François Bordeyne
email: francois.bordeyne@sb-roscoff.fr

Aline Migné

Dominique Davoult

Metabolic activity of intertidal Fucus spp. communities: evidence for high aerial carbon fluxes displaying seasonal variability

Metabolic activity of intertidal Fucus spp. communities: evidence for high aerial carbon fluxes displaying seasonal variability

INTRODUCTION

Communities dominated by canopy-forming Phaeophyceae are particularly widespread along intertidal rocky shores of temperate regions. Such communities are usually considered to be very productive, although their metabolism has rarely been studied at the community scale [START_REF] Golléty | Benthic metabolism on a sheltered rocky shore: Role of the canopy in the carbon budget[END_REF][START_REF] Tait | Primary productivity of intertidal macroalgal assemblages: comparison of laboratory and in situ photorespirometry[END_REF]). Yet, these communities are constituted of numerous species [START_REF] Rindi | Composition and spatio temporal variability of the epiphytic macroalgal assemblage of Fucus vesiculosus Linnaeus at Clare Island, Mayo, western Ireland[END_REF][START_REF] Kersen | Epiphytes and associated fauna on the brown alga Fucus vesiculosus in the Baltic and the North Seas in relation to different abiotic and biotic variables: Epiphytes and associated fauna on brown alga[END_REF]Migné et al. 2015b), giving rise to many interactions. For instance, numerous trophic relationships occur in these systems [START_REF] Golléty | Complexity of the food web structure of the Ascophyllum nodosum zone evidenced by a δ13C and δ15N study[END_REF][START_REF] Duarte | Effects of alga Fucus serratus decline on benthic assemblages and trophic linkages at its retreating southern range edge[END_REF], as well as competition for light and space [START_REF] Underwood | Experimental ecology of rocky intertidal habitats: what are we learning?[END_REF]. Such interactions could lead to large variability in the metabolism of primary producers. They must for instance synthesize defence molecules against grazers, epibionts and/or biofilms [START_REF] Brock | Chemical inhibition of barnacle larval settlement by the brown alga Fucus vesiculosus[END_REF][START_REF] Wahl | Marine Hard Bottom Communities: Patterns, Dynamics, Diversity, and Change[END_REF] or are subjected to a reduced light due to epibionts, leading to a reduction of photosynthetic activity [START_REF] Sand-Jensen | Effect of epiphytes on eelgrass photosynthesis[END_REF][START_REF] Oswald | The effect of encrusting Bryozoans on the photosynthetic activity of Fucus Serratus L[END_REF]). On the contrary, [START_REF] Tait | Dynamics of productivity in naturally structured macroalgal assemblages: importance of canopy structure on light-use efficiency[END_REF] demonstrated that the structural differences of primary producers, but also their location in the community assemblage could lead to an enhancement of the global primary production. Therefore, all these interactions play a fundamental role and need to be accounted for when investigating community metabolism, as highlighted by Migné et al. (2015a).

These communities also experiment large changes in abiotic conditions at various temporal scales. The most drastic change occurs at the tidal scale, due to the alternation of immersion and emersion periods. Although usually thought as stressful for intertidal organisms, emersion periods can, however, be a very useful time for algae to do photosynthesis [START_REF] Golléty | Benthic metabolism on a sheltered rocky shore: Role of the canopy in the carbon budget[END_REF], with some of them exhibiting the major part of their production when emersed [START_REF] Quadir | The effects of emergence and submergence on the photosynthesis and respiration of marine macrophytes[END_REF]). Thus, investigations of intertidal communities' metabolic activity should not neglect the low tide periods.

The study of [START_REF] Golléty | Benthic metabolism on a sheltered rocky shore: Role of the canopy in the carbon budget[END_REF] is to our knowledge the first one dealing with in situ metabolic activity of Phaeophycean dominated communities under emersed conditions. This pioneer study showed that primary production and respiration rates of the sheltered midintertidal Ascophyllym nodosum community followed a seasonal trend related to changes in light availability and temperature, and that the canopy and its epibionts were responsible for the huge majority of community' carbon fluxes.

Following the principles of the intertidal zonation of canopy-forming species and the general relationship between diversity of marine species and tidal level [START_REF] Raffaelli | Intertidal ecology[END_REF][START_REF] Molina-Montenegro | Positive associations between macroalgal species in a rocky intertidal zone and their effects on the physiological performance of Ulva lactuca[END_REF], it could be expected that the tidal level at which the communities are established is of paramount importance in controlling the magnitude and the seasonal variability of their metabolism, as well as the contribution (and its seasonal variability) of the canopy to the total metabolism. Furthermore, the hydrodynamism to which the communities are exposed and specific environmental conditions (e.g. nutrient depletion), could as well have an influence on these parameters. In the current context of harvesting of Phaeophyceae along European West coasts [START_REF] Jacob | Etats des lieux de la filière des macro-algues en Europe[END_REF]), it appears crucial to better understand the year-round dynamic of the communities they form, but also the way these canopy-forming species contribute to this dynamic.

In addition, some essential information on algae-environment interactions could be obtained considering the elemental composition of canopy-forming Phaeophyceae, which was shown to reflect both assimilation and use of nutrients and organic compounds by the algae [START_REF] Chapman | Seasonal growth in Laminaria longicruris: Relations with dissolved inorganic nutrients and internal reserves of nitrogen[END_REF][START_REF] Chapman | Seasonal growth in Laminaria longicuris: Relations with reserve carbohydrate storage and production[END_REF]. This appears to be particularly attractive in intertidal systems since algae have to rely on their own nitrogen reserves when emerged [START_REF] Pedersen | Nutrient control of estuarine macroalgae: growth strategy and the balance between nitrogen requirements and uptake[END_REF].

Mid-and low-mid intertidal communities dominated respectively by canopy-forming algae

Fucus vesiculosus Linnaeus and Fucus serratus Linnaeus are particularly widespread along European coasts [START_REF] Jueterbock | Climate change impact on seaweed meadow distribution in the North Atlantic rocky intertidal[END_REF]). This study focused on the metabolic activity of these two adjacent communities, using in situ carbon fluxes measurements performed at the onset of emersion periods, under saturating irradiance. We hypothesized that (1) while following temporal variations, their gross primary production and respiration remained substantial all over the year, that (2) canopies consistently acted as the major contributor of total carbon fluxes in the communities, with this contribution following seasonal pattern, and that (3) the nitrogen and organic carbon contents of the canopies also varied with seasons and environmental conditions. Finally, we also hypothesized that (4) the lower shore community exhibited a higher metabolic competitiveness than the upper shore community, in accordance with the principles of vertical zonation of canopy species.

MATERIALS & METHODS

Study site

This study was performed on F. vesiculosus and F. serratus communities established on an intertidal semi-sheltered boulder reef located in front of the Station Biologique de Roscoff (Brittany, France) (48°43.743'N, 3°59.407'W), in the southwest part of the English Channel.

On this reef, these communities form dense and multi-layer assemblages which frequently cover 100% of the substratum, making it an ideal place to study their metabolism. This shore is subjected to semi-diurnal tidal cycle with maximal range of about 9 m. It presents a typical vertical distribution of communities dominated by canopy-forming Phaeophyceae, where F. vesiculosus community is characteristic of the mid-intertidal (3.0 to 4.0 m above chart datum, corresponding to an annual emerged time of 20-35%) while F. serratus community is characteristic of the low mid-intertidal (2.5 to 3.0 m above chart datum, corresponding to an annual emerged time of 15-20%).

Environmental conditions

This site, located in the temperate part of Northern hemisphere, displays a seasonal day:night cycle varying from 9:15 in late December to 16:8 in late June. It is also subjected to seasonal variations of irradiance and temperature. The weather station of Brest-Guipavas (located at approx. 40 km of our study site) provided monthly maximal instantaneous irradiance (Maxirr, in W m -2 ) and monthly mean air temperature (Tair, in °C) datasets (October 2012-December 2013). The SOMLIT network (sampling site: Estacade, Roscoff, located at approx. 600 m of our study site) provided monthly mean seawater temperature (Tseawater, in °C) and monthly means seawater nutrient concentrations (NH4 + , NO3 2-, NO2 -and PO4 3-, in µmol L -1 ) datasets (October 2012 -December 2013).

Benthic metabolism measurements and sampling

Measurements of benthic metabolism of F. vesiculosus and F. serratus communities were performed once a month from October 2012 to December 2013, with the exception of December 2012. They were always done at the onset of emersion of a spring tide (around midday) to avoid a desiccation effect on metabolic rates. At each occasion, three intra-site replicates (2 to 5 m distance between each) of a 0.09 m² rock area were haphazardly selected for both communities (only two replicates in November 2013 for F. serratus community). A replicate included one to several Fucus thalli (> 25 cm long) with the associated community, which was essentially made-up of Fucus epibionts (i.e. algae and sessile invertebrates directly attached to the Fucus, see [START_REF] Wahl | Marine Hard Bottom Communities: Patterns, Dynamics, Diversity, and Change[END_REF] and biofilms, as well as sub-canopy and encrusting macroalgae, rocky and sedimentary microphytobenthos and invertebrates, including the mobile ones on Fucus fronds. Each area was then enclosed in a custom-built incubation chamber (17.7 L) made of clear Plexiglas, connected to an infrared CO2 gas analyzer (LiCor Li-820) in closed air circuit. Air motion of 1 L min -1 was ensured by a pump in this closed circuit. CO2 fluxes measurements were performed at the rock-air interface, as described by [START_REF] Migné | A closed-chamber CO2-flux method for estimating intertidal primary production and respiration under emersed conditions[END_REF], at ambient light and in darkness by covering up the chambers. These measurements allowed estimating the total community net primary production (NPPt) and the total community respiration (CRt), respectively, assuming that CO2 fluxes measured in darkness after abrupt transition from ambient light reflected the respiration activity of the communities (del Giorgio and Williams 2005). Incubations were sufficiently short (i.e. 5 -10 min) to avoid feedback of temperature and gas concentration changes on metabolic rates.

Total community gross primary production (GPPt) was calculated as GPPt = NPPt + CRt. CO2 fluxes were calculated from recorded data using the slope of CO2 concentration (µmolCO2 molair -1 ) against time (min). Therefore, GPPt and CRt were expressed in carbon unit (mgC m -2 h -1 ) assuming a molar volume of 22.4 L mol -1 at standard temperature and pressure and a molar mass of 12 gC molCO2 -1 . Finally, the community metabolic balance was calculated each month for both communities as the ratio of CRt to GPPt.

After performing this first set of light and dark incubations, Fucus thallus and the associated epibionts and biofilms were removed from the incubation chambers, while mobile fauna being on the fronds was left inside of the chambers with the remaining organisms (including algae and invertebrates). A second set of light and dark incubations was performed on this residual community, in order to estimate residual community net primary production (NPPr) and residual community respiration (CRr). Residual community gross primary production (GPPr) was calculated following the formula used previously (GPPr = NPPr + CRr). These measurements allowed to determine the contribution of the canopy to the carbon fluxes of the community (GPPcan = GPPt -GPPr; CRcan = CRt -CRr).

Removed canopies were brought back to the laboratory to measure their dry masses after drying for 48 h at 60 °C. Dry masses were used to calculate the respiration rate of canopies (Rcan/DMcan) and their productivity (GPPcan/DMcan) expressed in mgC gDM -1 h -1 .

Incident photosynthetically available radiation (400-700 nm) was recorded (LiCor QuantumSA-190, µmol photons m -2 s -1 ) close to the incubation chambers. Mean values during measurements at ambient light varied from 465 µmol photons m -2 s -1 in December 2013 to 2094 µmol photons m -2 s -1 in June 2013. Measurements were thus assumed to be done under saturating irradiance, based on the average value of 291 µmol photons m -2 s -1 given for the onset of light saturation of coastal macroalgal communities [START_REF] Middelboe | Highly predictable photosynthetic production in natural macroalgal communities from incoming and absorbed light[END_REF].

Carbon and nitrogen content

Just before drying the removed canopies as mentioned previously, samples (≈ 1g of fresh weight) were taken from Fucus fronds of each replicate, to assess their C and N contents.

These sampling were done from January to December 2013. Samples were rinsed with distilled water before being dried (60°C, 48 h) and ground to a fine powder using a mortar and pestle. C and N mass percentages were determined at the Station Biologique de Roscoff using a Flash EA 1112 CHN analyzer (ThermoFinningan) calibrated against nicotinamide and acetanilide reference materials.

Statistical analysis

Seasonal patterns of GPPt, CRt, CRt/GPPt, GPPcan/DMcan, Rcan/DMcan, C and N contents were highlighted by fitting a sinusoidal curve to the monthly mean values:

𝑦 = 𝑎 + 𝑏 𝑠𝑖𝑛( 2𝜋 365 𝑥 + 𝑐)
where y is the predicted value of the considered parameter, and x the time in days. F-test was used to test the fit, using R software, version 3.1.2 (R Core Team, 2014).

Comparisons of the two communities (community metabolism, canopy metabolism and C and N contents) were done using Wilcoxon signed rank test on the mean values for each month, performed with the R software, version 3. 1.2 (R Core Team, 2014).

Correlations between monthly maximal instantaneous irradiance and PAR recorded during light incubations, and between N contents and seawater N-nutrients pool, were tested with Pearson coefficient using the R software, version 3.1.2 (R Core Team, 2014).

For both communities, the relationship between GPPt or CRt and environmental variables was analysed by linear multiple regression (Best procedure and AIC selection criteria) with pvalues obtained by permutation, using PRIMER software, version 6.1.12 associated with PERMANOVA+ for PRIMER, version 1.0.2. Predictor variables of GPPt consisted of monthly maximal instantaneous irradiance, monthly means of air and seawater temperature and of nutrients seawater concentrations (NO3 -, NO2 -, NH4 + and PO4 2-), and canopy dry mass.

Predictor variables of CRt consisted of monthly means of air and seawater temperature, and canopy dry mass.

RESULTS

Environmental conditions

Monthly

Benthic metabolism

Fucus vesiculosus community exhibited a gross primary production (GPPt, mean ± SE) ranging from 341.6 ± 41.0 mgC m -2 h -1 in March 2013 to 962.9 ± 195.4 mgC m -2 h -1 in June 2013 and respiration (CRt, mean ± SE) ranging from 102.8 ± 27.1 mgC m -2 h -1 in February 2013 to 562.7 ± 117.1 mgC m -2 h -1 in August 2013. GPPt of the Fucus serratus community ranged from 440.0 ± 80.6 mgC m -2 h -1 in January 2013 to 1104.4 ± 101.9 mgC m -2 h -1 in August 2013 and CRt ranged from 74.3 ± 7.9 mgC m -2 h -1 in January 2013 to 475.9 ± 65.8 mgC m -2 h -1 in August 2013 (Fig 3). Both GPPt and CRt of F. vesiculosus and F. serratus communities followed a seasonal pattern significantly fitted by sinusoidal curves (Table 1), with minimal values occurring during winter and maximal values occurring during summer, even though a metabolic depression was observed in July. While they followed the same seasonal fluctuations, the F. vesiculosus community exhibited significantly lower GPPt than F.

serratus community (20% lower in average) but significantly higher CRt (25% higher in average) (Table 2). The metabolic balance (i.e. CRt/GPPt) was always lower than 1 during our measurements under saturating light conditions. It followed a significant seasonal pattern too (Table 1) with minimal values in winter and maximal values during late summer and early fall (from 0.23 to 0.73 for F. vesiculosus community and from 0.16 to 0.45 for F. serratus community) (Fig 4). The metabolic balance was significantly higher for the F. vesiculosus community (Table 2), in agreement with the observed differences for both GPPt and CRt of these communities.

The variability of GPPt was best explained by NO3 -seawater concentration and DMcan taken together for the F. vesiculosus community whereas it was best explained by Maxirr, Tair and all seawater nutrient concentrations for the F. serratus community (Table 3). The variability of CRt was best explained by the three predictor variables taken together (Tair, Tseawater and DMcan) for the F. vesiculosus community whereas it was best explained by Tair alone for the F.

serratus community (Table 3).

The metabolism of these two communities was consistently dominated by the canopies (i.e.

the dominant species and their epibionts) since they accounted for the huge majority of the global CO2 fluxes (77-97% of CRt and 83-95% of GPPt). Nevertheless, no seasonal pattern was observed for the relative contribution of canopies, neither for respiration nor for primary production (F-test, p > 0.05). The productivity (GPPcan/DMcan, mean ± SE) of F. vesiculosus canopy ranged from 0.172 ± 0.029 mgC gDM -1 h -1 in November 2013 to 0.473 ± 0.072 mgC gDM -1 h -1 in June 2013 and its respiration rate (Rcan/DMcan, mean ± SE) from 0.043 ± 0.010 mgC gDM -1 h -1 February 2013 to 0.217 ± 0.053 mgC gDM -1 h -1 in June 2013. The productivity of F. serratus canopy ranged from 0.083 mgC gDM -1 h -1 in November 2013 to 0.740 ± 0.112 mgC gDM -1 h -1 in April 2013 and its respiration rate from 0.040 ± 0.008 mgC gDM -1 h -1 in November 2012 to 0.240 ± 0.027 mgC gDM -1 h -1 in June 2013 (Fig 5). As for whole communities, both productivity and respiration rate of the canopies followed a significant seasonal pattern (Table 1), except for the F. vesiculosus productivity (F-test, p > 0.05). Finally, the productivity of F. vesiculosus was significantly lower than the one of F. serratus (30% lower in average). No significant difference was found for respiration rate (Table 2).

Carbon and nitrogen contents

C and N contents measured from Fucus thallus samples exhibited a seasonal pattern significantly fitted by sinusoidal curves (Fig 6, Table 1). For both Fucus species, C content was the lowest in autumn and the highest in summer, ranging from 37.5 to 39.7% of DM for F. vesiculosus and from 37.8 to 41.7% of DM for F. serratus. On the contrary, N content was the lowest in summer and the highest in spring, ranging from 0.88 to 2.39% of DM for F.

vesiculosus and from 0.99 to 2.16% of DM for F. serratus (Fig 6). F. serratus held significantly higher C content but lower N content than F. vesiculosus (Table 2). N content was significantly correlated with seawater N-nutrient pool, both for F. vesiculosus (r = 0.694, p = 0.012) and F. serratus (r = 0.665, p = 0.018).

DISCUSSION

By measuring in situ metabolism of intertidal Fucus vesiculosus and F. serratus communities under saturating irradiance, this study highlights that, whatever the season, these communities display a substantial metabolic activity at the beginning of emersion periods. Our results of metabolic activity overlapped those previously obtained for Ascophyllum nosodum community (188-846 mgC m -2 h -1 for GPPt and 122-616 mgC m -2 h -1 for CRt, [START_REF] Golléty | Benthic metabolism on a sheltered rocky shore: Role of the canopy in the carbon budget[END_REF]. Consequently, in such latitudes, intertidal Phaeophycean-dominated communities appear to be photosynthetically active and efficient throughout the year, even though measurements were performed under emerged conditions, which are often considered to be very stressful (Wahl et al. 2011, and references therein). This study also confirms that Phaeophycean-dominated communities are the most productive systems of intertidal habitats.

Indeed, F. vesiculosus and F. serratus communities displayed considerable higher year-round mean GPPt (in mgC m -2 h -1 ) than those obtained in studies using the same in situ measurement method, i.e. 7 to 9 times higher than a Zostera noltei bed [START_REF] Ouisse | Seasonal variations of community production, respiration and biomass of different primary producers in an intertidal Zostera noltii bed (Western English Channel, France)[END_REF]), 15 to 20 times higher than a muddy-sand community [START_REF] Migné | In situ measurements of benthic primary production during emersion: seasonal variations and annual production in the Bay of Somme (eastern English Channel, France)[END_REF]) or 35 to 44 times higher than a sandy beach community [START_REF] Hubas | Factors controlling benthic metabolism during low tide along a granulometric gradient in an intertidal bay (Roscoff Aber Bay, France)[END_REF]. All these studies were conducted at the community scale, integrating the interactions between species and their responses to a broad range of local conditions, especially of incident light [START_REF] Middelboe | Highly predictable photosynthetic production in natural macroalgal communities from incoming and absorbed light[END_REF][START_REF] Tait | Dynamics of productivity in naturally structured macroalgal assemblages: importance of canopy structure on light-use efficiency[END_REF]Migné et al. 2015a), and accounting for the contribution of biofilms which is known to be highly significant, both for autotrophic and heterotrophic activities (Golléty and Crowe

2013).

As expected, both GPPt and CRt exhibited a seasonal pattern, with minima in winter and maxima in summer, in each community. Such seasonal pattern is in agreement with the general idea that light availability and temperature act as the major drivers of community metabolism [START_REF] Davison | Environmental effects on algal photosynthesis: Temperature[END_REF][START_REF] Golléty | Benthic metabolism on a sheltered rocky shore: Role of the canopy in the carbon budget[END_REF][START_REF] Ouisse | Seasonal variations of community production, respiration and biomass of different primary producers in an intertidal Zostera noltii bed (Western English Channel, France)[END_REF][START_REF] Kemp | Metabolic balance between ecosystem production and consumption[END_REF].

However, inorganic seawater nutrient concentrations also act as a key factor of metabolic regulation of marine systems. Photosynthesis can be limited by low nutrient concentrations which affect algal growth and lead to changes in the structure and the food webs of the communities [START_REF] Bosman | The influence of coastal upwelling on the functional structure of rocky intertidal communities[END_REF]. In this study, a gross primary production depression was observed in July for both F. vesiculosus and F. serratus communities and matched with the lowest records in seawater nutrient concentrations close to the study site, suggesting that a nutrient limitation has occurred during this period. The same depression in primary production was observed in summer for the A. nodosum community [START_REF] Golléty | Benthic metabolism on a sheltered rocky shore: Role of the canopy in the carbon budget[END_REF]), also matching with the lowest seawater nutrient concentrations (SOMLIT network data), strengthening this nutrient limitation hypothesis. Furthermore, the multiple correlations analyses showed that the variability of GPPt was best explained by incident light, air temperature and seawater nutrient concentrations in F. serratus community, and by NO3 - concentration and canopy dry mass in F. vesiculosus community. Surprisingly, neither incident light nor temperature appeared in the best model of regressions for GPPt of F. vesiculosus community. It can be hypothesized that as the F. vesiculosus community is located upper on the shore than the F. serratus community, it has a more restricted access to seawater nutrients, inducing a higher susceptibility to nutrient limitation, and particularly to nitrates, which dominate the nutrient pool.

The GPPt depression observed in July could also be related to the highest irradiances as an excess of light could act as significant stress for photo-autotrophic organisms (e.g. [START_REF] Huppertz | Photoinhibition of photosynthesis in the marine brown alga Fucus serratus as studied in field experiments[END_REF][START_REF] Häder | Photoecophysiology of marine macroalgae[END_REF][START_REF] Bischof | Effects of ultraviolet radiation on photosynthesis and related enzyme reactions of marine macroalgae[END_REF]. However, [START_REF] Creis | Constitutive or inducible protective mechanisms against UV-B radiation in the brown alga Fucus vesiculosus? A study of gene expression and phlorotannin content responses[END_REF] have demonstrated that the exposition of F. vesiculosus individuals to UV-B conditions approximately two times more important than the maximal conditions occurring in Brittany only led to a slight reduction in the maximal efficiency of PSII after 1 and 4 weeks of chronic exposure. In addition, as we were working at the community scale, only the upper layer of the community (mainly composed of Fucus spp.) was exposed to high light, and could have suffer from it. This layer acted as a protective layer for the other components of the community, which benefited from the attenuation of incident light and could have maintained a high metabolic activity. The canopy biomass appeared to be a good predictor of metabolic variations of the F. vesiculosus community GPPt. Binzer and Sand-Jensen (2002) demonstrated that at high density, photosynthetic tissues have the ability to fully use incident light. This would imply an optimal distribution and utilisation of incident light among all photosynthetic tissues in the community. Nevertheless, at very high canopy density, photosynthetic activity could be limited under high light due to uneven distribution of photons (Binzer and Sand-Jensen 2002).

During our measurements, F. vesiculosus and F. serratus communities always performed as autotrophic entities, with a CRt/GPPt ratio strictly lower than 1. Interestingly, this metabolic balance fluctuated according to a seasonal pattern, as did photosynthesis and respiration. Such pattern could be related to the seasonal fluctuations of the biomass of these communities, as the metabolic balance was maximal in summer when maximal biomass accumulation occurred. Indeed, community gross primary production is limited at high canopy density while it respiration rate is linearly related to its density (Binzer and Sand-Jensen 2002).

Finally, this seasonal variation implies that metabolic balance has to be used with caution if measured punctually, particularly when dealing with the annual carbon budget of an ecosystem.

By removing F. vesiculosus and F. serratus canopies, we showed that they were responsible for the great majority of CO2 fluxes (77-97% of CRt and 83-95% of GPPt). This confirms that the role of canopy-forming macroalgae in the community is not restricted to facilitation for associated species, but is also fundamental in the community metabolism, all over the year.

No seasonal pattern emerged however, even though diversity and abundance of epibionts and others species, like the opportunistic ones, are known to exhibit a strong seasonal variability (e.g. [START_REF] Rindi | Composition and spatio temporal variability of the epiphytic macroalgal assemblage of Fucus vesiculosus Linnaeus at Clare Island, Mayo, western Ireland[END_REF]. Being substantial productive entities, canopy-forming algae can control the community processes they dominate, to constitute one of the major suppliers of organic carbon for coastal food webs [START_REF] Paine | Trophic control of production in a rocky intertidal community[END_REF][START_REF] Golléty | Benthic metabolism on a sheltered rocky shore: Role of the canopy in the carbon budget[END_REF]. By this way, the global current decline of canopy-forming macroalgae (Airoldi and Beck 2007; [START_REF] Duarte | Recent and historical range shifts of two canopy-forming seaweeds in North Spain and the link with trends in sea surface temperature[END_REF]) and the predicted shift of European F. vesiculosus and F. serratus communities northward [START_REF] Jueterbock | Climate change impact on seaweed meadow distribution in the North Atlantic rocky intertidal[END_REF][START_REF] Jueterbock | Thermal stress resistance of the brown alga Fucus serratus along the North-Atlantic coast: acclimatization potential to climate change[END_REF] are likely to have dramatic effects on the dynamics of coastal ecosystems [START_REF] Thompson | Rocky intertidal communities: past environmental changes, present status and predictions for the next 25 years[END_REF].

As major metabolic component of their communities, the F. vesiculosus and F. serratus canopies rationally exhibited high productivity and respiration rate (in mgC gDM -1 h -1 ). These rates were found to be in the same range as those obtained for other slow-growing

Phaeophyceae (e.g. [START_REF] Quadir | The effects of emergence and submergence on the photosynthesis and respiration of marine macrophytes[END_REF][START_REF] Williams | High and dry: Variation in net photosynthesis of the intertidal seaweed Fucus gardneri[END_REF][START_REF] Golléty | Benthic metabolism on a sheltered rocky shore: Role of the canopy in the carbon budget[END_REF]). Yet, some previous studies on Fucus spp. indicated productivity 2 to 10 times higher than our results [START_REF] Brinkhuis | Photosynthesis and respiration of exposed saltmarsh fucoids[END_REF][START_REF] Maberly | Contribution of air and water to the carbon balance of Fucus spiralis[END_REF][START_REF] Kawamitsu | Photosynthesis and carbon storage between tides in a brown alga, Fucus vesiculosus[END_REF] whereas respiration rates were quite similar. These high productivities might be explained by the use of fucoids segments under laboratory conditions, contrasting with our in situ conditions, where the canopies consisted of multi-layer assemblages subjected to strong selfshading, at least for the basal layers. Furthermore, canopies were considered as a multispecific complex, composed of Fucus thalli associated with their epibionts (e.g. Chlorophyta, Rhodophyta, Bryozoa, Tunicates, etc.) and biofilms. These species probably acted as productivity inhibitors due to light attenuation, at least for the heterotrophic ones, as demonstrated by [START_REF] Sand-Jensen | Effect of epiphytes on eelgrass photosynthesis[END_REF] on eelgrass leaves. Considering this, our results appeared to be realistic about what really happened on the field. As for whole communities, a seasonal pattern of productivity and respiration rate was observed, except for F. vesiculosus productivity, confirming the previous hypothesis about metabolic drivers. The C contents of both species followed a clear seasonal pattern, as those well described for Laminaria species (e.g. [START_REF] Gagné | Seasonal patterns of growth and storage in Laminaria longicruris in relation to differing patterns of availability of nitrogen in the water[END_REF][START_REF] Gevaert | Carbon and nitrogen content of Laminaria saccharina in the eastern English Channel: biometrics and seasonal variations[END_REF], with the highest C content in summer, when C assimilation by photosynthesis exceeds C requirements, leading to a storage of carbohydrates [START_REF] Gomez | Seasonal changes in C, N and major organic compounds and their significance to morpho-functional processes in the endemic Antarctic brown alga Ascoseira mirabilis[END_REF][START_REF] Lehvo | Growth of Fucus vesiculosus L.(Phaeophyta) in the northern Baltic proper: energy and nitrogen storage in seasonal environment[END_REF]) and the lowest in winter when photosynthesis was not sufficient to supply C requirements, leading to a gradual use of carbohydrate reserves [START_REF] Hatcher | An annual carbon budget for the kelp Laminaria longicruris[END_REF][START_REF] Lehvo | Growth of Fucus vesiculosus L.(Phaeophyta) in the northern Baltic proper: energy and nitrogen storage in seasonal environment[END_REF]. Any variation in C content due to reproduction investment was detected while both species are known to exhibit seasonal trend in their reproductive strategy [START_REF] Knight | A biological study of Fucus vesiculosus L. and F. serratus L[END_REF][START_REF] Brenchley | Carbon and nitrogen allocation patterns in two intertidal fucoids: Fucus serratus and Himanthalia elongata (Phaeophyta)[END_REF]). Yet, this reproductive process is known to be spread over several months [START_REF] Knight | A biological study of Fucus vesiculosus L. and F. serratus L[END_REF], with resources investment rarely exceeding 55% and fertile individuals found year-round [START_REF] Brenchley | Carbon and nitrogen allocation patterns in two intertidal fucoids: Fucus serratus and Himanthalia elongata (Phaeophyta)[END_REF]. The N contents also followed seasonal variations, which are well documented in the literature too (e.g. Asare and Harlin 1983;[START_REF] Carlson | Seasonal variation in growth, reproduction and nitrogen content of Fucus vesiculosus L. in the Öresund, Southern Sweden[END_REF][START_REF] Brenchley | Carbon and nitrogen allocation patterns in two intertidal fucoids: Fucus serratus and Himanthalia elongata (Phaeophyta)[END_REF][START_REF] Young | Seasonal variations in nitrate reductase activity and internal N pools in intertidal brown algae are correlated with[END_REF]). For both species, N content was highest in winter and lowest in summer, and was significantly correlated with seawater N-nutrient concentrations, as already observed by [START_REF] Young | Seasonal variations in nitrate reductase activity and internal N pools in intertidal brown algae are correlated with[END_REF]. This is also consistent with the N turnover rate of approximately 2 to 3 weeks exhibited by F. vesiculosus [START_REF] Viana | Experimental assessment of the macroalgae Ascophyllum nodosum and Fucus vesiculosus for monitoring N sources at different time-scales using stable isotope composition[END_REF]. High seawater nutrient availability in winter allowed a substantial N assimilation and storage by Fucus species. Stored N is gradually used to support an active growth during spring and summer, when N-seawater is depleted. Nutrients limitation of metabolic activity, supposed to occur at community scale, is strengthened by N contents values. Indeed, [START_REF] Pedersen | Nutrient control of estuarine macroalgae: growth strategy and the balance between nitrogen requirements and uptake[END_REF] have calculated a critical N tissue content of 1.71% of DM to support maximum growth rate for F. vesiculosus.

Lower contents were observed during several months in our F. vesiculosus and F. serratus

individuals. Yet, we could expect a seasonal fluctuation of critical N concentration and/or N-requirements for these species, which could restrict N-limitation during summer months, when both N tissue and seawater nutrient concentrations were the lowest.

This study allowed the comparison of the metabolism, at the beginning of the emersion period and under saturating irradiance, of two adjacent communities along the vertical zonation. The upper community exhibited the lowest GPPt and the highest CRt, and as a consequence, the highest metabolic balance. The canopy productivity was also lower for F. vesiculosus than for F. serratus. This is consistent with the C content values, significantly lower in F. vesiculosus than in F. serratus. Differences in C content were particularly pronounced during summer and fall, when a higher net primary production for F. serratus was observed. [START_REF] Moebus | Rehydration of desiccated intertidal brown algae: release of dissolved organic carbon and water uptake[END_REF] have evidenced that lower C contents in upper species could be a consequence of a higher organic C release during low tide in response to a higher desiccation degree. Finally, F. serratus appeared to be more competitive than F. vesiculosus in a metabolic point of view, in agreement with the vertical zonation rules [START_REF] Raffaelli | Intertidal ecology[END_REF]. The latter species has probably achieved more efficient, but costly, protecting mechanisms to deal with abiotic stresses, such as temperature or desiccation, to the detriment of its competitiveness. This could cause high differences in the annual carbon balance of adjacent communities and needs to be investigated in details further.

Conclusion and perspectives

This study showed that the two widespread Fucus vesiculosus and F. serratus communities exhibited high rates of both gross primary production and respiration all over a year, confirming their importance in the intertidal area. Nevertheless, as our measurements were achieved at the beginning of emersion for each community, further investigations need to be performed, using complementary methods, to survey the metabolism of these intertidal communities during tidal and daily cycles. This is essential to assess accurately the annual carbon balance of such Phaeophycean-dominated systems to bring some insights about their functional ecology, but also to carry on such measurements with the prospect to get fundamental information about ecosystem functioning modifications due to the large and complex changes that occur on the biosphere [START_REF] Staehr | The metabolism of aquatic ecosystems: history, applications, and future challenges[END_REF].

AKNOWLEDGMENTS

The authors thank all the EFEB team members and the students for their help on field experiments. We also thank Cédric Leroux for performing the carbon and nitrogen spectral Table Table 2: Wilcoxon signed rank test results (V, p) for differences between Fucus vesiculosus and Fucus serratus in mean community gross primary production (GPPt), community respiration (CRt), metabolic balance (CRt/GPPt), canopy productivity (GPPcan/DMcan), canopy respiration rate (Rcan/DMcan), C content (% C), N content (% N).

Table Table 3:

  photosynthetically available radiation during light incubations (data not shown) were

Fig. 1 FigureFig. 2

 12 Fig. 1 Monthly maximal instantaneous irradiance, expressed in W m -2 , and monthly mean of

Figure

  Figure

Fig. 3

 3 Fig. 3 Mean (± SE) community gross primary production (GPPt) and respiration (CRt), both expressed in mgC m -2 h -1 , as a function of time, for Fucus vesiculosus (a) and Fucus serratus

Figure

  Figure

Fig. 4

 4 Fig. 4 Mean (± SE) metabolic balance, calculated as CRt/GPPt, as a function of time, for

Figure

  Figure

Fig. 5

 5 Fig. 5 Mean (± SE) gross canopy productivity (GPPcan/DMcan) and canopy respiration rate (Rcan/DMcan), both expressed in mgC gDM -1 h -1 , as a function of time, for Fucus vesiculosus (a)

Figure

  Figure

Fig. 6

 6 Fig. 6 Mean (± SE) C content (a) and N content (b), both expressed in % of DMcan, as a

Figure

  Figure

Table 1 :

 1 Sinusoidal curve parameters (n, a, b, c and R²) and F-test significant level (*: p < 0.05; **: p < 0.01; ***: p < 0.001) for Fucus vesiculosus and Fucus serratus community gross primary production (GPPt), community respiration (CRt), metabolic balance (CRt/GPPt), canopy productivity (GPPcan/DMcan), canopy respiration rate (Rcan/DMcan), C content (% C), N content (% N).

  Results of linear multiple regressions(R², p) analyzing relationships between GPPt or CRt and environmental variables, for both community. Maxirr = monthly maximal instantaneous irradiance; Tair = monthly mean air temperature; Tseawater = monthly mean seawater temperature; NO3 -= monthly mean nitrates concentration in seawater; NO2 -= monthly mean nitrites concentration in seawater; NH4 + = monthly mean ammonium concentration in seawater; PO4 2-= monthly mean phosphates concentration in seawater; DMcan = canopy dry mass.

  analyses, and the SOMLIT network for performing seawater temperature and nutrient concentrations measurements. Three anonymous reviewers are thanked for constructive comments. This work benefited from the support of the French Government run by the National Research Agency and with regards to the investment expenditure programme IDEALG ANR-10-BTBR and was partly funded by the Brittany region.

	ambient nitrate concentrations. Plant Cell Environ 30:764-774. doi: 10.1111/j.1365-
	3040.2007.01666.x
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	Airoldi L, Beck MW (2007) Loss, status and trends for coastal marine habitats of Europe. In:
	Gibson RN, Atkinson RJA, Gordon JDM (eds) Oceanography and Marine Biology,
	Vol 45. Taylor & Francis Group, Boca Raton, pp 345-405
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	10.1111/j.0022-3646.1983.00254.x
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Table


Laura Gajdzik

Gilles Lepoint

David Lecchini

Bruno Frédérich
email: bruno.frederich@ulg.ac.be

Comparison of isotopic turnover dynamics in two different muscles of a coral reef fish during the settlement phase

Keywords: Acanthuridae, stable isotopes, development, ontogeny, metamorphosis, starvation, surgeonfish Acanthuridae, isótopos estables, desarrollo, ontogenia, metamorfosis, inanición, surgeonfish

The temporal variation in carbon and nitrogen isotopic compositions (noted as δ 13 C and δ 15 N) was investigated in the convict surgeonfish (Acanthurus triostegus) at Moorea (French Polynesia). Over a period of 24 days, juveniles were reared in aquaria and subjected to two different feeding treatments: granules or algae. The dynamics of δ 13 C and δ 15 N in two muscles (the adductor mandibulae complex and the epaxial musculature) having different functions were compared. At the end of experiments, a steady-state isotopic system in each muscle tissue was not reached. Especially for the algal treatment, we found different patterns of variation in isotopic compositions over time between the two muscles. The turnovers of δ 13 C showed opposite trends for each muscle but differences are mitigated by starvation and by the metamorphosis. Our study highlighted that the metabolism of coral reef fish may be subjected to catabolism or anabolism of non-protein precursors at settlement, inducing variation in isotopic compositions that are not linked to diet change.

INTRODUCTION

The life cycle of the majority of reef fishes is a complex process that comprises two main distinct phases, a pelagic larval stage corresponding to long-distance migrations of larvae and a demersal stage that includes juveniles as well as adults (Leis 2002, Lecchini and[START_REF] Lecchini | Synthèse sur l'influence des processus pélagiques et benthiques, biotiques et abiotiques, stochastiques et déterministes, sur la dynamique de l'autorecrutement des poissons coralliens[END_REF]. The larval phase ends with the coral reef settlement [START_REF] Leis | Pacific coral-reef fishes: The implications of behaviour and ecology of larvae for biodiversity and conservation, and a reassessment of the open population paradigm[END_REF]. The shift from a pelagic to a benthic environment represents a period of transition during which fishes undergo morphological, physiological and behavioural changes (e.g. [START_REF] Mccormick | Delayed metamorphosis of a tropical reef fish (Acanthurus triostegus): A field experiment[END_REF]Makey 1997, Lecchini 2005). This major event in the life history of reef fishes is generally called metamorphosis [START_REF] Mccormick | Post-settlement transition in coral reef fishes: Overlooked complexity in niche shifts[END_REF] and may correspond to rapid variations in internal stable isotope compositions [START_REF] Herzka | Changes in isotopic composition of red drum (Sciaenops ocellatus) larvae in response to dietary shifts: Potential applications to settlement studies[END_REF]. Thus, comparing carbon and nitrogen isotopic compositions during that phase might provide information about physiology and tissue turnover.

During the last few decades, stable isotopes have emerged as common tools for investigating food webs, trophodynamics and trophic ecology by defining trophic levels and identifying the diets of consumers (e.g. [START_REF] Lepoint | Applications of C and N stable isotopes to ecological and environmental studies in seagrass ecosystems[END_REF][START_REF] Carassou | Assessment of fish trophic status and relationships by stable isotope data in the coral reef lagoon of New Caledonia, southwest Pacific[END_REF]). The isotopic turnover can be defined as the rate at which elemental isotopes in the diet are incorporated into the consumer [START_REF] Tieszen | Fractionation and turnover of stable carbon isotopes in animal tissues: Implications for δ 13 C analysis of diet[END_REF]. Various studies have compared the rates of isotopic turnover between different types of tissues in fishes. For example, [START_REF] Olsen | Changes in fatty acid composition and stable isotope signature of Atlantic cod (Gadus morhua) in response to laboratory dietary shifts[END_REF] found that the isotopic turnovers in plasma, muscle and heart reached a steady state faster than in the liver of the Atlantic cod Gadus morhua. The turnover of isotopes may also vary greatly among fish species. For example, the d 13 C and d 15 N values of red drum (Sciaenops ocellatus) larvae indicated that carbon and nitrogen both have rapid turnovers that matched the very high growth rate of young fish because the isotopic compositions shift in 1-2 days and stabilize within 10 days [START_REF] Herzka | Changes in isotopic composition of red drum (Sciaenops ocellatus) larvae in response to dietary shifts: Potential applications to settlement studies[END_REF]. A study conducted on the winter flounder (Pseudopleuronectes americanus) indicated that a stable system could occur within a period of 16 days in recently metamorphosed fish [START_REF] Bosley | Estimating turnover rates of carbon and nitrogen in recently metamorphosed winter flounder Pseudopleuronectes americanus with stable isotopes[END_REF]. On the other hand, a one-year study revealed that the isotopic equilibrium with food sources could take several months in 2.5-year-old Coregonus nasus [START_REF] Hesslein | Replacement of sulfur, carbon, and nitrogen in tissue of growing broad whitefish (Coregonus nasus) in response to a change in diet traced by δ 34 S, δ 13 C, and δ 15 N. Can[END_REF]). These studies have provided reliable data about isotopic turnovers and allow comparisons among species. However few studies have focused on changes in isotope compositions during the growth of coral reef fishes correlated with diet shifts (but see [START_REF] Frédérich | Trophic Niche Width, Shift, and Specialization of Dascyllus aruanus in Toliara Lagoon, Madagascar[END_REF][START_REF] Frédérich | Mismatch between shape changes and ecological shifts during the post-settlement growth of the surgeonfish, Acanthurus triostegus[END_REF] and information about the dynamics of isotopic turnover during the settlement phase of tropical reef fishes is unavailable.

In studies devoted to the trophic ecology of tropical reef fishes (e.g. [START_REF] Carassou | Assessment of fish trophic status and relationships by stable isotope data in the coral reef lagoon of New Caledonia, southwest Pacific[END_REF][START_REF] Frédérich | Trophic niches of thirteen damselfishes (Pomacentridae) at the Grand Récif of Toliara, Madagascar[END_REF][START_REF] Gajdzik | The mangrove forests as nursery habitats for the ichthyofauna of Mida Creek (Kenya, East Africa)[END_REF], the epaxial musculature is usually sampled for the isotopic analyses. To our best knowledge, no study has compared the ontogenetic dynamics of isotopic turnover from different muscles in a coral reef fish during the transition phase from the pelagic to the benthic environment. Here, we compared the variation of isotopic composition in two muscles having different functions: the adductor mandibulae complex, which generates jaw-closing movements during feeding, and the epaxial musculature involved in swimming activities [START_REF] Vandewalle | Analyse des mouvements potentiels de la région céphalique du goujoun, Gobio gobio (L.) (Poissons, Cyprinidae)[END_REF][START_REF] Lauder | Evolution of the feeding mechanism in primitive actinopterygian fishes: A functional anatomical analysis of Polypterus, Lepisosteus, and Amia[END_REF]. In grazing reef fishes (e.g. acanthurids and scarids), there is a drastic ontogenetic shift correlated with a change in prey consumed between settling larvae and adults that corresponds to a diet change from zooplanktivory to benthivory [START_REF] Frédérich | Mismatch between shape changes and ecological shifts during the post-settlement growth of the surgeonfish, Acanthurus triostegus[END_REF]. This probably impacts the metabolism of each muscle in a different manner. For example, the activity of the adductor mandibulae complex should be more intense in juveniles and adults as they graze on algae in comparison with larvae feeding on zooplanktonic preys [START_REF] Sampey | Glimpse into guts: overview of the feeding of larvae of tropical shorefishes[END_REF]. Therefore, the growth of both muscles could reflect different isotopic dynamics related to their development and functional demands.

The present study explored experimentally the dynamics of carbon and nitrogen stable isotopes during the first 24 days of post-settlement in an algivorous coral reef fish, Acanthurus triostegus. We specifically targeted two muscles having different functions: the adductor of mandibulae involved in feeding activity and the epaxial musculature used for locomotion. Our objectives were to compare the dynamics of isotopic turnover in each muscle type when fish were fed with different diets (artificial granules vs algae). We expected variation in the patterns of isotopic turnover between muscles and between diet treatments. We hypothesized that the turnover of both isotopes in the adductor mandibulae would be more rapid than in epaxial musculature, especially for the algal treatment. Indeed, the juvenile oral morphology should become shaped for grazing and algal treatment would stimulate morphological adaptations for biting [START_REF] Frédérich | Mismatch between shape changes and ecological shifts during the post-settlement growth of the surgeonfish, Acanthurus triostegus[END_REF]. According to the metamorphosis hypothesis, when reef fishes undergo morphological and physiological changes in a short period of time, we expected carbon and nitrogen composition in each tissue to approximate the equilibrium with the isotopic composition of their diets at the end of the experiments (i.e. 24 days).

MATERIALS AND METHODS

Target species

The convict surgeonfish, Acanthurus triostegus (Linnaeus, 1758), was chosen as a model to carry out such a study given its widespread distribution in the Indo-Pacific region [START_REF] Randall | A contribution to the biology of the convict surgeonfish of the Hawaiian Islands, Acanthurus triostegus sandoicensis[END_REF]. At Moorea (French Polynesia), the larval stage is about 44 to 60 days, after which larvae colonize the reef at night and settle directly on shallow sandy areas with coral slabs (McCormick 1999, Lecchini and[START_REF] Lecchini | Spatial repartition and ontogenetic shifts in habitat use by coral reef fishes (Moorea, French Polynesia)[END_REF]. When sexual maturity is reached (standard length, SL=95 mm), adults move towards the barrier reef community, where they usually form large feeding schools [START_REF] Randall | A contribution to the biology of the convict surgeonfish of the Hawaiian Islands, Acanthurus triostegus sandoicensis[END_REF]. Larvae feed on zooplankton while juveniles and adults graze on algae [START_REF] Frédérich | Mismatch between shape changes and ecological shifts during the post-settlement growth of the surgeonfish, Acanthurus triostegus[END_REF].

Sampling and experiment

Sample collection was carried out in September-October 2009 in the lagoon of Moorea (17°30'S, 149°50'W; French Polynesia; Fig. 1). Settling larvae of Acanthurus triostegus (n=81), also called "naïve" larvae, were obtained from nets fixed to the reef crest at dawn [START_REF] Lecchini | New perspectives to aquarium fish trade[END_REF]. The first surgeonfish larvae, which were captured all at once (n=17) and formed one cohort, were sacrificed and represent the t 0 in the following framework. The other larvae (n=64), caught during another retrieval of nets, form a second cohort which was divided into two equal parts. Each half was allocated to a feeding treatment (n=32 for treatment 1 and n=32 for treatment 2). For a comparison between treatment 2 and juveniles collected in the field, we used the isotopic data of the epaxial musculature from 91 individuals (SL, 22.45-55.25 mm) captured at the same location and same date, and published in [START_REF] Frédérich | Mismatch between shape changes and ecological shifts during the post-settlement growth of the surgeonfish, Acanthurus triostegus[END_REF]. Juveniles were caught with a seine net on the beach zone (Fig. 1). Throughout the paper, we use the term "larvae" for individuals collected with crest nets and the term "juveniles" for fish maintained in aquaria. Treatment 1 consisted of providing commercially available granules, especially adapted for coral reef fish (SERA marin granules). These granules are composed of 52% proteins, 8.1% fat content, 3.5% crude fibres, 6.5% inorganic matter and 5.1% moisture. In treatment 2, the food sources were algae growing on stones/rubbles and found near beach shores. They were a mix of filamentous algae, red algae (Polysiphonia spp.) and green algae (Enteromorpha spp.) and represent the putative natural food of this species during the juvenile phase [START_REF] Ogden | The role of herbivorous fishes and urchins in coral reef communities[END_REF]Lobel 1978, Frédérich et al. 2012).

Sampling treatment

The cohort of 64 larvae was equally separated into two aquaria of 200 L (100×50×40) filled with running sea water at a constant temperature of 26°C. Daylight simulation provided 12 h of light/darkness. In treatment 1, a total of 32 larvae were fed with granules three times per day. In treatment 2, the bottom of each aquarium was fully filled with stones or coral rubbles covered by turf algae, which were replaced daily. The experiments lasted 24 days, during which three to five individuals were randomly sampled every three days and then killed with an overdose of tricaine methanesulfonate (MS-222) according to ethical recommendations. All fish were measured and weighed. The SL was taken to the nearest millimeter with a Vernier caliper. Muscle tissue (0.5-2 cm 3 ) from the epaxial section of the fish body and of the adductor mandibulae complex were sampled.

Processing for stable isotopes analysis

Samples of both types of muscle tissues and food sources (i.e. granules and algae) were dehydrated for 48 h at 60°C before being ground into a homogenous powder. After grinding, samples containing carbonates (brushed algae) were placed for 24 h under a glass bell with fuming HCl (37%) (Merck KGaA © , for analysis quality) in order to eliminate calcareous material.

Samples were analysed on a V.G. Optima (Micromass) IR-MS coupled to an N-C-S elemental analyser (Carbo Erba). Routine measurements were precise to within 0.3% for both d 13 C and d 15 N values. Carbon and nitrogen isotope ratios were reported as per mil (‰) and expressed in the conventional delta notation (d 13 C, d 15 N) relative to the vPDB (Vienna Peedee Belemnite) standard and to atmospheric nitrogen standard, respectively. Certified materials were IAEA-N1 (d 15 N=0.4±0.2‰) and IAEA CH-6 (sucrose) (d 13 C=-10.4±0.2‰):

δX = [(Rsample/Rstandard) -1] × 1000
where X is either 13 C or 15 N and R is the ratio of the heavy to the light isotope.

Statistical analysis

At settlement (t 0 ), significant differences in composition of carbon and nitrogen between the two types of muscles were checked using a Student t-test when variances were normally distributed and homogeneous (Shapiro-Wilk test and Levene test, respectively) and a Welch test when unequal variances were found even after transformation. For each food treatment lasting from t 3 to t 24 , the relationship between δ 13 C, δ 15 N or elemental ratio of carbon to nitrogen (C/N) and time was investigated using two types of model:

(1) an exponential model plateau followed by one phase decay and (2) a linear regression model. The equation of the exponential model plateau followed by one phase decay is: (-vt) where δ t is the stable isotopic composition of either adductor mandibulae or the epaxial musculature at the time when fish were collected from the aquaria, δ i is the initial value before the diet switch, δ f is the final isotopic composition equilibrated to the new diet, v is a measure of the turnover rate and t is the time that the fish were in the experiment (day) [START_REF] Tieszen | Fractionation and turnover of stable carbon isotopes in animal tissues: Implications for δ 13 C analysis of diet[END_REF]. This model allows for the calculation of the "Trophic Enrichment Factor" (TEF) which is the arithmetic difference in the nitrogen isotope ratio of consumer and diet (δ 15 N=δ 15 N consumer -δ 15 N diet ).

δ t = δ f + (δ i -δ f )e
The equation for the linear regression model is:

Y=β+αX where β is the y-intercept and α the slope of the linear regression. In order to perform the second model, a coefficient of correlation (r) was first calculated with Pearson's product-moment method for parametric data and Spearman's rank correlation coefficient was used for non-parametric data. If the results were significant (p<0.05), a linear regression model was performed and the coefficient of determination was calculated (R 2 ). Normality of residuals of each model was tested using a Shapiro-Wilk test. If linear models were validated, analyses of covariance tests (ANCOVA) were performed to compare the dynamics of isotopic compositions between the two muscles. All statistical analyses were done with GraphPad Prism, version 5.0 [START_REF] Motulsky | Prism 5 Statistics Guide[END_REF].

RESULTS

Isotopic compositions in larvae (t 0 ) and food sources

At settlement (t 0 ), d 13 C values for the adductor mandibulae ranged from -20.73‰ to -19.55‰, d 15 N values from 15.29‰ to 18.62‰ and carbon (C) to nitrogen (N) ratios were about 3.66 to 4.21 (Table 1). Compositions for epaxial musculature varied from -18.14‰ to -17.23‰ for carbon and from 17.14‰ to 18.96‰ for nitrogen, and the C/N ratio ranged from 3.22 to 3. 2).

Effects of food treatments on fish growth

Acanthurus triostegus individuals fed with granules increased significantly in weight and size over time (linear regressions R 2 =0.64, p<0.05; R 2 =0.53, p<0.05, respectively; Fig. 2). Conversely, there was a significant negative correlation between weight and time for fish grazing on algae (linear regression R 2 =0.53, p<0.05; Fig. 2A). These fish did not increase in size during the experiment (t 6 , 23.85 mm and 0.53 g; t 24 , 22.50 mm and 0.31 g; Fig. 2B).

Treatment 1: patterns of isotopic variation in juveniles fed with granulated food

Results of linear regression models of both experiments are presented in Table 3. The turnover of d 13 C in the adductor mandibulae and in the epaxial musculature showed the same increasing linear dynamics (ANCOVA, test for common slopes, F 1,47 =0.0016, p>0.05; Fig. 3A). On the other hand, the d 15 N values were decreasing for both types of muscles but their dynamics were different. Nitrogen values in the adductor mandibulae complex decreased linearly over time (mean value of 15.56‰ at the end of the experiment). The isotopic composition in epaxial musculature of each individual fitted an exponential model, whose values reached a plateau at 14.76‰ (Table 3, Fig. 3B). This exponential model allowed the calculation of the TEF, which is 6.56‰. The C/N ratio remained quite stable over time for each type of muscle. For both muscles, no significant correlation was found between the C/N ratio and time (Table 3, Fig. 3C).

Treatment 2: patterns of isotopic variation in juveniles fed with algal food

When larvae were fed with turf algae, d 13 C values of both muscles showed opposite dynamics during the experiment (ANCOVA, test for common slopes: F 1,48 =31.45, p<0.05; Table 3). Values of d 13 C increased linearly towards food source for the adductor mandibulae (i.e. reaching less negative values) while they decreased for the epaxial musculature (Fig. 3D) but values for each muscle tended towards -18.64‰ and -18.77‰, respectively. For both types of muscles, d 15 N values did not change significantly over time (Table 3, Fig. 3E). The data did not fit exponential models (Table 3). The C/N ratio of the adductor mandibulae decreased linearly over time (r=0.41, p<0.05; Table 3, Fig. 3F). Conversely, no relationship was found between C/N ratio in epaxial musculature and time (Table 3).

Comparison with populations of juveniles from the field

No correlation was found between d 13 C or d 15 N and the SL of juvenile specimens from the field (p>0.05). Therefore, no model was estimated. However, the C/N ratio decreased linearly for both muscles (linear regression, adductor mandibulae Y=4.30-0.019X [R 2 =0.30; p<0.05] and epaxial musculature Y=3.28-0.004X [R 2 =0.39; p<0.05; Fig. 4]).

DISCUSSION

Natural variation in stable isotope compositions in settling larvae

At settlement (t 0 ), the isotopic compositions differ significantly between muscles and this tissue-specific variation can be explained by physiological factors. Indeed, white muscles are less variable in d 13 C and d 15 N than red muscles [START_REF] Pinnegar | Differential fractionation of δ 13 C and δ 15 N among fish tissues: implications for the study of trophic interactions[END_REF]. The adductor mandibulae of fish have a mix of fast (i.e. white muscle) and slow fibres (i.e. red muscle) [START_REF] Hernandez-Lagunas | Zebrafish narrowminded disrupts the transcription factor prdm1 and is required for neural crest and sensory neuron specification[END_REF], while a thin superficial layer of red fibres covers the main white fibres making epaxial musculature [START_REF] Rowlerson | Comparative study of myosins present in the lateral muscle of some fish: species variations in myosin isoforms and their distribution in red, pink and white muscle[END_REF]. Therefore, the amount of red fibres between the adductor mandibulae and the epaxial musculature in acanthurid larvae may be different and might significantly influence their isotopic and elemental compositions. A variation of the discrimination factors between muscle tissues can also explain this difference of isotopic compositions [START_REF] Caut | Variation in discrimination factors (δ 15 N and δ 13 C): The effect of diet isotopic values and applications for diet reconstruction[END_REF].

The C/N ratios are relatively high in A. triostegus larvae and juveniles (i.e. >3.2) (Table 1, Figs 3C, F and4), suggesting the presence of carbon-rich compounds in muscle tissues [START_REF] Sweeting | Effects of chemical lipid extraction and arithmetic lipid correction on stable isotope ratios of fish tissues[END_REF]. This probably comes from the lipid-rich planktonic diet of larvae, which allows them to store enough fat during the pelagic larval phase. In a natural population, this ratio decreased significantly when fish grew, meaning that lipid stores are used during the settlement phase (Fig. 4, [START_REF] Mccormick | Effects of feeding history on the growth characteristics of a reef fish at settlement[END_REF].

Unreached isotopic equilibrium after 24 days post-settlement

Many studies have revealed that the isotopic equilibrium can be reached after highly variable periods of time among fish species. Results of a feeding experiment on larvae of the Pacific blue fin tuna (Thunnus orientalis) showed that nitrogen values of fish approximated the isotopic composition of the food source after 20 days [START_REF] Tanaka | Relationship between prey utilization and growth variation in hatchery-reared Pacific bluefin tuna, Thunnus orientalis (Temminck et Schlegel), larvae estimated using nitrogen stable isotope analysis[END_REF]. In other experiments on larvae of the catfish Clarias gariepinus and Ictalurus punctatus, results of isotopic compositions reached the "plateau" phase between 4 to 10 weeks, respectively (Enyidi et al. 2013, Filbrun and[START_REF] Filbrun | Stable isotopes reveal live prey support growth of juvenile channel catfish reared under intensive feeding regimens in ponds[END_REF]. These studies were conducted on similar developmental stages (i.e. the larval stage) but tuna and catfishes did not undergo any drastic diet shift during ontogeny. Conversely, flatfishes observe a heavy metamorphosis and a diet shift from a pelagic food web towards a demersal habitat during growth. In the winter flounder (Pseudopleuronectes americanus) stable isotopes revealed that this diet shift occurred over a short period of 16 days [START_REF] Bosley | Estimating turnover rates of carbon and nitrogen in recently metamorphosed winter flounder Pseudopleuronectes americanus with stable isotopes[END_REF]. In the present study, the isotopic equilibrium for A. triostegus was not reached after 24 days post-settlement although most marine ecologists agree that coral reef fishes observe a metamorphosis around this phase. Consequently, this study should probably be re-done over two or three months to define the turnover time.

Effects of food treatment on isotopic dynamics in the adductor mandibulae and the epaxial musculature

According to varied functional demands between treatments, we expected to observe differences in isotopic dynamics between muscles. Indeed, we hypothesized a higher isotopic turnover for the adductor mandibulae complex than for the epaxial musculature, especially when fish are grazing on algae. Conversely, we expected the same turnover for the epaxial musculature in both treatments because the swimming activity did not vary.

When A. triostegus were fed on granules, the adductor mandibulae complex and the epaxial musculature showed the same linear pattern of variation for the isotopic compositions of carbon (Fig. 3A and Table 3). Both tissues displayed a relatively slow turnover, as δ 13 C data followed linear models. Surprisingly, linear regressions moved in an opposite direction to the value of the food source (Fig. 3A). As granules are composed of different elements (see Materials and Methods section), we suggest that the studied surgeonfish assimilated only some components of this food. The value of the "real" food (i.e. the components really incorporated) might have a different isotopic composition from the value for the whole granule and that would explain this unexpected isotopic dynamics.

For δ 15 N, the dynamics differs between the adductor mandibulae and the epaxial musculature (Fig. 3B) although the nitrogen isotopic compositions tend to decrease over time. Accordingly with growth and weight gain (Fig. 2), these isotopic dynamics are representative of a situation in which food contains highenergy ingredients, allowing synthesis of new muscle fibres. The equilibrium for the adductor mandibulae was not reached at the end of the experiment (24 days), contrary to epaxial musculature values, which fitted an exponential model, allowing a TEF of 6.56‰ to be calculated for nitrogen. This value is higher than the one found in another algivorous surgeonfish, Acanthurus sohal (TEF=4.69‰; [START_REF] Mill | Explaining isotope trophic-step fractionation: why herbivorous fish are different[END_REF]) but close to other TEFs recorded for tropical herbivorous fishes (Mill et al. 2007, Hata andUmazawa 2011). Currently, such a high TEF for herbivorous species is the subject of controversy among ecologists using stable isotopes. Vari-ous studies have found that discrimination factors depend on the species, the trophic position of the species, diet isotopic ratio and tissue, and a better quality food is apparently correlated with lower δ 15 N enrichment [START_REF] Vanderklift | Sources of variation in consumer-diet δ 15 N enrichment: A meta-analysis[END_REF][START_REF] Caut | Variation in discrimination factors (δ 15 N and δ 13 C): The effect of diet isotopic values and applications for diet reconstruction[END_REF][START_REF] Wyatt | Variability in isotope discrimination factors in coral reef fishes: Implications for diet and food web reconstruction[END_REF]). However, Mill et al. (2007) demonstrated that high TEF was expected for herbivores, partly as a consequence of their very high ingestion rate. The observed TEF value of 6.56‰ might be partially determined by the selective assimilation of granule components (see above) or by a failure to properly model the isotope dynamics with the time-limited data.

Contrary to our hypothesis, muscle isotopic composition changed faster in the epaxial musculature than in the adductor mandibulae complex. This might be due to the fact that feeding on a more pelagic type of food (i.e. granules) does not require better-developed adductor mandibulae muscle than feeding on benthic algae [START_REF] Frédérich | Ontogenetic shape changes in Pomacentridae (Teleostei, Perciformes) and their relationships with feeding strategies: a geometric morphometric approach[END_REF]). Perhaps granules or the "absence of biting on the benthos" do not induce the expected morphological and physiological changes (e.g. the development of large adductor mandibulae and robust oral jaws) required to efficiently graze on turf algae [START_REF] Frédérich | Ontogenetic shape changes in Pomacentridae (Teleostei, Perciformes) and their relationships with feeding strategies: a geometric morphometric approach[END_REF][START_REF] Frédérich | Mismatch between shape changes and ecological shifts during the post-settlement growth of the surgeonfish, Acanthurus triostegus[END_REF].

When algae were provided to A. triostegus juveniles, the d 13 C turnovers were different between the two types of muscles. Values of d 13 C in the adductor mandibulae increased towards the mean value of isotopic composition in algae (i.e. -15.54‰) (Table 2). For the epaxial musculature, d 13 C values showed an opposite trend and shifted away from the food source value. This pattern suggests that epaxial musculature is probably used as a carbon source to favour the construction of a stronger jaw-closing system, increasing the efficiency to scrape algae. Moreover, the diet shift from "feeding on pelagic prey" to grazing could act as an environmental factor favouring or inducing morphological and physiological adaptations for algivory during ontogeny [START_REF] Frédérich | Mismatch between shape changes and ecological shifts during the post-settlement growth of the surgeonfish, Acanthurus triostegus[END_REF]. The decreasing C/N ratio in the adductor mandibulae (Fig. 3F) sustains the hypothesis of a high level of activity and lipid loss in this muscle complex compared with the unchanged C/N ratio during treatment 1 (Fig. 3C; [START_REF] Schmidt | Trophic relationships among Southern Ocean copepods and krill: Some uses and limitations of a stable isotope approach[END_REF][START_REF] Logan | Lipid corrections in carbon and nitrogen stable isotope analyses: Comparison of chemical extraction and modelling methods[END_REF].

Effects of starvation and metamorphosis on the dynamics of isotopic composition

During treatment 2, fish suffered from starvation, as suggested by their significant loss of weight (Fig. 2). Although we used the natural food source of A. triostegus (i.e. turf algae covering beach rocks or rubbles), the quantity of algae was dependent on the surface of the aquaria, inducing a limited amount of food source. In fact, starvation is a critical phase during which there is a continuous flow of molecules through different metabolic pathways, which are difficult to disentangle [START_REF] Ferron | An appraisal of condition measures for marine fish Larvae[END_REF]. The breakdown of proteins and other molecules (i.e. catabolism) also occurs, leading to isotopic changes that are not related to diet change [START_REF] Ferron | An appraisal of condition measures for marine fish Larvae[END_REF]. Therefore, fasting animals may differ in their stable isotope ratios from organisms fed ad libitum [START_REF] Guelinckx | Changes in δ 13 C and δ 15 N in different tissues of juvenile sand goby Pomatoschistus minutus: A laboratory diet-switch experiment[END_REF]). If diet did not contain enough carbohydrates, proteins can be catabolized in order to provide metabolic intermediates for the synthesis of other biologically important compounds [START_REF] Wilson | Utilization of dietary carbohydrate by fish[END_REF]. A study on the Atlantic salmon Salmo salar revealed that fasting individuals re-used their own body fat reserves by catabolism of stored lipid and protein reserves [START_REF] Doucett | Effects of the spawning migration on the nutritional status of anadromous Atlantic salmon (Salmo salar): Insights from stable-isotope analysis[END_REF]. Here, the catabolism of endogenous fat stored from epaxial musculature is plausible but not so obvious in view of the constant C/N ratio during the treatment 2 (Fig. 3F). On the other hand, the decreasing d 13 C values in the epaxial musculature might be due to the de novo synthesis (i.e. anabolism) of non-protein precursors such as carbohydrates for the development of other tissues such as the adductor mandibulae complex.

The C/N ratio dynamics were highly similar in "natural populations" and during algal treatment (Figs 3F and 4). This reinforces the assumption that A. triostegus may be subjected to catabolism or anabolism of non-protein precursors, inducing variation in isotopic compositions in different muscle types. This hypothesis is not strictly diet-specific but also physiologically due to internal metamorphosis during growth [START_REF] Maruyama | Change in stable nitrogen isotope ratio in the muscle tissue of a migratory goby, Rhinogobius sp., in a natural setting[END_REF][START_REF] Ankjaerø | Tissue-specific turnover rates and trophic enrichment of stable N and C isotopes in juvenile Atlantic cod Gadus morhua fed three different diets[END_REF]. In this study, it is rather difficult to separate the effects of starvation and metamorphosis on the isotopic turnover dynamics in each muscle. Through our experimentation, we can argue that ecologists have to be careful about the significance of isotopic compositions in settling coral reef fish. A tissue-specific trade-off between sensitivity to changes in resource use (i.e. diet) and resistance to internal changes linked to metamorphosis probably occurs [START_REF] Colborne | Effect of nutritional condition on variation in δ 13 C and δ 15 N stable isotope values in Pumpkinseed sunfish (Lepomis gibbosus) fed different diets[END_REF].

CONCLUSION

This study is the first one exploring the dynamics of carbon and nitrogen turnover from two different muscles during the first days of post-settlement in a coral reef fish. As expected, we found some different patterns of variation in isotopic compositions over time between muscles. When artificial granules were provided, the turnover of both isotopes was faster in the epaxial musculature than in the adductor mandibulae. For algal treatment, the carbon turnovers showed opposite trends for each muscle but this difference was probably mitigated by starvation. Finally, a steadystate system in each muscle tissue of A. triostegus was not reached after a diet shift, at the end of the 24-day experiment. Overall, our results reveal that the metabolism of settling reef fish may be subjected to catabolism or anabolism of non-protein precursors, inducing variation in isotopic compositions.

Fig. 1 .

 1 Fig. 1. -Map of Moorea (French Polynesia). The sampling sites are the same as in Frédérich et al. (2012).

  28. Isotopic compositions in the adductor mandibulae differed significantly from values in the epaxial musculature (Welch test, d 13 C, dF=23, p<0.05; Student t-test, d 15 N, dF=22; p<0.05; C/N, dF=15, p<0.05). For the food sources, granules showed more negative values of d 13 C and lower values of d 15 N than algae (Table

Fig. 2 .

 2 Fig. 2. -Variation of weight (A) and size (B) of Acanthurus triostegus over time during the experiments. Plotting weight vs time (A), for treatment 1 Y=0.57+0.266X and for treatment 2 Y=0.73-0.013X. Plotting standard length vs time (B), for treatment 1 Y=24.61+0.220X. Linear regressions are represented by straight filled lines.

Fig. 3 .

 3 Fig. 3. -Relationship between time (day) and isotopic values (d 13 C, d 15 N and C/N) in Acanthurus triostegus during treatment 1 (A, B, C) and treatment 2 (D, E, F). Linear regressions are represented by straight filled lines.

Fig. 4 .

 4 Fig. 4. -Relationship between size (standard length, SL) and carbon/ nitrogen (C/N) ratio in larvae and juveniles of Acanthurus triostegus from Moorea caught in 2009. Linear regression for the adductor mandibulae complex Y=4.30-0.0190X (R 2 =0.30; p<0.001) and for the epaxial musculature Y=3.28-0.004X (R 2 =0.39; p<0.05). Linear regression is represented by straight filled lines.

Table 1 .

 1 -Composition of d 13 C and d 15 N in 16 larvae of Acanthurus triostegus at reef settlement (t 0 ) from Moorea in 2009. Mean value and standard deviation (sd) are also presented.

			Adductor mandibulae		Epaxial musculature
	Individuals	Standard length (mm)	δ 13 C (‰)	δ 15 N (‰)	C/N	δ 13 C (‰)	δ 15 N (‰)	C/N
	1	24	-20.26	15.78	3.95	-17.75	18.96	/
	2	25.1	-19.55	/	3.66	-18.03	18.15	3.22
	3	23.8	-20.02	/	3.87	-17.83	18.42	/
	4	22.8	-20.45	16.7	4.1	-17.84	18.63	3.22
	5	23.7	-20.33	/	4.05	/	/	/
	6	24	-19.85	18.62	3.89	-18.14	18.78	/
	7	24.1	-19.59	18.3	3.87	-17.82	18.86	3.23
	8	23.4	-19.73	/	4	-17.70	17.46	3.22
	9	24.5	/	/	/	-17.91	18.65	/
	10	25.3	-19.79	16.74	4.16	-17.81	17.14	/
	11	25	-20.73	15.29	4.19	-18.03	17.66	/
	12	24.6	-19.9	/	3.88	-17.74	17.19	3.28
	13	24.8	-19.95	/	4.21	-17.74	18.63	3.23
	14	24	-19.76	18.14	3.68	-17.84	17.93	/
	15	24.3	-19.92	17.35	4.05	-17.89	18.83	3.2
	16	24.8	-19.56	16.97	3.89	-17.23	18.39	/
	Mean±sd		-19.96±0.34	17.01±1.13	3.72±1.00	-17.82±0.20	18,24±0.62	3.23±0.20

Table 2 .

 2 -Carbon and nitrogen isotope compositions (mean±sd) of food sources: algae growing on coral rubbles collected on the fringing reefs (Moorea) and granulated food. Standard deviations (sd) are only presented for algal food.

	Food sources	δ 13 C (‰)	δ 15 N (‰)
	Granulated food	-20.60	8.20
	Algae	-15.54±2.46	13.53±0.83

Table 3 .

 3 -Results of models: type of model, coefficient of determination (R 2 ) and p-value for d 13 C, d 15 N and carbon (C) and nitrogen (N) ratio of Acanthurus triostegus caught at Moorea in 2009. Only means of intercept and slope are presented. AM and EM refer to the adductor mandibulae complex and epaxial musculature, respectively; ns, "non-significant"; *, p-value<0.05.

	Treatments	Y vs X	Muscle	Exponential model: plateau followed by one phase decay	Linear regression model	R 2	P
		δ 13 C vs time	AM EM	ns ns	Y=-19.30+0.022X Y=-17.89+0.022X	0.21 0.44	* *
	Exp. 1: Granules	δ 15 N vs time	AM EM	ns Y=14.76+3.998e -(0.061x3.789)	Y=18.45-0.137X ns	0.42 0.43	* *
		C/N vs time	AM EM	ns ns	ns ns	ns ns	ns ns
		δ13C vs time	AM EM	ns ns	Y=-19.38+0.036X Y=-17.61-0.032X	0.49 0.32	* *
	Exp. 2: Algae	δ15N vs time C/N vs time	AM EM AM EM	ns ns ns ns	ns ns Y=3.61-0.010X ns	ns ns 0.41 ns	ns ns * ns

ACKNOWLEDGEMENTS

L.G., B.F. and G.L. are respectively a research fellow, a postdoctoral researcher and an associate researcher of the Fonds National de la Recherche Scientifique of Belgium (F.R.S-FNRS). We would like to thank Serge Planes, Yannick Chancerelle, Pascal Ung and Benoit Espiau (CRIOBE, Moorea, French Polynesia) for helping to collect the fishes and for providing hospitality and laboratory facilities. This study was supported by grants from ANR (ANR-06-JCJC 0012-01) and the CRISP programme (Coral Reef Initiative in the South Pacific-C2A). This paper is MARE paper number 299.