# From reproductive behaviour to responses to predators: Ocean acidification does not impact the behaviour of an herbivorous marine gastropod

Sabine Roussel, Manon Coheleach, Sophie Martin, Rob Day, Aicha Badou, Sylvain Huchette, Philippe Dubois, Arianna Servili, Fanny Gaillard, Stéphanie Auzoux-Bordenave

► To cite this version:

Sabine Roussel, Manon Coheleach, Sophie Martin, Rob Day, Aicha Badou, et al. From reproductive behaviour to responses to predators: Ocean acidification does not impact the behaviour of an herbivorous marine gastropod. Science of the Total Environment, 2024, 907, pp.167526. 10.1016/j.scitotenv.2023.167526. hal-04298084

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From reproductive behaviour to responses to predators: ocean acidification does not impact the behaviour of an herbivorous marine gastropod.

Sabine ROUSSEL¹*, Manon COHELEACH¹, Sophie MARTIN², Rob DAY³, Aicha BADOU⁴,
Sylvain HUCHETTE⁵, Philippe DUBOIS⁶, Arianna SERVILI⁷, Fanny GAILLARD²,
Stéphanie AUZOUX-BORDENAVE⁸

¹. Université de Brest, CNRS, IRD, Ifremer, LEMAR, Plouzané F-29280, France

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2. UMR 7144 "Adaptation et Diversité en Milieu Marin" (AD2M), CNRS/SU, Station Biologique de Roscoff, Roscoff Cedex 29680, France

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3. School of Biological Sciences, University of Melbourne, Parkville, Vic., Australia

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4. Direction Générale Déléguée à la Recherche, l'Expertise, la Valorisation et l'Enseignement (DGD REVE), Muséum National d'Histoire Naturelle, Station marine de Concarneau, Concarneau 29900, France

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5. France Haliotis, Kerazan, Plouguerneau 29880, France

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6. Laboratoire de Biologie Marine, Université Libre de Bruxelles, Brussels CP160/15, 1050, Belgium

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7. IFREMER, Université de Brest, CNRS, Plouzané IRD, LEMAR, F-29280, France

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8. BOREA, MNHN/CNRS/SU/IRD, Muséum National d'Histoire Naturelle, Station Marine de Concarneau, Concarneau 29900, France

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*Corresponding author: Sabine Roussel

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Tel.: 33 (0) 2 98 01 70 43

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E-mail address: sabine.roussel@univ-brest.fr

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28 Ocean acidification (OA), which reduces ocean pH and leads to substantial changes in seawater
29 carbonate chemistry, may strongly impact organisms, especially those with carbonate skeletons.

30 In marine molluscs, while the physiological effects of OA are well known, with a reduction of
31 growth and shell calcification, there are few studies on behavioural effects. A large marine
32 gastropod, *Haliotis tuberculata*, was exposed to ambient (pH~T~ 8.0) or low pH (pH~T~ 7.7) during
33 a 5-month experiment. Because animal fitness can be affected through various behavioural
34 changes, a broad spectrum of behavioural parameters was investigated, including situations
35 involving no stress, responses to predators, righting to evaluate indirectly the level of energy
36 reserves, and finally, reproductive behaviour. In addition, we measured the expression profile
37 of the GABA A-like and serotonin receptor genes, often described as central neuromodulators
38 of sensory performance and behaviour and known to be affected by OA in molluscs. No
39 significant effect of low pH as compared to ambient pH was observed on abalone behaviour for
40 any of these behavioural traits or gene expressions after either one week or several months of
41 exposure to OA. The significance tests were corroborated by estimating the size of pH effects.
42 The behaviour of this mollusc appears not to be affected by pH decrease expected by the end
43 of the century, suggesting some resilience of the species to OA at the adult stage. This is
44 probably related to the ecological niche of this abalone, where important pH variations can be
45 observed at tidal, diurnal or seasonal scales.

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47 KEYWORDS: ocean acidification, behaviour, predator, diurnal rhythm, reproductive
48 behaviour, abalone, *Haliotis tuberculata*

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## 1. Introduction

Rising atmospheric carbon dioxide ($CO_2$) reduces ocean pH and causes a shift in seawater carbonate chemistry (Orr et al., 2005; Doney et al., 2009). The average ocean surface water pH is expected to decrease at least by 0.3 pH unit at the end of the century if annual $CO_2$ emissions continue to increase and are not stabilised rapidly (scenarios with high and very high greenhouse gas concentrations, SSP3 -7.0 and SSP5 – 8.5) (IPCC, 2021). These changes will take place at an unprecedented rate for marine organisms (Gattuso et al., 2015) threatening calcifying species such as corals and molluscs (Hofmann et al., 2010; Kroeker et al., 2013).

One way animals may respond to a changing environment is through modifications of their behaviour (Nagelkerken et Munday, 2016). Ocean acidification (OA) affects a plethora of marine animal behavioural traits. A majority of the studies have been done on fish behaviour, focusing specifically on predator-prey interactions: larval, juveniles or adult fishes become less risk averse, and are even attracted to the odour of predators. In addition, a reduction of the ability to find settlement sites and to find food or prey was reported (reviews from Briffa et al. 2012; Nagelkerken and Munday 2016; Ashur et al. 2017). Results are much less consistent in marine invertebrates, with contrasting results depending on taxon, stage, $pCO_2$ exposure and the behaviour studied (Clements et Comeau, 2019). Some experiments have reported negative effects on predator avoidance (Manriquez et al., 2014; Jellison et al., 2016; Manriquez et al., 2016), predation rate (Dodd et al., 2015), foraging performance (Leung et al., 2015; Horwitz et al., 2020; Park et al., 2020), shelter use (Park et al., 2020), swimming behaviour (Gravinese et al., 2019), response to flow (Cohen-Rengifo et al., 2019), righting time (Manriquez et al., 2013) or decision-making (de la Haye et al., 2011). However, others found no detectable effects on the ability to right (Schram et al., 2014; McCarthy et al., 2020), capacity to detect the position
of the prey/food (Manriquez et al., 2014), escape ability (Schram et al., 2014) or motility (Jellison et al., 2016). Some even observed increased avoidance behaviour to predators (Bibby et al., 2007), increased activity (Watson et al., 2017) or righting ability (Manriquez et al., 2016).

Several factors may explain these contrasted effects. The difference in the reduction of pH might be a factor explaining the contrasted results. In some experiments, molluscs were exposed to a pH reduction similar (0.3-0.4 pH unit) to the one expected at the end of century in scenarios with intermediate or high greenhouse gas concentrations in subtidal environment (Manriquez et al., 2014; McCarthy et al., 2020; Park et al., 2020) while in other experiments, pH reduction was stronger with fluctuations up to pH observed in intertidal rock pools, estuaries or during upwelling processes (Dodd et al., 2015; Jellison et al., 2016; Manriquez et al., 2016). Some species may have been subjected to greater pH fluctuations in estuarine and tidepool environments than the overall level expected by the end of the century, and thus have evolved resilience. Another explanation is the difference between experiments of exposure time to low pH: some short-term experiments have exposed species to low pH for only a few hours or days (for a review, see Briffa et al. 2012, Clements and Comeau 2019), while other exposure times involved a few weeks (Bibby et al., 2007; Schram et al., 2014) or several months (Manriquez et al., 2013; Manriquez et al., 2014). In addition, most of these studies were based on only few behavioural variables, which probably are not representative of the complex behavioural responses of an animal facing environmental stress.

*Haliotis spp.* are calcifying gastropods in the order Vetigastropoda, and some are among the largest marine herbivores in the world. Most species live on rocky shores, in narrow crevices or under boulders, in low-tidal zone and shallow subtidal environments. On the Atlantic coast of France, *H. tuberculata* is naturally exposed to variable seawater pH for several hours during
emersion of spring tides in rock pools of the low tide zone. In rock pools of the mid-tidal zone, pH~T~ can decrease down to 7.5 at night and increase up to 10 during day-light periods (Legrand et al., 2018). However, most of the individuals are found in subtidal zones in a much less variable pH environment. For instance, in the Bay of Brest, one of this species natural habitats, pH~T~ ranges between 7.9 in autumn up to 8.2 during winter and spring periods (Qui-Minet et al., 2018).

With this study, we aimed to elucidate the effects of OA using a high greenhouse gas emission scenario (IPCC, 2021) on a wide range of key behavioural parameters that determine the persistence of this mollusc found worldwide in coastal area. An experimental approach with different time-scales and extensive behavioural measurements was undertaken. The tests were performed one week and up to five months after the start of OA exposure. In addition to stress responses under different pH conditions, unstressed abalone behaviour was studied in the experimental aquariums, in the absence of any handling stress. Because reproductive behaviour directly impacts the fitness of a species, spawning behaviour was also measured under ambient and decreased pH. Furthermore, because the effects of OA on behaviour might be a consequence of chemosensory impairment, altered central processing impairment, or both (Ashur et al., 2017), we measured the expression profile of two genes involved in neurosensory transmission (the GABA A-like receptor and the serotonin receptor). The consequences of OA on behaviour of marine species has recently been quite controversial (Clark et al., 2020). To take into the “decline effect” in OA impacts reported on fish behaviour (i.e. the decrease in significant negative effects found in recent studies compared to initial studies), the estimated size effect was calculated for each test performed in the present study (Clements et al., 2022) to highlight the magnitude of any difference, if any was present.
## 2. Materials and methods

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### 2.1. Abalone collection and experimental set-up

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In January 2017, 3.5-year-old *H. tuberculata* were collected from an offshore sea-cage structure at the France Haliotis abalone farm (48°36′50N, 4°36′3W; Plouguerneau, Brittany, France) containing 600 individuals (48.5 ± 4.2 mm shell length, 16.2 ± 4.4 g shell weight). During the sea-rearing procedure, fresh algae, mainly composed of a mixture of *Palmaria palmata*, *Laminaria digitata* and *Saccharina latissima*, were collected on the shore and provided *ad libitum* to each sea-cage once a month. Abalone were brought to the France Haliotis land-based facilities in the sea-cage in less than one hour, ensuring minimum stress during transport and minimum handling. After gently detaching the abalone from their support with a spatula, each abalone was measured and weighed. Because abalone are mostly nocturnal foragers (Roussel et al., 2020), a phosphorescent tag was glued to the shell with cyanoacrylate gel to record their night-time behaviour. In addition, numbered Hallprint® tags were glued to the shell to identify the abalone individually in tanks.

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The individuals were randomly distributed into ten 45 L experimental rearing tanks (1 x w x h, 50 x 30 x 35 cm) equipped with baked clay hiding places (n = 26 abalone per tank) and the abalone were fed twice every week with macroalgae. A baked clay ring weight was used to hold the algae on the bottom of the tank, opposite the hiding place (Figure 1). The daily light versus dark regime was adjusted following the seasonal cycle (9 : 15 hr in February, 10 : 14 in March, 11 : 13 in April, 12 : 12 in May, and 13 : 11 in June). Temperatures were adjusted monthly from 12.5 C in January to 18.5 C in June to follow the natural seasonal change. To avoid stressful conditions during light/dark changes, a transition of light level was programmed
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over 30 min during dawn and dusk using a dimmer (Gold Star, Besser Elektronik, Italy).

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Abalone were conditioned in the laboratory for three weeks before the start of the experiment
under ambient pH/pCO₂ conditions.

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A detailed description of the experimental set-up can be found in Avignon et al. (2020). Briefly, five tanks were randomly assigned to each of two pH treatments: a control condition corresponding to the local seawater pH (pH<sub>T</sub> of 8.0, corresponding to a pCO<sub>2</sub> of ~460 µatm) and a lower pH value (pH<sub>T</sub> of 7.7, pCO<sub>2</sub> of ~1000 µatm) corresponding to the projected decrease of -0.3 pH units under climate change scenario SSP3-7.0 (IPCC, 2021). Experimental design recommendations in OA research (Cornwall et Hurd, 2016) were followed by using header tanks (Figure 1) with independent CO<sub>2</sub> bubbling and pH control as well as conversion to pH units on the total scale (pH<sub>T</sub>) after calibration with Tris/HCl and 2-aminopyridine/HCl buffers (Dickson, 2010).

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<figure><img src="image_2.png" /></figure>

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<caption>**Fig. 1:** Experimental apparatus showing the experimental rearing tank and header tank where pCO₂ was adjusted to the target pH.</caption>

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## 2.2. Control and monitoring of pH and carbonate parameters

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In February 2017, pH values in the header tanks for the low pH treatments were gradually

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decreased over 6 days by 0.05 pH units / day until pH~T~ 7.7 was reached. Thereafter, the pH

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treatments were maintained for 5 months between February and the end of June 2017. In the

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five CO₂-enriched header tanks, pCO₂ was adjusted by bubbling CO₂ (Air Liquide, France)

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through electro-valves controlled by a pH-stat system (IKS Aquastar, Germany). Experimental

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tanks were continuously supplied with seawater from the header tanks with a minimum of 15

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l·h⁻¹. Total alkalinity (A~T~) of seawater was measured monthly on 50-mL samples taken from

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each experimental tank according to the method described in Avignon et al. (2020).

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Calculations of pCO₂, dissolved inorganic carbon (DIC), HCO₃⁻, CO₃²⁻ concentrations, and

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saturation state of aragonite (Ωar) and calcite (Ωca) are detailed in Avignon et al. (2020).

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## 2.3. Behavioural test procedures

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A trained observer carried out behavioural analyses for the diurnal, hiding, righting, predator

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and foot contact tests described below, using the Observer program (Observer©XT, Noldus).

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It was not possible to do blinded analyses as the abalone were identified with individual tags.

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The diurnal rhythm, feed intake and foot contact tests, involving no or mild stress, were done

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in the experimental rearing tanks using all the abalone in the tank. Tests were performed at the

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same pH/pCO₂ as those of the treatment just after reaching the target pH to measure acute stress

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responses to pH decrease (week 1, W1), and several months after exposure started to measure

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chronic stress effects (three, four or five months, respectively - M3, M4, M5) (see below for

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details).
Responses to acute stress situations such as the righting, hiding and predator tests were done in an aquarium or a raceway in the laboratory next to the experimental facility. Aquarium or raceway was cleaned 3 times and renewed with clean water before testing each individual abalone. It was filled with seawater taken from the abalone buffer tank from the same treatment. Four abalone from each experimental tank were tested one week after reaching the target pH (W1). Different abalone were tested for each behavioural test to avoid handling bias. The same individuals were tested again after 3 months of pH exposure (M3). A 3-month period is sufficiently long to allow a recovery from the experimental procedure for abalone. The four abalone from the same tank were tested consecutively and returned to their tank after all were tested to avoid confronting other abalone with stress cues. Treatments were tested alternatively but in a random sequence of the five replicate tanks.

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205 **2.3.1. Diurnal rhythm**

Diurnal rhythm was studied the first 48h after reaching the target pH (W1, using 26 abalone per tank and n = 5 tanks per treatment) and after 3 months of exposure to low pH (M3, using a minimum of 16 abalone per tank and n = 5 tanks per treatment). Measures were done with no handling stress: all experimental aquariums were continuously videotaped with 3 digital cameras (TS-WD6001HPSC, Sygonix Gmbh, Germany), linked to a 24h-recording device (TVVR 40021, Abus, Germany). Videos were recorded over 48-h for all the aquariums and analysed using scan sampling with the Observer program. Every 10 minutes, the number of abalones moving (i.e. the number that had changed position between two scan samplings), the number of abalones eating algae (less than 2 cm from the algae with small movements of the algae observed), and the number of abalones in the open zone (not under the baked clay hiding place or next to the algae) were recorded. From these data, the following variables were
calculated per aquarium: time spent moving (min.abalone⁻¹.day⁻¹), time spent feeding (min.abalone⁻¹.day⁻¹) and time spent in open zone (min.abalone⁻¹.day⁻¹).

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## 2.3.2. Feed intake

Feed intake was measured over the first 3 months, two or three times a week (28 measures per tank, n = 5 tanks per treatment). At each food distribution, fronds of *Palmaria palmata* were delicately dried on absorbent paper, weighed and then placed in the clay ring opposite to the hiding place. *P. palmata* is the most appropriate alga for *H. tuberculata* growth (Roussel et al., 2019a). The remaining algae were removed, dried with the same method and weighed. Two additional samples of this alga were placed in two extra aquariums submitted to normal and low pH but without abalone. The quantity of wet algae (in mg) ingested per gram of wet abalone in each aquarium was calculated, taking into account the degradation of the algae under each treatment in the extra aquariums. Weights of abalone in each aquarium were estimated using initial weight before acclimation, W1 and M2 measurements (Avignon et al., 2020).

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## 2.3.3. Foot contact test

This test was performed 4 days after reaching the target pH (W1, n = 105 abalone per treatment) and after 3 months of exposure (M3, n = minimum 83 abalone per treatment). The test involved gently touching the abalone resting (relaxed foot with epipodium visible) with a finger on the outer mantle edge border, and running the finger clockwise around the abalone in 10 s while the abalone were in their tanks. The test stopped once the abalone returned to their initial resting position (or after 90 s if they did not return). The following variables were measured: number of abalones doing a swivelling movement after touching (swivelling movement of the abalone shell from one side to the other, often observed to break the predator grip), number of abalones changing position after touching (epipodium i.e. appendages along the foot with sensory organs

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or / and foot contraction) and number of abalone changing of position and returning to their initial position before the end of the observation period.

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### 2.3.4. Righting test

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A glass aquarium (w × l × h, 20 × 35 × 20 cm) was used, filled with 5 l of seawater. Individual abalone were placed on their back in the centre of the aquarium. The time to right was measured from the time the abalone were placed in the aquarium on their shell until they had fully turned over. The number of attempts to turn over (defined as the number of times the abalone placed its foot on the bottom and contracted its muscle) was also recorded. If an abalone did not successfully turn over after a delay of 4 min, a time to right of 4 min was recorded for this abalone.

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### 2.3.5. Hiding test

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A glass aquarium (w × l × h, 20 × 35 × 20 cm), filled with 5 l of seawater, and with a halogen light above, was equipped with a baked clay shelter positioned at one end of the aquarium. Abalone were placed on their foot at the side of the test aquarium opposite the shelter. The time until the first movement and the time before complete hiding were measured, with a maximum time of 15 min if an abalone did not move into the shelter.

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### 2.3.6. Starfish predator test

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Starfish *Marthasterias glacialis* (20 cm width) were collected from the offshore abalone sea cages of France Haliotis, placed in a 30 L aquarium and fed with dead, non-experimental abalone twice a week during the experimental test period. A raceway (l × w × h, 2.5 × 0.4 × 0.15 m) filled with 50 l of seawater was used, with a 10 × 10 cm square grid printed on the bottom and on the side of the raceway. An abalone was placed in the centre of the raceway on

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its foot. When the abalone had a semi-relaxed or relaxed foot attached to the bottom of the tank, the starfish was held in contact with the abalone foot for 10 s. While the starfish touched the abalone, any protective swivelling movement of the abalone shell was recorded as well as any mucus release, any turn-around behaviour and any movement directly away from the starfish.

The number of abalone that performed these four escape behaviours was recorded. The time until the first movement was measured. In addition, the time spent moving, the number of squares crossed as well as the time to reach the edge of a circle of radius 20 cm away from the predator were recorded during the 5 min period of the test.

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### 2.3.7. Spawning behaviour

The spawning behaviour was measured after 5 months of exposure (M5) in the spawning room of France Haliotis. A LED light covered with a red filter (Medium red, Rosco Supergel) allowed us to observe abalone behaviour while not disturbing them during the spawning process. On the day of spawning induction, a maximum of 4 female and 4 male abalone were selected per tank out of the 12 abalone available (i.e. abalone used for the righting, hiding and predator abalone tests). Sex-ratios were equalised as far as possible. In total, 34 abalone for pH~T~ 7.7 and 37 abalone for pH~T~ 8.0 were studied. Abalone were placed individually in 5 L buckets with continuous water renewal. Spawning induction was performed by shining ultraviolet (UV) light and simultaneously heating the filtered seawater fed into the buckets from 17°C to 21°C over the course of 1h. Two buffer tanks were used connected to the 5 L buckets: one tank at pH~T~ 8.0 and one tank at pH~T~ 7.7 to match the treatment applied to the abalone. The circulation of UV irradiated water was stopped as the abalone started to spawn. Abalone were allowed to spawn for 5.5 h from the start of the experiment. The time of active preparation movement (active movement in the bucket, crawling to the top of the bucket or a swivelling behaviour) as well as the duration of spawning (ejection of the gametes into the water column) and the number of

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<figure><img src="image_3.png" /></figure>

abalone spawning was recorded. Effect of pH on other reproduction parameters are presented
in Avignon et al. (2020).

<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>

## 2.4. Gene expression analysis

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

The expression profiles of selected genes were analysed in the head of one to two individual
abalone per aquarium (n = 9 for pH 8.0 and n = 10 for pH 7.7) after four months (M4) of
exposure to ambient and low pH. The GABA A-like receptor and the serotonin (5-HT) receptor
genes were selected because of their putative functions in the neuroendocrine regulation of
behaviour (notably locomotion, memory and learning) in gastropods. The methods of gene
expression analysis are described in Avignon et al. (2020).

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

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<figure><img src="image_26.png" /></figure>

<figure><img src="image_27.png" /></figure>
317 **Table 1**

318 Specific primers used for gene expression analysis in *Haliotis tuberculata*: Genbank accession
319 number, primer sequences and reference.

<table><thead><tr><th>Gene</th><th>Sequence 5'-3'</th><th>Reference</th></tr></thead><tbody><tr><td>GABA<br>receptor</td><td>F-<br>GCACACCGAGTGTGAGAAGA<br>R-<br>CAACACTGAATGTGGCGAAC</td><td>(Auzoux-<br>Bordenave et al.,<br>2022)</td></tr><tr><td>Serotonin<br>receptor</td><td>F-<br>GCTACCACGACGACCATATC<br>R-<br>CCTTTCACGCTTTAGTTCCA</td><td>(Auzoux-<br>Bordenave et al.,<br>2022)</td></tr><tr><td>18S</td><td>F-<br>GGTTCCAGGGGAAGTATGGT<br>R-<br>AGGTGAGTTTTCCCGTGTTG</td><td>Gaume et al.</td></tr><tr><td>EF1</td><td>F-<br>ATTGGCCACGTAGATTCTGG<br>R-<br>GCTCAGCCTTCAGTTTGTCC</td><td>Gaume et al.</td></tr></tbody></table>

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322 2.5. Ethical notes
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All the abalone used for behavioural experiments were returned to the commercial sea-cages at the end of the experiment. At the end of the tests, starfish were released in the field where they were collected.

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## 2.6. Statistical analysis

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All statistical analyses were performed with R software (R Core Team, 2015). Where continuous data from individual abalone were recorded (Righting test, hiding test, starfish predator test, spawning behaviour, gene expression), differences between treatments were tested using linear mixed models with the lmerTest package (Kuznetsova et al., 2017) based on the methods described by Winter (2013). This model used the pH as a fixed factor and aquarium as a random factor. In addition, the abalone length was added as covariate for behavioural tests.

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For the diurnal rhythm, a mixed model was used with pH and days as a fixed factor, and the aquarium as a random factor. Statistical analysis was performed separately for the data obtained after reaching the target pH (W1) and after several months of low pH exposure (M3, M4 or M5 tests). The denominator degrees of freedom and F statistic were computed using Satterthwaite's method. The normality of the residuals was verified with Shapiro's test and homogeneity of variance with Levene's test. When assumptions of homogeneity of variance and normal distribution of residuals were not confirmed, the data were log, inverse or square root transformed. If normality of residuals was not verified after data transformations, values from individual abalone were averaged by aquarium, and aquariums were used as the replicates (n = 5 per treatment) in a Mann-Whitney U test (also called Wilcoxon rank sum test). If homogeneity of variance could not be verified after data transformations, a Welch's test was applied using averaged values per tank. Differences were considered significant at *p* < 0.05, and a trend at *p* < 0.10. Data are presented as means of treatments ± standard error unless otherwise indicated. Where the data recorded were counts (i.e. foot contact test), contingency tables were used and

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a Pearson's chi-square test was performed or a Fisher's exact test if there were less than 5 expected counts per cell. No aquarium effect could be included when using contingency table analysis.

Effect size and effect size variances were estimated for the continuous and ratio-type behavioural variables of this experiment using the methodology of Clements et al. (2022) to evaluate how the difference between the two treatments was large. A logarithmic transformed response ratio was calculated using the formula:

$$ \ln RR = \ln $$

where $X_{pH7.7}$ and $X_{pH8}$ are the average measured response in each pH treatment.

Effect size variance was calculated as

$$ v = \frac{(S_{pH7.7})^2}{n_{pH7.7} X_{pH7.7}} + \frac{(S_{pH8})^2}{n_{pH8} X_{pH8}} $$

where S and n are the standard deviation and sample size for pH7.7 and pH8 treatment, and $X_{pH7.7}$ and $X_{pH8}$ are the average measured response in each experimental and control treatment.

For continuous variables, behavioural data were averaged per tank to take into account potential tank effects (sample size, n = 5 per treatment). For binomial data (such as the number of abalone that changed their position), percentages were calculated per tank (n = 5 per treatment) and used to calculate the effect size variance and effect size.

## 3. Results

### 3.1. *Behavioural tests*

#### *Diurnal rhythm and feed intake*
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No significant effect of decreased pH was observed at either W1 or M3 for the time spent moving (W1: F<sub>1,8</sub> = 0.259, p = 0.624; M3: F<sub>1,8</sub> = 2.183, p = 0.178, mixed model analysis), the time spent feeding (W1: F<sub>1,8</sub> = 3.416, p = 0.102; M3: F<sub>1,8</sub> = 0.966, p = 0.354, mixed model analysis), and the time spent in the open area of the tank floor (W1: F<sub>1,8</sub> = 0.564, p = 0.474; M3: F<sub>1,8</sub> = 2.504, p = 0.152, mixed model analysis) (Table 2). No significant effect of the covariate abalone length was found. Similarly, the feed intake was not significantly affected by the pH during the first 3-months (W<sub>1,8</sub> = 0.083, p = 0.780, Welch's test, Table 2).

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**Table 2**

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Behaviour responses of adult Haliotis tuberculata exposed to ambient pH (pH<sub>T</sub> 8.0) and low pH (pH<sub>T</sub> 7.7). Diurnal behaviour was observed by scan sampling during a 48-h period after 2 days (W1, 26 abalone per tank, 5 tanks per treatment) and after 3 months of exposure (M3, n = 17-18 abalone per tank, 5 tanks per treatment). Feed intake was measured twice a week during the first three months of exposure. Mixed model analysis and lsmeans ± S.E.M, except otherwise stated.

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<table><thead><tr><th>pH</th><th>pH 8.0</th><th>pH 7.7</th><th>F<sub>1,8</sub> / F<sub>w</sub></th><th>P</th></tr></thead><tbody><tr><td colspan="5"><em>W1 diurnal behaviour (min. abalone<sup>-1</sup>.day<sup>-1</sup>)</em></td></tr><tr><td>Time spent moving</td><td>60.0 ± 6.09</td><td>55.7 ± 6.09</td><td>0.259</td><td>0.624</td></tr><tr><td>Time spent feeding</td><td>19.6 ± 2.22</td><td>13.8 ± 2.22</td><td>3.416</td><td>0.102</td></tr><tr><td>Time spent in open zone</td><td>56.1 ± 9.49</td><td>46.0 ± 9.49</td><td>0.564</td><td>0.474</td></tr><tr><td colspan="5"><em>M3 diurnal behaviour (min.abalone<sup>-1</sup>.day<sup>-1</sup>)</em></td></tr><tr><td>Time spent moving</td><td>49.3 ± 5.44</td><td>37.9 ± 5.44</td><td>2.183</td><td>0.178</td></tr><tr><td>Time spent feeding</td><td>37.0 ± 6.71</td><td>27.6 ± 6.71</td><td>0.966</td><td>0.354</td></tr><tr><td>Time spent in open zone</td><td>54.1 ± 6.28</td><td>40.1 ± 6.28</td><td>2.504</td><td>0.152</td></tr></tbody></table>
Feed intake during a 3-month period

Quantity of algae ingested per gram of 31.0 ± 0.70 30.7 ± 0.70 0.083 0.780
abalone (mg algae. g⁻¹ abalone. day⁻¹) δ

δ Welch's test, mean ± s.e.

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391
Table 3

392 Behavioural responses of abalone Haliotis tuberculata exposed for 1 week (W1), three months (M3) or five months (M5) to ambient pH (pH<sub>T</sub> 8.0)

393 or to low pH (pH<sub>T</sub> 7.7) (chi-square or mixed model F test unless otherwise stated). Results are lsmeans ± s.e.m if mixed model analysis, otherwise

395 counts or means ± s.e.

396

<table><thead><tr><th rowspan="2">Tests</th><th colspan="4">W1</th><th colspan="4">M3</th></tr><tr><th>pH 8.0</th><th>pH 7.7</th><th>F/χ<sup>2</sup>/W</th><th>p</th><th>pH 8.0</th><th>pH 7.7</th><th>F/χ<sup>2</sup>/W</th><th>p</th></tr></thead><tbody><tr><td><em>Foot contact test</em></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Number of abalone doing a swivelling movement<sup>δ</sup></td><td>3 out of 105</td><td>3 out of 105</td><td>0<sup>δ</sup></td><td>1<sup>δ</sup></td><td>14 out of 83</td><td>7 out of 86</td><td>2.96</td><td>0.086</td></tr><tr><td>Number of abalone changing of position after touching</td><td>63 out of 105</td><td>61 out of 105</td><td>0.078</td><td>0.779</td><td>54 out of 83</td><td>54 out of 86</td><td>0.094</td><td>0.759</td></tr><tr><td>Number of abalone returning to their initial position</td><td>42 out of 105</td><td>38 out of 105</td><td>0.389</td><td>0.533</td><td>49 out of 83</td><td>48 out of 86</td><td>0.179</td><td>0.672</td></tr><tr><td><em>Righting test</em></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Time to right (s)</td><td>75.2 ± 15.21</td><td>91.9 ± 15.21</td><td>0.965</td><td>0.332<sup>β</sup></td><td>62.5 ± 20.92</td><td>108.3 ± 20.65</td><td>1.61</td><td>0.239<sup>β</sup></td></tr><tr><td>Number of attempts to turn over</td><td>1.65 ± 0.42</td><td>1.85 ± 0.32</td><td>17</td><td>0.396<sup>γ</sup></td><td>2.2 ± 0.79</td><td>3.4 ± 0.53</td><td>20.50</td><td>0.115<sup>γ</sup></td></tr><tr><td><em>Hiding test</em></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>
<table><tbody><tr><td>Time until the first movement (s)</td><td>131.3 ± 34.96</td><td>47.1 ± 34.96</td><td>1.24</td><td>0.297 <sup>β</sup></td><td>28.4 ± 4.81</td><td>23.2 ± 4.71</td><td>0.34</td><td>0.564 <sup>β</sup></td></tr><tr><td>Time before complete hiding (s)</td><td>406.0 ± 81.86</td><td>370.6 ± 80.56</td><td>16</td><td>0.548 <sup>γ</sup></td><td>172.5 ± 39.74</td><td>156.5 ± 38.74</td><td>0.61</td><td>0.439 <sup>β</sup></td></tr><tr><td>Predator test</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Number of abalone performing the four escape behaviours</td><td>2 out of 20<br>45.9 ± 5.61</td><td>3 out of 20<br>31.3 ± 5.61</td><td>0.23<br>3.95</td><td>0.633 <sup>δ</sup><br>0.082 <sup>β</sup></td><td>10 out of 18<br>45.0 ± 29.39</td><td>6 out of 20<br>30.0 ± 5.00</td><td>2.54<br>7</td><td>0.111<br>0.309 <sup>γ</sup></td></tr><tr><td>Time until the first movement (s)</td><td>216.0 ± 16.20</td><td>223.9 ± 8.15</td><td>14</td><td>0.841 <sup>γ</sup></td><td>192.5 ± 13.30</td><td>190.1 ± 14.04</td><td>11</td><td>0.841 <sup>γ</sup></td></tr><tr><td>Time spent moving (s)</td><td>4.9 ± 0.79</td><td>6.5 ± 0.79</td><td>1.92</td><td>0.173</td><td>7.6 ± 1.13</td><td>7.0 ± 1.07</td><td>0.026</td><td>0.872 <sup>β</sup></td></tr><tr><td>Number of squares crossed</td><td>197.7 ± 19.57</td><td>171.2 ± 19.57</td><td>1.47</td><td>0.233 <sup>β</sup></td><td>160.6 ± 45.72</td><td>133.4 ± 39.51</td><td>9</td><td>0.548 <sup>γ</sup></td></tr><tr><td>Time to reach 20 cm far from the predator (s)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td colspan="8" style="text-align: center;">M5</td></tr><tr><td>Spawning behaviour</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Number of abalone spawning</td><td>31 out of 37</td><td>30 out of 34</td><td>0.29</td><td>0.737 <sup>δ</sup></td><td></td><td></td><td></td><td></td></tr><tr><td>Time of preparation (min)</td><td>187.6 ± 18.49</td><td>178.1 ± 18.79</td><td>0.12</td><td>0.735</td><td></td><td></td><td></td><td></td></tr><tr><td>Time of spawning (min)</td><td>245.1 ± 8.00</td><td>232.0 ± 10.46</td><td>0.96</td><td>0.360 <sup>β</sup></td><td></td><td></td><td></td><td></td></tr></tbody></table>

<footnote>δ Fisher's exact test</footnote>

<footnote>γ Wilcoxon rank sum test with continuity correction</footnote>

<footnote>β mixed model with log, inverse or square root transformations</footnote>
398 Foot contact test

399 There was no difference between the numbers of abalone swivelling after touching under the
two pH condition at W1 ($\chi^2 = 0, df = 1, p = 1$, Fisher's exact test), although a trend was observed
400 at M3, with less abalone swivelling in the pH 7.7 treatment compared to those exposed to pH
401 8.0 at M3 ($\chi^2 = 2.96, df = 1, p = 0.086$, Pearson's chi-square test). The number of abalone
402 changing position after touching did not differ significantly between the treatments (W1: $\chi^2 =$
403 0.078, df = 1, p = 0.779; M3: $\chi^2 = 0.094, df = 1, p = 0.759$, Pearson's chi-square test), nor did
404 the number of abalone changing position and returning to their initial position before the end
405 of the observation period (W1: $\chi^2 = 0.389, df = 1, p = 0.533$; M3: $\chi^2 = 0.179, df = 1, p = 0.672$,
406 Pearson's chi-square test, Table 3).
407

408

409 Righting test

410 No significant difference was observed for the time to right (W1: $F_{1,38} = 0.965, p = 0.332$, mixed
model analysis with inverse transformation; M3: $F_{1,8.2} = 1.61, p = 0.239$, mixed model analysis
411 with log transformation), nor for the number of attempts to turn over (W1: W = 17, p = 0.396;
412 M3: W = 20.5, p = 0.115, Mann-Whitney U test - Wilcoxon rank sum test, Table 3) between
413 the two pH treatment.
414

415

416 Hiding test

417 The time until the first movement did not differ significantly between pH treatments (W1: $F_{1,8}$
= 1.24, p = 0.297, mixed model analysis with log transformation; M3: $F_{1,37} = 0.34, p = 0.564$,
mixed model analysis with square root transformation), nor did the time before complete hiding
418
419 (W1: W = 16, p = 0.548, Mann-Whitney U test - Wilcoxon rank sum test; M3: $F_{1,37} = 0.61, p$
420 = 0.439, mixed model analysis with inverse transformation, Table 3).
421

422
423 *Starfish predator test*

424 After the first week, there was a trend towards faster response in the lower pH treatment: these
425 abalone moved faster after the contact with the starfish (W1: F₁,₈ = 3.95, p = 0.082, mixed
426 model analysis with inverse transformation) but there was no significant difference at M3 (W
427 = 7, p = 0.309, Wilcoxon rank sum test). Similarly, there were no significant differences in the
428 time spent moving (W1: W = 14, p = 0.841, Wilcoxon rank sum test; M3: W = 11, p = 0.841,
429 Wilcoxon rank sum test), the numbers of squares crossed (W1: F₁,₃₈ = 1.92, p = 0.173, mixed
430 model analysis; M3: F₁,₃₆ = 0.026, p = 0.872, mixed model analysis with square root
431 transformation), the times to reach 20 cm away from the predator (W1 : F₁,₃₈ = 1.47, p = 0.233,
432 mixed model analysis with log transformation; M3: W = 9, p = 0.548, Wilcoxon rank sum test)
433 and the number of abalone performing the four escape behaviours (W1: χ² = 0.23, df = 1, p =
434 0.633, Fisher's exact test; M3: χ² = 2.54, df = 1, p = 0.111, Pearson's chi-square test).
435

435

436 *Spawning behaviour*

437 The number of abalone spawning (χ² = 0.24, df = 1, p = 0.623, Pearson's chi-square test), the
438 time of active preparation movement (F₁, 7.5 = 0.29, p = 0.603, mixed model analysis) and the
439 time before spawning (F₁, 7.1 = 1.26, p = 0.298, mixed model analysis with inverse
440 transformation) were all similar between the two treatments (Table 3).
441

441

442 *3.2. Gene expression*

443 The gene expressions of the serotonin receptors and GABA A-like receptors were not
444 significantly different between the two treatments (serotonin receptor: F₁,₁₇ = 0.032, p = 0.860
445 mixed model analysis with log transformation; GABA A-like receptor: F₁,₁₇ = 0.366, p = 0.553,
446 mixed model analysis) (Figure 2).

447

(A)

(B)
<figure><img src="image_28.png" /></figure>

<figure><img src="image_29.png" /></figure>

448

449

450

451

**Fig. 2:** Gene expression of the (A) GABA A-like and (B) serotonin receptors of adult Haliotis tuberculata exposed for 4 months to ambient pH (pH~T~ 8.0) or to low pH (pH~T~ 7.7). Lsmeans ± S.E.M.

452

453

### 3.3. Estimated effect size

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Calculation of estimated effect sizes showed that, if any difference was present for the behavioural tests, these differences would be small (Figure. 3): 33 estimated effect sizes were below 0.5 out of the 36 variables measured in total for the different periods. In addition, estimated effect sizes were inconsistently positive or negative depending of the test and period of measure.

459
Fig. 3. Estimated size effect and variances of *Haliotis tuberculata* studied during a foot contact test, a righting test, a hiding test, a predator test, a 48-h diurnal rhythm measured in housing tank, stimulated for spawning, or sampled for serotonin and GABA A-like gene expression. Abalone were tested 1 week (white column) and several months (three, four or five months, grey column) after exposure to ambient pH (pH<sub>T</sub> 8.0) or low pH (pH<sub>T</sub> 7.7). Estimated size effects and variances were calculated following Clements et al (2022) 's methodology, using the number of tanks (n = 5 per treatment) as sample size.

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<figure><img src="image_30.png" /></figure>

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<figure><img src="image_31.png" /><figcaption>PREDATOR TEST</figcaption></figure>

<figure><img src="image_32.png" /></figure>

472

<figure><img src="image_33.png" /></figure>

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<figure><img src="image_34.png" /></figure>

<figure><img src="image_35.png" /></figure>

<figure><img src="image_36.png" /></figure>
476

## 4. Discussion

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The effect of OA was studied on a large range of behaviour traits that could impact survival and fitness of the abalone *H. tuberculata*. The reduction by 0.3 pH unit from ambient pH did not significantly modify responses to predators, righting and hiding abilities, spawning behaviour, feeding behaviour or any measured diurnal activity pattern, after one week of exposure as well as after several months of exposure. These tests have already been used for testing other factors in previous studies, and were sensitive measures for evaluating stress of abalone (Lachambre et al., 2017a; Lachambre et al., 2017b; Roussel et al., 2019b). In addition, no change in relative expression was reported for the two genes involved in the neuroendocrine control of behaviour. Calculation of the estimated size effect showed that almost 90% of the behavioural variables measured had an estimated effect size (InRR) between 0.5 and -0.5. Even if there were some effects of the pH treatments, they were small and non-consistent, with abalone performing better in pH 8.0 or in pH 7.7 depending on the tests.

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### *Effects on behaviour*

Because interactions between the prey and their predators constitute an integral part of the ecology and evolution of marine organisms, as well as the structure and function of communities, we focused first on prey-predator responses, with a simulation of an attack with the foot contact test and a direct exposure to a predator. When exposed to a starfish, *H. tuberculata* responds with stereotyped behaviours: it does a swivelling movement, releases some mucus, turns around and flees thereafter (Roussel et al., 2019b). Similar behavioural patterns were reported for other species such as *Haliotis rubra* (Day et al., 1995). The responses to the predator were similar between the pH treatments, indicating little effect of a lower pH on predator detection and escape responses. These results strongly differ from those obtained in
marine fishes, in which an impairment of prey-predator responses due to OA was reported
(reviews by Briffa et al., 2012; Nagelkerken and Munday, 2016; Ashur et al. 2017). In other
gastropods, the effects of pH on prey-predator responses are variable (Clements et Comeau,
2019). Low pH had a negative effect on the response to a predator in a rocky shore species,
*Concholepas concholepas*, by disrupting predator-avoidance behaviour in juveniles reared at
pH~T~ 7.7 during five months (Manriquez et al., 2014) and affecting predator-escape response in
juveniles exposed to pH~T~ 7.5 during 3 months in comparison to juveniles exposed to current-
day levels at pH~T~ 7.7-7.85 (Manriquez et al., 2016). In contrast, some studies found an increased
avoidance in *Littorina littorea* exposed to predator cues when kept at very low pH~NBS~ (6.6 for
low pH vs 8.0 for ambient pH) (Bibby et al., 2007), and no effect of 6-week exposure to pH~T~
7.8 on the maximum escape speed of the limpet *Nacella concinna* when exposed to starfish
(Schram et al., 2014).

Due to its habitat preference, in subtidal and intertidal areas, *H. tuberculata* can be exposed
to waves and surges (Clavier et Chardy, 1989). For many benthic invertebrates, the ability to
right after detachment by strong wave action reduces vulnerability to predation or unwanted
spatial transport. Hiding is another key behaviour to avoid predation that is crucial for
individual survival, especially for juveniles, for which higher mortalities are reported in the
natural environment (Shepherd et Breen, 1992). In the present study, adult abalone exposed to
ambient pH and low pH took similar times to find a hiding place, to right themselves and to
escape when facing a starfish. As for prey-predator effects, the absence of effects of OA on the
hiding response of the abalone contrasts with the significant effects found on other gastropods.
An experiment performed on the caenogastropod *Concholepas concholepas* found that
juveniles exposed for 83 days to lower pH values (pH~T~ 7.8 and 7.6) were faster to right
themselves compared to juveniles exposed to ambient pH~T~ of 8.0 (Manriquez et al., 2013). A
full-factorial experiment combining pH and temperature stress on the common limpet *Nacella*
concinna and the vetigastropod snail *Margarella antarctica* found a non-significant difference in the proportions of limpets displaying the capacity to right, but a significant temperature-pH interaction effect on the mean righting times of snails (Schram et al., 2014). In abalone, the righting time is a reliable indirect indicator of its energetic status and later survival (Lachambre et al., 2017b). Metabolic rates and immune function measured on other abalone from the same experiment were not significantly affected by lowered pH in spite of significant effects on shell growth and calcification (see Avignon et al. 2020 for physiological and calcification measures).

This lack of effects on overall metabolism corresponds with the absence of effect on the righting behaviour.

We found no evidence that OA modified the quantity of algae ingested during the 3-month exposure of abalone to a reduction of 0.3 pH unit. In addition, there was no detectable difference in diurnal rhythm, including feeding duration, during 48h observations of a large number of individuals (130 abalone per treatment for W1 and at least 83 abalone for M3). Few experiments have measured the long-term effect of acidification on feed intake of herbivores. Results in the literature are contradictory for OA, even for the same taxon (Nagelkerken et Munday, 2016).

Our result suggests that short term (one week) as well as long term exposure (3 months) to seawater acidification of 0.3 pH unit does not modify the foraging capacity or the feed intake of *H. tuberculata*. Our measurement of diurnal rhythm in a "non-stressful" environment, adapted to the biology of the animals (the presence of a hiding place, access to algae and the potential for active foraging behaviour) would be expected to provide valuable information about daily behaviours that would likely occur in a natural context. In addition, the lack of effects did not result from a stress bias due to a new environment, because abalone had at least 3 weeks of acclimation to the novel environment before the measurements.

Reproduction is one of the crucial functions that ensure the persistence of populations and communities. Abalone are gonochoric species, with external fertilisation occurring in the
seawater column (Shepherd et Laws, 1974; Bilbao et al., 2010). Synchronisation of gamete releases among individuals within a population is important for fertilisation success. Abalone submitted to OA over 5 months had the same spawning behaviour as adult abalone reared in ambient pH: the number of abalone spawning as well as the time to preparatory behaviour and time before spawning were similar between the pH treatments. However, successful reproduction will depend not only on the behaviour but also on gonad development and gonad maturity. Dissection of other individuals from the same experiment after 4 months of exposure to pH treatments (Avignon et al., 2020) showed that the gonad weight was significantly lower for abalone exposed to pH~7.7~, suggesting some disruption in reproduction. To fully understand the effects of OA on abalone reproduction, a multi-parameter approach using reproductive behaviour and physiology as well as larval survival is clearly necessary.

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563 *Effect on gene expression*

Gene expression profiling has already been used in a panel of organisms exposed to near-future pH scenarios. The hypothesis was that marine organisms exposed to acidified seawater would present compensatory changes in behavioural processes that would be reflected by gene expression changes. In this context we aimed to detect gene expression changes in targeted genes involved in the processes of neurotransmission and central regulation of behavior when the abalone *H. tuberculata* was exposed to OA conditions. Recent evidence shows that GABA and serotonin signaling play major roles in the nervous systems of gastropod mollusks, notably in the modulation of motor control and cognitive processes such as memory and learning (Miller, 2019; Aonuma et al., 2020). The GABA A receptor has often been described as responsible for the sensory impairment at a central level observed in fish exposed to acidification (Nilsson et al., 2012). However, no modification of the GABA A receptor or serotonin receptor in the head of these adult abalone was observed. This is in contrast to the
576

effects of low pH<sub>NBS</sub> 7.85 compared to pH<sub>NBS</sub> 8.17 for 2-5 days on a caenogastropod, the stromb
*Gibberulus gibbosus*, which produced disrupted predator escape function (Watson et al., 2014).

577

This effect appeared to be due to malfunction of GABA A-type receptors in the stromb, as the gabazine blocker of these receptors restored the predator escape behaviour. Our results in abalone indicate that long term exposure to decreased pH predicted by the “business as usual” scenario for the end of the century does not significantly impact the expression of their two main signalling receptors. Interestingly, GABA and serotonin signalling are implied in a wide range of cognitive functions as the central transmission of sensorial signals and locomotion. This is in line with the lack of effects of acidification on the tested behaviour (notably the diurnal rhythms, predator responses and hiding activity).

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*Why no effects?*

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The most plausible hypothesis to explain the lack of significant effects on adult abalone
behaviour is the evolution of resistance to variable environmental pH. This might be due to the
ecological niche of abalone, in intertidal and/or subtidal areas, where diel and seasonal variation
in coastal pCO₂ can often far exceed near-future projections. In Brittany, *H. tuberculata*
populations in the subtidal zone can experience pH variation from 7.9 up to 8.2 seasonally (Qui-
Minet et al., 2018). In mid-intertidal pools, the fluctuations are even more extreme, with daily
pH variations from 7.5 up to 10 in a few hours. These variations are due to community
respiration with CO₂ release at night and photosynthesis with CO₂ uptake during daylight in
pools that are isolated from the adjacent ocean during low tides, with supplementary variations
according to the season and period of the day (Legrand et al., 2018). The degree of variability
a species encounters is an important consideration to understand biological responses to OA
(Waldbusser et Salisbury, 2014).
Pollution and OA can influence animal behaviours in three ways (Briffa et al 2012): disruption of metabolic processes, reduction of the ability to gather information from the surrounding environment to make subsequent decisions, and limitation of the ability of an organism to avoid polluted locations. To be able to evaluate the metabolism and energetic balance, energetic input should be measured by measuring feed intake as well as energy expenditure. We showed that feed intake measured during 3 months was very similar between the treatments. In addition, foraging activity and locomotion was similar for hundreds of individuals video-recorded in their rearing mesocosms, probably indicating no increased energetic expenditure in otherwise unstressed situations.

In addition to behavioural measurements, other functions were studied such as basal metabolism, showing that it was not impacted by decreased pH in adult *H. tuberculata* (Avignon et al., 2020). The metabolic process can also be disrupted due to elevated costs for maintaining acid-base balance (Pörtner et al., 2004). In adult *H. tuberculata*, our previous studies also found that abalone did not compensate for a seawater pH decrease of 0.3 unit during the first two months of exposure, but started to acclimate after four months, as suggested by the compensation of their extracellular pH (Avignon et al. 2020). In addition, there was no detectable disruption of the ability to gather information and then to make decisions in the abalone. The similar response to starfish predator cues among pH treatments, the similar time to find a hiding place and the similar response time after stimulation to show preparatory behaviour for reproduction or before spawning showed that the ability to detect chemical cues and temperature change was not impaired in those abalone exposed to low pH. These results were confirmed by the lack of difference in gene expression of the GABA-A neurotransmitter receptor, a major inhibitory receptor implicated in various behavioural pathways (Ashur et al 2017).
Behavioural impairment might also be observed in animals that cannot avoid low pH. The ability to cope with environmental changes will be different depending on the animal mobility. Adult abalone, in contrast to fish, have limited mobility. They rely on strong attachment to the substratum by the muscular foot to avoid most predation. Because of this reduced mobility and the pH variability in their ecological niche, abalone have probably been selected for resilience to variations in pH, at least for a few hours per day. However, the shell integrity of marine molluscs is essential to protect themselves from predators such as rays and crabs and is involved in attachment to the substrate in some gastropods (Shepherd et Breen, 1992). In *H. tuberculata*, adult shell mechanical properties as well as biomineral architecture were greatly impacted by a pH reduction of 0.3 unit after several months of exposure (Avignon et al., 2020), suggesting that OA might reduce protection from predators and resistance to hydrodynamic forces, potentially impacting wild abalone populations. Even if abalone did not change their prey-predator responses when facing OA, the more fragile shell might potentially jeopardize wild populations already threatened by overfishing and environmental perturbations.

## 5. Conclusion

Biological responses to OA are thought to depend on a number of physiological and life-history traits at larval, juvenile and adult stages. The results of the present study demonstrated that the behaviour of adult *H. tuberculata* is not impacted by an experimental pH decrease of 0.3 pH unit, suggesting that the adult stage is probably the most robust stage from a behavioural point of view in comparison to larval or juvenile stages (Auzoux-Bordenave et al., 2020). Because abalone naturally experience pH variations in their low-intertidal or high-subtidal environment, the species apparently has developed a relative resilience to this environmental stress. However, the net fitness outcome and the capacity of organisms to survive and persist under OA is a
complex trade-off between behaviours and other biological processes and should be monitored
over a long term period (i.e at least several months). Although no behavioural impairment
occurred, a reduction of 0.3 pH unit impacted other physiological functions such as calcification
and reproduction in adult *H. tuberculata* (Avignon et al., 2020). Together, these results highlight
that a multicriteria approach should be applied when studying the effect of acidification on
marine species.

656

657 ACKNOWLEDGEMENTS

658

659 The authors thank Ewan Harney for his generous help in the identification of *H. tuberculata*
660 specific GABA A-like receptor and serotonin receptor sequences.

661 This work was supported by the program "Acidification des Océans" (ICOBio project) funded
662 by the Fondation pour la Recherche sur la Biodiversité (FRB) and the Ministère de la Transition
663 Ecologique et Solidaire (MTES), and the French LabexMER program (OASYS project).

664

665

666

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