Fanny Noisette
email: fanny.noisette@live.fr

François Bordeyne

Dominique Davoult

Sophie Martin

Affiliations

Assessing the physiological responses of the gastropod Crepidula fornicata to predicted ocean acidification and warming

Keywords: calcification, coastal system, invasive species, metabolism, mollusk, pCO 2, temperature

Organisms inhabiting coastal waters naturally experience diel and seasonal physicochemical variations. According to various assumptions, coastal species are either considered to be highly tolerant to environmental changes or, conversely, living at the thresholds of their physiological performance. Therefore, these species are either more resistant or more sensitive, respectively, to ocean acidification and warming. Here, we focused on Crepidula fornicata, an invasive gastropod that colonized bays and estuaries on northwestern European coasts during the 20 th century. Small (< 3 cm in length) and large (> 4.5 cm in length), sexually mature individuals of C. fornicata were raised for 6 months in three different pCO 2 conditions (390, 750 and 1400 µatm) at four successive temperature levels (10, 13, 16 and 19°C). At each temperature level and in each pCO 2 condition, we assessed the physiological rates of respiration, ammonia excretion, filtration and calcification on small and large individuals. Results show that, in general, temperature positively influenced respiration, excretion and filtration rates in both small and large individuals. Conversely, increasing pCO 2 negatively affected calcification rates, leading to net dissolution in the most drastic pCO 2 condition (1400 µatm) but did not affect the other physiological rates. Overall, our results indicate that C. fornicata can tolerate ocean acidification, particularly in the intermediate pCO 2 scenario. Moreover, in this eurythermal species, moderate warming may play a buffering role in the future responses of organisms to ocean acidification.

Introduction

Predictions indicate that coastal ecosystems will be strongly affected by ocean acidification and warming, currently two of the most prominent anthropogenic processes influencing marine life [START_REF] Harley | The impacts of climate change in coastal marine systems[END_REF]. Due to the increase in atmospheric CO 2 partial pressure (pCO 2 ), pH in surface waters is predicted to decline by 0.06 to 0.32 units and sea surface temperatures to increase by 1.0 to 3.7°C by the end of the century, depending on the Intergovernmental Panel on Climate Change (IPCC) representative concentration pathway considered [START_REF] Stocker | Climate Change[END_REF]. Modifications in seawater carbonate chemistry due to ocean acidification lead to a decrease in carbonate ion concentrations (CO 3 2-) [START_REF] Orr | Anthropogenic ocean acidification over the twenty-first century and its impact on calcifying organisms[END_REF]) and a reduction in the calcium carbonate saturation state (Ω), which regulates the thermodynamics of calcium carbonate (CaCO 3 ) precipitation [START_REF] Feely | Ocean acidification: present conditions and future changes in a high-CO 2 world[END_REF]. In estuarine and coastal waters, pH is more variable than in the open ocean due to intense biological and biogeochemical processes [START_REF] Andersson | Ocean acidification: setting the record straight[END_REF]. In these habitats, ocean acidification and warming will shift the baselines, exacerbate natural variations in pH and temperature, and probably threaten the communities living there [START_REF] Waldbusser | Ocean acidification in the coastal zone from an organism's perspective: multiple system parameters, frequency domains, and habitats[END_REF].

Mollusks constitute a major taxonomic group in estuarine and coastal waters in terms of community structure and ecosystem functioning [START_REF] Gutiérrez | Mollusks as ecosystem engineers: the role of shell production in aquatic habitats[END_REF]. Because most marine mollusk taxa accumulate significant amounts of CaCO 3 to form protective external shells, they may be sensitive to the changes in pH and carbonate chemistry induced by ocean acidification (for review, see [START_REF] Gazeau | Impacts of ocean acidification on marine shelled molluscs[END_REF], although recent studies have shown that some species could be resistant to elevated pCO 2 [START_REF] Range | Calcification, growth and mortality of juvenile clams Ruditapes decussatus under increased pCO 2 and reduced pH: Variable responses to ocean acidification at local scales?[END_REF][START_REF] Ries | Marine calcifiers exhibit mixed responses to CO 2 -induced ocean acidification[END_REF]. Along with direct impacts on calcification, high CO 2 concentrations may also have indirect effects on metabolism by disturbing the extracellular acid-base equilibrium, leading to general internal acidosis [START_REF] Melzner | Physiological basis for high CO 2 tolerance in marine ectothermic animals: pre-adaptation through lifestyle and ontogeny?[END_REF]). These potential shifts in acid-base homeostasis have the potential to change organisms' energy balance [START_REF] Pörtner | Synergistic effects of temperature extremes, hypoxia, and increases in CO 2 on marine animals: from Earth history to global change[END_REF].

In mollusks, the effects of elevated pCO 2 and/or decreased pH alone are highly species-specific (see review in [START_REF] Gazeau | Impacts of ocean acidification on marine shelled molluscs[END_REF], and depend on species sensitivity and any existing compensation mechanisms [START_REF] Michaelidis | Effects of long-term moderate hypercapnia on acid-base balance and growth rate in marine mussels Mytilus galloprovincialis[END_REF]). To better estimate future ocean acidification effects on mollusk species, various physiological processes have been studied in bivalves and gastropods such as respiration [START_REF] Beniash | Elevated level of carbon dioxide affects metabolism and shell formation in oysters Crassostrea virginica[END_REF][START_REF] Bibby | Ocean acidification disrupts induced defences in the intertidal gastropod Littorina littorea[END_REF]), excretion [START_REF] Fernandez-Reiriz | Physiological energetics of juvenile clams Ruditapes decussatus in a high CO 2 coastal ocean[END_REF]; Liu and He 2012), feeding [START_REF] Fernandez-Reiriz | Tolerance of juvenile Mytilus galloprovincialis to experimental seawater acidification[END_REF][START_REF] Marchant | Short-term exposure to hypercapnia does not compromise feeding, acid-base balance or respiration of Patella vulgata but surprisingly is accompanied by radula damage[END_REF], immune response [START_REF] Bibby | Effects of ocean acidification on the immune response of the blue mussel Mytilus edulis[END_REF][START_REF] Matozzo | First evidence of immunomodulation in bivalves under seawater acidification and increased temperature[END_REF]) and protein or enzyme production [START_REF] Matozzo | Can the combination of decreased pH and increased temperature values induce oxidative stress in the clam Chamelea gallina and the mussel Mytilus galloprovincialis?[END_REF][START_REF] Tomanek | Proteomic response to elevated pCO 2 level in eastern oysters, Crassostrea virginica: evidence for oxidative stress[END_REF]. However, few studies have simultaneously assessed the responses of more than three physiological processes to ocean acidification and warming. The concomitant increase in seawater temperature and pCO 2 are likely to affect mollusk metabolism because, in addition to changes in gas solubility and the proportion of carbon species [START_REF] Zeebe | History of seawater carbonate chemistry, atmospheric CO 2 , and ocean acidification[END_REF], temperature also strongly affects physiological and biochemical reactions [START_REF] Cossins | Estimation of filtering rate from clearance of suspensions[END_REF]. Because warming can modulate the metabolism responses to ocean acidification [START_REF] Ivanina | Interactive effects of elevated temperature and CO 2 levels on energy metabolism and biomineralization of marine bivalves Crassostrea virginica and Mercenaria mercenaria[END_REF][START_REF] Melatunan | Effects of ocean acidification and elevated temperature on shell plasticity and its energetic basis in an intertidal gastropod[END_REF], investigations of both pH and temperature effects are valuable for understanding the responses of mollusks in the future ocean.

One of the most abundant and widespread shelled mollusks on the French northwestern Atlantic and Channel coasts is the slipper limpet Crepidula fornicata, Linnaeus 1758 [START_REF] Blanchard | Spread of the slipper limpet Crepidula fornicata (L. 1758) in Europe. Current state and consequences[END_REF]. This gastropod native to the northeastern American coast was introduced in Europe at the end of the 19 th century, mainly via oysters imported for farming [START_REF] Blanchard | Origine et état de la population de Crepidula fornicata (Gastropoda Prosobranchia) sur le littoral français[END_REF]. It then colonized European coasts from southern Sweden to southern France, becoming invasive in some places [START_REF] Blanchard | Spread of the slipper limpet Crepidula fornicata (L. 1758) in Europe. Current state and consequences[END_REF]. C. fornicata lives in shallow sites, especially in bays and estuaries where it can reach very high densities of more than 1000 individuals per m 2 [START_REF] Blanchard | Origine et état de la population de Crepidula fornicata (Gastropoda Prosobranchia) sur le littoral français[END_REF]. This species is known to be highly robust to environmental stress, in particular temperature and salinity [START_REF] Diederich | Thermal tolerance of Crepidula fornicata (Gastropoda) life history stages from intertidal and subtidal subpopulations[END_REF][START_REF] Noisette | Metabolic responses to temperature stress under elevated pCO 2 in the slipper limpet Crepidula fornicata[END_REF], parameters that have diel and seasonal variations in these coastal habitats. Established C. fornicata populations have largely affected biodiversity and ecosystem functioning in terms of sediment modifications [START_REF] Ehrhold | The role of Crepidula proliferation in the modification of the sedimentary tidal environment in Mont-Saint-Michel Bay (The Channel, France)[END_REF], changes in faunal assemblages [START_REF] De Montaudouin | Does the slipper limpet (Crepidula fornicata, L.) impair oyster growth and zoobenthos biodiversity? A revisited hypothesis[END_REF]) and trophic structure [START_REF] Chauvaud | Long-term variation of the Bay of Brest ecosystem: benthic-pelagic coupling revisited[END_REF]. This species also affects benthic biogeochemical cycles by enhancing filtration, metabolic activities, CaCO 3 production, and the recycling of nutrients and dissolved carbon back into the pelagic ecosystem [START_REF] Martin | Respiration, calcification, and excretion of the invasive slipper limpet, Crepidula fornicata L.: Implications for carbon, carbonate, and nitrogen fluxes in affected areas[END_REF][START_REF] Martin | Benthic community respiration in areas impacted by the invasive mollusk Crepidula fornicata[END_REF][START_REF] Ragueneau | Direct evidence of a biologically active coastal silicate pump: ecological implications[END_REF] Although C. fornicata is likely highly tolerant to environmental fluctuations, the combined effects of decreased pH and increased temperature may push this species away from its physiological optimum. Thus the objective of this work was to quantify the respiration, ammonia excretion, filtration and calcification responses of small and large specimens of C. fornicata in different temperature and pCO 2 conditions. Investigating the physiology of this key engineer in some coastal ecosystems in a context of climate change is one way to better understand the sensitivity of this species and its potential future ecological impact. In Morlaix Bay (2009 to 2011), phytoplankton groups (> 5µm), the most important food resource of C. fornicata [START_REF] Decottignies | Exploitation of natural food sources by two sympatric, invasive suspension-feeders: Crassostrea gigas and Crepidula fornicata[END_REF], were mainly dominated by planktonic diatoms in concentrations varying between 10 to 300 cells mL -1 (depending on the season) and dinoflagellate species that were found at lower abundances (ca. 25 cells mL -1 ; Leroy 2011).

Methods

Sampling site and in situ conditions

Biological material

C. fornicata forms stacks of several individuals in which each individual adheres to the dorsal surface of the shell of the subjacent partner in the stack. It is a protandrous hermaphrodite, meaning that the small individuals at the top of the stacks are generally males and the large ones at the bottom, females [START_REF] Coe | Sexual phases in Crepidula[END_REF]. After sampling, stacks were brought directly to the Station Biologique de Roscoff where they were kept in natural, unfiltered seawater for 6 weeks at a temperature gradually lowered to 10°C, reflecting the seasonal drop in temperature between autumn and winter. Sexually mature individuals (more than 1 cm in length) were selected and separated into two class sizes: small individuals (29.5 ± 0.9 mm length) from the top of the stack and larger ones (45.4 ± 0.6 mm length) from the bottom.

They were separated from the stack and individually labeled with tags glued on their shell.

Empty subjacent shells, whose soft tissue was removed, served as substratum for the sampled live individuals. Other empty shells whose size was similar to that of the substratum shell of live individuals were also selected for flux corrections (see part "Metabolic rates and O:N ratios" below). All the shells were gently brushed to remove epibionts without altering periostracum layer.

Length (in mm), volume (in mL) and tissue dry weight (DW in g) of the live individuals were determined for each incubated specimen at the end of the whole experiment.

Length was measured with calipers, volume was estimated as the volume of seawater moved when individual was immersed and DW was determined after drying fresh samples at 60°C for 48 h.

Experimental conditions

Single small and large individuals, along with their substratum shell, were randomly distributed into nine 10 L aquaria with 10 individuals of each class size per each aquarium.

Empty shells were also distributed into nine other 10 L aquaria (4 shells per aquarium). At the beginning of the experiment, pH was gradually decreased over 2 weeks by 0.02 pH unit per day from 8.1 until the different pH treatments were reached. C. fornicata individuals and empty shells were then subsequently held for 24 weeks (12 January to 27 June 2012) in three pCO 2 treatments selected according to the recommendations in [START_REF] Barry | Atmospheric CO 2 targets for ocean acidification perturbation experiments[END_REF]: (1) 390 µatm (pH T = 8.07) represented current pCO 2 , (2) 750 µatm (pH T = 7.82) corresponded to the elevated pCO 2 level predicted by the IPCC for the end of the century (Solomon et al. 2007) and (3) 1400 µatm (pH T = 7.56) represented a pCO 2 five-fold higher than preindustrial pCO 2 (280 µatm) also predicted for 2100 [START_REF] Stocker | Climate Change[END_REF]. pCO 2 was adjusted by bubbling CO 2 -free air (current pCO 2 ) or pure CO 2 (elevated pCO 2 ) in three 100 L header tanks supplied with unfiltered seawater pumped directly from the foot of the Station Biologique de Roscoff.

Each of the three pCO 2 treatments had six replicate 10 L aquaria, three for live organisms and three for empty shells. They continuously received CO 2 -treated seawater at a rate of 9 L h -1 (i.e. a renewal rate of 90% h -1 ) from the header tanks. pCO 2 was monitored and controlled by an offline feedback system (IKS Aquastar, Karlsbad, Germany) that regulated the addition of gas in the header tanks. The pH values of the IKS system were adjusted from daily measurements of pH T in the 18 aquaria using a pH meter (826 pH mobile, Metrohm AG, Herisau, Switzerland) calibrated with Tris HCl and 2-aminopyridine HCl buffers (Dickson et al. 2007).

In each pCO 2 treatment, temperature was raised from 10 to 19°C with an incremental step of 3°C. The first three temperature levels (10 to 16°C) simulated the natural change in temperature from winter to summer in Morlaix Bay whereas the last level (19°C) corresponded to a temperature increase of 3°C predicted for the end of the century (Solomon et al. 2007). C. fornicata individuals were held for three weeks at each temperature before carrying out the metabolic measurements (see below). This acclimation time was long enough to overcome the immediate stress response [START_REF] Meistertzheim | Identification of differentially expressed genes of the Pacific oyster Crassostrea gigas exposed to prolonged thermal stress[END_REF]. Temperature was maintained at (1) 10°C (1 st trial period) from 16 January to 12 February 2012; (2) 13°C (2 nd trial period) from 27 February to 25 March 2012; (3) 16°C (3 rd trial period) from 9 April to 6 May 2012, and (4) 19°C (4 th trial period) from 21 May to 27 June 2012. Between two temperature levels, temperature was gradually increased by 0.2°C day -1 over two weeks. The 18 aquaria were placed in thermostatic baths in which temperature was regulated to within ± 0.2°C using submersible 150 to 250 W heaters controlled by the IKS system. Three independent 10 L aquaria named "control" were maintained at 10°C under ambient pH (with no pCO 2 control) until the end of the experiment in order to estimate a potential bias on metabolism induced by the mesocosm experiment over time. Each aquarium contained 10 small and 10 large slipper limpets on their substratum shell and was supplied with the same seawater sourced from the header tanks. They were kept in a thermostatic bath regulated at 10°C by an aquarium chiller (TC5, TECO®, Ravenna, Italy).

In addition to the natural phytoplankton found in the unfiltered seawater, all slipper limpets were fed twice a week with a stock solution composed of the diatom Chaetoceros gracilis (~ 15 × 10 6 cells mL -1 ) and the dinoflagellate Isochrysis affinis galbana (~ 26 × 10 6 cells mL -1 ); 400 mL of this microalgal mix was added to each aquarium at each feeding.

Seawater flow was stopped for 2 h when organisms were fed and filtering actively. During this feeding time, pH variation did not exceed 0.05 units.

Individuals that did not adhere to their substratum shell and that showed no reaction when their foot was stimulated were counted as dead and removed from the tanks. Mortality reached only 8% at the end of the experiment among all pCO 2 conditions.

Seawater parameter monitoring

Seawater parameters were monitored throughout the experiment. pH T and temperature were recorded daily in each of the 21 aquaria (18 + 3 controls) using a pH meter (826 pH mobile, Metrohm AG, Herisau, Switzerland) as described above. Total alkalinity (A T ) was measured at each trial period by 0.01 N HCl potentiometric titration on an automatic titrator (Titroline alpha, Schott SI Analytics, Mainz, Germany). Salinity was also measured at each trial period with a conductimeter (LF 330/ SET, WTW, Weilheim, Germany). Seawater carbonate chemistry, i.e. dissolved inorganic carbon (DIC), pCO 2 and the saturation state of aragonite (Ω Ar ) were calculated for each pCO 2 level and temperature with CO 2 SYS software (Lewis and Wallace 1998) using constants from [START_REF] Mehrbach | Measurement of apparent dissociation-constants of carbonic-acid in seawater at atmospheric-pressure[END_REF] refitted by [START_REF] Dickson | A comparison of the equilibrium constants for the dissociation of carbonic acid in seawater media[END_REF].

Metabolic rates and O:N ratios

Metabolic rates were assessed at each temperature level after a four-day starvation period and after the shells were gently cleaned to remove biofilm-forming organisms. Two small and two large individuals were selected per aquarium. They were incubated individually in 185 mL (small) and 316 mL (large) acrylic chambers (Engineering & Design Plastics Ltd, Cambridge, UK) filled with seawater from their respective aquaria. They were put on a plastic grid above a stirring bar (speed 100 rpm.), which ensured water homogeneity. Chambers were placed in their original aquaria for incubation to keep the temperature constant. Incubations were carried out in dark for 2 to 10 h, depending on temperature and limpet size, to maintain oxygen saturation above 80% until the end of the incubation. At each temperature period, empty shell incubations were carried out to correct individual rates for fluxes related to the substratum shell. Blank incubations containing only seawater from the aquarium also helped to correct fluxes for any microbiological activity in seawater.

Oxygen concentrations were measured at the beginning and the end of the incubation period with a non-invasive fiber-optics system and reactive oxygen spots attached to the inner wall of the chambers (FIBOX 3, PreSens, Regensburg, Germany). Spots were calibrated at the beginning of each trial period with 0% and 100% oxygen buffers. Seawater was sampled for ammonium (NH 4 + ) concentration and A T measurements with 100 mL syringes at the beginning of the incubation, directly in the aquaria just after the chambers were closed, and at the end of the incubation, in the incubation chamber itself. Samples were filtered through 0.7 μm Whatman GF/F filters into 100 mL glass bottles and fixed with reagent solutions for ammonium or poisoned with mercuric chloride (0.02% vol/vol; [START_REF] Dickson | Guide to best practices for ocean CO 2 measurements[END_REF]) for A T measurements. Vials were stored in the dark pending analysis. NH 4 + concentrations were then determined using the Solorzano method [START_REF] Solorzano | Determination of ammonia in natural waters by the phenolhypochlorite method[END_REF] based on spectrophotometry at a wavelength of 630 nm (spectrophotometer UV-1201V, Shimadzu Corp, Kyoto, Japan). A T (in µEq L -1 ) values were determined by 0.01 N HCl potentiometric titration on an automatic titrator (Titroline alpha, Schott SI Analytics, Mainz, Germany) and by using the Gran method (non-linear least-squares fit) applied to pH values from 3.5 to 3.0 [START_REF] Dickson | Guide to best practices for ocean CO 2 measurements[END_REF].

Respiration (in µmol O 2 g -1 DW h -1 ; equation [1]) and excretion (in µmol NH 4 + g -1 DW h -1 ; equation [2]) were directly calculated from oxygen and ammonium concentrations, [

1] R = ΔO 2 ×V Δt × DW [2] E = ΔNH 4 + ×V Δt × DW [3] G n = - (ΔA T -ΔNH 4 + ) × V) 2 × Δt × DW
where ΔO 2 (in µmol O 2 L -1 ) is the difference between initial and final O 2 concentrations; Δ NH 4 + (in µmol NH 4 + L -1 ) is the difference between initial and final NH 4 + concentrations; ΔA T is the difference between initial and final total alkalinity (µmol Eq L -1 ); V (in L) is the volume of the chamber minus C. fornicata volume; Δt (in h) is the incubation time and DW (in g) is the soft tissue dry weight of incubated C. fornicata.

In addition, oxygen consumption of the individuals maintained at 10°C during the experiment were assessed on six small and six large individuals at each trial period, following the technique described above. These "controls" tested if mesocosm conditioning induced metabolic stress over time.

The O:N ratio, which corresponds to the amount of oxygen consumed for nitrogen excreted, was calculated from respiration and excretion rates except for the experiments run at 10°C for which rates were too low to obtain significant data. Generally, the O:N ratio is considered a common indicator of the proportion of the three metabolic substrates (carbohydrates, lipids and proteins) used in energy metabolism [START_REF] Mayzaud | O: N atomic ratio as a tool to describe zooplankton metabolism[END_REF].

The atomic ratio of oxygen uptake and excreted nitrogen was calculated following the equation [4] based on [START_REF] Thomsen | Moderate seawater acidification does not elicit long-term metabolic depression in the blue mussel Mytilus edulis[END_REF]:

[4]: O:N = R / E
where R is the respiration rate used as a proxy of the quantity of oxygen consumed by the individual and E, the excretion rate representing the concentration of nitrogen excreted.

Filtration rates

At each trial period, the filtration rate of three small and three large slipper limpets per pCO 2 condition (i.e. 1 individual per size per aquarium) was determined by calculating clearance rates (Coughlan 1969). To do so, 10 and 20 mL of a microalgae mix (C. gracilis, T.

affinis galbana, 1:1) were added to the small and large chambers (same as for metabolic measurements), respectively, using a 10 mL syringe equipped with a thin tube. The mean initial concentration of the mix was 1 200 000 ± 310 000 cell mL -1 . In parallel, control incubations containing only microalgae were carried out to check that phytoplankton cells did not multiply significantly during the incubation. Water from the chambers was sampled with the syringe every 15 min until the water became totally clear (around 2 h). Samples were immediately fixed with 25% glutaraldehyde and frozen at -80°C pending analyses [START_REF] Marie | Flow cytometric analysis of phytoplankton, bacteria and viruses[END_REF]). The number of microalgal cells in each sample was then determined on 200 µL aliquots using flux cytometry (Cell Lab Quanta TM , SC, Beckman Coulter, USA). Filtration rates (F, in mL SW g -1 DW min -1 ) were calculated following equation [5]:

Statistical analyses

All statistical analyses were performed using the R software, version 2.15.0 (R Core Team 2013). Normality and homoscedasticity were checked using Kolmogorov-Smirnov's test and Levene's test, respectively, before each statistical test. Spatial pseudoreplication effect was first tested by considering "aquarium" as a random factor (p-value < 0.05). Then, statistical analyses were simplified to two-way ANOVAs with repeated measurements on the same individual through the four trial periods (different temperature levels) separately for small and large individuals. These analyses were performed for the four physiological rates (respiration, excretion, calcification and filtration) and the O:N ratio, assuming pCO 2 and temperature as fixed factors. Student-Newman-Keuls (SNK) post hoc tests were applied to identify differences among treatments with a confidence level of 95% when ANOVA showed significant results. In parallel, any changes in the respiration rate of individuals constantly maintained at 10°C through time were assessed using a non-parametric Friedman test for repeated measurements, separately for small and large slipper limpets. All results are given as mean ± standard error (SE).

Results

Seawater parameters

The mean temperature and carbonate chemistry parameters among the pCO 2 and temperature conditions are presented in Table 1. Temperature was stable at each trial period with a variability lower than 0.5°C. The different pCO 2 levels remained close to the selected values of 390, 750 and 1400 µatm except at 19°C where all pCO 2 increased from the baseline (+ 100-200 µatm). A T ranged from 2365 ± 2 to 2422 ± 2 µEq kg -1 . Ω Ar decreased by less than 1 only in the 1400 µatm pCO 2 condition. Salinity varied between 34.2 ± 0.1 and 35.1 ± 0.1 among the different pCO 2 and temperature levels with no effect of the temperature increase on salinity.

Respiration, excretion and O:N ratio

Respiration and excretion rates changed significantly with temperature, but not with pCO 2 , in small and large individuals (Figure 1, Table 2). After pooling results for all pCO 2 conditions , mean respiration rates in small C. fornicata increased from 3.78 µmol O 2 g -1 DW

h -1 at 10°C to 11.76 µmol O 2 g -1 DW h -1 at 19°C. In large individuals, the lowest mean respiration rate was recorded at 10°C (4.82 µmol O 2 g -1 DW h -1 ) whereas rates did not differ from 13 to 19°C with a mean value of 11.50 µmol O 2 g -1 DW h -1 . Oxygen fluxes measured on empty shells represented only 4% of the whole organism fluxes measured and decreased only slightly with temperature.

Mean excretion rates calculated among pCO 2 conditions for small C. fornicata individuals gradually increased from 0.15 µmol NH 3 g -1 DW h -1 at 10°C to 1.47 µmol NH 3 g -1 DW h -1 at 19°C. Excretion rates of large individuals showed a parabolic trend with an increase from 10°C (0.16 µmol NH 3 g -1 DW h -1 ) to 16°C (1.34 µmol NH 3 g -1 DW h -1 ) followed by a decrease at 19°C (0.74 µmol NH 3 g -1 DW h -1 ). The ammonium fluxes of empty shells represented less than 1% of the fluxes estimated for whole organisms and were higher at 10°C than at the other temperature levels (rates practically nil).

O:N ratios varied greatly, ranging from 2.86 to 31.68 with a mean value of 12.91 ± 0.56. They varied with pCO 2 or temperature according to size (Table 2, Figure 2). In small C. fornicata individuals, O:N ratios were the highest at 750 µatm and similar between 380 and 1400 µatm. In large individuals, the O:N ratios varied with temperature and were significantly higher at 16°C.

Filtration

Temperature significantly affected filtration rates in both small and large individuals (Figure 1, Table 2). In small C. fornicata, mean filtration rates among pCO 2 were similar between 10 and 16°C (25.50 mL g -1 DW min -1 ), but increased at 19°C (54.30 mL g -1 DW min - 1 ). pCO 2 alone did not affect the filtration rate but the interaction of pCO 2 and temperature was significant (Table 2, p-value < 0.001). At 19°C, filtration rates increased significantly with the increase in pCO 2 . In large individuals, mean filtration rates increased gradually from 10°C

(5.43 mL g -1 DW min -1 ) to 19°C (25.78 mL g -1 DW min -1 ) without any effect of pCO 2 conditions.

Calcification

Calcification rates were significantly affected by pCO 2 increase in both small and large individuals but not by temperature (Figure 1, Table 2). Pooling all temperature levels together, mean calcification rates were similar at pCO 2 of 390 µatm (1.88 and 1.63 µmol CaCO 3 g -1 DW h -1 in small and large individuals, respectively) and 750 µatm (1.02 and 0.60 µmol CaCO 3 g -1 DW h -1 in small and large, respectively), but significantly lower at 1400 µatm pCO 2 (-2.53 and -1.77 µmol CaCO 3 g -1 DW h -1 in small and large individuals, respectively). In the highest pCO 2 condition (1400 µatm), net calcification rates were negative, corresponding to dissolution. Although the interaction between pCO 2 and temperature was not significant for either small or large limpets, pCO 2 response appeared to vary as a function of temperature, particularly at 1400 µatm. In this drastic pCO 2 condition, organisms globally dissolved at 10, 13 and 16°C and calcified (or dissolved less) at 19°C.

Calcification rates decreased with the decrease in the mean aragonite saturation state (Ω Ar ) which correlated with pCO 2 increase (Figure 3). When Ω Ar decreased below the threshold of 1, calcification rates were always negative reflecting a dissolution process. At the 750 and 1400 µatm pCO 2 conditions, Ω Ar was higher at 19°C than at the other temperature levels because the saturation state increases with temperature.

Mesocosm controls

In the aquaria maintained at 10°C throughout the entire experiment, temperature was stable over the first weeks of the experiment and slowly increased from 8 April to the end of the experiment until reaching a mean of 12.4°C between 21 April and 15 June because we had technical problems with the chiller (Table 1). Respiration in small individuals showed high variation over time (Figure 4, white bars) but no time effect was detected (Friedman test, df = 3, χ² = 6.6, p = 0.086, n = 6). Conversely, respiration rates of large individuals increased throughout the experiment (Figure 4, gray bars) with a significant time effect (Friedman test, df = 3, χ² = 9.4, p = 0.024, n = 6).

Discussion

An increase in temperature affected three of the four physiological processes assessed on small and large C. fornicata individuals. In particular, respiration and ammonia excretion rates clearly increased along the tested temperature gradient. In contrast, increases in pCO 2 affected only net calcification of the slipper limpets. Interestingly, the coupled effect of temperature and pCO 2 improved the rate of calcification in the most drastic conditions, particularly in small individuals.

Temperature effect

The respiration (0.6 -34.6 µmol O 2 g -1 DW h -1 ) and excretion rates (-2 -4.4 µmol NH 3 g -1 DW h -1 ) measured at 390 µatm pCO 2 in small and large C. fornicata individuals ranged metabolic rates recorded in situ in the Bay of Brest in northwestern France (4 to 45 µmol O 2 g -1 DW h -1 and 0.5 to 2.3 µmol NH 3 g -1 DW h -1 ; [START_REF] Martin | Respiration, calcification, and excretion of the invasive slipper limpet, Crepidula fornicata L.: Implications for carbon, carbonate, and nitrogen fluxes in affected areas[END_REF]. Both rates increased with temperature in small and large individuals regardless of pCO 2 . Although respiration rates gradually increased with temperature in small C. fornicata individuals, they only increased from 10°C to 13°C, remaining stable at higher temperatures in large C. fornicata. This increase is a common response due to the rate-enhancing effects of temperature on physiological and biochemical reactions in ectotherms [START_REF] Cossins | Estimation of filtering rate from clearance of suspensions[END_REF]. The intensity of respiratory and excretory processes were also dependent of body size. The respiration and excretion rates of small individuals were higher than those of large individuals because the metabolic rate (per unit biomass) decreases with increasing individual size [START_REF] Parsons | Biological oceanographic processes[END_REF][START_REF] Bertalanffy | Metabolic types and growth types[END_REF]. Small individuals have higher energy consumption because they grow faster than the large individuals (Von [START_REF] Bertalanffy | Basic concepts in quantitative biology of metabolism[END_REF]).

The filtration rates measured in small and large C. fornicata fall into the range of maximum feeding rates calculated by [START_REF] Newell | Adjustment of components of energy-balance in gastropod Crepidula fornicata in response to thermal acclimation[END_REF] in C. fornicata between 11 and 20°C (18 to 41 mL g -1 min -1 ; 15°C acclimated individuals). Rates were higher in small than in large individuals because, again, small organisms feed more actively per unit body mass [START_REF] Sylvester | Filtration rates of the invasive pest bivalve Limnoperna fortunei as a function of size and temperature[END_REF]. Filtration rates increased with temperature as previously described in other studies [START_REF] Newell | Adjustment of components of energy-balance in gastropod Crepidula fornicata in response to thermal acclimation[END_REF]. In small individuals, rates were constant between 10 and 16°C and increased only at 19°C while they increased regularly with temperature in the large individuals. In Calyptraeidae, small individuals -i.e. males with low mobility -utilize two feeding strategies: grazing with radula and filtration with gills [START_REF] Navarro | Grazing-filtration as feeding mechanisms in motile specimens of Crepidula fecunda (Gastropoda : Calyptraeidae)[END_REF]. Therefore, small individuals may have supplemented their diet between 10 and 16°C by grazing. For the increased energy requirements at 19°C, small slipper limpets may also increase their filtration rate to meet these supplementary needs. In large sedentary individuals (usually females), filtration is the only feeding mechanism [START_REF] Navarro | Grazing-filtration as feeding mechanisms in motile specimens of Crepidula fecunda (Gastropoda : Calyptraeidae)[END_REF] and filtration rate increases with temperature to help cover the higher energy needs.

Surprisingly, temperature did not affect calcification rates although an increase was expected in response to the increase in metabolism and energy requirements [START_REF] Martin | Respiration, calcification, and excretion of the invasive slipper limpet, Crepidula fornicata L.: Implications for carbon, carbonate, and nitrogen fluxes in affected areas[END_REF]. Because mollusk shell production is an energetically costly process [START_REF] Gazeau | Impacts of ocean acidification on marine shelled molluscs[END_REF], the absence of any change in calcification rates may be due to food limitation during the experiment, especially at elevated temperatures (16 and 19°C). At these temperatures, providing additional food only twice a week may not have been sufficient to support maximal individual shell growth under pH stressful conditions. If food had been provided more regularly and/or in higher quantities, C. fornicata calcification may not be potentially restricted and individuals may have better mitigated the effect of high pCO 2 [START_REF] Thomsen | Food availability outweighs ocean acidification effects in juvenile Mytilus edulis: laboratory and field experiments[END_REF]. Future experiments should include measuring integrated shell growth at each temperature level to determine the food effect more completely.

Mesocosm experiments cannot perfectly reproduce in situ conditions such as natural diet or tidal cycles. This may lead to an increased stress for the organisms grown in these systems [START_REF] Bibby | Effects of ocean acidification on the immune response of the blue mussel Mytilus edulis[END_REF]). The mesocosm effect on organisms was tested through O 2 consumption measurements in individuals kept ca constant temperature throughout the experiment ("controls"). The respiration rates did not change over time in small individuals, whereas the respiration in large individuals increased slightly in correlation with a +2°C temperature increase from the beginning to the end of the experiment, because of technical problems with the chiller. Although food may have constituted a bias, particularly in the oneoff calcification response to temperature, the absence of strong changes in respiration rates in "controls", unexceptional metabolic rates ranging those measured in situ and very low mortality during the experiment (only 8%) all suggest the absence of any acute mesocosm effect on the other physiological traits of C. fornicata.

pCO 2 effect

In contrast to temperature, pCO 2 did not affect C. fornicata respiration or excretion rates regardless of size. Other studies have underlined a lack of any pCO 2 effect on bivalve and limpet respiration [START_REF] Dickinson | Interactive effects of salinity and elevated CO 2 levels on juvenile eastern oysters, Crassostrea virginica[END_REF][START_REF] Fernandez-Reiriz | Tolerance of juvenile Mytilus galloprovincialis to experimental seawater acidification[END_REF][START_REF] Marchant | Short-term exposure to hypercapnia does not compromise feeding, acid-base balance or respiration of Patella vulgata but surprisingly is accompanied by radula damage[END_REF], although some mollusk species exposed to high pCO 2 levels have shown metabolic depression (i.e. decrease in oxygen uptake) to compensate -albeit often drastic -pCO 2 increases [START_REF] Michaelidis | Effects of long-term moderate hypercapnia on acid-base balance and growth rate in marine mussels Mytilus galloprovincialis[END_REF][START_REF] Navarro | Impact of medium-term exposure to elevated pCO 2 levels on the physiological energetics of the mussel Mytilus chilensis[END_REF]. ). Thus, the potential for metabolic resistance of C. fornicata to elevated pCO 2 is likely due to another effective acidosis-buffering system, such as the increase in internal HCO 3 -concentrations [START_REF] Gutowska | Acid-base regulatory ability of the cephalopod (Sepia officinalis) in response to environmental hypercapnia[END_REF][START_REF] Michaelidis | Effects of long-term moderate hypercapnia on acid-base balance and growth rate in marine mussels Mytilus galloprovincialis[END_REF] or higher H + excretion [START_REF] Melzner | Physiological basis for high CO 2 tolerance in marine ectothermic animals: pre-adaptation through lifestyle and ontogeny?[END_REF][START_REF] Pörtner | Synergistic effects of temperature extremes, hypoxia, and increases in CO 2 on marine animals: from Earth history to global change[END_REF].

Similarly to the respiration and excretion processes, filtration rates did not change as a function of pCO 2 in either small or large C. fornicata in our study. Filtration responses with respect to pCO 2 depend most of the time on the presence of metabolic depression [START_REF] Fernandez-Reiriz | Physiological energetics of juvenile clams Ruditapes decussatus in a high CO 2 coastal ocean[END_REF]Liu and He 2012;[START_REF] Navarro | Impact of medium-term exposure to elevated pCO 2 levels on the physiological energetics of the mussel Mytilus chilensis[END_REF]. The absence of variation in filtration rates at the different pCO 2 levels indicates that the quantity of food ingested by C.

fornicata did not vary either. Food is known to interact with other stressors, such as pCO 2 , and significantly influence metabolic responses [START_REF] Melzner | Food supply and seawater pCO 2 impact calcification and internal shell dissolution in the blue mussel Mytilus edulis[END_REF][START_REF] Pansch | Habitat traits and food availability determine the response of marine invertebrates to ocean acidification[END_REF].

Quality or quantity changes in the diet can even worsen the condition of invertebrates [START_REF] Berge | Effects of increased sea water concentrations of CO 2 on growth of the bivalve Mytilus edulis L[END_REF][START_REF] Vargas | CO 2 -driven ocean acidification reduces larval feeding efficiency and change food selectivity in the mollusk Concholepas concholepas[END_REF]. Although our microalgal mix did not perfectly match the natural diet of C. fornicata [START_REF] Barillé | Feeding responses of the gastropod Crepidula fornicata to changes in seston concentration[END_REF][START_REF] Decottignies | Exploitation of natural food sources by two sympatric, invasive suspension-feeders: Crassostrea gigas and Crepidula fornicata[END_REF]), the diatoms and dinoflagellate microalgae provided in the experiment correspond to the main taxa present in Morlaix Bay, assuming a nutritional quality close to the natural diet. However, we cannot assure that the quantity of food was not a limiting factor in our experiment. To be sure that microalgae supplied would not represent a bias, the slipper limpets should be fed ad libitum which represented a technical issue on a 6 month experiment.

In our study, net calcification was similar between 390 and 750 µatm pCO 2 and strongly decreased at 1400 µatm pCO 2 regardless of size, which is a common response in mollusks [START_REF] Beniash | Elevated level of carbon dioxide affects metabolism and shell formation in oysters Crassostrea virginica[END_REF][START_REF] Melatunan | Effects of ocean acidification and elevated temperature on shell plasticity and its energetic basis in an intertidal gastropod[END_REF][START_REF] Range | Calcification, growth and mortality of juvenile clams Ruditapes decussatus under increased pCO 2 and reduced pH: Variable responses to ocean acidification at local scales?[END_REF]. This pattern contrasts with that reported in [START_REF] Ries | Marine calcifiers exhibit mixed responses to CO 2 -induced ocean acidification[END_REF], with a parabolic response in C. fornicata calcification with the highest rates observed at 600 µatm pCO 2 . The stability of calcification rate at 750 µatm pCO 2 (compared to 390 µatm pCO 2 ) may be due to the biological control of the calcification process and/or the presence of the periostracum, the organic layer covering the crystalline layers of the shell. This organic layer has been predicted to play a great role in maintaining shell integrity of mollusks in elevated pCO 2 conditions [START_REF] Ries | Marine calcifiers exhibit mixed responses to CO 2 -induced ocean acidification[END_REF]) and to protect them from dissolution in CaCO 3 -undersaturated waters [START_REF] Huning | Impacts of seawater acidification on mantle gene expression patterns of the Baltic Sea blue mussel: implications for shell formation and energy metabolism[END_REF].

Moreover, mollusks may be able to maintain extrapallial fluid in chemical conditions favoring CaCO 3 precipitation at the calcification site, even if external seawater pCO 2 is high [START_REF] Hiebenthal | Effects of seawater pCO 2 and temperature on shell growth, shell stability, condition and cellular stress of Western Baltic Sea Mytilus edulis (L.) and Arctica islandica (L.)[END_REF]. Regulation of enzymes involved in the calcification process, such as chitinase [START_REF] Cummings | Ocean acidification at high latitudes: potential effects on functioning of the antarctic bivalve Laternula elliptica[END_REF] or carbonic anhydrase [START_REF] Ivanina | Interactive effects of elevated temperature and CO 2 levels on energy metabolism and biomineralization of marine bivalves Crassostrea virginica and Mercenaria mercenaria[END_REF], may also help maintain calcification in high pCO 2 conditions . In our study, at 1400 µatm, calcification rates dropped, perhaps due to physiological changes in the internal acid-base balance affecting shell deposition [START_REF] Waldbusser | Biocalcification in the eastern oyster (Crassostrea virginica) in relation to long-term trends in Chesapeake Bay pH[END_REF] or to an eroded and/or damaged periostracum (pers. obs.).

Degradation of this protective layer may lead to higher vulnerability of the shell to external dissolution processes [START_REF] Range | Seawater acidification by CO 2 in a coastal lagoon environment: effects on life history traits of juvenile mussels Mytilus galloprovincialis[END_REF][START_REF] Ries | Marine calcifiers exhibit mixed responses to CO 2 -induced ocean acidification[END_REF]), which occurs not only in dead shells but also in live animals [START_REF] Harper | Are calcitic layers an effective adaptation against shell dissolution in the Bivalvia?[END_REF]. Furthermore, chemical dissolution increased with an increase in pCO 2 and a correlated decrease in Ω Ar ; the combined effect led to a decrease in net calcification rates observed in both small and large C. fornicata individuals at high pCO 2 conditions.

Combined effects of temperature and pCO 2

In the range of pCO 2 and temperatures tested, the interaction of these two variables had no negative effect on C. fornicata respiration and excretion rates. As a eurythermal species even coping with high temperature in some bays during summer (e.g. Bassin d'Arcachon in southwestern France; De [START_REF] De Montaudouin | Does the slipper limpet (Crepidula fornicata, L.) impair oyster growth and zoobenthos biodiversity? A revisited hypothesis[END_REF], C. fornicata can have an optimal temperature of 19°C or higher [START_REF] Diederich | Thermal tolerance of Crepidula fornicata (Gastropoda) life history stages from intertidal and subtidal subpopulations[END_REF][START_REF] Noisette | Metabolic responses to temperature stress under elevated pCO 2 in the slipper limpet Crepidula fornicata[END_REF].

Thus, 19°C may not constitute a real thermal stress and not transgress the metabolic optimal threshold for this species. Increase in temperature is predicted to enhance sensitivity to high pCO 2 levels beyond the optimal temperature of the species and close to its upper limit of thermal tolerance [START_REF] Pörtner | Physiology and climate change[END_REF]. However, at the cold side of a species optimal temperature, warming can increase resilience to ocean acidification [START_REF] Gianguzza | Temperature modulates the response of the thermophilous sea urchin Arbacia lixula early life stages to CO 2 -driven acidification[END_REF].

Therefore, an increase in temperature may actually improve tolerance to pCO 2 increases in C.

fornicata.

Calcification rates of both small and large C. fornicata showed a positive trend with temperature in the most drastic pCO 2 conditions (1400 µatm). Temperature-mediated increases in metabolism and feeding rates may potentially offset reductions in calcification rates caused by high pCO 2 conditions [START_REF] Melzner | Food supply and seawater pCO 2 impact calcification and internal shell dissolution in the blue mussel Mytilus edulis[END_REF][START_REF] Thomsen | Food availability outweighs ocean acidification effects in juvenile Mytilus edulis: laboratory and field experiments[END_REF]. In addition to this physiological effect, moderate warming can mediate the effects of ocean acidification by the chemical effect on seawater chemistry [START_REF] Kroeker | The role of temperature in determining species' vulnerability to ocean acidification: a case study using Mytilus galloprovincialis[END_REF]. Temperature affects CO 2 solubility in seawater as well as the equilibrium coefficients governing carbonate chemistry [START_REF] Millero | The marine inorganic carbon cycle[END_REF]. As shown in our study, the saturation state of aragonite was greater in warmer water than in colder water for a given pCO 2 , thereby enhancing calcification and reducing the dissolution processes in the high pCO 2 conditions. These results highlight the importance of considering the physiological and geochemical interactions between temperature and carbonate chemistry when interpreting species' vulnerability to ocean acidification. A better understanding of how warming influences species' responses to high pCO 2 levels through both direct (e.g. increases in metabolic rates) and indirect pathways (e.g. changes in carbonate chemistry) is thus necessary.

Conclusion

A trade-off between stressors may affect the physiology of organisms in an unexpected way [START_REF] Kroeker | The role of temperature in determining species' vulnerability to ocean acidification: a case study using Mytilus galloprovincialis[END_REF]). In our case, C. fornicata appeared to be able to tolerate slight increases in pCO 2 but its calcification was affected by drastic conditions with a positive effect of temperature, thereby mitigating any ocean acidification effects. This outcome highlights the need of multistressor studies to understand the future of marine species in a context of climate change in which different physico-chemical factors vary in different ways.

Furthermore, our results indicate that some species can be highly tolerant to future pCO 2 increases. C. fornicata tolerance likely stems from mechanisms that allow it to acclimate or adapt to environmental fluctuations in its habitat [START_REF] Clark | Identification of molecular and physiological responses to chronic environmental challenge in an invasive species: the Pacific oyster, Crassostrea gigas[END_REF], because species living in environments with large abiotic variations tend to have high phenotypic plasticity, allowing them to survive in stressful conditions [START_REF] Somero | The physiology of climate change: how potentials for acclimatization and genetic adaptation will determine "winners" and "losers[END_REF]. This capacity to resist decreases in pH may reinforce the ecological role of C. fornicata populations in the ecosystems in which they are established, even under projected future conditions anticipated due to climate change.

Tables Table 1: Mean seawater temperature and parameters of the carbonate system in each pCO 2 treatment (3 aquaria per treatment) and at each trial period (i.e. temperature level). The pH T (pH on the total scale) and total alkalinity (A T ) were measured whereas the other parameters were calculated. Mean A T calculated for each trial period (n = 3 for controls 10°C and 19 < n < 30 for other condition pCO 2 conditions) and pCO 2 condition was used for the calculations.

pCO 2 , CO 2 partial pressure; DIC, dissolved inorganic carbon and Ω Ar , saturation state of seawater with respect to aragonite.

C

  . fornicata stacks were collected by SCUBA divers on 30 November 2011, in Morlaix Bay (northwestern Brittany, France), at the "Barre des Flots" site (3°53.015'W; 48°40.015'N) at approximately 11 m depth. No temporal series of abiotic parameters were available for this exact location. However, variations in the physico-chemical parameters (surface measurements) at a station (called Estacade), located approximately 10 km from the Barre des Flots site, were obtained from the Service d'Observation des Milieux LITorraux (SOMLIT) between 2010 and 2013, with a sampling step of 15 days. Between October 2010 and March 2013, temperature varied between 8.1°C (January 2011) and 16.5°C (August 2011) with mean values (± SE) of 10.1 ± 0.2°C in winter, 12.7 ± 0.4°C in spring and 15.8 ± 0.02°C in summer.

  respectively. Net calcification (in µmol CaCO 2 g -1 DW h -1 ; equation [3]) was estimated using the alkalinity anomaly technique[START_REF] Smith | Carbon-dioxide and metabolism in marine environments[END_REF] based on a decrease in A T by 2 equivalents for each mole of CaCO 3 precipitated (Wolf-Gladrow et al. 2007). As ammonium production increases alkalinity in a mole-per-mole ratio (Wolf-Gladrow et al. 2007), the alkalinity variation was corrected by the ammonium flux to calculate CaCO 3 fluxes.

  where[Ci] and[Cf] (in cell mL -1 ) were respectively the initial and final cell concentrations in the chamber water; V (in L) is the volume of the chamber minus individual C. fornicata volume; Δt (in h) is the incubation time and DW (in g) is the tissue dry weight of the individual incubated.

  Responses of ammonia excretion to high pCO 2 in mollusks are also specific: increase in ammonia excretion can occur under elevated pCO 2[START_REF] Fernandez-Reiriz | Physiological energetics of juvenile clams Ruditapes decussatus in a high CO 2 coastal ocean[END_REF][START_REF] Langenbuch | Changes in metabolic rate and N excretion in the marine invertebrate Sipunculus nudus under conditions of environmental hypercapnia identifying effective acid-base variables[END_REF][START_REF] Range | Impacts of CO 2 -induced seawater acidification on coastal Mediterranean bivalves and interactions with other climatic stressors[END_REF]) while some bivalves show opposing trends (Liu and He 2012;[START_REF] Navarro | Impact of medium-term exposure to elevated pCO 2 levels on the physiological energetics of the mussel Mytilus chilensis[END_REF]). The increase in ammonia excretion under increased pCO 2 conditions can be interpreted as an internal pH regulatory mechanism, sometimes based on protein catabolism[START_REF] Fernandez-Reiriz | Tolerance of juvenile Mytilus galloprovincialis to experimental seawater acidification[END_REF][START_REF] Thomsen | Moderate seawater acidification does not elicit long-term metabolic depression in the blue mussel Mytilus edulis[END_REF]. In our study, neither change in excretion rates nor in O:N ratios calculated were detected between the 390 and 1400 µatm conditions. This similarity indicates that potential intracellular pH regulation of C. fornicata was not induced by enhancing protein metabolism[START_REF] Fernandez-Reiriz | Tolerance of juvenile Mytilus galloprovincialis to experimental seawater acidification[END_REF]

FiguresFigure 1 :

 1 Figures

Figure 2 :

 2 Figure 2: O:N ratios for the three pCO 2 treatments (shaded in grey) at 13, 16 and 19°C for small and

Figure 3 :

 3 Figure 3: Mean net calcification rates as function of aragonite saturation state, in the three pCO 2

Figure 4 :

 4 Figure 4: Respiration rates in the control treatment (10°C) for the different trial periods (i.e. temperature

  Figure 1 :

Figure 3 :

 3 Figure 3:

Table 2 :

 2 Summary of two-way repeated measurements ANOVAs followed by Student-Newman-Keuls post hoc tests testing the effect of pCO 2 , temperature and their interaction on Crepidula fornicata physiology. Numbers in bold indicate significant p-values and values with different letters are significantly different at p < 0.05.

			Temperature pH T		pCO 2		A T		DIC		Ω Ar
			(°C)				(µatm)		(µEq kg -1 )		(µmol C kg -1 )
		n	Mean	SE	Mean	SE	Mean	SE Mean	SE Mean	SE Mean	SE
	1 st trial period (10°C)
	390 µatm	23	9.7	0.2	8.14	0.01 322	7	2365	2	2138	4	2.47	0.04
	750 µatm	23	9.8	0.2	7.82	0.01 729	19	2368	2	2270	4	1.33	0.03
	1400 µatm	23	9.5	0.2	7.55	0.03 1486	75	2376	2	2366	11	0.78	0.08
	control 10°C	40	9.2	0.2	8.19	0.02 288	17	2370	3	2115	8	2.73	0.07
	2 nd trial period (13°C)
	390 µatm	27	12.9	0.2	8.12	0.02 356	25	2418	2	2167	8	2.76	0.07
	750 µatm	27	13.0	0.1	7.81	0.01 781	20	2416	2	2304	3	1.48	0.03
	1400 µatm	27	12.8	0.1	7.53	0.01 1557	43	2422	2	2405	4	0.82	0.02
	control 10°C	41	11.0	0.1	8.18	0.01 297	12	2419	2	2152	5	2.88	0.05
	3 rd trial period (16°C)
	390 µatm	28	15.9	0.1	8.08	0.01 376	10	2379	5	2126	5	2.80	0.05
	750 µatm	28	16.1	0.1	7.82	0.00 748	8	2369	5	2238	2	1.66	0.01
	1400 µatm	28	16.0	0.1	7.55	0.01 1492	19	2380	5	2345	2	0.94	0.01
	control 10°C	42	11.4	0.1	8.23	0.01 253	6	2376	4	2083	5	3.13	0.05

Acknowledgments

The authors thank the "Marine Operations and Services Department" at the Station Biologique de Roscoff for the underwater sampling. We also thank the "Multicellular Marine Models" staff, and especially Ronan Garnier, for providing microalgae and their help for building the aquarium system. In addition, we are grateful to SOMLIT (Service d'Observation en Milieu LITtoral, INSU-CNRS) for providing the abiotic parameter datasets. We are also grateful to Roseline Edern for her help with cytometry flux analysis. We really appreciated the editor and reviewers' helpful and constructive comments which greatly improved this manuscript. This work was supported by the CALCAO project, which received funding from the Brittany Regional Council, and contributed to the ''European Project on Ocean


Anca Tefana Florea

Electrochemical affinity sensors for biomedical, food and environmental applications

Dr. Christopher A. Mills Directeurs de these: Prof. Abdelhamid Errachid-El-Salhi Dr. Nadia Zine "I am not young enough to know everything"

Oscar Wilde Dedicated to my family I would like to express my deepest gratitude to my supervisors Prof. Dr. Robert Sãndulescu and Dr. Nicole Jaffrezic Renault for the opportunity to work in their excellent research groups. Their constant support, valuable time, constructive advices and positive attitude and confidence towards me have been wholehearted and made me believe in myself as a researcher.

I owe a big debt of gratitude to Dr. Cecilia Cristea for her friendship, continuous support, confidence in me and precious advices in both my work and life.

I am greatly appreciative to prof. Dr. Giovanna Marrazza from Univeristy of Florence for her guidance and support and to dr. Andrea Ravalli for his valuable advices and help.

I also thank the comitee for the advices and help in improving my thesis.

I owe special thanks to all my collegues and friends from the Department of Analytical Chemistry Cluj, from ISA Lyon and "Ugo Shiff" Department Florence, who contributed not only to work but also to making it fun. There are countless people that have been helpful throughout time and with whom I shared great experiences, so I apologize in advance for forgetting naming some of them. Thanks to Bogdan, Andreea, Mihaela, Lumi, Bibi, Oana, whose advices, friendship and support was essential to my progress. I thank Coco, Rebecca and Diego for their friendship and cheerfulness and for making Florence home for me. I am greatly thankful to Mamis for the support, friendship and good spirit all along the way. I am grateful to fam. Lacroix for their hospitality during my staying in Lyon. I would like to thank to all doctorants in ISA Lyon, Elena, Sylvia, Nedjla, Mohamed, Vika, Katya etc, for the memorable experiences in and outside work in Lyon. To ZhenZhong, Huy and Madiha for their collaboration and help regarding MIP sensors. Special thanks to Alvaro for his friendship and encouragement. I also thank Eli for her friendship, cheerfulness and inspiration.

I would like to thank all my friends for their support and understanding.

Finnaly, I am deeply thankful to my parents, my brother, my sister in law and to my whole family for their endless love, encouragement and support. Words truly can not express how lucky I feel to have had them every step along the way.

STATE OF THE ART 1. General aspects of affinity sensors

Types of biorecognition element

Antibodies

Applications of electrochemical immunosensors for cancer biomarkers detection

Table I.

Table II.

Gold nanoparticles

Classification

Non covalent

Covalent:

Preparation of molecularly imprinted polymers

Molecularly imprinted polymers in sensors

Immobilization of primary antibody 1.2.2.2. Blocking of free binding sites 1.2.2.3. Capturing the MUC1 protein 1.2.2.4. Binding of secondary antibody and third antibody labelled with alkaline phosphatase

Protocol of aptamer based bioassay

Immobilization of the primary aptamer 1.2.3.2. Blocking of free binding sites 1.2.3.3. Capturing the MUC1 protein 1.2.3.4. Binding of secondary aptamer and labelling with streptavidin alkaline phosphatase

Analysis of human serum samples by the aptamer based bioassay

Electrochemical measurements with screen printed arrays 1.3. Results and discussion

Optimization of experimental parameters of aptamer based bioassay

Table IV.

Optimization of experimental parameters for antibody based bioassay

Selectivity studies of the bioassay

Serum samples analysis

Results and discussion

Principle of electrochemical aptamer based biosensor

Optimization of experimental parameters

Characterization of AuNPs graphite SPE based aptasensor by EIS

Materials and methods

Chemicals and instrumentation p

Experimental techniques used for fabrication and characterization of the molecularly imprinted sensors

Steps in the construction of the MIP sensors

Preparation of the functionalized AuNPs p

Electrodeposition of molecularly imprinting films onto the gold electrodes

Electrochemical measurements on MIP and NIP modified electrodes

Surface characterization by atomic force microscopy (AFM)

Analysis of real samples

Results and discussion

Electrochemical preparation of MIP and NIP based sensors

Electrochemical and morphological characterization of MIP and NIP films

Optimization of experimental conditions

Optimization of experimental parameters for GMT imprinted sensor

Optimization of experimental parameters for TC imprinted sensor

Optimization of experimental parameters for Gly imprinted films

Anca tefana Florea

Table X.

Reproducibility and reusability of the imprinted sensor

Tabel XI.

Fig

  Fig. 1.

Fig

  Fig. 4.

  Fig. 5.

Fig

  Fig. 6.

Fig

  Fig. 7.

Fig

  Fig. 8.

Fig

  Fig. 10.

Fig

  Fig. 11.

Fig

  Fig. 12.

Fig

  Fig. 13.

Fig. 15

 15 Fig. 15

Fig

  Fig. 16.

  Fig. 17.

Fig. 21

 21 Fig. 21

Fig

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  Fig. 23.

  Fig. 24.

  Fig. 28.

Fig

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Fig

  Fig. 32.

  Fig 34.

  Fig. 35.

Capteurs électrochimiques d'affinité applique dans l'analyse biomédicale, sécurité alimentaire et environnementale Mots clés INTRODUCTION STATE OF THE ART

	1.	General aspects of affinity sensors
	1.2. General classification
	1.3. Voltamperometric sensors
	1.4. Impedimetric sensors
	1.5. Single use sensors
	1.6. Immobilization of the bioelements
	2.	Immunosensors and aptasensors
	2.1. Definitions
	2.2. General classification
	2.3. Types of format
	2.3.1. Competitive immunosensors
	2.3.2. Non competitive immunosensors
	2.4. Types of biorecognition element
	2.4.1. Antibodies
	2.4.2. Aptamers
	2.5. Immobilization of antibodies/aptamers
	2.5.1. Physical adsorption
	2.5.2. Entrapment
	2.5.3. Covalent Binding
	2.5.4. Oriented Binding
	2.6.

Applications of electrochemical immunosensors for cancer biomarkers detection 3. Nanomaterials for the design of amperometric sensors

		4.1. Definition and general principle
		4.2. Classification
		Preparation of molecularly imprinted polymer
		Molecularly imprinted polymers in sensors
		3.1. Magnetic beads
		3.2. Gold nanoparticles
	4.	Molecularly imprinted polymers

PERSONAL CONTRIBUTIONS 1. Optimized bioassays for Mucin1 detection in serum samples

	1.1. Introduction
	1.2. Materials and Methods
	1.2.1. Chemicals and instrumentation
	1.2.2. Protocol of antibody based bioassay
	1.2.3. Protocol of aptamer based bioassay
	1.2.3.1. Immobilization of the primary aptamer
	1.2.3.2. Blocking of free binding sites
	1.2.3.3. Capturing the MUC1 protein
	1.2.

3.4. Binding of secondary aptamer and labelling with streptavidin alkaline phosphatase 1.2.4. Analysis of human serum samples by the aptamer based bioassay 1.3. Results and discussion 1.3.1. Optimization of experimental parameters of aptamer based bioassay 1.3.2. Optimization of experimental parameters for antibody based bioassay 1.3.3. Detection of MUC1 in buffered solutions 1.3.4. Selectivity studies of the bioassay 1.3.5. Serum samples analysis 1.4. Conclusions 2.

Label free MUC1 aptasensor based on electrodeposition of gold nanoparticles on screen printed electrodes

	2.3.3. Characterization of AuNPs graphite SPE based aptasensor by EIS
	2.3.4. Quantitative detection of MUC1 protein
	2.4. Conclusion
	3.
	2.1. Introduction
	2.2. Experimental
	2.2.1. Chemicals and instrumentation
	2.2.2. Preparation of AuNPs modified SPE
	2.2.3. Immobilization of the aptamer and interaction with the protein
	2.3. Results and discussion
	2.3.1. Principle of electrochemical aptamer based biosensor
	2.3.2. Optimization of experimental parameters

MIP based sensors for the detection of various analytes of environmental, biomedical and food safety interest

	4. General conclusions LR ABBREVIATIONS mAb
	5. Originality of the thesis MUC1
	Ab MUC16 REFERENCES MUC4	INTRODUCTION
	Ag MIP 3.1. Introduction Apt MMOF 3.2. Materials and methods AP mRNA 3.2.1. Chemicals and instrumentation A (n) MW 3.2.2. Experimental techniques used for fabrication and characterization of the molecularly imprinted sensors 3.2.2.2. Preparation of the functionalized AuNPs pAb cDNA 3.2.2.1. Steps in the construction of the MIP sensors BSA ODN CE NIP via p
	3.2.2.3. Electrodeposition of molecularly imprinting films onto the gold CDR PATP Para
	electrodes CNT PCR
	3.2.2.4. Electrochemical measurements on MIP and NIP modified rAb CV
	electrodes DEA RE
	3.2.2.5. Surface characterization by atomic force microscopy (AFM) DNA RNA
	3.2.2.6. Analysis of real samples DNT SAMs
	3.3. Results and discussion DPV SCE
	3.3.1. Electrochemical preparation of MIP and NIP based sensors DPSV SEM
	3.3.2. Electrochemical and morphological characterization of MIP and NIP dT SPE E2 SPCs 3.3.3. Electrochemical behaviour and recognition ability of MIP films towards EDTA SPR the target analytes films EIS SWV 3.3.4. Optimization of experimental conditions ELISA TC 3.3.4.1. Optimization of experimental parameters for GMT imprinted sensor GC TNT
	3.3.4.2. Optimization of experimental parameters for TC imprinted sensor GC/MS TRIS Tris
	GCE WE
	3.3.4.3. Optimization of experimental parameters for Gly imprinted films Gly WHO
	GMT QD
	3.3.5. Analytical performance of the imprinted and non imprinted polymer HPLC QCM
	Ig
	3.3.6. Analyses of real samples LBL
	3.3.7. Reproducibility and reusability of the imprinted sensor LOD
	3.3.8. Stability studies LOQ
	3.4. Conclusions LSV

Table V .

 V

Table V . Chapitre 2 Contexte 2. Label free MUC1 aptasensor based on electrodeposition of gold nanoparticles on screen printed electrodes 2.1. Introduction . 2.2. Experimental 2.2.1. Chemicals and instrumentation 2.2.2. Preparation of AuNPs modified SPE 2.2.3. Immobilization of the aptamer and interaction with the protein

 V

Table VIII Table IX .

 VIIIIX

	Anca tefana Florea
	o

Acknowledgements

There are more people that I can count who helped making this work possible. I owe a huge debt of gratitude to my supervisors, my coworkers, my family and my friends.