization protocol is perfectly suited for advanced optical imaging.

The protein-dot array on glass can also serve as a flat stamp from which the protein nano-dots can be readily transferred to the surface of a soft polymer like PDMS. The process is the inverse of conventional micro-contact printing and is summarized in Fig. 1d. First, the PDMS substrates are prepared by sandwiching non-reticulated PDMS (base mixed with desired amount of cross-linkers) between a glass coverslip which serves as support and a smooth passivated glass slide, separated by glass spacers of desired thickness (typically 140 microns). The PDMS is then cured and the passivated slide is removed to yield the substrate which comprises a glass coverslip supporting a thin film of PDMS with very smooth surface. This PDMS surface is then manually put in contact with the previously prepared protein-master. On separation, the protein dots are seen to have been transferred to the PDMS surface. The stiffness of the PDMS can be controlled by controlling the amount of cross-linkers used. An estimated stiffness of 30 KPa, 140 KPa and 3 MPa was produced by choosing base to cross-linker ratio of 50:1, 35:1 and 10:1 respectively. 31,32 Below we refer to these as soft, medium and stiff substrates. Fig. 6 shows representative fluorescence images of protein nano-dots transferred from masters fabricated with 2  m diameter microspheres onto the stiff, medium and soft PDMS substrates. It is apparent that the protein dots on PDMS retain the essential features of the master (corresponding to Fig. 2a) for all three stiffness presented here. The pitch of the transferred protein pattern on the PDMS (1930  40 nm, 1820  40 nm and 1860  50 nm for stiff, medium and soft respectively) closely corresponds to that of the master (1920  30 nm). The lateral size of the dots, corresponding to the full width at half maximum of the intensity line profiles from the images (390  20 nm, 480  30 nm and 390  10 nm for stiff, medium and soft respectively) is slightly larger than the size of the dots on the master (310  30 nm). This probably happens due to deformability of the PDMS substrate as well as the imprecise nature of manual transfer.

We have successfully transferred patterns from masters fabricated using both silica and polystyrene beads of different size. The observed patch size and inter patch distance on masters at different steps (measured from optical and AFM images) and of the protein nano-dots on PDMS substrates (measured from optical images) are summarized in Table 1b.

Here we have reported a simple cost-effective strategy for large scale patterning of protein nano-dots on hard and soft substrates. The technique can be easily implemented as a bench top setup, bypassing many of the current limitations on fabricating nanoscale masters, which rely on expensive and specialized facilities and absolutely clean environment. Our approach uses only commercially available reagents and chemicals, thus avoiding complicated chemical synthesis or nano-fabrication required for gold-nano dot based approaches. Since the transfer is from one planar surface to another, many of the problems associated with microcontact printing of very small features are absent. Such functionalized PDMS substrates hold out the promise of mechanosensing studies combined with nano-scale surface stimulation of living cells. We believe that this generic method will lead to a popular route for bench-top patterning of bioactive molecules over a large area on hard as well as soft substrates for exploring the molecular, cellular and biophysical basis of cell behavior at the nanoscale. microspheres (PS) concentration 2.53% and 2.61% respectively, or 2  m and 500 nm silica microspheres concentration 10% and 9.83% respectively (). The PS colloidal suspensions were used directly; silica colloidal suspensions were diluted into 1:5 and 1:8 ( / ) with ultrapure water for 2  m and 500 nm diameter respectively.

A glass platform with edge angle of 4 o was set up on an ordinary laboratory bench under ambient conditions. Next, a glass coverslip, thoroughly cleaned as described above, was set on the platform at an angle of 13 o such that the colloidal bead suspension spread under gravity. The edge angle of glass coverslip was decreased to 4 o when the spreading front of the suspension reached the bottom of glass coverslip. Uniform spreading was further facilitated by turning around that coverslip so that the bottom and top parts of the coverslip were exchanged. In absence of this action, most of microspheres in the suspension move to the bottom of glass coverslip and aggregate into multilayered clusters. The decrease of edge angle and exchange between bottom and top parts of the coverslip causes a back-flow of the suspension which carries some of the microspheres back from bottom to disperse and fills the defects in the monolayer formed during the first spreading step.

To determine the optimum volume of aqueous solution needed to wet the slide, we initially spread a drop of water instead of suspension on the cleaned glass coverslip. Through measuring the spreading time of the front edge of water arriving at the bottom of coverslip, we found that a volume of 40 -60  L is suitable for optimal wetting (Table SI1). 45  L is the volume used consistently throughout our experiments. This provides, on one hand enough time for particle absorption before draining, and on the other hand, leaves enough time before evaporation for inducing a back-flow as described above.

We optimized the concentration of beads in the suspension for the optimal volume determined above. Dilution of 1:2, 1:4, 1:5 and 1:6 (v suspension /v water ) were carried out. In case of 2  m silica particles, when the dilution was less than 1:4, large areas of bead multi-layers were obtained. On the other hand, when the dilution was more than 1:6, lot of defects were observed. A dilution of 1:5 was found to be optimal and was chosen. The procedure was repeated for each type of bead to find the optimal spreading condition. To further functionalize with protein, the PEG grafted coverslip, with the mask removed, was incubated in 300  L of 10  g/ml BSA in PBS. For proof-of-principle pilot experiments, fluorescent BSA was used whereas for tests with cells, BSA coupled to biotin was used. In the latter case, further incubation with 2  g/ml Texas-Red coupled neutravidin in PBS buffer solution followed by incubation in mono-biotinylated anti-CD3 (OKT3 kind gift from Rajat Varma, NIH) was performed. Each incubation lasted 30 minutes at room temperature. Excess protein was removed by exchanging the buffer against protein free PBS in a series of typically ten washing steps.

Preparation of PDMS substrates. Protein nano-dot arrays on glass were prepared as described above, choosing the concentration of the fluorescent BSA solution for the final incubation as 20  g/ml. After verification of the quality of the nano-dot array on the glass master by epi-fluorescence microscopy, the glass supported PDMS sheet prepared as above were put in contact with the glass master. Uniform pressure was applied manually to effectuate the transfer. The protein nano-dot pattern was readily transferred to the PDMS surface. The substrates were then imaged in epi-fluorescence to asses the quality of transfer.

Polydimethylsiloxane

Imaging of patterns and data analysis.

Epi-fluorescence images of the protein nano-dot arrays were acquired on an inverted optical microscope (Axio Observer, Zeiss, Germany) equipped with an EM-CCD camera (Andor, UK)

and high magnification objective (100, NA 1.4, Zeiss, Germany). Images were analyzed with Observation was performed on a Zeiss inverted microscope equipped with an Andor EMCCD camera. Reflection interference Contrast Microscopy (RICM) was performed with a 63x NA=1.25 Antiflex oil immersion objective (Zeiss, Germany) and using 540 nm green line of a fluorescence excitation light source (X-cite, Canada) . Total Internal Reflection Fluorescence microscopy (TIRF-M) was performed using an Argon laser filtered at 488 nm in excitation using a 100x, NA=1.45 oil immersion objective (Zeiss, Germany). IRM (Interference Reflection Microscopy) was performed with the same objective using a mercury lamp illumination filtered at 546 nm and a semi-reflecting mirror. For observation through the PDMS layer (data not shown), a NA=1.40 oil immersion objective was used instead to exploit longer working distance.

See Ref. 29 of main text for information on RICM and IRM.

Alternative protocol for PEG grafting.

In this protocol, the PEG molecular chain was covalently grafted on to glass surface through bonding of carboxyl group of PEG-COOH to amide group at the terminal of APTES (Fig. SI 2).

By activating the amide group into activated ester with EDC (1-(3-Dimethylaminopropyl)-3ethylcarbodiimide hydrochloride), the amide group could readily bind to the carboxyl group in the presence of slight excess of NMM (N-methylmorpholine). Since the activated ester is not stable, protective NHS (N-Hydroxysuccinimide) was added into the reactive system to extend the life of activated ester and allow for full grafting APTES. All reagents were from Sigma, France.

  with Fig. 2, it is seen that whereas in Fig 2, the interior of the imaged circles is lower than the surrounding sea of APTES, in Fig. 4, the interior of the circles is higher than the surrounding sea of PEG. Comparison with epi-fluorescence images clearly indicate that the extra material corresponds to the fluorescent protein.

  (Fig 5b). After spreading, the cells were fixed, stained for T-cell receptor (TCR) molecules and imaged in TIRF (Fig 5c) and IRM 29 (Fig 5d) mode. IRM and RICM images show

Figure 1 .

 1 Figure 1. Schematic representation of the fabrication strategy for developing large-scale protein nanopatterns on soft substrates (a) self-assembly of the colloidal bead mask on glass substrate, (b) deposition of APTES from vapor phase through the mask and functionalizing with PEG molecules, (c) functionalization with protein, and (d) transfer of the protein nano-dots onto soft, glass supported PDMS. The pitch "d", as measured from AFM and fluorescence imaging is equal to the diameter of the beads used to fabricate the masks. The patch size " ω" depends weakly on the bead diameter and could be varied from 80 to 200 nm.

Figure 2 .

 2 Figure 2. (a) Tapping mode AFM image of glass after grafting APTES (mask: 1  m diameter beads ); (b) corresponding height profile of the line drawn in (a); (c) zoom-in 3D image of a single circle; (d) correspondingprofile. Red dash line indicates the height of APTES molecule grafted on the surface, which is 1.5 +/-0.2 nm as measured from >10 motifs and is consistent with the grafting of a monolayer of APTES.

Figure 3 .

 3 Figure 3. Epi-fluorescence images of protein patch arrays fabricated with different types of masks. (a) fluorescent-BSA (mask: 2  m silica beads); (b) fluorescent-BSA (mask: 1  m PS beads); (c) Neutravidin bound to BSA-biotin (mask: 500 nm silica beads). (d), (e) and (f) are corresponding plot of the intensity profiles. (g and h): BSA patterns fabricated with 6  m PS beads at room temperature -RT (g) or pretreated at 110 o C (h); (i) corresponding intensity profiles. Insets display the Fourier transforms of the corresponding images to emphasize the ordering of the lattice.

Figure 4 .

 4 Figure 4. (a) Tapping mode AFM image of protein nano-dots fabricated through depositing 300  L of 10  g/ml BSA/PBS solution, using 2  m diameter beads for mask; (f) corresponding intensity profile of the line drawn in (e). Red and blue dashed lines indicate the height of PEG grafted on APTES and protein adsorbed on glass respectively.The height difference of about 3 nm indicates adsorption of multiple layers of proteins on glass.

Figure 5 .

 5 Figure 5. Spreading of Jurkat cell on functionalized glass substrate (mask from 2  m silica beads) (a) epifluorescence image of neutravidin dots functionalized with anti-CD3 and surrounded by a sea of non-fluorescent PEG; (b) a single Jurkat cell imaged in RICM (c) TIRF-M image of another cell, on the same substrate as a ; (d) corresponding IRM image.

Figure 6 .

 6 Figure 6. Epi-fluorescence images of protein nano-dot arrays on hard (a), medium (b): and soft (c) PDMS layers. Insets display the Fourier transforms.; (d), (e) and (f) are corresponding intensity profiles of the lines drawn. The pitch and the size (HWFM) are indicated.

Figure 1 .

 1 Figure 1. Schematic representation of the fabrication strategy for developing large-scale protein nanopatterns on soft substrates (a) self-assembly of the colloidal bead mask on glass substrate, (b) deposition of APTES from vapor phase through the mask and functionalizing with PEG molecules, (c) functionalization with protein, and (d) transfer of the protein nano-dots onto soft, glass supported PDMS. The pitch "d", as measured from AFM and fluorescence imaging is equal to the diameter of the beads used to fabricate the masks. The patch size " ω" depends weakly on the bead diameter and could be varied from 80 to 200 nm.

Figure 2 .

 2 Figure 2. (a) Tapping mode AFM image of glass after grafting APTES (mask: 1  m diameter beads ); (b) corresponding height profile of the line drawn in (a); (c) zoom-in 3D image of a single circle; (d) corresponding profile. Red dash line indicates the height of APTES molecule grafted on the surface, which is 1.5 +/-0.2 nm as measured from >10 motifs and is consistent with the grafting of a monolayer of APTES.

Figure 3 .

 3 Figure 3. Epi-fluorescence images of protein patch arrays fabricated with different types of masks. (a) fluorescent-BSA (mask: 2  m silica beads); (b) fluorescent-BSA (mask: 1  m PS beads); (c) Neutravidin bound to BSA-biotin (mask: 500 nm silica beads). (d), (e) and (f) are corresponding plot of the intensity profiles. (g and h): BSA patterns fabricated with 6  m PS beads at room temperature -RT (g) or pretreated at 110 o C (h); (i) corresponding intensity profiles. Insets display the Fourier transforms of the corresponding images to emphasize the ordering of the lattice.

Figure 4 .

 4 Figure 4. (a) Tapping mode AFM image of protein nano-dots fabricated through depositing 300  L of 10  g/ml BSA/PBS solution, using 2  m diameter beads for mask; (f) corresponding intensity profile of the line drawn in (e).Red and blue dashed lines indicate the height of PEG grafted on APTES and protein adsorbed on glass respectively.The height difference of about 3 nm indicates adsorption of multiple layers of proteins on glass.

Figure 5 .

 5 Figure 5. Spreading of Jurkat cell on functionalized glass substrate (mask from 2  m silica beads) (a) epifluorescence image of neutravidin dots functionalized with anti-CD3 and surrounded by a sea of non-fluorescent PEG; (b) a single Jurkat cell imaged in RICM (c) TIRF-M image of another cell, on the same substrate as a ; (d) corresponding IRM image.

Figure 6 .

 6 Figure 6. Epi-fluorescence images of protein nano-dot arrays on hard (a), medium (b): and soft (c) PDMS layers. Insets display the Fourier transforms.; (d), (e) and (f) are corresponding intensity profiles of the lines drawn. The pitch and the size (HWFM) are indicated.

Fig. SI 1

 1 Fig. SI 1 shows typical examples of masks visualized by optical microscopy in transmitted

Figure SI 1 .Table SI 1 :

 11 Figure SI 1. Top row: Optical microscopy (transmission bright field) images of colloidal bead

  Sylgard 184 (PDMS) was purchased from Dow Corning, USA and Trichloro(1H,1H,2H,2H-perfluorooctyl)silane (fluoro-silane) from Sigma, France. The PDMS elastomer base and the curing agent was mixed in the desired ratio -10:1, 35:1 and 50:1 (w base /w agent ), to obtain hard, medium and soft PDMS respectively (see Ref. 31 and 32 of main text). The PDMS substrates were fabricated by curing the base/curing agent mixture between two glass coverslips, separated using glass spacers of about 140 micron, and baked at 60 o C for 16 hours. One of the coverslips was destined to act as the support for the PDMS sheet. The other coverslip was silanized with fluoro-silane (from vapor phase in a closed desiccator under vacuum at room temperature for 30 minutes) prior to contact with uncured PDMS. After curing a drop of 2-propanol was injected into the gap between the coverslips to facilitate their separation. On separation, PDMS sheets of thickness corresponding to the spacers, supported on glass and with one smooth surface were obtained. The glass supported PDMS substrates were ultrasonicated in ultrapure water (3 times 10 minutes) with repeated flushing and blow-drying with nitrogen to eliminate the propanol. Finally, dried PDMS sheets were desiccated at ~ 40KPa vacuum at room temperature overnight to remove the residual propanol on PDMS surface.

Fiji [Nature Methods 9 ( 7 )

 97 : 676-682]/ImageJ v1.47 (http://imagej.nih.gov/ij/) and IgorPro (WaveMatrics, USA) software packages. For the intensity traces presented (Fig.3 and 5of main text), the camera offset was subtracted from the data prior to analysis: no other modification was done to the raw data (no background subtraction, for example). The reported size of the dots correspond to half width at full maximum. Statistics over at least 3 samples and 10 dots are presented. The error-bars are standard deviations.Tapping mode AFM measurements were carried out on a NTEGRA system (NT-MDT, Russia): in air after grafting APTES and in PBS buffer solution (pH = 7.4), after depositing protein. Silicon tips (NSC35, MicroMash, Bulgaria) with a typically resonance frequency of 120 kHz and less than 10 nm tip radius were used. AFM images were analyzed using NTEGRA Imaging Analysis 2.1.2 software packages. Images were flattened following standard practice.Statistics over at least 10 dots are presented. The error-bars are standard deviations.Cell culture, marking and cell imaging.Jurkat E6 T-lymphocytes were cultivated in RPMI complete medium supplemented with 10% Foetal Bovine Serum and with 1% GlutaMAX (Invitrogen). Last splitting was realized 24h before experiments to ensure a concentration of 0.6 million cells/mL at the moment of the experiment. Cells were deposited on the substrates in Hepes-BSA buffer (20mM HEPES pH 7.2, 137 mM NaCl, 5 mM KCl, 0.7 mM Na 2 HPO 4 , 6 mM D-glucose, 2 mM MgCl 2 , 1 mM CaCl 2 , 0.025% BSA) and incubated during 15 min at 37° C 5% CO 2 . Fixation was performed 15 min after deposition with 2% paraformaldehyde during 10 min at 37° C and followed by extensive rinsing with PBS. For fluorescent experiments, the cells were labeled by incubation with 1 µg/ml of Alexa 488 labeled antibody directed against the T-Cell receptor (anti Vbeta8, Ebiosciences) during 60 min, followed by extensive rinsing.

Figure SI 2 .

 2 Figure SI 2. Schematic representation of the strategy used to bind PEG-COOH molecules to

Table 1a .

 1a The size of APTES and protein nano-dots on glass as measured by AFM or epi-

			fluorescence (fluo)*.
	Bead	APTES (AFM)	Protein (AFM)	Protein (fluo)
	Size/Type	(nm)	(nm)	(nm)
	6  m Polystyrene			750  60
	  m Silica	190  10	150 ± 15 nm	310 ± 30
	  m Polystyrene	120  10	120 ± 20	390 ± 20
	  nm Silica	60  10	80 ± 10	240 ± 10

Table 1b

 1b AFM data are the averaged values of at least 10 motifs; Fluorescence data, as measured from FWHM of intensity profiles are for at least 10 dots for 3 samples.  indicates data not available. Error bars correspond to standard deviation.

			PDMS
	Glass
		10 : 1 (3 MPa)	35 : 1 (140 KPa)	50 : 1 (30 KPa)
	310 ± 30 nm	390 ± 10 nm	480 ± 30 nm	390 ± 20 nm

. The size of protein nano-dots (fabricated with   m Silica beads as mask) on PDMS substrates measured by epi-fluorescence *.



Acknowledgments: We thank Loïc Magiera-Millé for pilot experiments on colloidal bead masks, Herve Dallaporta and Frank Thibaudau for fruitful discussions, Anne Marie Lellouch and Sophie Cadra for help with T-cells and Rajat Varma for gift of monobiotinylated OKT3. KS and FP acknowledge funding from region-PACA (grant no. 2011_06554). The research leading to these results has also received funding from the European Research Council under FP/2007-2013/ ERC Grant Agreement n. 307104.

Experimental Section

Preparation of glass substrates.

Glass coverslips (thickness = 170 microns, Assistent, Karl Hecht KG, Germany), 24  24 mm, were cleaned according to the following protocol: ultrasonication in 2% ( / ) aqueous solution of Hellmanex (Sigma, France) for 20 minutes, rinsing 10 times with ultrapure water (resistivity of 18.2 M  cm, Elga, UK), again ultrasonication in 2% Hellmanex solution for 20 minutes; ultrasonication in ultrapure water (2 times 20 minutes) with repeated rinsing; finally blow-drying with nitrogen stream. Note that this procedure renders the glass ultra hydrophilic (contact angle less than 5 o ). Such high hydrophilicity is essential for the subsequent step of mask formation.

Simpler cleaning procedures, for example with organic solvents or piranha solution were found to be insufficient.

Fabrication of colloidal bead masks.

Suspensions of colloidal microspheres (beads) with different diameters and made either of polystyrene (PS) or of Silica were purchased from Polysciences, Germany and were were washed