# Grazers increase the sensitivity of coralline algae to ocean acidification and warming

Erwann Legrand, Pascal Riera, Mathieu Lutier, Jérôme Coudret, Jacques

Grall, Sophie Martin

► To cite this version:

Erwann Legrand, Pascal Riera, Mathieu Lutier, Jérôme Coudret, Jacques Grall, et al.. Grazers increase the sensitivity of coralline algae to ocean acidification and warming. Journal of Sea Research (JSR), 2019, 148, pp.1-7. 10.1016/j.seares.2019.03.001. hal-02360270

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1 **Title:** Grazers increase the sensitivity of coralline algae to ocean acidification and warming

2 Erwann LEGRAND¹*, Pascal RIERA¹, Mathieu LUTIER¹, Jérôme COUDRET¹, Jacques

3 GRALL², Sophie MARTIN¹

4 ¹Sorbonne Université, CNRS - UMR7144 – EFEB – Station Biologique de Roscoff, 29680

5 Roscoff, France

6 ²UBO, IUEM, Place Nicolas Copernic, 29280 Plouzané, France

7 *Corresponding author: Erwann Legrand

8 Tel/Fax: +33 (0)2 98 29 23 23/24

9 Email: erwann.legrand@sb-roscoff.fr

10 **Key-words:** global change; algal-herbivore interactions; CO₂; temperature; calcareous algae;

11 grazing
12 Abstract

13 Coralline algae are expected to be adversely impacted by ocean acidification and warming.

14 Most research on these algae has involved experiments on isolated species, without

15 considering species interactions, such as grazing. This myopic view is challenging because

16 the impact of climate change on coralline algae will depend on the direct impacts on

17 individual coralline species and the indirect effects of altered interactions with other species.

18 Here, we tested the influence of grazing on the response of the coralline alga *Lithothamnion*

19 *corallioides* to near-future ocean acidification and warming. Two three-month experiments

20 were performed in the winter and summer seasons in mesocosms under crossed conditions of

21 pCO₂ (ambient and high pCO₂) and temperature (ambient and + 3°C) in the presence and

22 absence of grazers. In the winter, *L. corallioides* photosynthesis decreased with rising

23 temperature in the presence of grazers, while calcification increased. It is likely that increased

24 calcification may act as a structural protection to prevent damage from grazing. However,

25 increasing calcification rates in the presence of grazers may be detrimental to other

26 physiological processes, such as photosynthesis. In the summer, *L. corallioides* primary

27 production, respiration, and calcification were higher in the presence of grazers than in their

28 absence. Light calcification rates were reduced under high pCO₂ in the presence of grazers

29 only. Moreover, dark calcification rates were more adversely affected by pCO₂ increase in the

30 presence of grazers. Through their feeding activity, grazers may alter the structural integrity

31 of thalli and increase the sensitivity of coralline algae to ocean acidification. Our results

32 indicate that both season and grazing play a key role in the response of *L. corallioides* to

33 acidification and warming. Seasonal variations and species interactions are thus critical to

34 consider to make ecologically relevant predictions of the effects of future environmental

35 changes.

36
## 1. Introduction

Over the past 250 years, oceans have absorbed approximately one-third of atmospheric carbon dioxide (CO₂), reducing the surface water pH of 0.1 units and inducing significant changes in the water carbonate chemistry (Sabine et al. 2004). The oceans have also assimilated an important part of Earth's additional heat, increasing sea surface temperature about 0.7°C over the last 100 years (Gattuso et al. 2015). The present atmospheric CO₂ level of 400 parts per million volume (ppmv) is likely to reach 936 ppmv by the end of the 21st century, under the “business as usual” scenario (RCP 8.5) (Pörtner et al. 2014). This phenomenon could lead to an ocean pH decrease of 0.33 units and a sea surface temperature increase of 2.7°C by the end of this century (Bopp et al. 2013).

Since the last decade, the scientific community's interest in understanding how the projected ocean acidification and warming will impact marine organisms has greatly increased (Kroeker et al. 2013; Riebesell and Gattuso 2015; Yang et al. 2016), with a specific interest regarding marine calcifiers. Despite recent studies showed the ability of some calcareous species to cope with ocean acidification (Leung et al., 2017; DeCarlo et al., 2018), most of them are considered vulnerable (Doney et al. 2009). Among calcifying species, red calcareous coralline algae (Corallinaceae, Rhodophyta) are expected to be adversely impacted by global warming and ocean acidification. They are thought to be among organisms the most vulnerable to ocean acidification due to the high solubility of their magnesium calcite skeleton (Andersson et al. 2008; Haese et al. 2014).

To date, most research has focused on the response of coralline algae at the species scale under the influence of ocean acidification and warming (Martin and Gattuso 2009; Martin et al. 2013; Noisette et al. 2013; Hofmann and Bischof 2014) and studies examining the species interactions in a context of global change remain still poorly documented (Legrand et al. 2017). Species interactions are a key element of ecosystems functioning and are likely to play
an important role in the response of species in a context of climate change (O'Connor *et al.* 2011; Hansson *et al.* 2012; Kroeker *et al.* 2012). Several studies have documented the importance of grazing in the control of algal biomass (Sousa *et al.* 1981; Cloern 2001; Guillou *et al.* 2002) or the stimulation of their productivity (Littler *et al.* 1995; Cerda *et al.* 2009).

Despite this, the influence of grazing on the physiological response of macroalgae to climate change remains poorly understood. We hypothesized here that the presence of grazer may act as an additional pressure on maerl and increase its vulnerability to ocean acidification and warming.

The study of Legrand *et al.* (2017) tested the response of assemblages of free-living coralline algae *Lithothamnion corallioides*, epiphytic fleshy algae, and main grazer species (gastropods and sea urchins) under crossed conditions of pCO₂ (ambient and high pCO₂) and temperature (ambient and + 3°C). This research suggested that grazers have both indirect (regulation of *L. corallioides* epiphytic competitors) and direct (effect on *L. corallioides* structural integrity) impacts on *L. corallioides* physiology, driving its response to climate change (Legrand *et al.* 2017). However, these results did not allow us to understand the specific influence of grazing on the response of coralline algae to global change. Therefore, we investigated here the physiological response of the coralline alga *L. corallioides*, to ocean acidification and warming in the presence and the absence of grazer. The results in the presence of grazers were presented in Legrand *et al.* (2017). As the response of species to climate change is also known to vary depending on seasonal changes in environmental factors (Godbold and Solan 2013), we examined the impact of grazers on *L. corallioides* physiology both in winter and summer conditions.
## 2. Materials and methods

### 2.1. Biological material

Thalli of the maerl species *L. corallioides* Crouan and Crouan, 1867 were collected using a naturalist dredge (width: 1 m, height: 0.2 m, net: 1.5 m long) from a maerl bed from the bay of Brest, France (Anse du Roz, 48°19'56N 04°19'56W). Thalli of *L. corallioides* without any apparent epiphyte were selected. Thalli were not cleaned in order to keep epiphytes spores that could be present on their surface. The three main species of grazers living in maerl beds were also sampled: the two gastropods species *Gibbula magus* Linnaeus, 1758 and *Jujubinus exasperatus* Pennant, 1777 and the urchin species *Psammechinus miliaris* Müller, 1771 (Grall et al. 2006). For each species, only medium sized individuals were selected (Table 1).

Samples were collected on January 24, 2015 (winter conditions) and September 15, 2015 (summer conditions). At each season, 1 kg of living maerl, 500 g of dead thalli of *L. corallioides*, 40 individuals of *G. magus*, 40 individuals of *P. miliaris*, and 80 individuals of *J. exasperatus*, were randomly selected and transported in seawater tanks to the Roscoff Marine Station.

<table><caption>**Table 1.** Mean sizes (± 1 standard deviation) of the two gastropods species *Gibbula magus* (shell diameter, n = 40) and *Jujubinus exasperatus* (shell height, n = 80) and the urchin species *Psammechinus miliaris* (test diameter, n = 40) maintained during the two three-month experiments conducted in winter (March to June 2015) and summer (September to December 2015) conditions.</caption><thead><tr><th>Species</th><th></th><th>Winter experiment</th><th>Summer experiment</th></tr></thead><tbody><tr><td><em>Gibbula magus</em></td><td>shell diameter (cm)</td><td>2.51 (± 0.19 cm)</td><td>2.07 (± 0.22 cm)</td></tr><tr><td><em>Jujubinus exasperatus</em></td><td>shell height (cm)</td><td>0.81 (± 0.13 cm)</td><td>0.93 (± 0.15 cm)</td></tr><tr><td><em>Psammechinus miliaris</em></td><td>test diameter (cm)</td><td>1.42 (± 0.23 cm)</td><td>2.00 (± 0.15 cm)</td></tr></tbody></table>
## 2.2. *Experimental set-up*

The experimental design was that described in Legrand *et al.* (2017). Two three-month long experiments were conducted in winter (March to June 2015) and summer (September to December 2015) conditions. 20 artificial assemblages were created and randomly assigned to 20 15-L aquaria, as described in Legrand *et al.* (2017). Each assemblage was composed of 45 g of living *L. corallioides*, two *G. magus* individuals, two *P. miliaris* individuals and four *J. exasperatus* individuals. In addition, 20 g of living *L. corallioides* were added in each aquarium in a part separated from the grazers by a grid. The living maerl density in the parts of the aquarium with and without grazers was similar, corresponding to about 1 kg m⁻². In order to have more realistic assemblages, dead thalli of *L. corallioides* were also added in both parts (20 g and 9 g, respectively; about 400 g m⁻²). This ratio is consistent with natural ratio (Hily *et al.* 1992). Algae and grazers were then acclimated to the laboratory conditions for 7 days. After the acclimation period, pH was gradually decreased by 0.05 units per day over 7 days through CO₂ bubbling, until reaching required experimental values. Similarly, temperature was increased by 0.5°C per day. At each season, two pCO₂ conditions were tested and crossed with two temperature conditions to examine the interactive effect of pCO₂ and temperature. Therefore, four crossed conditions were tested:

1) ambient pCO₂ and ambient temperature (control, A-pCO₂; T)

2) high pCO₂ and ambient temperature (H-pCO₂; T)

3) ambient pCO₂ and high temperature (A-pCO₂; T + 3°C)

4) high pCO₂ and high temperature (H-pCO₂; T + 3°C)

Ambient pCO₂ conditions (A-pCO₂) were determined according to *in situ* winter (7.98) and summer (8.06) mean pH<sub>T</sub> (pH on the total scale) monitored above maerl beds in the Bay of Brest (from Martin, unpublished data). Elevated pCO₂ (H-pCO₂) corresponded to the “business-as-usual” scenario predicted for the end of the century, with a pH decrease of -0.33
units (Bopp *et al.*, 2013). Ambient temperature (T) corresponded to *in situ* winter (10.0°C) and summer (17.1°C) conditions in the Bay of Brest recorded by SOMLIT (from 2003 to 2014), and high temperature (T + 3°C) was determined according to the “business-as-usual” scenario predicted for 2100 (Bopp *et al.*, 2013). The temperature and the pH were controlled by an offline feedback system (IKS Aquastar, Karlsbad, Germany) using heaters and CO₂ bubbling, respectively (Legrand *et al.*, 2017). The regulation was made in four 100 L header tanks, continuously supplied with filtered (5 µm) natural seawater (waterflow rate of 150 L h⁻¹). Monitoring of seawater parameters within aquaria were described in Legrand *et al.* (2017).

Irradiance was set to the mean *in situ* daily irradiance at 5 m depth in the Bay of Brest according to Martin *et al.* (2006). It was 30-40 µmol photons m⁻² s⁻¹ in winter and 90-100 µmol photons m⁻² s⁻¹ in summer. The light was provided by two or four 80 W fluorescent tubes (JBL Solar Ultra Marin Day, JBL Aquaria, Nelson, New Zealand) above the aquaria under a 10/14 h or 14/10 h light/dark photoperiod, for winter or summer conditions, respectively.

## 2.3. *Metabolic measurements*

After each three-months experiment, *L. corallioides* was cleaned of epiphytes. Physiological measurements were performed through incubations in 185 mL acrylic respirometry chambers (Engineering & Design Plastics Ltd, Cambridge, UK), both in the parts with grazers and without grazers. Incubations were carried out in the light and in the dark under constant temperature. 10 to 15 g of *L. corallioides* were placed on a plastic grid above a stirring bar. The stirring bar in the chambers ensured the seawater was well mixed. Incubations were conducted during the day and lasted from 1 to 2 h according to the season and the light or dark conditions.
Net primary production (light incubations) and respiration rates (dark incubations) were calculated by measuring oxygen concentration at the beginning and at the end of incubations.

Oxygen concentration measurements were carried out using a non-invasive optical fiber system (FIBOX 3, PreSens, Regensburg, Germany). Reactive spots were calibrated with 0% and 100% buffer solutions. The 0% buffer solution was prepared by dissolving 1 g of sodium sulfite ($Na_2SO_3$) in 100 mL of seawater. The 100% buffer solution was prepared by bubbling air into 100 mL of seawater using an air-pump for 20 min to obtain air-saturated seawater. Net primary production (NPP, in µmol $O_2$ gDW$^{-1}$ h$^{-1}$, eq. 1), respiration (R, in µmol $O_2$ gDW$^{-1}$ h$^{-1}$, eq. 1) and gross primary production (GPP, in µmol $O_2$ gDW$^{-1}$ h$^{-1}$, eq. 2) rates were calculated as:

$$ \text{NPP or R} = \frac{\Delta O_2 \times V}{\Delta t \times DW} \quad (\text{Eq. 1}) $$

$$ \text{GPP} = \text{NPP} - R \quad (\text{Eq. 2}) $$

where $\Delta O_2$ is the difference between the initial and final oxygen concentrations (µmol $O_2$ L$^{-1}$), V the volume of the chamber (L), $\Delta t$ the incubation time (h), and DW the dry weight of the organisms incubated (g). The dry weight was obtained after 48 h at 60°C.

Control incubations containing only seawater were carried out to correct oxygen fluxes from any biological activity in seawater. Oxygen fluxes calculated in control chambers were subtracted from oxygen fluxes of chambers containing algae.

Seawater samples were taken in each aquarium at the beginning of incubations and in the chambers at the end of incubations for measurements of total alkalinity ($A_T$) and ammonium ($NH_4^+$), as described in Legrand et al. (2017). $A_T$ was obtained from the method of Dickson et al. (2007), using open-cell titration (automatic titrator; TitroLine alpha, Schott SI Analytics, Mainz, Germany). $NH_4^+$ concentrations were determined using spectrophotometry (630 nm;
spectrophotometer UV-1201V, Shimadzu Corp, Kyoto, Japan) according to the Solorzano method (Solorzano, 1969). Light and dark calcification rates ($G_l$ or $G_d$, in µmol CaCO$_3$ gDW$^{-1}$ h$^{-1}$, eq. 4) were calculated using the alkalinity anomaly technique (Smith and Key, 1975) and corrected from $NH_4^+$ fluxes (Gazeau *et al.* 2015) as:

$$G_l \text{ or } G_d = \frac{(-\Delta A_T + \Delta NH_4^+) \times V}{2 \times \Delta t \times DW} \quad (\text{Eq. 4})$$

where $\Delta AT$ is the difference between the initial and final total alkalinity concentrations (µeq L$^{-1}$) and $\Delta NH_4^+$ is the difference between the initial and final ammonia concentrations.

## 2.4. Chlorophyll a analysis

At the end of the experiments, chlorophyll a content was measured in *L. corallioides* thalli collected in each aquarium. Samples were immediately frozen at -20°C pending analyses.

Then samples were freeze-dried and crushed into powder using a mortar, in the dark. An aliquot of 0.15 g of powder was precisely weighed and suspended in 10 mL of 90% acetone and stored in the dark at 4°C for 12 h. Samples were then centrifuged at 4000 rpm. The supernatant was collected, and absorbance was measured at 630, 647, 664, and 691 nm.

Chlorophyll a concentration (µg gDW$^{-1}$) was calculated from Ritchie (2008).

## 2.5. Data analysis

Statistical analyses were carried out using the free software R 3.2.2 version (©The R Foundation for Statistical Computing). The effect of grazing, temperature and $pCO_2$ on the net and gross primary production, respiration, light and dark calcification and chlorophyll a content of *L. corallioides* was tested for each season, using three-way ANOVAs.

# 3. Results

In the summer, gross primary production rates (GPP) were significantly higher in the presence of grazers (+ 33%), while no effect of grazing was detected in the winter (Fig. 1a,b; Table 2).

In the winter, *L. corallioides* net primary production (NPP) and GPP are significantly affected
201 by the interaction between grazing and temperature and the interaction between pCO₂ and
202 temperature (Fig. 1a,c; Table 2; Supplementary material S1a,c). In the summer, increased
203 temperature reduced NPP (Fig. 1d; Table 2).

204 The presence of grazers significantly increased respiration rates (R) in the summer (+ 88%),
205 while no effect was observed in the winter (Fig. 1e,f; Table 2). *L. corallioides* R was reduced
206 under elevated temperature in the winter (Table 2). In the summer, an antagonistic effect of
207 pCO₂ and temperature was detected on R (Table 2; Supplementary material S2a).

208 In the summer, an interaction of grazing and temperature was detected, with an increase in
209 chlorophyll *a* content observed under elevated temperature in the presence of grazers, while a
210 decrease occurred in their absence (Table 2; Supplementary material S2b).
<figure><img src="image_2.png" /><figcaption>**Figure 1.** Winter and summer gross (a and b, respectively) and net (c and d, respectively) primary production and respiration (e and f, respectively) of *L. corallioides* in the different pCO₂ (A-pCO₂ = Ambient pCO₂; H-pCO₂ = High pCO₂) and temperature (T = Ambient temperature; T+3°C = High temperature) treatments, after being maintained three months in the presence (black) and absence (white) of grazers. Results are presented as means ± SE (n = 5). Results "with grazers" come from the work of Legrand *et al*. (2017).</figcaption></figure>
**Table 2.** Analysis of variance results for the effects of grazing (presence/absence), temperature (T; ambient/elevated) and pCO₂ (ambient/elevated) on gross and net primary production, respiration, chlorophyll *a* content, and light and dark calcification rates of *L. corallioides* in the winter and summer. Statistical analyses were performed using 3-way crossed ANOVAs. Significant p-values are shown in

<table><thead><tr><th>Gross production</th><th>Net production</th><th>Respiration</th><th>Chlorophyll a</th><th>Light calcification</th><th>Dark calcification</th></tr><tr><th>GPP</th><th>NPP</th><th>R</th><th></th><th>G<sub>l</sub></th><th>G<sub>d</sub></th></tr></thead></table>

bold (α = 0.05). Degrees of freedom = 1
<table><thead><tr><th></th><th>F</th><th>p</th><th>F</th><th>p</th><th>F</th><th>p</th><th>F</th><th>p</th><th>F</th><th>p</th></tr></thead><tbody><tr><td><strong>WINTER</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Grazing</td><td>0.7</td><td>0.42</td><td>0.5</td><td>0.47</td><td>0.3</td><td>0.59</td><td>1.5</td><td>0.23</td><td>19.0</td><td><strong>&lt; 0.001</strong> ↗</td></tr><tr><td>T</td><td>0.1</td><td>0.80</td><td>0.6</td><td>0.45</td><td>9.5</td><td><strong>0.004</strong> ↘</td><td>0.7</td><td>0.41</td><td>10.6</td><td><strong>0.003</strong> ↗</td></tr><tr><td>pCO<sub>2</sub></td><td>2.5</td><td>0.12</td><td>2.8</td><td>0.11</td><td>0.1</td><td>0.72</td><td>0.7</td><td>0.42</td><td>3.3</td><td>0.077</td><td>100.7</td><td><strong>&lt; 0.001</strong></td></tr><tr><td>Grazing x T</td><td>4.6</td><td><strong>0.041</strong></td><td>5.2</td><td><strong>0.030</strong></td><td>0.1</td><td>0.70</td><td>3.6</td><td>0.067</td><td>0.3</td><td>0.59</td><td>5.4</td><td><strong>0.027</strong></td></tr><tr><td>Grazing x pCO<sub>2</sub></td><td>1.2</td><td>0.29</td><td>1.2</td><td>0.28</td><td>0.1</td><td>0.71</td><td>0.3</td><td>0.61</td><td>0.2</td><td>0.66</td><td>14.2</td><td><strong>&lt; 0.001</strong></td></tr><tr><td>pCO<sub>2</sub> x T</td><td>6.1</td><td><strong>0.019</strong></td><td>9.9</td><td><strong>0.004</strong></td><td>1.1</td><td>0.29</td><td>0.4</td><td>0.55</td><td>0.5</td><td>0.49</td><td>0.0</td><td>0.95</td></tr><tr><td>Grazing x T x pCO<sub>2</sub></td><td>0.9</td><td>0.36</td><td>0.6</td><td>0.43</td><td>0.6</td><td>0.45</td><td>0.0</td><td>0.91</td><td>1.9</td><td>0.18</td><td>4.1</td><td>0.052</td></tr><tr><td><strong>SUMMER</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Grazing</td><td>27.2</td><td><strong>&lt; 0.001</strong> ↗</td><td>2.6</td><td>0.12</td><td>179.6</td><td><strong>&lt; 0.001</strong> ↗</td><td>5.0</td><td><strong>0.033</strong></td><td>77.1</td><td><strong>&lt; 0.001</strong></td><td>157.4</td><td><strong>&lt; 0.001</strong></td></tr><tr><td>T</td><td>0.6</td><td>0.44</td><td>5.2</td><td><strong>0.031</strong> ↘</td><td>19.5</td><td><strong>&lt; 0.001</strong></td><td>0.0</td><td>0.92</td><td>1.8</td><td>0.19</td><td>5.4</td><td><strong>0.028</strong></td></tr><tr><td>pCO<sub>2</sub></td><td>0.6</td><td>0.43</td><td>0.4</td><td>0.52</td><td>0.0</td><td>0.95</td><td>0.2</td><td>0.69</td><td>0.3</td><td>0.58</td><td>96.6</td><td><strong>&lt; 0.001</strong> ↘</td></tr><tr><td>Grazing x T</td><td>0.2</td><td>0.64</td><td>0.3</td><td>0.61</td><td>1.0</td><td>0.33</td><td>7.4</td><td><strong>0.011</strong></td><td>2.2</td><td>0.15</td><td>5.8</td><td><strong>0.022</strong></td></tr><tr><td>Grazing x pCO<sub>2</sub></td><td>0.1</td><td>0.77</td><td>0.3</td><td>0.62</td><td>0.3</td><td>0.58</td><td>2.5</td><td>0.12</td><td>8.6</td><td><strong>0.006</strong></td><td>1.9</td><td>0.18</td></tr><tr><td>pCO<sub>2</sub> x T</td><td>0.7</td><td>0.43</td><td>2.0</td><td>0.17</td><td>4.3</td><td><strong>0.048</strong></td><td>0.2</td><td>0.70</td><td>0.2</td><td>0.70</td><td>0.8</td><td>0.38</td></tr><tr><td>Grazing x T x pCO<sub>2</sub></td><td>0.5</td><td>0.48</td><td>0.0</td><td>0.84</td><td>1.9</td><td>0.18</td><td>0.0</td><td>0.84</td><td>1.9</td><td>0.18</td><td>1.1</td><td>0.31</td></tr></tbody></table>
<figure><img src="image_3.png" /><figcaption>**Figure 2.** Chlorophyll a content (mean ± SE, n = 5) of *L. corallioides* thalli maintained in the presence (black) and the absence of grazers (white) in the different pCO₂ (A-pCO₂ = Ambient pCO₂; H-pCO₂ = High pCO₂) and temperature (T = Ambient temperature; T+3°C = High temperature) treatments. Results "with grazers" come from the work of Legrand *et al*. (2017).</figcaption></figure>
227 The presence of grazer increased the light calcification rates ($G_l$) in the winter (+ 50%; Fig.3a)
228 and this positive effect was amplified in the summer (+ 106%; Fig.3b). In winter conditions,
229 $L. corallioides$ $G_l$ was significantly higher under elevated temperature (Table 2). In the
230 summer, an interactive effect of grazing and $pCO_2$ was detected on $G_l$, with an increase in $G_l$
231 observed under high $pCO_2$ in the absence of grazer, while a decrease was evidenced in their
232 presence (Table 2; Supplementary material S2c).

233
234 Grazing alone increased dark calcification rates ($G_d$) in the summer (+ 41%; Fig. 3c,d). In the
235 winter, the positive effect of increased temperature on $G_d$ was more pronounced in the
236 presence of grazers (Table 2; Supplementary material S1e). Furthermore, the decline in $G_d$
237 under high $pCO_2$ was more important in the presence of grazers (Table 2; Supplementary
238 material S1f). In the summer, increased $pCO_2$ alone significantly decreased $G_d$ (Table 2).

239 Grazing and temperature have an interactive effect on $G_d$ in the summer, as the decline in $G_d$
240 was mainly observed under elevated temperature in the absence of grazers.
<figure><img src="image_4.png" /><figcaption>**Figure 3.** Winter and summer light (a and b, respectively) and dark (c and d, respectively) calcification of *L. corallioides* in the different pCO₂ (A-pCO₂ = Ambient pCO₂; H-pCO₂ = High pCO₂) and temperature (T = Ambient temperature; T+3°C = High temperature) treatments, after being maintained three months in presence (black) and absence (white) of grazers. Results are presented as means ± SE (n = 5). Results "with grazers" come from the work of Legrand *et al*. (2017).</figcaption></figure>
248 **4. Discussion**

249 The present findings revealed that the response of *L. coralloides* to ocean acidification and
250 warming is highly related to changes in seasons and grazing conditions. Within natural
251 ecosystems, grazers are often associated with an important ecological function, exerting a
252 control on the composition and abundance of plant biomass (Hairston *et al.* 1960; Guillou *et
253 al.* 2002; Bonaviri *et al.* 2011). In the present study, weak effects of grazing were detected on
254 gross primary production, respiration, chlorophyll *a* content, and dark calcification of *L.
255 corallioides* in the winter. In their study, Legrand *et al.* (2017) evidenced a lower metabolic
256 rate and feeding activity of the gastropod *G. magus* and the urchin *P. miliaris* in winter,
257 compared with summer conditions. The reduced activity of these species may decrease the
258 grazing pressure on *L. corallioides* and thus the impact on its physiology. Moreover, the
259 reduced metabolism of *L. corallioides* in the winter may also partly explain the relative lack
260 of significant effect of grazing in this condition. Conversely in the summer, changes in gross
261 primary production, respiration, chlorophyll *a* content, and calcification appeared mainly
262 related to grazing. Positive grazer-algal interaction has often been evidenced between grazers
263 and coralline algae (Steneck 1982, 1983; Littler *et al.*, 1995, Wai and Williams 2005), grazing
264 acting as a “service” for algae by removing algal competitors (Steneck 1982; Carpenter 1986;
265 Littler *et al.*, 1995; Wai and Williams, 2005). Grazers may also remove a part of non-
266 photosynthetic tissues and senescent cells present on the thallus surface (Wegeberg and
267 Pueschel 2002). This increases light availability for underlying photosynthetic cells and
268 enhances primary production (Littler *et al.* 1995; Silliman and Zieman 2001). The presence of
269 compensatory mechanisms in *L. corallioides* may also increase the primary production in
270 response to grazing (Lamberti and Moore, 1984; Wai and Williams, 2005). The present
271 results also highlighted an increase in calcification in response to grazing, which may act as a
structural protection for the algae (Steneck 1983; Hay et al. 1994; Littler et al. 1995; Rahman and Halfar 2014).

Several studies evidenced that moderated temperature rise usually enhanced photosynthesis, respiration and calcification in coralline algae (see review by Martin *et al.* 2013). The present data suggested that the relationship between temperature and physiological processes may be more complex, depending on the season and grazing condition. In the winter, increased temperature enhanced *L. corallioides* photosynthesis in the absence of grazers, while a decline was observed in their presence. On the other hand, calcification was positively affected by increased temperature, especially in the presence of grazers. In several coralline algae, photosynthesis involves carbon concentration mechanisms to convert bicarbonates (HCO₃⁻) to CO₂ for Rubisco using carbonic anhydrase enzyme (Giordano *et al.* 2005; Hofmann *et al.* 2012). Hofmann *et al.* (2012) suggested that this enzyme may also be involved in the calcification process to convert CO₂ into HCO₃⁻ and then carbonates (CO₃²⁻). It is likely that the increase in carbonic anhydrase activity with temperature may help algae to increase calcification in the winter and to maintain its structural protection in the presence of grazers. However, the allocation of carbonic anhydrase to the calcification process may be detrimental to photosynthesis, as these two processes may thus be concurrent (Martin *et al.* 2013). This hypothesis would be consistent with the decline of photosynthesis observed under elevated temperature in the presence of grazers.

In the winter, the negative effect of pCO₂ rise on dark calcification was exacerbated in the presence of grazers. In the summer, the pCO₂ rise positively affected light calcification, while a negative effect was observed in the presence of grazers. In coralline algae, carbonate precipitation occurs in the cell walls, excepted for reproductive and epithelial cells, which are located just below the thallus surface (Irvine and Chamberlain 1994). A part of epithelial cells was likely to be removed by grazers (Steneck 1982), making calcified cells more exposed to
297 the external environment. When grazing and pCO₂ increase are combined, calcified cells
298 would thus be more exposed to dissolution, reducing light calcification rates. Moreover,
299 calcification is generally considered as a structural defense in calcareous algae (Hay et al.
300 1994). Thus, ocean acidification is likely to affect their structural integrity increasing their
301 vulnerability to grazing (Johnson and Carpenter 2012; Ragazzola et al. 2012). In the urchin
302 *Paracentrotus lividus*, Asnaghi et al. (2013) also evidenced an increase in coralline
303 consumption under high pCO₂. This process may be essential in controlling the response of *P.*
304 *lividus* to ocean acidification through a modulation of carbonate uptake (Asnaghi et al., 2013).
305 In a context of ocean acidification, the presence of grazers may thus increase the sensitivity of
306 *L. corallioides*. Despite this, the sensitivity of *L. corallioides* to ocean acidification is likely to
307 be weakened when increased pCO₂ is combined with increased temperature in the presence of
308 grazers, which differs from the conclusions of other studies (Anthony et al., 2008; Martin and
309 Gattuso, 2009).

310 In conclusion, the present findings provide evidence that both season and grazing have a
311 major impact on the physiology of *L. corallioides* and drive its response to ocean acidification
312 and warming. In the winter, the ability of *L. corallioides* to enhance its calcification under
313 elevated temperature may act as an important process to maintain the structural integrity of
314 thalli in the presence of grazers. However, the metabolic cost of maintaining calcification to
315 face grazing may be detrimental to other physiological processes, such as photosynthesis. In
316 the summer, grazers potentially moderated epiphytic development, decreasing the light and
317 nutrients competition induced by fast growing turf algae. Our results also evidenced that *L.*
318 *corallioides* calcification was adversely affected when grazing was combined with increased
319 pCO₂ in the summer. Through their feeding activity, grazers may alter the structural integrity
320 of thalli and increase the sensitivity of coralline algae to ocean acidification. These results
321 provide insights that grazers may have an important function in the response of coralline algae
322 in the context of climate change. Field and laboratory experiments considering both seasonal
323 variations and the response of species in multi-species assemblage are therefore critical to
324 make ecologically relevant predictions of the effects of future environmental changes.

325 **Acknowledgements**

326 The authors thank the CRBM (Center of Marine Biologic Resources) of the Station
327 Biologique de Roscoff for its kind permission to use their premises for the duration of the
328 experiments. We are grateful to Olivier Bohner for his field and laboratory assistance and for
329 the help for system maintenance. We also thank Murielle Jam for samples freeze-drying. We
330 acknowledge the crew of the oceanographic vessel Albert Lucas for its help with species
331 collection. We also thank the SOMLIT (Service d'Observation en Milieu LITtoral, INSU-
332 CNRS) program for the temperature data sets provided. This work was supported by the
333 Brittany Regional Council, the French national EC2CO program ("Écosphère Continentale et
334 Côtière", project MAERLCHANGE) and the French National Research Agency via the
335 "Investment for the Future" program IDEALG (n° ANR-10-BTBR-04).

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Winter conditions

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485

486 S1. Interaction plots for the effects of grazing, temperature and pCO₂ on the metabolism of *L.*

487 *corallioides* in winter conditions. Plots were done when an interactive effect of at least two

488 factors was detected (grazing, temperature and/or pCO₂) using 3-way ANOVA (p-value in

489 bold).
Summer conditions

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S2. Interaction plots for the effects of grazing, temperature and pCO₂ on the metabolism of *L.*

*corallioides* in summer conditions. Plots were done when an interactive effect of at least two

factors was detected (grazing, temperature and/or pCO₂) using 3-way ANOVA (p-value in

bold).