Chloe Carbonne
email: chloe.carbonne@imev-mer.fr

Nuria Teixidó

Billy Moore

Alice Mirasole

Thomas Guttierez

Jean-Pierre Gattuso

Steeve Comeau

Two temperate corals are tolerant to low pH regardless of previous exposure to natural CO2 vents

Keywords: ocean acidification, CO2 vents, temperate, Mediterranean, coral, Cladocora caespitosa, Astroides calycularis, calcification, tolerance, low pH

Ocean acidification is perceived to be a major threat for many calcifying organisms, including scleractinian corals. Here we investigate (1) whether past exposure to low pH environments associated with CO2 vents could increase corals tolerance to low pH and (2) whether zooxanthellate corals are more tolerant to low pH than azooxanthellate corals. To test these hypotheses, two Mediterranean colonial corals Cladocora caespitosa (zooxanthellate) and Astroides calycularis (azooxanthellate) were collected from CO2 vents and reference sites and incubated in the laboratory under present-day (pH on the total scale, pHT 8.07) and low pH conditions (pHT 7.70). Rates of net calcification, dark respiration and photosynthesis were monitored during a six-month experiment. Monthly net calcification was assessed every 27 to 35 d using the buoyant weight technique, whereas light and dark net calcification was estimated using the alkalinity anomaly technique during 1 h incubations. Neither species showed any change in net calcification rates, respiration, and photosynthesis regardless of their environmental history, pH treatment and trophic strategy. Our results indicate that C. caespitosa and A. calycularis could tolerate future ocean acidification conditions for at least 6 months. These results will aid in predicting species' future responses to ocean acidification, and thus improve the management and conservation of Mediterranean corals.

Introduction

Ocean acidification describes the shift in carbonate chemistry caused by the uptake of anthropogenic CO2 by the ocean [START_REF] Caldeira | Anthropogenic carbon and ocean pH[END_REF]Wickett 2003, Gruber et al. 2019). It causes an increase in dissolved inorganic carbon and bicarbonate ion concentrations, along with a concurrent decrease in pH, carbonate ion concentration and its associated saturation state (Ω; [START_REF] Orr | Anthropogenic ocean acidification over the twenty-first century and its impact on calcifying organisms[END_REF]. Calcifying species are perceived to be particularly vulnerable to ocean acidification (Kroeker et al. 2013a). For example, corals are particularly sensitive to changes in carbonate chemistry as their skeleton is made of aragonite, a metastable form of calcium carbonate that is less stable than calcite (Erez et al. 2011, Foster and[START_REF] Foster | Skeletal mineralogy of coral recruits under high temperature and pCO2[END_REF]. As a result, coral calcification is expected to decrease as seawater acidity increases [START_REF] Gattuso | Photosynthesis and calcification at cellular, organismal and community levels in coral reefs: A review on interactions and control by carbonate chemistry[END_REF].

Coral calcification decreases by on average 22% under pH values expected by the end of the century under the high-CO2 emissions scenario (RCP 8.5 from AR5 of ICPP; [START_REF] Chan | Sensitivity of coral calcification to ocean acidification: a meta-analysis[END_REF]. However, a range of contradictory species-and experience-specific responses have been described (e.g. [START_REF] Kornder | Thresholds and drivers of coral calcification responses to climate change[END_REF], with some species such as massive Porites spp. being tolerant to ocean acidification [START_REF] Fabricius | Losers and winners in coral reefs acclimatized to elevated carbon dioxide concentrations[END_REF]. This resistance to ocean acidification was also described in several temperate and cold-water corals (e.g. [START_REF] Rodolfo-Metalpa | Response of the temperate coral Cladocora caespitosa to mid-and long-term exposure to pCO2 and temperature levels projected for the year 2100 AD[END_REF]. Such tolerance is seen in many studied parameters such as net calcification rate, respiration, photosynthetic physiology, calcifying fluid pH (McCulloch et al. 2012a[START_REF] Wang | Resilience of the temperate coral Oculina arbuscula to ocean acidification extends to the physiological level[END_REF]. According to [START_REF] Varnerin | Recruits of the temperate coral Oculina arbuscula mimic adults in their resilience to ocean acidification[END_REF] even juveniles from the temperate Oculina arbuscula were only slightly affected by ocean acidification.

A central question when assessing the future of corals in a high-CO2 world is whether they have the capacity to acclimatize and further adapt to ocean acidification. This question remains unanswered as corals are generally long-lived organisms for which it is extremely challenging to study acclimatization and adaptation processes over several generations. Acclimatization refers to reversible phenotypic changes limited by genotype, occurring under naturally varying conditions by a physiological readjustment of the organism's tolerance levels [START_REF] Edmunds | Acclimatization in tropical reef corals[END_REF]. In contrast, adaptation refers to changes in the genetic composition of a population by natural selection, in which traits will be passed on to the next generation [START_REF] Savolainen | Ecological genomics of local adaptation[END_REF]. In a recent study, [START_REF] Cornwall | A coralline alga gains tolerance to ocean acidification over multiple generations of exposure[END_REF] conducted a oneyear experiment showing that coralline algae have the capacity to acclimatize to low pH after seven generations of exposure. Replicating such a study on corals is virtually impossible as they are long-lived organisms which only reach sexual maturity after several years. CO2 vents therefore offer a unique opportunity to investigate the response of corals to ocean acidification over long periods of time. In such systems, volcanic CO2 bubbles from the seafloor which acidifies the surrounding seawater and creates an environment, with carbonate chemistry conditions that mimic those expected in the future [START_REF] Hall-Spencer | Volcanic carbon dioxide vents show ecosystem effects of ocean acidification[END_REF][START_REF] Camp | The future of coral reefs subject to rapid climate change: lessons from natural extreme environments[END_REF][START_REF] González-Delgado | The importance of natural acidified systems in the study of ocean acidification: what have we learned?[END_REF]. By investigating individuals, species, and communities along transects radiating from the sources of CO2, it is possible to substitute time for space. It must be pointed out, however, that CO2 vents are imperfect windows into the future because they generally do not mimic other climate-related changes such as ocean warming. Furthermore, it is not possible to disentangle any changes in the mean versus variability of seawater pH [START_REF] Hall-Spencer | Volcanic carbon dioxide vents show ecosystem effects of ocean acidification[END_REF]. Overall, these systems exhibit significant decreases in trophic complexity and biodiversity, as well as a major decrease in the abundance of calcifying organisms (Kroeker et al. 2013b[START_REF] Linares | Persistent natural acidification drives major distribution shifts in marine benthic ecosystems[END_REF][START_REF] Teixidó | Functional biodiversity loss along natural CO2 gradients[END_REF]. Corals living in the vicinity of CO2 vents have been exposed to low and high variability pH conditions for an extended period of time (years to decades). They are therefore ideal models to investigate acclimatization and adaptation to ocean acidification [START_REF] Fantazzini | Gains and losses of coral skeletal porosity changes with ocean acidification acclimation[END_REF][START_REF] Foo | The carbon dioxide vents of Ischia, Italy, a natural system to assess impacts of ocean acidification on marine ecosystems: an overview of research and comparisons with other vent systems[END_REF]. Populations or individuals living in these variable and more extreme environments are hypothesized to be more tolerant to suboptimal environmental conditions such as acidification (e.g. [START_REF] Cornwall | Resistance of corals and coralline algae to ocean acidification: Physiological control of calcification under natural pH variability[END_REF]).

CO2 vents have been used for transplant experiments between acidified and nearby reference sites with ambient pH and no vent activity. For example, a decline in calcification was described in the zooxanthellate corals Cladocora caespitosa and Balanophyllia europaea transplanted to a CO2 vent site in Ischia [START_REF] Rodolfo-Metalpa | Coral and mollusc resistance to ocean acidification adversely affected by warming[END_REF]. The skeletal growth of Porites astreoides living in a natural pH gradient showed no acclimatization to ocean acidification despite their life-long exposure to low pH [START_REF] Crook | Reduced calcification and lack of acclimatization by coral colonies growing in areas of persistent natural acidification[END_REF]. Few laboratory studies have focused on temperate scleractinian corals from natural CO2 vents despite the relevance of studying the tolerance of temperate coral populations to ocean acidification [START_REF] Trotter | Quantifying the pH "vital effect" in the temperate zooxanthellate coral Cladocora caespitosa: Validation of the boron seawater pH proxy[END_REF][START_REF] Teixidó | Ocean acidification causes variable trait-shifts in a coral species[END_REF]. Such studies are important for understanding and elucidating how these species will respond to the suboptimal pH conditions projected for the end of this century.

The symbiosis between scleractinian corals and endosymbiotic algae, commonly referred to as zooxanthellae, has been studied thoroughly [START_REF] Davy | Cell biology of cnidarian-dinoflagellate symbiosis[END_REF]. Photosynthetic products from zooxanthellae contribute significantly to the energy budget of the scleractinian host [START_REF] Allemand | Coral Calcification[END_REF][START_REF] Muller-Parker | Interactions between corals and their symbiotic algae[END_REF] and can play an important role in calcification [START_REF] Erez | Coral calcification under ocean acidification and global change[END_REF][START_REF] Inoue | A simple role of coral-algal symbiosis in coral calcification based on multiple geochemical tracers[END_REF]). However, not all corals harbor zooxanthellae (azooxanthellate corals). [START_REF] Gibbin | Intracellular pH and its response to CO2-driven seawater acidification in symbiotic versus non-symbiotic coral cells[END_REF] demonstrated that zooxanthellae regulate intracellular pH under acidified conditions, whereas azooxanthellate corals can suffer from reduced intracellular pH which can lead to intracellular acidosis. It can thus be expected that photosynthesis may enhance calcification by (1) providing energy for this energetically costly process and (2) by increasing pH at the site of calcification [START_REF] Allemand | Coral Calcification[END_REF]. In a transplant experiment in the CO2 vents of Panarea, [START_REF] Prada | Ocean warming and acidification synergistically increase coral mortality[END_REF] showed that the calcification of two azooxanthellate corals Leptopsammia pruvoti and Astroides calycularis decreased at low pH, whereas calcification of the zooxanthellate coral B. europaea was unaffected. A similar result was observed in pH-controlled aquaria by [START_REF] Ohki | Calcification responses of symbiotic and aposymbiotic corals to near-future levels of ocean acidification[END_REF] as polyps of Acropora digitifera with symbionts showed higher calcification rates than polyps without symbionts under low pH, suggesting that symbionts confer tolerance to ocean acidification. However, in a study led by McCulloch et al. (2012b), azooxanthellate coldwater corals presented higher intracellular pH regulation than shallow-water zooxanthellate corals under acidification. As it is not truly known whether or not symbionts promote the maintenance of elevated pH at the site of calcification, it is interesting to compare zooxanthellate and azooxanthellate corals as they may display contrasting responses to ocean acidification.

The present study focuses on two Mediterranean long-lived corals of key relevance for conservation, the zooxanthellate species C. caespitosa (Linnaeus, 1767) and the azooxanthellate species A. calycularis (Pallas, 1766). Cladocora caespitosa is a unique zooxanthellate reef-building coral that provides a valuable ecosystem service in the Mediterranean Sea which is lacking large bioconstructions [START_REF] Kersting | Cladocora caespitosa bioconstructions in the Columbretes Islands Marine Reserve (Spain, NW Mediterranean): distribution, size structure and growth[END_REF].

Astroides calycularis is commonly found in the southwestern Mediterranean Sea and can be highly abundant in low light, shallow rocky habitats [START_REF] Zibrowius | The "Southern" Astroides calycularis in the Pleistocene of the northern Mediterranean-An indicator of climatic changes (Cnidaria, scleractinia)[END_REF]. These two scleractinian corals naturally occur at two newly discovered CO2 vent systems along the coast of Ischia, Italy. In situ data obtained by [START_REF] Teixidó | Ocean acidification causes variable trait-shifts in a coral species[END_REF] showed variable responses in skeletal structure and growth patterns of A. calycularis, with colonies from the CO2 vent site characterized by smaller size, fewer polyps, and less porous, denser skeletons. Thus, these two scleractinian coral species grown under elevated pCO2 environments are great model systems for assessing tolerance to acidification. Here, two hypotheses are tested: (1) past exposure to low pH conditions confers tolerance to ocean acidification and (2) the coral-algal symbiosis modulates this tolerance. To test these hypotheses, corals from natural CO2 vent systems and reference sites were exposed to present-day (pH on the total scale, pHT ~ 8.07) and low pH conditions (pHT ~7.70) during a six-month long laboratory experiment, in which calcification, respiration and photosynthesis rates were assessed.

Material and Methods

Study sites

We studied populations of the corals Cladocora caespitosa and Astroides calycularis that naturally occur at two distinct CO2 vent sites with low pH, and two reference sites with ambient pH and no vent activity at the island of Ischia, Italy (Fig. S1 Supplementary materials). Cladocora caespitosa occurs near a CO2 vent site adjacent to rocky reefs at about 10 m depth (Chiane del Lume, hereafter Vent 1), whereas A. calycularis occurs in another CO2 vent site between 1 -2 m depth (Grotta del Mago, a semi-submerged cave hereafter Vent 2, where CO2 vents occur at 5 m depth). Vent gas compositions at Vent 1 and Vent 2 were predominantly CO2 (mean ± SE: 93.9 ± 0.6 for Vent 1, 93.4 ± 1.0 for Vent 2), with undetectable levels of hydrogen sulfide (< 0.0002 %), and did not elevate the temperature (for more information on gas measurements, see [START_REF] Teixidó | Ocean acidification causes variable trait-shifts in a coral species[END_REF]. The reference sites without any visible vent activity and exhibiting ambient pH were located: i) approximately 100 m west from Vent 1 for C.caespitosa (hereafter Ambient 1, 10 m depth) and ii) 1.5 km away from Vent 2 for A. calycularis (San Pancrazio, an overhang, hereafter Ambient 2, 1-2 m depth) (Fig. S1 Supplementary materials).

Coral field surveys

The C. caespitosa abundance was quantified using 12 quadrats of 50 cm x 50 cm, which were randomly placed along the rocky reef at 10 m depth at the two sites (n = 24 quadrats in total).

Abundance was assessed by counting the number of colonies found in each quadrat. In addition, size of colonies was determined by measuring the major colony diameter with a plastic ruler (with an error of ± 1 cm) [START_REF] Peirano | Biomass, carbonate standing stock and production of the mediterranean coral Cladocora caespitosa (L.)[END_REF]. Major colony diameter was selected as the best colony size descriptor [START_REF] Kersting | Cladocora caespitosa bioconstructions in the Columbretes Islands Marine Reserve (Spain, NW Mediterranean): distribution, size structure and growth[END_REF]). The A. calycularis abundance was quantified using 12 photo-quadrats (25 x 25 cm) between 1 and 2 m depth in the two other sites (n = 24 quadrats in total, Vent 2, Ambient 2). Size frequency-distribution was calculated by counting the number of polyps of each colony and each colony was then pooled into one of five size classes (I: 1-5 polyps: II: 6-10 polyps; III: 11-15 polyps; IV: 16-20 polyps; V: > 20 polyps). The survey's depth for C. caespitosa and A. calycularis corresponded to the depth range at which colonies were sampled for the experiment (see below). Abundance and sizefrequency data for A. calycularis have been described previously in [START_REF] Teixidó | Ocean acidification causes variable trait-shifts in a coral species[END_REF].

In situ pHT and carbonate chemistry associated with CO2 vents and ambient pH sites SeaFET TM Ocean pH sensors (Satlantic) were deployed to quantify variation in pH at the two CO2 vent and the two ambient pH sites. Measurements were taken every 15 minutes. One sensor was deployed at the Vent 1 site from May 7 to 21, 2019, and one sensor was deployed at the Ambient 1 site during the same period. At the semi-submerged cave (Vent 2), sensors were deployed in May-June 2019 (before summer) and in September 2018 (after summer). Dates of deployment for Vent 2 were from September 8 to September 24, 2018 and from May 30 to June 18, 2019 at Vent 2. One sensor was deployed in the reference area Ambient 2 during the same period. Before deployment, the SeaFETs were calibrated with ambient pH water in the aquarium facilities at the Center Villa Dohrn (Ischia, Italy). The mean offset between calibration samples and calibrated SeaFET pH was ± 0.002 units, indicating high quality pH dataset. Discrete water samples were collected using Niskin bottles to measure the carbonate system parameters during the pH sensor deployment. Samples for total alkalinity (AT) were collected using standard operating protocols. The HCl (0.1 M) titrant solution was calibrated against certified reference materials distributed by A.G. Dickson (CRM,Batches #153,#171,and #177). Precision of the AT measurements of CRMs was < 2 and < 1 μmol kg -1 from nominal values. AT and pHT were used to determine the remaining carbonate system parameters at in situ temperature and depth of each sampling period in the R package seacarb v3.2.12. For full details of pH sensors deployment, calibration, and total alkalinity standard operating protocols, see [START_REF] Teixidó | Ocean acidification causes variable trait-shifts in a coral species[END_REF].

Coral collection

A total of 81 colonies of C. caespitosa and A. calycularis were randomly sampled for the aquarium experiment. Fourteen (Vent 1) and sixteen (Ambient 1) colonies of C. caespitosa were collected at 10 m depth; twenty-seven (Vent 2) and fourteen (Ambient 2) colonies of A. calycularis were collected from 1 to 2 m depth. A portion of approximately 3 x 3 cm was gently dislodged from each colony using a hammer and chisel. They were placed in coolers filled with seawater changed regularly and maintained in Ischia for two days prior to transportation to the aquarium facilities of the Laboratoire d'Océanographie de Villefranche, France. The duration of the transfer was less than 10 h; colonies were wrapped in seawatersoaked paper tissues. The colonies were cleaned, tagged with a number stuck under it using water-resistant epoxy glue, and maintained at an ambient pHT of ~8.05 during 2 weeks.

Experimental set-up and treatments

Colonies of C.caespitosa and A. calycularis were maintained in two constant pH treatments during six months (202 days), one "present-day" treatment with a pHT = 8.08 ± 0.01 (mean ± SE, n = 348, total number of weekly pHT measures in each 12 present-day experimental tank) and one "low pH" treatment with a pHT = 7.72 ± 0.01 (mean ± SE, n = 348, total number of weekly pHT measures in each 12 low pH experimental tank) corresponding to the pH values expected by the end of the century under the RCP 8.5 CO2 emissions scenario. More specifically, 14 colonies from Vent 1 and 16 colonies from Ambient 1 of C. caespitosa and 27 from Vent 2 and 14 from Ambient 2 of A.calycularis were used for this experiment. The vent-low pH condition was used to test the acclimation or long-term tolerance of the colonies to low pH in the lab, after a life-time of exposure to in situ low pH. The ambient-low pH condition was used to test the short-term tolerance to low pH of colonies that have not been exposed to low pH before. The vent-present day condition tested the response to ambient pH of colonies exposed in the long-term to in situ low pH. The ambient-present day condition was the control. There were six independent 5 L experimental tanks for each of the four conditions (origin x pH treatment), for a total of 24 experimental tanks. Each experimental tank contained one to three colonies of each species, depending on their size, to get approximately the same total amount of polyps per tank (Tab. S1 Supplementary materials).

Seawater pumped from Villefranche Bay at 5 m depth was continuously flowing into six 25 L header tanks, used as water storage and to maintain gravity pressure for water flow into the experimental tanks. Three header tanks were maintained at pHT ~ 8.07 (present-day treatment) and the other three at pHT ~ 7.7 (low pH treatment). pH was controlled in the header tanks using pH controllers (APEX, Neptune Systems) which regulated the delivery of pure CO2. Each header tank gravity-fed four experimental four-liter tanks at a rate of 100 ml min -1 (Fig. 1).

Light was provided by 89 W LED light bars (Aquaristik, Aqualumix). Irradiance gradually increased from 0 at 06:00 to a maximum irradiance of 180 µmol photons m -2 s -1 between 11:00 and 14:00, and gradually decreased to 0 at 18:00. According to the attenuation coefficients of PAR in Villefranche Bay calculated in [START_REF] Martin | Response of Mediterranean coralline algae to ocean acidification and elevated temperature[END_REF], the maximum irradiance levels used in the study (180 μmol photons m -2 s -1 ) is within the irradiance range observed at 10 m, where Cladocora caespitosa can be found in the Mediterranean Sea (154 to 449 μmol photons m -2 s -1 ). As A. calycularis were collected in a semi-submerged cave and an overhang, dark plastic bags covered one side of the experimental tanks to shade colonies from direct light. The 24 experimental tanks were kept in 6 water baths under controlled temperature following Ischia's natural changes (Fig. S2 Supplementary materials, APEX, Neptune Systems). Submersible pumps (NEWA) provided water motion in each experimental tank. Three times a week, each tank was provided with a 30 mL solution of freshly hatched brine shrimps (Artemia sp.) for feeding.

Health status of experimental colonies

With pictures at the start of the experiment, each colony has been classified into two categories: colonies with loss of coenosarc (tissue between polyps) and colonies with tissue connecting their polyps. The number of dead polyps per colony (complete disappearance of tissue in the calice) was counted by comparing the pictures of the same colony at the start and the end of the experiment.

Carbonate chemistry in experimental tanks

pH in the header and experimental tanks was measured weekly using a handheld pH-meter (826 pH mobile, Metrohm) calibrated with a TRIS buffer (batch #T33 provided by A.

Dickson, Scripps Institution of Oceanography, USA) before each set of measurements. Total alkalinity (AT) of each experimental and header tank was measured weekly during the first four weeks in order to ascertain whether conditions were altered by metabolic activity.

Subsequently, and for the remainder of the experiment, AT was measured every month in four randomly selected tanks and one header. AT was determined by potentiometric titration using a Metrohm 888 Titrando following the method of [START_REF] Dickson | SOP 3a Determination of total alkalinity in sea water using an open-cell titration[END_REF]). Titrations of certified reference material (Batch #186) provided by A. Dickson were used to assess the accuracy of the measurements and were within 6.5 µmol kg -1 of the reference value. Ischia's temperatures are between 0.87 to 2.67°C higher than in the Bay of Villefranche during the studied period.

Water pumped on the Bay of Villefranche was heated to be as close as possible to Ischia's

Six-month and monthly calcification

Calcification rate was assessed using the buoyant weight technique [START_REF] Davies | Short-term growth measurements of corals using an accurate buoyant weighing technique[END_REF]. Weighing was done every 27 to 35 days. Changes in wet weight were converted to dry weight using the following equation: Dark respiration, gross photosynthesis and short-term calcification Dark respiration, net photosynthesis and short-term calcification were determined during less than 1 h incubations in 500 ml transparent perspex chambers, placed in a temperaturecontrolled water bath to maintain a constant temperature. Incubations were carried out a month after the start of the experiment (hereafter "start") and a month before the end of the experiment (hereafter "end") at the same temperature of 20°C. Magnetic stirrers were used to provide water motion in the incubation chambers. Each colony was transferred to an individual chamber filled with water from the experimental tanks from which the corals were taken for the incubation. A blank incubation with no coral colony and filled with water from the same header tank was performed for each four experimental tanks. Incubations lasted 30 to 60 min depending on the magnitude of the metabolic activity. To measure oxygen uptake during respiration, colonies were maintained in darkness by covering the chamber with an opaque plastic bag and incubations started after a 10 min acclimation period. Production of oxygen by photosynthesis was only measured on colonies of C. caespitosa under an irradiance of 180 µmol photons m 2 s -1 . Oxygen saturation within the chambers was measured every 5 s using a fiber optic oxygen sensor (PreSens, OXY-4 mini), which was calibrated at 0% saturation in an oxygen-free solution created by saturating seawater with sodium sulfite, and at 100% saturation in an oxygen-saturated seawater obtained by bubbling ambient air during 5 min. The difference in oxygen saturation between the beginning and end of the incubation was converted to O2 mg L -1 using the gas_O2sat function of the marelac R package [START_REF] Soetaert | marelac : Tools for aquatic sciences[END_REF]. Oxygen consumption was then normalized by coral surface area, incubation time and chamber volume to obtain the rates of respiration (negative in dark conditions) and net photosynthesis (oxygen production in excess of the respiratory demand under light conditions). The rate of gross photosynthesis was calculated by subtracting the respiration rate in dark to the rate of net photosynthesis, with the assumption that respiration rate is the same under light and dark conditions.

Water samples were collected at the beginning and end of the incubation for each colony for calcification determination using the alkalinity anomaly technique [START_REF] Smith | Carbon dioxide and metabolism in marine environments[END_REF].

This method relies on the stoichiometric relationship between total alkalinity and calcification, where the precipitation of one mole of CaCO3 lowers the total alkalinity by two moles. Alkalinity anomalies were normalized by the coral surface area, incubation time, and chamber volume to calculate light and dark calcification rates. The alkalinity anomaly method was only applied to the zooxanthellate C. caespitosa.

Data analysis

Biological surveys: A generalized linear mixed model was used to test for differences in abundance (poisson family) as a function of site (fixed factor, 2 levels) and quadrat as a random effect. Chi-square contingency tables were used to compare the size-frequency distributions between sites. Laboratory experiment: The assumptions of normality and equality of variance were evaluated through graphical analyses of residuals using QQ plot functions in R software. Mean values of the colonies in the experimental tank were used as a replicate for six-month calcification. Each incubation was treated as a statistical replica because they were done in individual chambers on single individuals. The effect of header tanks was first tested with an ANOVA and since its significance was negligible (p > 0.05), it was not considered in subsequent analyses. A two way-ANOVA was then used to test for differences in net calcification caused by the origin (vent or ambient pH sites) and the pH treatments (present-day or low pH) for both C. caespitosa and A. calycularis. For net calcification as a function of time, a three-way ANOVA was applied, with origin, pH treatment, and time as fixed factors. Tukey's post hoc tests were conducted when significant differences were detected. Three-way repeated measure ANOVA was used for dark respiration, gross photosynthesis and short-term calcification since the same colonies of C. caespitosa and A. calycularis were used at the start and end of the incubations. Results have been reported as means ± standard error of the mean (SE) hereafter, unless otherwise noted.

Results

In situ environmental conditions and populations of corals

Water carbonate chemistry and in situ monitoring of seawater pHT (pH on the total scale) at the CO2 vent and reference sites with ambient pH showed mean pHT = 7.91 at Vent 1, pHT = 7.91 at Vent 2, pHT = 8.05 at Ambient 1, and pHT = 7.97 at Ambient 2, respectively (Tab. 1).

The field surveys showed significant lower abundance of C. caespitosa at Vent 1 compared to Ambient 1 (1.8 ± 0.16 colonies versus 3.3 ± 0.35, Z = 2.2, p < 0.02, Fig. S3 Supplementary materials). No large colonies of more than 21 cm were found at Vent 1 (Fig. S1, Supplementary materials). This reduction of size was also observed for A.calycularis at Vent 2 (92 % had up to 10 polyps versus 72 % in Ambient 2), which differed significantly between sites (χ 2 = 76.7, p<0.0001). Abundance of A. calycularis was lower in Vent 2 (67.1 ± 10.3 at Vent 2 versus 80.3 ± 14.5 at Ambient 2, Z = 2.65, p < 0.08). Ambient 2 Ambient pH 37.9 1 ± 0 (37.9, 37.9), n=7

26.4 2 ± 1 (25.9, 27.0), n=1691 with a maximum value of 25.17 ± 0.15 °C in August (mean ± SE, n = 24, Fig. S2 Supplementary materials). The "present-day" treatment was maintained at a mean pHT of 8.06 ± 0.06 (n = 347) whereas "low pH" treatment had a pHT of 7.73 ± 0.09 (n = 348). Salinity was 37.8 ± 0.5 (n = 25).

Colonies of Astroides calycularis had physical differences depending on their site of origin.

85% (23/27 colonies) of vent and 14% (2/14 colonies) of ambient colonies did not present coenosarc (i.e. tissue between polyps). Furthermore, by comparing photos from the beginning and end of the experiment, it was observed that none of the A. calycularis colonies from ambient suffered polyp death, while three vent colonies showed 2/5, 1/7 and 4/9 dead polyps (number of dead polyps / total number of polyps in the colony). No polyp loss was observed in Cladocora caespitosa.

Six-month, monthly and short-term calcification rates

The rates of six-month calcification of C. caespitosa and A. calycularis were not affected by the origin and the pH treatment (Fig. 2; Tab. S3 Supplementary materials; Two-way ANOVA, F1,20= 1.778, P = 0.2 for C. caespitosa, F1,20= 0.13, P = 0.72 for A. calycularis).

No statistically significant effect of origin and pH treatment on calcification rate was found in C. caespitosa over time (Fig. 3A; F1,110= 1.919, P = 0.17). However, different calcification rates with time were found for A. calycularis (Fig. 3B, F1,112= 26.2, P < 0.001), with significantly higher calcification measured during the first month of the experiment (1.0 ± 0.2 mg CaCO3 cm -2 d -1 ). The mean monthly calcification rate was 0.2 ± 0.1 mg CaCO3 cm -2 d -1

during the following months of experiment.

Dark respiration and gross and net photosynthesis

Net photosynthesis of C. caespitosa was not affected by origin and pH treatment (Fig. 5A; F1,40= 2.36, P = 0.134 and F1,40= 1.24, P = 0.272, respectively), but was different between the beginning and the end of the experiment (Fig. 5A; F1,40= 13.14, P < 0.001). Gross photosynthesis of C. caespitosa did not exhibit any statistically significant difference between origin, pH treatment and time (Fig. 5B; F1,24= 0.17, P = 0.683). Mean gross photosynthesis for all pH treatments was similar at the start and end of the experiment (0.024 ± 0.001 vs 0.020 ± 0.002 mg O2 cm -2 h -1 , n = 30). As light and dark respiration may differ, gross photosynthesis measurements are uncertain, and most likely underestimated.

Dark respiration rate of C. caespitosa was not significantly different between origin and pH treatment (Fig. 5C, F1,24= 0.008, P = 0.93 and F1, 24= 3.09, P = 0.091, respectively), but was significantly different between the beginning and end of the experiment (F1,24= 9.45, P = 0.005). In June, mean dark respiration rates for all conditions (origin and pH treatment) was -0.0079 ± 0.0003 mg O2 cm -2 h -1 (n = 30) while in November it was -0.0097 ± 0.0006 mg O2 cm -2 h -1 (n = 28), which represents a 23% of increase in dark respiration.

At the start of the experiment, the dark respiration of A. calycularis did not differ between origin or pH treatment (Fig. 5D, F1,37= 0.32, P = 0.58). At the end of the experiment (Fig. 5D), a significant difference between site of origin was observed (F1,36= 18.5, P < 0.0001), with corals from the ambient site having a mean dark respiration of -0.038 ± 0.003 mg O2 cm - 2 h -1 (n = 13) whilst colonies from the CO2 vent site had a rate of -0.087 ± 0.008 mg O2 cm -2 h - 1 (n = 27). The overall (all treatments) mean dark respiration rate were also different at the start and at the end of the experiment, with a lower mean dark respiration at the start (-0.026 ± 0.002 vs -0.071 ± 0.007 mg O2 cm -2 h -1 ; F1,36= 52.3, P < 0.0001).

Discussion

During this six-month laboratory experiment, we tested whether colonies of the zooxanthellate coral Cladocora caespitosa and the azooxanthellate coral Astroides calycularis growing in naturally acidified waters were more tolerant to low pH than colonies from ambient pH sites or acclimated. In contrast to our initial hypothesis, calcification rates and metabolic responses did not change for any coral species no matter the site of origin and pH treatment. Therefore, our results suggest that both species, independently of environmental history and trophic strategy, can tolerate low pH conditions for six months, under controlled conditions in aquaria. In contrast, in situ surveys revealed lower abundance and smaller sizes for both corals at CO2 vent sites compared with ambient sites.

The hypothesis that corals from vent sites would be less impacted by low pH stems from reports that organisms experiencing highly variable or low pH in their natural habitat may be more tolerant to ocean acidification [START_REF] Cornwall | Resistance of corals and coralline algae to ocean acidification: Physiological control of calcification under natural pH variability[END_REF]. Corals living in suboptimal environmental conditions may possess physiological and potentially genetic mechanisms that confer tolerance to such environmental conditions [START_REF] Camp | The future of coral reefs subject to rapid climate change: lessons from natural extreme environments[END_REF][START_REF] Teixidó | Ocean acidification causes variable trait-shifts in a coral species[END_REF]. For example, microhabitats experiencing periodic temperature extremes are potential habitats for heat tolerant corals [START_REF] Schoepf | Limits to the thermal tolerance of corals adapted to a highly fluctuating, naturally extreme temperature environment[END_REF]), yet these habitats can exhibit a mosaic of populations, harboring species previously reported with high to low thermal tolerance [START_REF] Ainsworth | Climate change disables coral bleaching protection on the Great Barrier Reef[END_REF] ). The calcification rates of A.

calycularis measured here are also of the same order of magnitude to those reported by [START_REF] Movilla | Annual response of two Mediterranean azooxanthellate temperate corals to low-pH and hightemperature conditions[END_REF] (0.39 ± 0.04 and 0.47 ± 0.06 mg CaCO3 cm -2 d -1 , respectively). In contrast to temperate and cold-water corals, tropical corals tend to be more affected by ocean acidification, as they exhibit a mean decrease in calcification of 10-25% when exposed to the pCO2 expected by the end of the century under the high-CO2 emissions scenario [START_REF] Chan | Sensitivity of coral calcification to ocean acidification: a meta-analysis[END_REF][START_REF] Cornwall | Global declines in coral reef calcium carbonate production under ocean acidification and warming[END_REF]. Most tropical corals investigated so far are fast calcifiers, growing in warm waters, therefore as seawater pH declines, they may be unable to maintain these energetically costly fast rates of calcification [START_REF] Comeau | Fast coral reef calcifiers are more sensitive to ocean acidification in short-term laboratory incubations[END_REF]. In contrast, temperate and cold-water corals are slower calcifiers with lower energy requirements for mineralization, thus making them more likely to maintain constant calcification rates at lower pH [START_REF] Rodolfo-Metalpa | Response of the temperate coral Cladocora caespitosa to mid-and long-term exposure to pCO2 and temperature levels projected for the year 2100 AD[END_REF][START_REF] Comeau | Fast coral reef calcifiers are more sensitive to ocean acidification in short-term laboratory incubations[END_REF]. [START_REF] Ries | A nonlinear calcification response to CO2-induced ocean acidification by the coral Oculina arbuscula[END_REF], also suggested that the effect of acidification on the calcification of temperate corals, such as Oculina arbuscula, may manifest at a lower pH threshold compared to tropical corals. The capacity of C. caespitosa and A. calycularis to control the carbonate chemistry at their site of calcification could explain this tolerance to low pH. For example, using the geochemical proxy δ 11 B it was shown that the temperate coral Balanophyllia europaea at the CO2 vents of Panarea Island (Italy) can maintain constant calcifying fluid pH when exposed to pH ranging from 8.07 to 7.74 [START_REF] Wall | Linking internal carbonate chemistry regulation and calcification in corals growing at a Mediterranean CO2 vent[END_REF]. This confirms the capacity of some corals to maintain internal pH homeostasis and utilize this as a way of calcifying under reduced seawater pH [START_REF] Comeau | Resistance to ocean acidification in coral reef taxa is not gained by acclimatization[END_REF].

From this study no conclusions can be drawn as to whether colonies from the vent sites are acclimated to low pH, as colonies from both vent sites and the ambient sites were not affected by low pH in the laboratory. However, controlling the transport of calcium and inorganic carbon to the calcifying medium and producing the skeletal organic matrix are all energydemanding processes [START_REF] Allemand | Coral Calcification[END_REF][START_REF] Wall | Linking internal carbonate chemistry regulation and calcification in corals growing at a Mediterranean CO2 vent[END_REF], therefore the maintenance of calcification rates under these treatments may have come at a cost. For example, [START_REF] Wall | Linking internal carbonate chemistry regulation and calcification in corals growing at a Mediterranean CO2 vent[END_REF] suggest that the inactivation of some calicoblastic cells could compensate for the energy required to regulate internal pH and maintain calcification rates under low seawater pH, but at the cost of increased skeletal porosity. This is supported by a study that showed a constant growth rate of the temperate coral B. europaea in acidified conditions at the expense of skeletal density [START_REF] Fantazzini | Gains and losses of coral skeletal porosity changes with ocean acidification acclimation[END_REF], which in return increases the sensitivity to bioerosion and susceptibility to damage [START_REF] Crook | Reduced calcification and lack of acclimatization by coral colonies growing in areas of persistent natural acidification[END_REF]. Skeletal porosity was not measured in the present study. However, increased sensitivity to bioerosion can also be linked to the tissue coverture of the skeleton. [START_REF] Rodolfo-Metalpa | Response of the temperate coral Cladocora caespitosa to mid-and long-term exposure to pCO2 and temperature levels projected for the year 2100 AD[END_REF] observed that following transplantation to low pH conditions, skeleton dissolution was observed in Cladocora caespitosa which had exposed skeleton, and not in Balanophyllia europaea which had less exposed skeleton. In our study, we observed that most of the A. calycularis colonies collected at the CO2 vent sites did not possess coenosarc (i.e. coral tissue between polyps) and exhibited polyp death during our experiment. These phenomena were also observed in natural colonies of A. calycularis [START_REF] Teixidó | Ocean acidification causes variable trait-shifts in a coral species[END_REF]) and the corals Pocillopora damicornis and Oculina patagonica when incubated at low pH [START_REF] Kvitt | Breakdown of coral colonial form under reduced pH conditions is initiated in polyps and mediated through apoptosis[END_REF]. The increased sensitivity to bioerosion of A. calycularis and C. caespitosa with exposed skeleton could explain in part the field observation.

Field observations confirmed the difference between the population of the CO2 vent and ambient sites, as both corals were less abundant and had smaller colonies at the vent sites (Fig. S3 Supplementary materials, [START_REF] Teixidó | Ocean acidification causes variable trait-shifts in a coral species[END_REF]. These contrasting results between field and laboratory observations, could be explained by differences in pH variability, which is much greater in the field with short periods of very low pH. Alternatively, other environmental parameters such as food availability or biological interactions (e.g., competition with algae) may contribute to these differences. pH variability at the CO2 vent sites can be very high, as pHT can range from 6.70 to 8.13 in a day [START_REF] Foo | The carbon dioxide vents of Ischia, Italy, a natural system to assess impacts of ocean acidification on marine ecosystems: an overview of research and comparisons with other vent systems[END_REF][START_REF] Teixidó | Ocean acidification causes variable trait-shifts in a coral species[END_REF], whereas pHT was very stable during our pH-controlled laboratory experiment (pHT = 7.72 ± 0.01 for low pH treatment). It may be that no differences in calcification and photosynthesis were observed in the laboratory, due to the fact that both corals are slowgrowing and long-lived species that have had a lifetime to acclimatize to their environmental pH. Therefore, the six-month time scale of the experiment may not have been enough to trigger any visible variation. However, even if our results did not show any statistical difference between treatments during the laboratory experiment, different trends in calcification between conditions can be observed for A. calycularis. The colonies from ambient-low pH condition seem to have lower calcification rate than the control colonies of ambient-present day condition (Fig. 2), suggesting a possible impact of low pH on calcification in the short term. The same trend is observed with the colonies from the vent site, as the vent-low pH presents reduced calcification rate compared to the vent-present day condition. Moreover, calcification was similar in the vent-low pH and the control ambientpresent day conditions. This trend suggests a possible acclimation to low pH in corals growing at the CO2 vent site Zooxanthellate and azooxanthellate corals were both used in the present study to assess the role that photosynthesis could play in mitigating the effects of ocean acidification on calcification. In zooxanthellate corals, light-enhanced calcification describes the relationship between calcification, photosynthesis and irradiance [START_REF] Gattuso | Photosynthesis and calcification at cellular, organismal and community levels in coral reefs: A review on interactions and control by carbonate chemistry[END_REF][START_REF] Allemand | Coral Calcification[END_REF]. Four main mechanisms explaining light-enhanced calcification were proposed: photosynthesis could (1) supply energy to the animal through the translocation of photosynthates, (2) increase pH at the site of calcification by using CO2, (3) remove phosphate, which is poison to carbonate crystallization, and (4) facilitate the production of the organic matrix which serves as a scaffold in which CaCO3 is precipitated [START_REF] Allemand | Coral Calcification[END_REF]). Here, short-term calcification of C. caespitosa was higher in light than in the dark, with a mean light:dark calcification ratio of 2.38 (See Tab. S4 Supplementary materials), which is close to the mean ratio of 3.0 obtained by [START_REF] Gattuso | Photosynthesis and calcification at cellular, organismal and community levels in coral reefs: A review on interactions and control by carbonate chemistry[END_REF]. However, neither light nor dark calcification rates differed with experimental pH conditions, thus we cannot conclude that photosynthesis did provide better tolerance to ocean acidification. Furthermore, no difference in the response of calcification to ocean acidification was observed between the zooxanthellate (C. caespitosa) and azooxanthellate species (A. calycularis). The data from the in situ survey at Ischia did not highlight a difference between the zooxanthellate and azooxanthellate corals either, as the size and abundance of colonies from CO2 vent sites were lower for both species. Contrastingly, [START_REF] Prada | Ocean warming and acidification synergistically increase coral mortality[END_REF] reported that the zooxanthellate coral B. europaea was unaffected by low pH conditions, whereas the azooxanthellate corals Leptopsammia pruvoti and A. calycularis showed a negative effect of pH on calcification.

However, the study of [START_REF] Prada | Ocean warming and acidification synergistically increase coral mortality[END_REF] was conducted in situ, on corals transplanted next to CO2 vents in Panarea Island, whereas our study is based on a laboratory experiment and field observations. Feeding could also explain this difference as it can mitigate the negative effects of environmental stressors on coral physiology [START_REF] Fox | Gradients in primary production predict trophic strategies of mixotrophic corals across spatial scales[END_REF]). In our experiment, corals were fed freshly hatched Artemia nauplii three-times a week therefore the demand of energy required to maintain calcification under low pH was likely sustained by heterotrophy [START_REF] Edmunds | Zooplanktivory ameliorates the effects of ocean acidification on the reef coral Porites spp[END_REF]. [START_REF] Kornder | Thresholds and drivers of coral calcification responses to climate change[END_REF] showed that in studies where corals were fed, the negative impacts of ocean acidification on corals were mitigated. Therefore, the fact that [START_REF] Movilla | Calcification reduction and recovery in native and non-native Mediterranean corals in response to ocean acidification[END_REF] observed a decrease in calcification at low pH in C. caespitosa may be explained by the fact that the colonies were only fed once per week.

No difference in photosynthesis was observed between site of origin or pH treatment. This aligns with the observation that there is a limited impact of environmentally relevant decreases in pH on photosynthesis [START_REF] Comeau | Effects of pCO2 on photosynthesis and respiration of tropical scleractinian corals and calcified algae[END_REF], and that low pH rarely causes bleaching [START_REF] Anthony | Ocean acidification causes bleaching and productivity loss in coral reef builders[END_REF]. Moreover, Mediterranean corals harbor Clade B Symbiodinium, that have growth rates, photosynthetic capacities and respiration rates that are unaffected by acidification [START_REF] Brading | Differential effects of ocean acidification on growth and photosynthesis among phylotypes of Symbiodinium (Dinophyceae)[END_REF]. Respiration was also not affected by the pH treatment in C. caespitosa, confirming previous observations for this species [START_REF] Rodolfo-Metalpa | Response of the temperate coral Cladocora caespitosa to mid-and long-term exposure to pCO2 and temperature levels projected for the year 2100 AD[END_REF], and other coral species [START_REF] Comeau | Effects of pCO2 on photosynthesis and respiration of tropical scleractinian corals and calcified algae[END_REF]. However, colonies of A.

calycularis from the vent site exhibited a higher rate of respiration at the end of the experiment (November) for both low and present-day pH treatments. This result contrasts with studies that show a decrease in respiration at lower pH (e.g. [START_REF] Comeau | Fast coral reef calcifiers are more sensitive to ocean acidification in short-term laboratory incubations[END_REF], or no impact on respiration [START_REF] Rodolfo-Metalpa | Response of the temperate coral Cladocora caespitosa to mid-and long-term exposure to pCO2 and temperature levels projected for the year 2100 AD[END_REF][START_REF] Comeau | Effects of pCO2 on photosynthesis and respiration of tropical scleractinian corals and calcified algae[END_REF]). However, an increase in respiration could be due to the increased energetic demand associated with maintaining calcification rates at lower pH [START_REF] Comeau | Effects of pCO2 on photosynthesis and respiration of tropical scleractinian corals and calcified algae[END_REF]. For example, a decrease in seawater pHT of 0.7 units requires an additional 1-2 kJ of energy mol -1 of CaCO3 precipitated (McCulloch et al. 2012a). For zooxanthellate corals, this energy is less than 1% of the energy provided by photosynthesis, however, for azooxanthellate corals such as A.

calycularis, respiration is the only way to up-regulate this energy demand. Moreover, the increase of respiration from colonies of vent sites of A. calycularis could be partly due to the smaller size of colonies at the vent site than at the ambient site [START_REF] Edmunds | Size-dependent physiological responses of the branching coral Pocillopora verrucosa to elevated temperature and pCO2[END_REF].

Although the corals studied here showed tolerance in the laboratory to pH conditions expected by 2100, it is a fragile equilibrium that can be disrupted by the inclusion of changes in other

Figure 1 .

 1 Figure 1. Experimental set-up used to test the effects of origin (ambient vs vent site) and pH treatment (present day, pHT = 8.07 ± 0.01 and low pH, pHT = 7.72 ± 0.01) on the coral C. caespitosa and A. calycularis. The experimental set-up was repeated 3 times, resulting in 6 header tanks, and 24 experimental tanks in which corals were randomly assigned.

  temperatures. Salinity at Ischia and in the Bay of Villefranche-sur-Mer are similar. Salinity data were gathered from the salinity recorded weekly in the Bay of Villefranche by the Service d'Observation Rade de Villefranche, SO-Rade, of the Observatoire Océanologique and the Service d'Observation en Milieu Littoral, SOMLIT/CNRS-INSU. pHT, temperature, AT and salinity were used to calculate the other carbonate chemistry parameters using the R package seacarb (Gattuso et al. 2020) (Tab. 1, Tab. S2 Supplementary materials).

  density of 2.93 g cm 3 . Six-month calcification rates corresponding to the entire experiment duration were determined as the change in dry weight between the first and the last weighing, normalized by the corals living tissue surface at the end of the experiment and the number of days of the experiment (202 days). Regular rates of calcification over time were normalized to the surface of the corals and to the number of days between weighing (~30 d). Surfaces were determined by the aluminum foil technique for C. caespitosa[START_REF] Marsh | Primary productivity of reef-building calcareous red algae[END_REF]) because these 3D colonies were impossible to analyze with 2D pictures. A. calycularis surfaces were measured by photographic analysis with the software ImageJ for (IMAGEJ, NIH US Department of Health and Human Services) as they had flat shape. Surface measurements were completed at the start and end of the experiment. Marginal changes in surface area were observed between the beginning and end of the experiment.

Figure 2 .

 2 Figure 2. Six-month calcification rates of C. caespistosa and A. calycularis collected from vent and ambient sites exposed to the two pH treatments (pHT = 8.08 ± 0.01, present day; pHT = 7.72 ± 0.01, low pH) during the entire duration of the experiment (202 days). Panel A) shows Cladocora caespitosa and panel B) shows Astroides calycularis. Dots represent the mean calcification rates of colonies per experimental tank and boxes represent the median and the 25 and 75% quartiles. The color of the box indicates the origin and the treatment. n = 6 per pH treatment and origin.

Figure 3 .

 3 Figure 3. Calcification rates of C. caespistosa and A. calycularis from vent and ambient sites under the two pH treatments (pHT = 8.08 ± 0.01, present day; pHT = 7.72 ± 0.01, low pH) over time (every 27 to 35 days). Values are means ± SE, Panel A) Cladocora caespitosa and B) Astroides calycularis. The color of the dots and lines indicates the origin and the treatment. We had a technical problem with day 139 weighing which is therefore not represented here. Numbers below arrows between dots represent the number of days between measurements. n = 6 per pH treatment and origin.

Figure 4 .

 4 Figure 4. Short-term calcification rates for Cladocora caespitosa from vent and ambient sites under the two pH treatments (pHT = 8.08 ± 0.01, present day; pHT = 7.72 ± 0.01, low pH) in the light and dark at the start (27 th of June 2019) and end of the experiment (24 th of October 2019). Values are means ± SE. Rates were calculated using alkalinity anomaly method following short term incubations (< 1 h) at 20°C. Panel A) and C) under light conditions, B) and D) under dark conditions. Dots represent the calcification rates of the coral colonies and boxes represent median and 25 and 75% quartiles. The color of the box indicates the origin and the treatment. n = 5 to 8 per pH treatment and origin.

Figure 5 .

 5 Figure 5. Net and gross photosynthesis and dark respiration rates of C. caespitosa and A. calycularis from the vent and ambient sites under the two pH treatments (pHT = 8.08 ± 0.01, present day; pHT = 7.72 ± 0.01, low pH) at the start (27 th of June 2019) and end of the experiment (24 th of October 2019). Values are means ± SE, Panel A) Cladocora caespitosa's net photosynthesis, B) Cladocora caespitosa's gross photosynthesis, C) Cladocora caespitosa's dark respiration. D) Astroides calycularis' dark respiration. The color of the bars indicates the origin and the treatment. n = 6 to 15 per pH treatment and origin.

TABLE 1 . Measured and estimated seawater physiochemical parameters at the CO2 vent sites, reference areas with ambient pH and both pH treatment in the experimental tanks for salinity (S), temperature (T), total alkalinity (AT), dissolved inorganic carbon (CT), pHT, pCO2, calcite (Ωc) and aragonite (Ωa) saturation.

 1 Values are means ± SD with 25 th and 75 th percentiles. Calculated concentrations of CT, pCO2, Ωc and Ωa are shown.

	1: Parameters measured

  . Such physiological mechanisms and intraspecific variability may exist in C. caespitosa and A. calycularis which highlights the importance of studying the influence of suboptimal environmental conditions on tolerance to stressors. Previous research has shown a decrease in calcification when corals were transplanted from ambient pH sites to CO2 vents (Rodolfo-Metalpa et al. 2011; Prada et al. 2017). However, previous studies on temperate/cold-water coral including C. caespitosa and A. calycularis have also shown a greater tolerance to ocean acidification than most tropical corals (see Tab.S5 Supplementary materials). Calcification rates reported in the present study agree with earlier studies. For C. caespitosa, Rodolfo-Metalpa et al. (2010) observed a yearly calcification rate of 0.51 mg CaCO3 cm -2 d -1 , which is similar to what we measured during

	this six-month experiment (0.43 ± 0.03 mg CaCO3 cm -2 d -1

Acknowledgements

This research was supported by the French Government through the National Research Agency -Investments for the Future ("4Oceans-Make Our Planet Great Again" grant, ANR-17-MPGA-0001). Thanks are due to the Service d'Observation Rade de Villefranche (SO-Rade) of the Institut de la mer de Villefranche and the Service d'Observation en Milieu Littoral (SOMLIT/CNRS-INSU) for their kind permission to use the Point B data. We thank Samir Alliouane for assistance in the laboratory and P. Sorvino (ANS Diving, Ischia) for assistance in the field.

environmental variables such as temperature that could lead to a greater impact on physiological processes and survival, as observed in the in situ survey and predicted by the negative trend on the calcification of A. calycularis. For example, marine heatwaves can lead to mass mortality of C. caespitosa and A. calycularis [START_REF] Rodolfo-Metalpa | Tissue necrosis and mortality of the temperate coral Cladocora caespitosa[END_REF] and are projected to be more frequent and severe in the future [START_REF] Galli | Marine heat waves hazard 3D maps and the risk for low motility organisms in a warming Mediterranean Sea[END_REF][START_REF] Smale | Marine heatwaves threaten global biodiversity and the provision of ecosystem services[END_REF].

Moreover, the impact of ocean acidification on reproduction as well as larval and juvenile survival, which can limit the persistence and dispersion of a species remains to be investigated. The present study contributes to our knowledge of Cladocora caespitosa and Astroides calycularis' ability to survive under future ocean acidification conditions. Such information will improve our ability to manage and conserve the biodiversity of Mediterranean calcifying species.

Author contributions


Jérôme Dejeu

Amélie Cot

Patrick Rougeot

Boris Lakard

Sophie Lakard

Michaël Gauthier

Development of New Sticky and Conducting Polymer Surfaces for MEMS Applications

Keywords: PANi, adhesion forces, micromanipulation, sticky surface, electropolymerisation

The development of new surfaces, highly sticky and conducting, is a great challenge in the field of microdevices fabrication, and more precisely for the 3D assembly of microcomponents. Such surfaces were prepared by electrochemical deposition of polyaniline films. The films prepared from a phosphoric acid solution were more adhesive than the ones obtained in other acids. Indeed, the adhesion of the polyaniline films prepared in H 3 PO 4 was found to be high (> 1 µN) and stable in time, as shown by AFM force measurements. The adhesion properties were correlated with the morphology, thickness and roughness of the polyaniline films, and the electrodeposition conditions (H 3 PO 4 concentration and electrodeposition time) were optimized. The adhesion properties of this original polymer films were found to be similar to those of the best commercial glues. The conductivity of the polyaniline film was also demonstrated as well as the possibility for the polyaniline films to switch reversibly from a non-adhesive to an adhesive behavior. A wide range of applications, in the field of telecommunications, bioengineering, and more generally speaking MEMS (microelectromechanical systems) can be envisaged for these materials.

Introduction

3D integration is a rapidly emerging and revolutionary technology, which vertically stacks and interconnects multiple materials and functional components to form highly integrated micronano systems. 3D integration aims to increase density and functionality per volume using silicon vias, to shorten interconnect length, to improve electrical performances, to strongly reduce chips power consumption, to integrate various functional layers based on different optimized process nodes, and to reduce the cost of such devices. This 3D integration is expected to lead to an industry paradigm shift due to its tremendous benefits. Recent works mention the fact that the demand for 3D stacking equipment will certainly grow exponentially in the following years. [START_REF] Hu | Die Stacking Is Happening[END_REF] One of the challenges of 3D integration is the electrical bonding between 3D thin stacked dies.

Current bonding methods require the application of mechanical pressure (wire bonding) or the use of thermal cycles (Solder Ball Grid Array -BGA). [START_REF] Yang | A review on reliability of electronic packaging in micro/nano manufacturing[END_REF][START_REF] Qin | Advances in Wire Bonding Technology for 3D Die Stacking and Fan Out Wafer Level Package in proc[END_REF] Both technologies generate mechanical stresses on the dies which will be particularly critical for thin dies. [START_REF] Ye | Challenges in Assembly and Reliability of Thin NAND Memory Die[END_REF] These bonding technologies have therefore limitations that can affect the overall yield and cost of 3D devices. To offer an alternative to conventional bonding technologies, we are proposing a new bonding principle which acts without thermal cycle (assembly at room temperature) and without mechanical pressure. Our strategy has several benefits for bonding and assembly which should allow the rapid fabrication of reliable products, without thermal or mechanical damage. This strategy is based on the chemical functionalization of objects by polymers, and more particularly by thin conducting polymer films deposited by electrochemical polymerization. Among the many existing conducting polymers, polyaniline (PANi) [START_REF] Trivedi | Handbook of Organic Conductive Molecules and Polymers[END_REF][START_REF] Stejskal | Preparation of a Conducting Polymer[END_REF] is a material of choice which exhibits high conductivity at both room [START_REF] Boara | Synthesis of Polyanilines with High Electrical Conductivity[END_REF] and high temperature [START_REF] Prokes | Polyaniline Prepared in the Presence of Various. Acids: 2. Thermal Stability of Conductivity[END_REF][START_REF] Trchova | FTIR Spectroscopic and Conductivity Study of the Thermal Degradation of Polyaniline Films[END_REF] due to the possibility to dope polyaniline films by both oxidation and protonation. PANi films are generally prepared from mono-or diacids such as HCl, [START_REF] Stejskal | Preparation of a Conducting Polymer[END_REF] Moreover, this polymer has already been used to develop nano and microrobots, [START_REF] Moo | Nano/Microrobots Meet Electrochemistry[END_REF][START_REF] Gao | Highly Efficient Catalytic Microengines: Template Electrosynthesis of Polyaniline/Platinum Microtubes[END_REF] nanomaterials 14 and smart systems. [START_REF] Kharade | Controlled Electrochemical Polymerization Strategies for Electroactive Polyaniline Thin Films[END_REF] More recently PANi films were used to develop an acetic acid vapor sensor, [START_REF] Turemis | ZnO/Polyaniline Composite Based Photoluminescence Sensor for the Determination of Acetic Acid Vapor[END_REF] to embed glucose oxydase [START_REF] German | Formation and electrochemical characterisation of enzyme-assisted formation of polypyrrole and polyaniline nanocomposites with embedded glucose oxidase and gold nanoparticles[END_REF] or in combination with poly(3,4ethylenedioxythiophene) (PEDOT) to enhance its electrochromic properties. [START_REF] Popov | Electrodeposited gold nanostructures for the enhancement of electrochromic properties of PANI-PEDOT film deposited on transparent electrode[END_REF][START_REF] Popov | Comparative Study of Polyaniline (PANI), Poly(3,4-ethylenedioxythiophene) (PEDOT) and PANI-PEDOT Films Electrodeposited on Transparent Indium Thin Oxide based Electrodes[END_REF] So, in this work, PANi films were prepared electrochemically in the presence of phosphoric acid whose anions were used as dopants. This allowed us to evidence that the use of phosphoric acid leads to polyaniline films having very original and interesting adhesive properties compared to polyaniline films prepared in other electrolytic solutions, in addition to a high conductivity.

Thanks to their remarkable adhesion and conduction properties, these electrodeposited polyaniline films are good candidates for applications in the field of 3D integration and microassembly.

Material and Methods

Polymer electrodeposition

Potentiostatic electropolymerization of polyaniline films was performed using a PGZ 301 to observe the surface. The wafer was tilted and viewed at an incidence angle of 40° or 35°: the same ion beam, with a current limited to 30 pA, was used as a scanning probe to image the crosssection of the film. Different layers of materials can be clearly identified due to good material contrast in the ion-induced secondary electron image. The thickness of the SiO 2 and polymer film can be measured directly from the cross-section view by compensating for the tilt angle, the apparent width of the layers being divided by the sinus of the tilt angle.

Effort of tearing

The measurements are performed on substrates coated with a polymer film: two substrates are perpendicular and a third one is deposited perpendicular to the first two. The contact area between the substrates is perpendicular (25 mm 2 on each side). The tests are carried to produce a force perpendicular to the plane formed by the substrates. The rod is held on a compliant system and is placed in the center of the perpendicular substrate. This force is measured with a precision balance (Mettler Toledo ML 3002).

Results and Discussion

Influence of the acid concentration and nature

Chronoamperometry technique was used to modify silicon surfaces by electropolymerization of aniline (0.4 M) from an acid electrolytic solution. The acid used was either a monoacid (hydrochloric acid, HCl, or nitric acid, HNO 3 ), a diacid (sulfuric acid, H 2 SO 4 ) or a triacid (phosphoric acid, H 3 PO 4 ) at 2 M. For all experiments, electropolymerization was carried out for 1 min. After that, adhesion force measurements were performed using an AFM microscope (Figure 1A). In Figure 1A), it appears that the adhesion forces are significantly different when polyaniline is formed from one acid or another. Indeed, it is noticeable that the adhesive force increases with the number of protons of the acid. More precisely, for a monovalent acid, the pull-off force is around -50 nN whereas it reaches -426 nN for H 2 SO 4 diacid, and less than -1 µN for H 3 PO 4 triacid. Moreover, for PANi films prepared in phosphoric acid, the exact value of the adhesive force can't be given precisely because an important deflection takes place which saturates the sensor. In fact, the conversion of the deflection signal cannot be performed because the sphere stuck to the end of the tip extremity is completely engulfed (see Figure SI 1 indicate an increase in pull-off force as the proton concentration increases in the electrolytic solution. Indeed, adhesive forces obtained for polyaniline films electrodeposited in 1 and 2 mol/L sulfuric acid (-575 and -397 nN, respectively) are strongly higher than those obtained in 0.5 mol/L sulfuric acid (-29 nN). Similarly, the adhesive forces measured in 1 and 2 mol/L phosphoric acid (both > -1000 nN) were strongly higher than those obtained in 0.5 mol/L solutions (-411 nN). In addition, it can be observed that the adhesion forces measured in phosphoric solutions were greatly higher than those obtained in sulfuric acid whatever the acid concentration. However, the quantity of protons incorporated present in the electrolyte solution is not the only explanation that could be given to explain the difference in the adhesion force values. Indeed, the morphology of the polymer film can also contribute to the adhesion behavior of the polymer films. That is why, AFM images of the polymer films grown in phosphoric acid were performed (Figure SI-2) leading to the conclusion that the roughness of the polymer film decreases when the acid concentration increases. This observation is consistent with the literature since it has already been demonstrated that a decrease of the roughness can lead to an increase of the adhesive force, as previously observed with polymer films [START_REF] Cot | Electrosynthesis and Characterization of Polymer Films on Silicon substrates for Applications in Micromanipulation[END_REF][START_REF] Dejeu | Morphological and Adhesive Properties of Polypyrrole Films Synthesized by Sonoelectrochemical Technique[END_REF] or structured surfaces. [START_REF] Dejeu | Nanostructured Nonadhesive Surfaces for Micro-and Nanomanipulation[END_REF][START_REF] Heim | Adhesion and Friction Forces between Spherical Micrometer-Sized Particles[END_REF][START_REF] Dejeu | Adhesion Control for Micro-and Nanomanipulation[END_REF] The crystallinity of the polymer films was also studied but all polyaniline films appeared amorphous and no significant difference was observed between the different polymer films (Figure SI-3).

Influence of the deposition time

To determine the influence of deposition time, the most promising polymer film to create adhesion between two surfaces was studied. Thus, polyaniline films obtained from 2 mol/L phosphoric acid solutions were prepared by applying a voltage of +2 V/SCE for 10 s, 30 s, 60 s, 120 s or 10 min. The adhesion force values and thickness of the resulting polymer films are summarized, in Table 2.

Deposition time (s)

Pull-off forces (nN) 10 s < -1500 30 s < -1500 60 s < -1000 120 s -61 600 s -130 Table 2. Influence of the electrodeposition time on the thickness and pull-off forces values of PANI films grown in H 3 PO 4 (2 mol/L).

Table 2 indicates that the adhesion properties of the polyaniline-modified surfaces are very high for low deposition times but decline when the deposition time increases. The most adhesive surfaces have pull-off forces (greater than 1 µN) which are ten times higher than the less adhesive ones. So, the deposition time is an important parameter to obtain adhesive surfaces. In order to correlate the adhesion properties of the films with their morphology, thickness and roughness, the polyaniline-modified surfaces were observed by scanning ion microscopy (SIM) and the thickness of the polyaniline films was measured. For this purpose, the polyaniline films were etched by irradiation using Ga + ions. After focused ion beam (FIB) irradiation, the wafer was tilted and viewed with the scanning ion microscope Figure 2. A significant difference in film thickness, morphology and microstructure can be observed in Figure 2. Indeed, a short electrodeposition time, 30 s, corresponds to the beginning of the film growth, therefore the film is not yet structured and looks like a gel and the thickness is 310 nm.

The surface is not covered with a homogeneous film but only with short oligomers and nucleation nodules with significant hydration. As the deposition time increases, the chain length of the electrodeposited polymer increases, so the film appears much more structured. Indeed, after 10 min, the thickness (2250 nm) and the roughness increased with the electrodeposition time which is consistent with an increase in the quantity of PANi deposited on the surface and with a decrease in the adhesive properties. The thickness measured were not unreaslistic with previous studies. Indeed, a thickness range from 75nm to 10µm was already measured by cyclic voltamperometry depending on the number of cycle, potential sweep rate, electrolyte and PANi concentration. [START_REF] Popov | Comparative Study of Polyaniline (PANI), Poly(3,4-ethylenedioxythiophene) (PEDOT) and PANI-PEDOT Films Electrodeposited on Transparent Indium Thin Oxide based Electrodes[END_REF][START_REF] Dejeu | Morphological and Adhesive Properties of Polypyrrole Films Synthesized by Sonoelectrochemical Technique[END_REF] It has been demonstrated that the adhesive properties of PANi films can be modulated by varying the electrodeposition time and the phosphoric acid concentration.

Switching properties and commercial comparison

Now, it could be interesting to determine whether the adhesion behavior of the polymer film can be easily switched from a low adhesive state to a higher one. That is why, a weakly-adhesive film was prepared from a 0.5 mol/L H 3 PO 4 solution (deposition time: 1 min), before being immersed in a 2 mol/L H 3 PO 4 solution for 1, 3 and 10 min (Figure 3A). In Figure 3A, a variation of the force measurement was observed. Indeed, the value of the normal deflexion increases with the immersion time. So the adhesion of the resulting PANi films was so great, after 3 or 10 minutes of immersion. The particular shape of the curve for 3 and 10 minutes of immersion (increasing of the signal and return to zero for a distance close to -65 µM) was explained by the fact that the laser left the photodiode due to the high adhesion force for the cantilever stiffness. Similarly, the peak observed at -25µm and -35µm respectively was due to the return of the laser on photodiode during the dissociation of the cantilever with the PANI film. This time of 3 or 10 min corresponds to the time necessary for the protons and counter-ions to migrate from the solution to the polymer film.

These results indicate that it is possible to tune the adhesion of the polymer film and thus it can be envisaged to switch reversibly from a non-adhesive to an adhesive state. After that, the adhesive ability of PANi-H 3 PO 4 films was compared with commercial materials such as Gel-Pak, which is an UV tape commonly used to glue microelectronic systems, in particular to facilitate their travel and protect them. These AFM force measurements were performed with a cantilever having a stiffness of 14 N/m (Figure 3B). The results are promising since the shape of the curves shows that PANi-H 3 PO 4 film is more adhesive than the Gel-Pak and only slightly less adhesive than the UV Tape.

Tearing force

To further characterize the adhesion force of the PANi film, experiments were conducted in air to measure the tearing force between two millimeters planes (Figure 4). In this aim, silicon substrates were modified with a PANi-H 3 PO 4 film (using the optimal parameters: 2 mol/L and 30 s). Two substrates are perpendicular and a third one is deposited perpendicular to the first two. The contact area between the substrates is perpendicular (25 mm 2 on each side). This experiment led to the measurement of an important peel strength (around 70 mN). After the measurement, the spacer (third surface perpendicular to the other two) was repositioned in the same place and the experiment was continued until the measured force decreased drastically. Good repeatability of the experiments was noted for the four first experiments. After that, the peel strength decreased. The same result was obtained in repeated trials. PANi-H 3 PO 4 films therefore have the property of being both adhesive and repositionable. This last property is of great interest for the manipulation of electronic components before packaging.

Applications

Micromanipulation

To validate this new adhesive surface in 3D integration of electronic components, micromanipulation and conductivity tests were performed, Figure 5 (and movie in supporting information).

The surface modified by a PANi-H 3 PO 4 film was captured with an air noddle and deposited on another surface. Using this protocol, 6 surfaces were stacked and moved together (Figure 5 and movie). Interestingly, the stacking can be easily deleted by a simple immersion of the whole system in water inducing a separation of the surface. for PANi electrodeposition, it could be useful to check whether the electrodeposited PANi films are doped with the anions obtained by dissolving phosphoric acid in water before measuring the conductivity of PANI-H 3 PO 4 films. Thus, to evidence the presence of anions in PANi films, Glow Discharge Optical Emission Spectroscopy (GDOES) experiments were performed (Figure SI-4). Whatever the deposition time, the presence of phosphorous elements into the film was demonstrated, thus confirming the doping of the PANi films. The area under the curve corresponding to a PANi film electrodeposited for 10 s is greater than the one obtained for the curves corresponding to higher electrodeposition times (1 min and 10 min). This means that the thinnest film contains the highest amount of anions. Thus, it is believed that the thinnest film is the film with the highest doping level, and it may lead to the highest conductivity. The higher incorporation of anions may also be an additional explanation for the sticky property of the PANi film and may participate in the unstructuring of the film.

After that, the conductivity of PANi-modified FTO (Fluorine doped Tin Oxide) surfaces (grown in 2 mol/L H 3 PO 4 at 2 V during 10 s) was measured using the Van der Pauw procedure (VDP). VDP procedure required four point-like contacts (nickel contacts coated by a thin layer of gold and denoted A, B, C and D) at the edge. In order to obtain the resistivity value of the polymer films, several independent measurements were necessary. Initially, applying the current I AB on the contact pair A-B, the voltage difference U CD between the contacts C and D was measured (the current must be sufficiently small to avoid Joule heating of the sample). It is suitable to repeat this measurement for the opposite current I′ AB =-I AB to eliminate voltage contributions whose polarity does not depend on current direction (namely contact potentials). [START_REF] Patois | Effect of various parameters on the conductivity of free standing electrosynthesized polypyrrole films[END_REF] We also used the FTO because the conductivity surface of the silica was sufficient. For the PANi-H 3 PO 4 film, a value of 1.86x10 4 S.m -1 was measured which is consistent with those already published in the literature. [START_REF] Boara | Synthesis of Polyanilines with High Electrical Conductivity[END_REF][START_REF] Ayad | Phosphoric Acid and pH Sensors based on Polyaniline Films[END_REF][START_REF] Blinova | Polyaniline Prepared in Solutions of Phosphoric Acid: powders, Thin Film, and Colloidal Dispersions[END_REF] Indeed, for a polypyrole film a conductivity between 2x10 3 and 3x10 5 S.m -1 was found depending on the monomer concentration, electrolyte salt, potential and area of the deposited film. [START_REF] Patois | Effect of various parameters on the conductivity of free standing electrosynthesized polypyrrole films[END_REF] The experimental value was also ten and one hundred times higher than the previously PANi film synthesized with 0.4M 28 and 2M 27 of H 3 PO 4 respectively. The conductivity on a gold surface functionalized with PANi-H 3 PO 4 film was also tested experimentally with two CMS diodes, Figure 6, to demonstrate the application of theses surfaces on the electronic domain. The two diodes were connected between two electrodeposited films. When a potential of 2 V was applied to the gold substrates coated by the polyaniline film, the CMS diodes switched on, Figure 6B. When no potential was applied the diode switched off, Figure 6A (see also Supporting Information for the movie). So, the polyaniline film developed in this study is highly adhesive, conductive and stable.

Concerning this last point, the film was stored in ambient air for a long period (nearly 3 years), and AFM force measurements were performed regularly. No change in the profile of the AFM curve was noticed even after three years. Similarly, the experimental setup, carried out to test the conductivity with CMS diodes, is still efficient, one year after its completion. So, this new film must find its place as a connector and as a conductive adhesive for micro-components. This conductive adhesive could find applications in 3D integration of electronic components since the connections performed were connections between millimeter or submillimeter plans.

Conclusion

In conclusion, the nano-joining of two micro-objects is possible thanks to a chemical modification of a surface by polyaniline films. This chemical modification is performed by electrochemical polymerization of aniline monomers from phosphoric acid solutions. The electrical and mechanical properties are enough satisfactory to guarantee the conductivity and the junction of micro-objects for applications in microelectronics, for example, and more generally for the assembly and connection of micro-objects. The object can be repositionable 4 times before the properties of the polyaniline film decrease. The reversibility of the link is also possible by immersing the microsystem in an acid solution to increase the adhesion or in ultrapure water to dissolve the film. Moreover, the stability of the film is important since the adhesive forces remain similar after 3 years. In the future, this new polymer film will betested in real conditions, to check the feasibility of its use in industry, in particular as a glue which could allow the assembly of (Bio)MEMS and microcomponents.

Figure 1 .

 1 Figure 1. Pull-off forces measurements obtained with polyaniline films prepared: A) in different

Figure 2 .

 2 Figure 2. SIM images and FIB etching of the PANi-H3PO4 films ([H3PO4] = 2 mol/L) for

Figure 3 .

 3 Figure 3. A) Influence of the immersion time in a 2 mol/L H 3 PO 4 solution of the PANi film

Figure 4 .

 4 Figure 4. Measurements of peel strength on PANi -H 3 PO 4 film (2 mol/L, electropolymerisation

ABFigure 5 .

 5 Figure 5. Micromanipulation of surfaces coated by PANI-H 3 PO 4 (2 mol/L, 30s): A) catching

Figure 6 .

 6 Figure 6. Conductivity test between stabilized and charged CMS diodes using PANi-H 3 PO 4

Table 1 .

 1 -1) in the polymer film, thus confirming the very adhesive character of the PANi-H 3 PO 4 film. The value obtained with the monoacid was concordant with values previously obtained by cyclic voltamperometry with PANi 20 film or with other conducting polymer such as PEDOT,[START_REF] Lupu | Morphological characterization and analytical application of poly(3,4-ethylenedioxythiophene)-Prussian blue composite films electrodeposited in situ on platinum electrode chips[END_REF] polypyrole[START_REF] Dejeu | Morphological and Adhesive Properties of Polypyrrole Films Synthesized by Sonoelectrochemical Technique[END_REF] or poly(pphenylenediamine).[START_REF] Cot | Electrosynthesis and Characterization of Polymer Films on Silicon substrates for Applications in Micromanipulation[END_REF] Since the best results were obtained with PANI+H 2 SO 4 and PANI-H 3 PO 4 films, the influence of the H 2 SO 4 and H 3 PO 4 concentrations was also tested by varying the acid concentrations from 0.5 mol/L to 2 mol/L (Figure1Band Table1). Influence of the acid concentration on the pull-off force values (nN) (stiffness of the

	Acid	H 2 SO 4			H 3 PO 4
	Concentration (mol/L)	0.5	1	2	0.5	1	2
	Pull-off (nN)	-29	-575	-397	-411	> -1000 > -1500
	cantilever: 0.3 N/m).
	Figure 1B and Table

ASSOCIATED CONTENT Supporting Information. The following files are available free of charge: Image of the AFM TIP during force spectroscopy on PANI-H3PO4 film, AFM PANI-H3PO4 film morphology, X-Ray Diffractometry, Glow Discharge Optical Emission Spectroscopy of PANI-H3PO4 as a function of the electrodeposited time, movie of micromanipulation and stacking of 6 surfaces and movie of CMS diodes adhesion and switch on when current applied through this new polymer surface. ST Institute, UMR CNRS 6174) for assistance (respectively Glow Discharge Optical Emission Spectroscopy and SIM/FIB experiments). This work was supported by the EU under FAB2ASM (contract FoF-NMP-2010-260079): Efficient and Precise 3D Integration of Heterogeneous Microsystems from Fabrication to Assembly, by Région Franche-Comté under FIMICAP (contract 2011C-07333): Fiabilisation du microassemblage et mise au point de capteurs intelligents par ingénierie des surfaces.

Funding Sources

EU under FAB2ASM (contract FoF-NMP-2010-260079), Région Franche-Comté (contract

2011C-07333)

ACKNOWLEDGMENT

The authors acknowledge Dr. V. Moutarlier (Université Bourgogne Franche-Comté, UTINAM Institute, UMR CNRS 6213) and Dr. R. Salut (Université Bourgogne Franche-Comté, FEMTO-

AUTHOR INFORMATION

Corresponding Author

*Jérôme Dejeu: jerome.dejeu@univ-grenoble-alpes.fr. Give contact information for the author(s) to whom correspondence should be addressed.

Author Contributions

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