Carlo Cattano
email: carlo.cattano@unipa.it

Joachim Claudet
email: joachim.claudet@gmail.com

Paolo Domenici
email: paolo.domenici@cnr.it

Marco Milazzo
email: marco.milazzo@unipa.it

Cattano Carlo

Domenici Paolo

Milazzo Marco

Living in a high CO2 world: a global meta-analysis shows multiple trait-mediated responses of fish to ocean acidification

Keywords: Behaviour, Calcification, Development, Fish-traits, Growth, Heterogeneity, Metabolism, Survival, Teleosts

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Living in a high CO2 world: a global meta-analysis shows multiple trait-mediated responses of fish to ocean acidification.

INTRODUCTION

By absorbing >25% of the carbon dioxide (CO 2 ) and >90% of the heat accumulating in the atmosphere, the ocean plays a key role in regulating the Earth's climate [START_REF] Gattuso | Contrasting futures for ocean and society from different anthropogenic CO 2 emissions scenarios[END_REF].

However, this occurs at a cost, as changes in the ocean's carbonate system resulting from increasing anthropogenic CO 2 emissionsa process termed ocean acidification (OA)have been progressing since the industrial revolution at a historically unprecedented rate [START_REF] Doney | Ocean acidification: the other CO 2 problem[END_REF][START_REF] Hönisch | The geological record of ocean acidification[END_REF]. OA is predicted to rise even more rapidly during this century, as average ocean PCO 2 (partial pressure CO 2 ) could reach ~1000 µatm [START_REF] Meinshausen | The RCP greenhouse gas concentrations and their extensions from 1765 to 2300[END_REF]) leading to 0.3 units reduction in pH [START_REF] Bopp | Multiple stressors of ocean ecosystems in the 21st century: projections with CMIP5 models[END_REF][START_REF] Stocker | Technical Summary[END_REF][START_REF] Pörtner | Ocean systems[END_REF][START_REF] Mcneil | Future ocean hypercapnia driven by anthropogenic amplification of the natural CO 2 cycle[END_REF], well beyond the threshold expected to be detrimental to many marine organisms [START_REF] Kroeker | Impacts of ocean acidification on marine organisms: quantifying sensitivities and interaction with warming[END_REF]. This may have far-reaching consequences for marine ecosystems, including biodiversity loss and changes to marine community structure (Nagelkerken & Connell, 2015).

International organizations warn that global economy could risk losing as much as $1 trillion USD annually if countries do not take urgent steps to slow down anthropogenic CO 2 emissions by the end of the century (CBD, 2014). Reflecting mounting concerns, research attempts to predict biological impacts of OA have gone through a striking increase in publishing rate (with more than 500 articles published in 2013), which is unparalleled by any other scientific field [START_REF] Riebesell | Lessons learned from ocean acidification research[END_REF].

Biological consequences of OA are fundamentally linked to the ecology and physiology of marine species [START_REF] Pörtner | Ocean systems[END_REF][START_REF] Heuer | Physiological impacts of elevated carbon dioxide and ocean acidification on fish[END_REF]. Existing quantitative syntheses have found that effects vary among life stages, species or broader taxonomic groups [START_REF] Hendriks | Vulnerability of marine biodiversity to ocean acidification: A meta-analysis[END_REF][START_REF] Kroeker | Meta-analysis reveals negative yet variable effects of ocean acidification on marine organisms[END_REF][START_REF] Harvey | Meta-analysis reveals complex marine biological responses to the interactive effects of ocean acidification and warming[END_REF][START_REF] Kroeker | Impacts of ocean acidification on marine organisms: quantifying sensitivities and interaction with warming[END_REF][START_REF] Wittmann | Sensitivities of extant animal taxa to ocean acidification[END_REF].

Fish were initially thought to be resilient to OA as they are able to defend their internal pH in a high CO 2 environment through active ion transport [START_REF] Claiborne | Acid-base regulation in fishes: cellular and molecular mechanisms[END_REF][START_REF] Brauner | Patterns of acid-base regulation during exposure to hypercarbia in fishes[END_REF][START_REF] Melzner | Physiological basis for high CO 2 tolerance in marine ectothermic animals: preadaptation through lifestyle and ontogeny?[END_REF]. Nevertheless, increased acid-base regulation activity may have energetic consequences that might affect physiology and development, especially in early life stages [START_REF] Ishimatsu | Fishes in high-CO 2 , acidified oceans[END_REF]. More recently, the compensatory responses that occur during acid-base regulation were found to influence fish calcification, behaviour and ion transport [START_REF] Nilsson | Near-future carbon dioxide levels alter fish behaviour by interfering with neurotransmitter function[END_REF][START_REF] Heuer | Physiological impacts of elevated carbon dioxide and ocean acidification on fish[END_REF]2016). Calcification in fish occurs in the inner ear for formation of otoliths, carbonated structures playing a role in sound detection, acceleration and orientation, and in the intestinal lumen, where calcium carbonate is precipitated to excrete excess calcium ions [START_REF] Heuer | Physiological impacts of elevated carbon dioxide and ocean acidification on fish[END_REF]. Fish potentially contribute with up to 45% of global calcium carbonate budget [START_REF] Wilson | Contribution of fish to the marine inorganic carbon cycle[END_REF], therefore playing an important role for ocean chemistry and carbon sequestration. Most notably, changes in the concentrations of acid-base relevant ions at higher CO 2 levels appear to impair the function of neurotransmitter receptors causing a range of behavioural changes [START_REF] Nilsson | Near-future carbon dioxide levels alter fish behaviour by interfering with neurotransmitter function[END_REF][START_REF] Hamilton | CO 2 -induced ocean acidification increases anxiety in Rockfish via alteration of GABAA receptor functioning[END_REF][START_REF] Heuer | Physiological impacts of elevated carbon dioxide and ocean acidification on fish[END_REF]. In fish, documented behavioural effects of OA involve impaired learning ability, sensory functions or decision-making, along with disrupted anti-predator responses, increased boldness, and altered homing behaviour (see [START_REF] Briffa | High CO 2 and marine animal behaviour: potential mechanisms and ecological consequences[END_REF][START_REF] Heuer | Physiological impacts of elevated carbon dioxide and ocean acidification on fish[END_REF]Clements & Hunt, 2015, for reviews). Several studies also documented that exposure to elevated CO 2 alters fish lateralization, the preference for left or right side during behavioural activity that confers benefits in terms of spatial orientation, reactivity, cognition and group coordination [START_REF] Domenici | Elevated carbon dioxide affects behavioural lateralization in a coral reef fish[END_REF]2014[START_REF] Jutfelt | Behaviour disturbance in a temperate fish exposed to sustained high-CO2 levels[END_REF].

Behavioural and sensory functions changes may affect key ecological processes such as predatorprey interactions, dispersal, settlement and habitat choice, with cascading implications at the community level [START_REF] Nagelkerken | Animal behaviour shapes the ecological effects of ocean acidification and warming: moving from individual to community-level responses[END_REF].

To date, the large majority of studies documenting effects of high levels of PCO 2 on fish have been performed in laboratory conditions, showing variable and sometime contradictory results or in some instances underestimating the potential ability of fish to acclimate and adapt to predicted OA in the long term [START_REF] Wittmann | Sensitivities of extant animal taxa to ocean acidification[END_REF]. Indeed, species sensitivity to OA may depend on some life history traits that could confer tolerance to the expected changes in CO 2 levels. For instance, species living in pelagic environments, where CO 2 concentrations are stable over time could be more sensitive to OA than coastal benthic species that usually experience high fluctuations of CO 2 concentrations due to the alternation of respiration and photosynthesis processes by reef organisms [START_REF] Munday | Climate change and the future for coral reef fishes[END_REF]Hoffman et al. 2011).

To date, existing quantitative generalisations of OA consequences for fish under different CO 2 projections have been controversial, with uncertainty associated with the small number of experiments conducted [START_REF] Harvey | Meta-analysis reveals complex marine biological responses to the interactive effects of ocean acidification and warming[END_REF][START_REF] Kroeker | Impacts of ocean acidification on marine organisms: quantifying sensitivities and interaction with warming[END_REF][START_REF] Wittmann | Sensitivities of extant animal taxa to ocean acidification[END_REF]. A more comprehensive picture of fish responses across different species characteristics and altered CO 2 concentrations is now possible, as research on the topic has progressed in the few last years.

Indeed, this represents an opportunity to test the robustness of the patterns found in previous studies and to highlight more general insights.

Here, we used a mixed-effects meta-analytical approach to investigate the effects of projected CO 2 for 2050-2070 and 2100 under intermediate or high emission scenarios (RCP 6 and 8.5, respectively;[START_REF] Meinshausen | The RCP greenhouse gas concentrations and their extensions from 1765 to 2300[END_REF][START_REF] Pörtner | Ocean systems[END_REF], on several response variables extracted from 75 studies (327 contrasts) on 44 species, from polar to tropical regions. We classified and analysed potential effects of OA according to two broad types of fish responses: (1) the eco-physiological responses (mortality, reproduction, metabolism, growth, calcification and yolk dimension); and (2) the behavioural responses (i.e. effects on fish behaviour and activity levels). To assess potential differences in responses, we also analysed such response variables both in short and longer term CO 2 exposure experiments, as well as under chronic exposure in natural CO 2 seeps.

In addition to this, since fish responses to OA can vary among ontogenetic stages and species traits, we also examined the effects of elevated CO 2 on the above response typologies across different life-stages, physiological types (i.e. stenohaline or euryhaline), climatic zones, and habitats (see Appendix S1 for details). Specific hypotheses associated to these different fish characteristics were the following. 1) Due to their inefficient system for ionic regulation, we expected fish early life stages (i.e. embryos, larvae and juveniles) to be more affected by elevated CO 2 levels than the adult stage. 2) Stenohaline species should be more sensitive than euryhaline fish, as these latter experience highly fluctuating PCO 2 environments during their life cycle. 3)

The eco-physiology and behaviour of tropical and polar species is more disrupted under OA conditions than that of temperate and sub-tropical ones, which evolved in a relatively more variable pH/CO 2 environment. 4) Similarly, being adapted to more stable CO 2 levels in offshore waters, pelagic species should be more sensitive than benthic and bentho-pelagic ones.

Several studies elucidated a clear relation between fish brain function, behaviour and ecological processes under expected OA levels [START_REF] Nilsson | Near-future carbon dioxide levels alter fish behaviour by interfering with neurotransmitter function[END_REF][START_REF] Chivers | Impaired learning of predators and lower prey survival under elevated CO 2 : a consequence of neurotransmitter interference[END_REF][START_REF] Lai | Altered neurotransmitter function in CO 2 -exposed stickleback (Gasterosteus aculeatus): a temperate model species for ocean acidification research[END_REF]. Documented fish behavioural changes in fish are a consequence of (upstream) neurosensory disruptions [START_REF] Heuer | Physiological impacts of elevated carbon dioxide and ocean acidification on fish[END_REF]2016), and may lead to altered (downstream) ecological processes (recruitment, predation or competition; [START_REF] Nagelkerken | Animal behaviour shapes the ecological effects of ocean acidification and warming: moving from individual to community-level responses[END_REF].

Therefore, we also assessed to what extent fish behavioural responses were related to neurosensory (cognition, audition, olfaction, vision, and a combination among olfaction and vision) and ecological (foraging, predation risk and habitat choice) categories under elevated CO 2 levels (see Appendix S1 for details).

MATERIAL AND METHODS

Data collection and selection criteria

We searched for studies testing the effects of altered PCO 2 levels on marine teleost fish published online or printed through to January 2017 using Google Scholar and ISI Web of Science with the combination of the following three keywords: ocean acidification, fish and CO 2 . We also used the reference lists of the retrieved papers. In addition, we searched for OA papers in the news stream managed by the Ocean Acidification International Coordination Centre (OA-ICC; http://news-oceanacidification-icc.org). Papers not reporting mean responses, error estimates and sample sizes in at least one control CO 2 condition and one elevated CO 2 treatment were discarded (in few instances, corresponding authors were contacted to obtain missing information).

As seawater carbonate chemistry varies regionally and between ecosystems [START_REF] Hofmann | High-Frequency Dynamics of Ocean pH: A Multi-Ecosystem Comparison[END_REF], control CO 2 levels used in the retrieved experiments spanned from 300 to 800 µatm PCO 2 .

Therefore, we considered for each study a PCO 2 range (ΔPCO 2 )expressed as the absolute difference between the PCO 2 values in the elevated and in the control CO 2 conditionthat was assigned to one of the following OA projections under intermediate or high emission scenarios (RCP 6 and 8.5, respectively;[START_REF] Meinshausen | The RCP greenhouse gas concentrations and their extensions from 1765 to 2300[END_REF]: (1) moderate PCO 2 expected by 2050-2070 following the RCP 8.5 (business-as-usual) scenario or expected by 2100 following the RCP 6 scenario (~500-700 µatm; ΔPCO 2 ≤350 µatm); (2) high PCO 2 expected by 2100 following the RCP 8.5 scenario or beyond 2100 following the RCP 6 scenario (~1000 µatm; ΔPCO 2 between 350 and 700 µatm) [START_REF] Meinshausen | The RCP greenhouse gas concentrations and their extensions from 1765 to 2300[END_REF]. We have not included in the analyses studies using PCO 2 levels beyond the predictions for 2100 under the business as usual scenario (i.e. PCO 2 > 1300 µatm), but a few studies (n=3) where control CO 2 conditions were above-average values as they reflected the conditions of specific ecosystems where acidification is predicted to be heavier (Hoffmann et al. 2011). Studies manipulating carbonate chemistry using acid addition were not considered as they do not mimic the expected changes in HCO 3 -concentrations and dissolved CO 2 .

We kept multi-species experiments only when data were independently collected. In the case of time series, we used only the final data point, except for mortality data, where the first time point of a time series was considered in order to maintain a conservative estimate and avoid bias of rearing fish in aquaria (e.g. [START_REF] Murray | Offspring sensitivity to ocean acidification changes seasonally in a coastal marine fish[END_REF]. When a single experiment reported several metrics related to the same response typology (e.g., length and weight to estimate growth) we randomly chose one metric to avoid autocorrelation. For studies designed to assess interactive effects of multiple stressors (for instance food availability, oxygen, salinity, and temperature along with PCO 2 ) we included only responses to altered PCO 2 and considered control levels for the other factors. We included data from trans-generational acclimation experiments when parents were reared under control CO 2 conditions. The data on survival were transformed to mortality estimates (= 1survival). For the calcification, we included only studies dealing with otolith formation (e.g. otolith area). To assess OA effects on fish metabolism, we analysed the resting metabolic rate (RMR, i.e. oxygen consumption at resting) and its proxies, and the metabolic scope (i.e. aerobic scope -MS, i.e. the difference between maximum and resting metabolic rates)

separately, as the directionality of each response under OA may be different. For instance, an increase of RMR may be related to an increase in the energy spent on acid base regulation and ventilation [START_REF] Perry | Acid-base balance and CO 2 excretion in fish: Unanswered questions and emerging models[END_REF], with this being potentially detrimental for the species performance as an increase in cost of acid-base regulation can shunt away the energy from growth [START_REF] Ishimatsu | Fishes in high-CO 2 , acidified oceans[END_REF]) by decreasing MS. By contrast, a reduction in MS results in less energy being available for life-history processes, such as growth, foraging activity and reproduction [START_REF] Pörtner | Physiology and climate change[END_REF] and can be due to a reduction in maximum metabolic rate even if RMR is not affected.

When assessing the fish behavioural response types to OA, we kept distinct those experiments showing behavioural changes (generally reported as reductions of behavioural performances, i.e. negative effects) from those experiments exhibiting changes in activity or boldness (which in turn often recorded increased effects, i.e. positive effects; but see [START_REF] Hamilton | CO 2 -induced ocean acidification increases anxiety in Rockfish via alteration of GABAA receptor functioning[END_REF] and split these into behaviour and activity, respectively. Importantly, the definitions of positive or negative effects (see also effect size calculation) apply to the directionality (above or below the null effect) of each response variable separately and may not reflect the actual fish performance outcome. As an example, in some experiments, fish may have exhibited either a behavioural (negative) change (i.e. a decreasing time percentage spent in the home site cue; We assessed how the following categories of life history traits could mediate the response of fish to OA: (1) life stage (egg-embryo, larvae, juvenile, or adult), ( 2) physiology (stenohaline or euryhaline), (3) climatic zone (polar, temperate, sub-tropical and tropical), and (4) adult habitat (benthic, bentho-pelagic and pelagic (see Appendix S1). In addition, behavioural contrasts were also categorised according to aspects of (5) the neurosensory system (i.e. cognition, audition, olfaction, vision and olfaction+vision when visual stimuli and olfaction cues were not kept separate in a given experiment) and ( 6) the ecological processes involved such as foraging (i.e., feeding activity and prey detection), predation risk and habitat choice (settlement and homing ability) (see Appendix S1). The hatchling stage, instead of the egg-embryo stage, was considered for categorising the behavioural, the neuro-sensory and the ecological contrasts. The habitat of eggs (benthic or pelagic) was also considered as a category when assessing OA effects on yolk .

The assignment of these different categories was made using Fishbase (www.fishbase.org) and where and are the variances associated with the means and , respectively, where and are the number of replicates in the impacted (elevated PCO 2 ) and control PCO 2 conditions, and where i, j, k, m and l are defined as above. All effect sizes were weighted, accounting for both the within-and among-study variance components.

Models were fitted and heterogeneity tests were run to assess how many fish traits (i.e., life stage, physiology, climatic zone, habitat, neurosensory system, ecological processes) could mediate the response of fish to OA. Models fitting and heterogeneity tests were done using the metaphor package [START_REF] Viechtbauer | Conducting Meta-Analyses in R with the metaphor Package[END_REF]) in R (R Core Team 2016).

RESULTS

Eco-physiological responses

Overall we found that rising CO 2 levels will significantly affect fish calcification (+5%), RMR (+7%) and yolk (-5%), while mortality, metabolic scope, reproduction and growth seemed to be unaffected (Fig. 1a, 2a, 3a; see Table S1,S2). Examining the effects of different CO 2 projections under the two RCP scenarios, we found that only high PCO 2 levels significantly increased calcification and RMR, by 6% and 8%, respectively, and decreased yolk area by 5% (Fig. 1a, 2a; 3a; see Table S1,S2).

However, we documented multiple adverse responses to ocean acidification when different fish traits were considered in our meta-analysis.

Trait-mediated effects of rising pCO 2 levels on fish mortality are reported in Fig. 1b (see Table S3, Fig. S1 for full results). Increased mortality was recorded for larvae exposed to high PCO 2 levels, whereas embryos, juveniles and adults were unaffected at both PCO 2 level ranges considered (Fig. 1b; see Table S3, Fig. S1). Effects of OA on mortality of stenohaline species were not evident, whereas euryhaline species exhibited higher mortality at high PCO 2 (+29%; Fig. 1b; see Table S3, Fig. S1). When considering climatic zones, mortality increased only for sub-tropical species exposed to high PCO 2 (+38%), and no effects were evident for polar, tropical and temperate species exposed at moderate and high PCO 2 levels (Fig. 1b; see Table S3, Fig. S1).

Mortality of pelagic fish increased at high PCO 2 levels, whereas benthic and benthopelagic species did not show significant effects at the two of PCO 2 level ranges considered (Fig. 1b; see Table S3, Fig. S1).

Growth of larvae significantly decreased by 4% at high PCO 2 , and no effect was observed at moderate PCO 2 (Fig. 1b; see Table S3, Fig. S2). This neutrality in growth response was also apparent on embryos and juveniles at both PCO 2 levels and on adults at high PCO 2 levels (Fig. 1b; see Table S3, Fig. S2). We did not find any OA effects on fish from different climatic zone, except for subtropical species that showed decreased growth (-14%) at high PCO 2 levels (Fig. 1b; see Table S3, Fig. S2). Growth of pelagic species significantly decreased by 9% at high PCO 2 conditions, whereas benthic and bentho-pelagic fish were neutrally affected by altered CO 2 (Fig. 1b; see Table S3, Fig. S2).

Calcification (otolith formation) responses to OA differed between fish life stages, physiologies, climatic zones, and habitats (Fig. 1b; see Table S3). Exposure to high PCO 2 levels significantly increased calcification of fish larvae (+6%), and of sub-tropical (+7%), temperate (+6%) and benthic (+6%) species (Fig. 1b; see Table S3, Fig. S3). In addition to this, the calcification of benthopelagic species was also affected but only one contrast was available for this analysis.

Fish reproduction and metabolic scope (MS) were unaffected at both PCO 2 levels, no matter the different fish traits considered (Fig. 2; see Table S3, Fig. S4,S6). Resting metabolic rate (RMR) increased in adult (+13%) and stenohaline (+8%) fish exposed to high PCO 2 levels.

Interestingly, polar species exposed to high CO 2 concentrations showed a RMR 33% higher than control conditions. Trait-mediated responses were also evident among fish habitats, as benthic fish increased their RMR at high PCO 2 levels, whereas benthopelagic and pelagic species resulted unaffected under elevated CO 2 conditions (Fig. 2b; see Table S3, Fig. S5).

We found that OA may cause an overall significant decrease in yolk size (Fig. 3a; see Table S1). When considering the effects of the different ranges of CO 2 , only high pCO 2 levels had a negative effect on yolk size. This result was evident in embryos (yolk size decreased by 7% at high pCO 2 levels) but not in larvae (Fig. 3b; see Table S3, Fig. S7 for full results). Stenohaline and benthic fish showed a negative effect on yolk size at high PCO 2 levels (-7% and -8%, respectively). When we compared data on fish species from different climatic zones, temperate species showed decreased yolk size only at moderate PCO 2 levels (but only one contrast was available for the analysis), whereas yolk of tropical species resulted to be affected under high PCO 2 levels. Offspring of benthic fish species showed decreased yolk size (-6%) after exposure to high CO 2 concentrations (Fig. 3b; see Table S3, Fig. S7).

Behavioural responses

Overall, OA conditions significantly and strongly affected behaviour (-54 %) and activity (+108 %) of fish (Fig. 4a, Table S1) and such responses did vary among PCO 2 levels (Fig. 4a, see Table S2). Moderate CO 2 concentrations decreased by 45% the behavioural performances of fish and increased their activity levels by 150% on average (Fig. 4a, see Table S2). At high PCO 2 levels fish displayed significant reductions of their behavioural performances (-60%), whereas no significant effects were evident on their activity (Fig. 4a, see Table S2). Behaviour of larvae was negatively affected at high PCO 2 levels, whereas juveniles showed significant reductions in behavioural performances at both PCO 2 levels considered (Fig. 4b; see Table S4, Fig. S8).

Behavioural disruptions were also evident for tropical species exposed at moderate and high CO 2 concentrations (Fig. 4b; see Table S4, Fig. S8). OA did not seem to affect the behaviour of subtropical and temperate species, but the number of available studies is too low to detect a clear response pattern (Fig. 4b; see Table S4, Fig. S8). No data on OA effects on the behaviour of polar species were available. When considering fish physiology and habitat traits, most of the contrasts revealed OA effects on stenohaline and benthic species, showing severe behavioural disruptions at both moderate and high PCO 2 levels (Fig. 4b; see Table S4, Fig. S8).

A significant 1.5-fold increase in juvenile fish activity (boldness) was recorded at moderate but not at high pCO 2 levels, whilst neutral effects were recorded for larval and adult stages at the two ranges of PCO 2 levels (Fig. 4b; see Table S4, Fig. S9). Fish activity increased only in stenohaline and benthic species exposed to moderate PCO 2 levels, whereas data on euryhaline, benthopelagic and pelagic species resulted understudied or not available (Fig. 4b; see Table S4, Fig. S9).

Significant alterations of fish behaviour mostly resulted from impaired olfaction, with on average 40% and 70% decreases at moderate and high PCO 2 levels, respectively (Fig. 5a; see Table S4). Fish behaviour was unaffected in experiments involving cognition and vision (Fig. 5a; see Table S4). Fish simultaneously exposed to visual and olfactory stimuli showed a decreased behavioural performance at moderate PCO 2 (only 2 contrasts included in the analyses) and no effects at high PCO 2 levels (Fig. 5a; see Table S4).

The observed fish behavioural impairments resulted in higher predation risk at both moderate (by 55% on average) and high (by 77%) PCO 2 levels. Feeding activity and detection of prey (i.e., the foraging activity of fishes) also decreased by 59% at high PCO 2 levels, but were unaffected at lower CO 2 concentrations (Fig. 5b; see Table S4). No effects on habitat choice were found for any of the PCO 2 levels considered.

Experimental CO 2 exposure time

Our analysis showed that fish sensitivity to OA scenarios differed according to experimental CO 2 exposure time and the PCO 2 ranges considered (Fig. 6; see Table S4). Short-term experiments at moderate PCO 2 levels did not affect any eco-physiological and behavioural responses, whereas short-term exposure to high PCO 2 levels significantly increased fish calcification (+6%) and mortality (+24%), and decreased growth (-3%) and behavioural performances (-60%) (Fig. 6; see Table S4). Long-term exposure at both moderate and high PCO 2 levels significantly increased fish activity/boldness (+232% and +2119%, respectively). High PCO 2 levels increased fish RMR (+20%) and decreased their behavioural performances (-58%). The effects of chronic exposure to elevated CO 2 concentrations were evident at moderate PCO 2 levels for behaviour (-88%) and for activity (+200%), no data are available for high PCO 2 levels .

DISCUSSION

New insights are gained from our findings by including the increasing number of studies published in recent years and the responses of fish to different CO 2 projections. This also allowed our analyses to identify a range of trait-based variations in fish sensitivity to OA, with ecophysiological and behavioural responses varying according to CO 2 scenarios, and fish ontogenetic phases and species characteristics. Specifically, we show that, if anthropogenic CO 2 emissions continue to rise both under the RCP 6 and RCP 8.5 scenarios, overall significant effects of elevated PCO 2 levels on fish metabolism, calcification (otolith formation), yolk consumption, behaviour and activity (boldness) are expected to occur in the next few decades. It is now wellestablished that under elevated CO 2 conditions, any ATP-demanding compensation activities for acid-base balance and enhanced transport of ions may incur elevated energetic costs, potentially leading to subtle fitness consequences for such response variables in marine fish [START_REF] Sokolova | Energy-Limited Tolerance to Stress as a Conceptual Framework to Integrate the Effects of Multiple Stressors[END_REF][START_REF] Heuer | Physiological impacts of elevated carbon dioxide and ocean acidification on fish[END_REF]. Hence, at the same time, we suggest that more subtle consequences of OA on mortality and growth of fish, as well as downstream ecological consequences, might occur.

There has been a clear call for addressing the important effects of species traits when making predictions about the impacts of environmental change (e.g. [START_REF] Estrada | Usefulness of species traits in predicting range shifts[END_REF]. Our work meets this call and sharpens the focus to fish. Previous meta-analyses did not take into account different fish traits and life stages instead focussing on a small amount of experiments available at that time [START_REF] Harvey | Meta-analysis reveals complex marine biological responses to the interactive effects of ocean acidification and warming[END_REF][START_REF] Kroeker | Impacts of ocean acidification on marine organisms: quantifying sensitivities and interaction with warming[END_REF][START_REF] Wittmann | Sensitivities of extant animal taxa to ocean acidification[END_REF][START_REF] Lefevre | Are global warming and ocean acidification conspiring against marine ectotherms? A meta-analysis of the respiratory effects of elevated temperature, high CO 2 and their interaction[END_REF]. As an example, it has previously been argued that OA studies on fish were biased by the high number of experiments carried out on tropical species [START_REF] Wittmann | Sensitivities of extant animal taxa to ocean acidification[END_REF], therefore suggesting that responses were predominantly focussed towards species with short life cycles, which are typical of this climate zone [START_REF] Leis | The biology, behaviour and ecology of the pelagic, larval stage of coral reef fishes[END_REF]. By analyzing the body of literature through to January 2017 and by including data on additional 30 non-tropical species and 115 contrasts, here we were able to provide a clearer picture and show that fish responses to elevated CO 2 levels may be CO 2 dependent and vary according to different fish characteristics and traits.

OA effects on marine fish are mediated by their life history traits and characteristics

We documented varying adverse responses when examining specific fish traits. In this regard, we did not find any specific fish characteristics which could potentially confer tolerance to fish under elevated CO 2 concentrations. These are important results as in addition to identifying the direction and quantifying the magnitude of these responses, we were able to show that, if fish traits are not taken into account, consequences of ocean acidification on marine animals might not be detected as in some cases species-specific responses could be antagonistic [START_REF] Esbaugh | Physiological implications of ocean acidification for marine fish: emerging patterns and new insights[END_REF].

Our study adds on previous evidence showing that larvae are the most sensitive life stage as they displayed increased mortality and calcification (i.e. otolith formation), and decreased growth at elevated CO 2 conditions (~1000 µatm PCO 2 ). The observed higher sensitivity to elevated PCO 2 levels for early life stages of fish might be linked to their high surface to volume ratio affecting diffusive processes and to their acid-base balance system not being fully developed, with ionic exchanges occurring across the skin of larvae and the yolk of embryos until their gills can develop (Munday et al. 2009a;[START_REF] Baumann | Reduced early life growth and survival in a fish in direct response to increased carbon dioxide[END_REF][START_REF] Hurst | Effects of ocean acidification on hatch size and larval growth of walleye pollock (Theragra chalcogramma)[END_REF].

Mortality at individual level affects population dynamics [START_REF] Munday | Replenishment of fish populations is threatened by ocean acidification[END_REF], which in turn can extend responses to higher community-and ecosystem-levels (Nagelkerken & Connell 2015). Since we only included experiments testing for the direct effects of elevated CO 2 on fish mortality, the increased mortality of larvae we recorded may be a conservative estimate, as more indirect effects can also occur leading to even higher mortality rates. As an example, we found that larval growth was significantly reduced at high CO 2 levels, and smaller larvae may exhibit a lower performance and survival in the wild as a result of their reduced swimming ability and less efficient predator avoidance (e.g. [START_REF] Miller | Larval size and recruitment mechanisms in fishes: toward a conceptual framework[END_REF], therefore leading to increased mortality through predation.

Heavily calcifying organisms like molluscs, echinoderms and corals are considered to be the most threatened marine taxa under OA [START_REF] Kroeker | Impacts of ocean acidification on marine organisms: quantifying sensitivities and interaction with warming[END_REF][START_REF] Wittmann | Sensitivities of extant animal taxa to ocean acidification[END_REF]. Fish are not considered calcifying organisms despite producing calcium carbonate in the inner ear for otolith formation or precipitating CaCO 3 in the intestinal lumen to reduce osmotic pressure, release calcium ions excess in the environment and thus facilitate water absorption [START_REF] Heuer | Physiological impacts of elevated carbon dioxide and ocean acidification on fish[END_REF]). Here we found a significant increase in the size of otoliths in larvae exposed to high PCO 2 levels expected to occur by 2050 (following the business-as-usual RCP8.5 scenario) or by 2100 (following the intermediate RCP6.0 scenario). Our results also showed that otolith calcification in both benthic and bentho-pelagic species may increase at high PCO 2 levels. Recent studies shed light on the potential mechanisms involved in increased otolith formation under OA.

Specifically, rising dissolved CO 2 concentrations in the ocean may lead to increased HCO 3 -levels in the fish plasma (which is used to compensate pH), while plasma PCO 2 levels remain elevated [START_REF] Esbaugh | Impacts of ocean acidification on respiratory gas exchange and acid-base balance in a marine teleost, Opsanus beta[END_REF]). It appears that plasma HCO 3 -import in the alkaline endolymph and the hydration of CO 2 in the saccular epithelium lead to increased HCO 3 -and hence to increased otolith size when fish are exposed to elevated CO 2 levels [START_REF] Heuer | Physiological impacts of elevated carbon dioxide and ocean acidification on fish[END_REF]. It is presently unknown whether larger otoliths may lower the ability of sound detection and the survival of fish in the wild, although we know that asymmetry between otoliths can be detrimental [START_REF] Gagliano | Dispersal without errors: symmetrical ears tune into the right frequency for survival[END_REF].

Despite the lack of effects of elevated CO 2 on the metabolism of fish early stages, adults experimentally exposed to high pCO 2 levels significantly increased their metabolism at rest.

Similar responses were detected on polar fish species. At present, some studies suggest that temperature effects lead to higher costs for pH regulation in cold-adapted eurytherms compared to polar stenotherms [START_REF] Pörtner | Climate Variability and the Energetic Pathways of Evolution: The Origin of Endothermy in Mammals and Birds[END_REF], however existing knowledge on OA effects on the metabolism of cold environment fish species is scant. Although an increase in RMR may be expected to result in a decrease in metabolic scope, our results show that MS is not affected by elevated CO 2 . This apparent contradiction could be due to either (1) a compensatory increase in maximum metabolic rate or (2) the fact that the number of studies on MS is much smaller than that for RMR and therefore the analysis for MS is less robust. A recent meta-analysis on warming and acidification effects on metabolism of marine organisms showed no significant effects of altered pCO 2 levels on resting metabolic rate and metabolic scope of teleost fish, highlighting high variability in the metabolic responses of fish [START_REF] Lefevre | Are global warming and ocean acidification conspiring against marine ectotherms? A meta-analysis of the respiratory effects of elevated temperature, high CO 2 and their interaction[END_REF]. In this regard, our analyses suggest that fish metabolic responses might be trait-mediated as we mostly found elevated CO 2 effects on RMR of benthic, stenohaline and polar species. Earlier studies hypothesised that exposure to high PCO 2 levels could lead to increased fish resting metabolism due to the costs associated with internal CO 2 balance, however mixed responses were found, with some studies confirming this assumption both in tropical (Munday et al., 2009b;[START_REF] Couturier | Speciesspecific effects of near-future CO 2 on the respiratory performance of two tropical prey fish and their predator[END_REF][START_REF] Ferrari | Interactive effects of ocean acidification and rising sea temperatures alter predation rate and predator selectivity in reef fish communities[END_REF] and non-tropical species [START_REF] Esbaugh | Respiratory plasticity is insufficient to alleviate blood acid-base disturbances after acclimation to ocean acidification in the estuarine red drum, Sciaenops ocellatus[END_REF][START_REF] Flynn | Ocean acidification exerts negative effects during warming conditions in a developing Antarctic fish[END_REF], whereas other studies revealed decreased resting metabolic rate after prolonged high-CO 2 exposure [START_REF] Rummer | Elevated CO 2 enhances aerobic scope of a coral reef fish[END_REF][START_REF] Pimentel | Impact of ocean acidification in the metabolism and swimming behavior of the dolphinfish (Coryphaena hippurus) early larvae[END_REF][START_REF] Heuer | Elevated CO 2 increases energetic cost and ion movement in the marine fish intestine[END_REF].

Fish reproductionindeed an energetically costly processwas unaffected by altered CO 2 levels, no matter which specific fish trait was considered. However, in this case, there were too few experiments to draw a clear picture of the effect of rising CO 2 on fish reproduction. In this regard, work on OA effects on fish reproduction per se should be a priority given its importance for population replenishment.

Marine habitats with different salinity levels also vary greatly in PCO 2 and thus euryhaline speciese.g. those fish having the ability to effectively osmoregulate across a broad range of salinitiesare likely to experience highly variable PCO 2 environments during their life cycle. This suggests these organisms (i.e. marine-brackish fish) can be pre-adapted to OA conditions.

Contrary to what we expected, we found a significantly higher mortality of euryhaline fish exposed to high PCO 2 levels. Mortality of stenohaline fish (which cannot tolerate a broad range of salinities) was unaffected by OA but these fish showed increased calcification rates (otolith formation) and metabolic rate at resting when exposed at high PCO 2 levels. To date, the impact of OA on fish osmoregulation at ecologically relevant CO 2 conditions is critically understudied [START_REF] Heuer | Physiological impacts of elevated carbon dioxide and ocean acidification on fish[END_REF].

Overall, we found a negative effect on yolk consumption (i.e. decreased yolk size) in fish exposed to elevated CO 2 . A decreased yolk size was evident after exposure to high PCO 2 levels for early developmental stages of benthic species, but not for pelagic ones. Similar to the trend proposed for stenohaline and euryhaline fish, some authors have hypothesised that embryos and larvae of pelagic spawning fish, which likely experience lower and more stable CO 2 conditions in the open ocean [START_REF] Hofmann | High-Frequency Dynamics of Ocean pH: A Multi-Ecosystem Comparison[END_REF], might be more susceptible to CO 2 increases compared to the offspring of fish laying eggs in the benthic environment, which in turn might be better adapted to natural CO 2 fluctuations due to the alternation of photosynthetic and respiration processes by primary producers [START_REF] Hofmann | High-Frequency Dynamics of Ocean pH: A Multi-Ecosystem Comparison[END_REF][START_REF] Munday | Ocean acidification does not affect the early life history development of a tropical marine fish[END_REF]. Contrary to these expectations, when categorising yolk size responses according to the habitat of developing embryos, negative (i.e. decreased yolk size) responses have been noted at high PCO 2 levels only in benthic spawning fish, but not in offspring with pelagic development. It is known that through maternal provisioning, females may adjust eggs characteristics (e.g. offspring yolk size) to environmental conditions [START_REF] Chambers | Environmental influences on egg and propagule sizes in marine fishes[END_REF] and that yolk consumption is affected by elevated CO 2 conditions during the embryonic development [START_REF] Chambers | Effects of elevated CO 2 in the early life stages of summer flounder, Paralichthys dentatus, and potential consequences of ocean acidification[END_REF].

Our meta-analysis also confirmed that OA could strongly influence the behaviour of tropical fish, particularly that of larvae and juveniles. Indeed, these responses were rather consistent at environmentally relevant PCO 2 levels expected by the next few decades. Importantly, the observed behavioural impairments were due to alterations of a range of different neurosensory functions. Olfaction was the most sensitive sensory function affecting fish behaviour even at PCO 2 levels expected in 2050. At the same time, in many experiments, fish reared in elevated CO 2 conditions were bolder, displaying up to 1.5-fold increase in their activity rates (e.g. less time spent in a shelter, or increased distance ventured) and therefore increased vulnerability to predators (e.g. [START_REF] Munday | Replenishment of fish populations is threatened by ocean acidification[END_REF].

As animals respond to environmental change through behavioural modifications, the downstream consequences of the observed behavioural and activity effects (particularly on larvae and juvenile fish) are expected to lower fish ecological performance (e.g. with respect to predatorprey dynamics, recruitment success, settlement, homing and habitat choice) and therefore to affect the outcomes at community level [START_REF] Nagelkerken | Animal behaviour shapes the ecological effects of ocean acidification and warming: moving from individual to community-level responses[END_REF]. Consistently, our quantitative findings indicate that the behavioural disruptions we identified could result in a higher predation risk and a lowered foraging activity, which in turn may lead to increased mortality and decreased growth, therefore decreasing the ability of a species to persist in a given community. Unexpectedly the settlement and homing ability (i.e., habitat choice) of fish was unaffected by rising CO 2 levels.

Certain effects, such as an increase in fish activity (boldness) and changes in fish lateralization, do not have an immediate bearing on ecological "fitness", because in nature they represent a trade off in which for example, bold individuals can find food faster [START_REF] Mamuneas | Bolder stickleback fish make faster decisions, but they are not less accurate[END_REF] but are more exposed to predation [START_REF] Ward | Correlates of boldness in threespined sticklebacks (Gasterosteus aculeatus)[END_REF] than shy individuals. Similarly, although a high degree of lateralization has been considered an advantage in terms of multitasking (escaping from predators and schooling; [START_REF] Bisazza | Enhanced schooling performance in lateralized fishes[END_REF][START_REF] Dadda | Does brain asymmetry allow efficient performance of simultaneous tasks?[END_REF][START_REF] Dadda | Behavioural asymmetry affects escape performance in a teleost fish[END_REF], Domenici et al., 2012), it was suggested to represent a trade off with the ability to deal equally well with stimuli of threats from all directions [START_REF] Vallortigara | Survival with an asymmetrical brain: Advantages and disadvantages of cerebral lateralization[END_REF]. In these cases, the effect of elevated CO 2 may be detrimental for fish because it alters the balance within the continuum of bold vs. shy and lateralized vs. non-lateralized individuals, with potential ecological consequences at population level [START_REF] Sih | Ecological implications of behavioural syndromes[END_REF].

Ocean acidification effects on fish under different experimental CO 2 exposure duration

To date, a great uncertainty associated with the consequences of different CO 2 projections on teleost fish, relies on whether such organisms will be able to cope with ongoing rapid changes in the carbonate chemistry of the ocean. Our analyses following different CO 2 exposure times from field-and lab-based experiments revealed that moderate PCO 2 levels have no effects on fish ecophysiology both in short-and long-term experiments, and that fish behavioural performances are disrupted particularly in chronically exposed fish populations. Despite consistent responses on fish calcification and behaviour, we found that high PCO 2 levels significantly affect mortality and growth of fish only after short-but not after long-term elevated CO 2 exposures. Indeed, our analyses suggests that further longer term and multi-generational experiments are needed to test for a potential adaptation of fish occurring at altered PCO 2 levels and we identify this issue as a critical gap of knowledge, which is further discussed in the next paragraph.

Future directions and conclusions

Acclimation and adaptation may have significant consequences for how marine organisms will respond to future high CO 2 oceans [START_REF] Sunday | Evolution in an acidifying ocean[END_REF] and some previous work argues that adaptation may be too slow for long-lived species [START_REF] Pörtner | Ocean systems[END_REF]. Due to the small number of experiments carried out so far, quantitative generalisations are not possible yet, and our investigation identified a critical need to increase the number of fish studies addressing such processes under OA. To date, experimental findings are not in line with conclusions made using fossil records and observations from paleo-studies which suggested that teleosts are very tolerant to elevated PCO 2 levels when compared to invertebrate taxa [START_REF] Wittmann | Sensitivities of extant animal taxa to ocean acidification[END_REF]. In this context, a few studies showed mixed potential for transgenerational acclimation (e.g. [START_REF] Allan | Parental effects improve escape performance of juvenile reef fish in a high-CO 2 world[END_REF][START_REF] Welch | Effects of elevated CO 2 on fish behaviour undiminished by transgenerational acclimation[END_REF] while others, examining OA effects on chronically CO 2 exposed fish populations (e.g. those from volcanic CO 2 seeps), documented changes in embryo metabolism, olfaction, spawning behaviour, and escape response [START_REF] Munday | Behavioural impairment in reef fishes caused by ocean acidification at CO 2 seeps[END_REF]Nagelkerken et al., 2015;[START_REF] Cattano | Effects of ocean acidification on embryonic respiration and development of a temperate wrasse living along a natural CO 2 gradient[END_REF][START_REF] Milazzo | Ocean acidification affects fish spawning but not paternity at CO 2 seeps[END_REF]. Although much progress has been made in designing experiments to assess how elevated PCO 2 can alter fish ecophysiology and behaviour, to date we are still far from understanding adaptive capacity to OA [START_REF] Munday | Elevated CO 2 affects the behavior of an ecologically and economically important coral reef fish[END_REF].

Given that additional environmental stressors will lead to dramatic ocean change in the futurei.e. increasing seawater temperature and more severe hypoxia are predicted to occur concomitantly to rising seawater CO 2their effects are unlikely to operate independently and there is a need to understand how the combined consequences of these stressors will affect the fitness of marine fish. [START_REF] Pörtner | Physiology and climate change[END_REF] have hypothesised additive and synergic effects of temperature and elevated CO 2 on individual performance, increasing the energy requirements for homeostasis regulation, and therefore, reducing the amount of energy available for other biological processes. Similarly, by constraining metabolism, predicted warming and hypoxia can be expected to contract the distribution of marine fish [START_REF] Deutsch | Climate change tightens a metabolic constraint on marine habitats[END_REF]. Importantly, there may be genetic correlations between phenotypic variation associated ocean acidification, warming and hypoxia that could potentially limit the rate of adaptation to these stressors when two or more of them occur simultaneously, and have antagonistic effects [START_REF] Munday | Elevated CO 2 affects the behavior of an ecologically and economically important coral reef fish[END_REF]. Identifying such constraints will be an important issue for studies on the adaptive potential of fishes to OA.

An additional unavoidable limitation of most OA studies on fish and other taxa is their unrepresentativeness of ecosystem effects since they do not take into account the complex species interactions acting at multiple levels of organisation in the real world. Yet, some attempts have succeeded in testing OA effects on key community ecological interactions (e.g. competition and predation) involving fish in their natural habitat [START_REF] Nagelkerken | Animal behaviour shapes the ecological effects of ocean acidification and warming: moving from individual to community-level responses[END_REF]. In this context, our study might also identify the need to expand the number of OA studies examining fish responses to community shifts and biogenic habitat modifications in naturally high CO 2 environments [START_REF] Munday | Behavioural impairment in reef fishes caused by ocean acidification at CO 2 seeps[END_REF][START_REF] Enochs | Ocean acidification enhances the bioerosion of a common coral reef sponge: implications for the persistence of the Florida Reef Tract[END_REF]Nagelkerken et al., 2015;[START_REF] Sunday | Ocean acidification can mediate biodiversity shifts by changing biogenic habitat[END_REF].

In summary, here we document negative effects on many fish eco-physiological and behavioural responses at CO 2 levels expected by the next few decades or by the end of this century depending on the CO 2 emission scenario considered. We suggest that some direct effectsparticularly on fish mortality and growthmay be underestimated as we found increased predation risk and decreased foraging, confirm that larvae are the most sensitive life stages (Munday et al., 2009a), and advise that downstream ecological consequences can be very likely.

Importantly, we do not find any traits potentially conferring fish tolerance to elevated CO 2 , and thus reject previous hypotheses that some specific fish traits (e.g., those species characteristics associated to highly fluctuating PCO 2 environments) may mitigate such responses. Many explanations for documented eco-physiological and behavioural responses of marine fish to elevated CO 2 rely on changes expected to occur in fish brain function [START_REF] Nilsson | Near-future carbon dioxide levels alter fish behaviour by interfering with neurotransmitter function[END_REF]) and acid-base compensation [START_REF] Heuer | Physiological impacts of elevated carbon dioxide and ocean acidification on fish[END_REF]. Likely, effects on fish will be heaviest where ocean acidification, warming, and hypoxia regionally coexist, indicating that studies like the one we present focussing on an individual environmental drivers of change maybe conservative.

However, some level of uncertainty remains over the long-term persistence of the observed effects on fish, particularly on some eco-physiological response variables. Given the importance of marine fish livelihoods in coastal communities, for ecosystem stability and food webs, it is fundamentally critical to slow down current rates of CO 2 emissions and deviate from the expected scenarios to mitigate OA effects on fish. and behavioural performance (behaviour and activity). Type of data as reported in Figure 1 legend.

Figure 1

 1 Figure 1 OA effects on fish eco-physiological responses (mortality, resting metabolic rate and

Figure 2

 2 Figure 2 OA effects on fish eco-physiological responses (reproduction, growth and calcification).

( a )

 a Overall and CO 2 dose-dependent responses and (b) summary of trait-mediated responses for each CO 2 projection relative to fish reproduction, growth, and calcification. Symbols and type of data as reported in Figure 1 legend , see also Figures S4-S6 for full results.

Figure 3

 3 Figure 3 OA effects on fish development (yolk, hatching time and embryonic abnormalities). (a)

Figure 5

 5 Figure 5 Links between fish neurosensory, behaviour and ecological processes. (a) Neurosensory

Figure 6

 6 Figure 6 Effects of different CO 2 exposure times (short-term, long-term and chronic) on fish eco-

  [START_REF] Devine | Rising CO 2 concentrations affect settlement behaviour of larval damselfishes[END_REF] or an increased (positive) activity (i.e. venturing at an increasing distance from a shelter;[START_REF] Munday | Elevated CO 2 affects the behavior of an ecologically and economically important coral reef fish[END_REF] under elevated CO 2 , although both types of response could theoretically lead to an increased mortality (hence decreased performance).When available, both metadata and/or raw data were obtained from the Pangaea data repository (http://www.pangaea.de). When not available, data were retrieved directly from the text or tables or with Data Thief III (B. Tummers, DataThief III 2006; http://datathief.org) to get data from figures.

For each type of response we assigned metrics to the following response variables: (1) mortality, growth, calcification, reproduction, metabolism [resting metabolic rate (RMR) and metabolic scope (MS)] and yolk (for the eco-physiological response types); (2) behaviour and activity (for the behavioural response types) (see Appendix S1).

ACKNOWLEDGEMENTS

We wish to thank Philip L. Munday and Benjamin P. Harvey for the insightful and constructive comments on the early draft of this manuscript. We also thank Jodie L. Rummer and Gabrielle M. Miller for providing data. This research was supported by FFR-A funds from University of Palermo to M.M and a PhD grant to C.C.

information available in each paper considered. Moreover, to evaluate the effects of different time of exposure to OA experimental conditions, we compared data from short-term experiments (≤4 weeks), from long-term experiments (>4 weeks) and from experiments conducted along natural CO 2 vents where fish with limited mobility are chronically exposed to elevated CO 2 concentrations (i.e. chronic exposure experiments) following Nagelkerken & Connell (2015).

Details on the studies we used in the meta-analysis along with response types, response variables, categories, category levels, metrics and effect sizes are reported in the supplementary Appendix S1.

Data analysis

We used a weighted, random-effects meta-analysis to quantify the response of fish to OA and assess how the studied categories can mediate this response. For each combination of response type i, response variable j, category k, category level m and study l, the effect size of the fish response to elevated pCO 2 levels was calculated as log-ratios [START_REF] Osenberg | Effect size in ecological experiments: the application of biological models in meta-analysis[END_REF]


Dr Petina L Pert
email: petina.pert@csiro.ausuggested

Lauric Thiault

Matthew I Curnock

Susanne Becken

Joachim Claudet

Nadine A Marshall
email: xxxxnadine.marshall00@gmail.com

Beauty and the reef: Evaluating the use of non-expert ratings for monitoring aesthetic values of coral reefs

Keywords: Aesethetic, value, beauty, appreciation, cultural ecosystem services, attitudes, perception, Great Barrier Reef

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Introduction

The reframing of nature by the Millennium Ecosystem Assessment report (Millennium Ecosystem Assessment, 2003), in particular, as providing goods and services, has enabled a mechanism by which the importance of natural places can be reconsidered [START_REF] Diaz | Linking functional diversity and social actor strategies in a framework for interdisciplinary analysis of nature's benefits to society[END_REF].

The aesthetic services that are provided by nature have become formally acknowledged and valued, where the provision of aesthetic services is strongly related to all other cultural services including recreation [START_REF] Ghermandi | Recreational, Cultural, and Aesthetic Services from Estuarine and Coastal Ecosystems[END_REF][START_REF] Junge | Aesthetic quality of agricultural landscape elements in different seasonal stages in Switzerland[END_REF]. Areas with conditions for recreation and leisure are often valuable due to their aesthetic and cultural heritage and identity qualities (e.g. [START_REF] Chan | Where are Cultural and Social in Ecosystem Services? A Framework for Constructive Engagement[END_REF][START_REF] Comberti | Ecosystem services or services to ecosystems? Valuing cultivation and reciprocal relationships between humans and ecosystems[END_REF][START_REF] Gee | Cultural ecosystem services in the context of offshore wind farming: A case study from the west coast of Schleswig-Holstein[END_REF][START_REF] Jobstvogt | Looking below the surface: The cultural ecosystem service values of UK marine protected areas (MPAs)[END_REF]). Likewise, areas with scenic beauty often provide inspiration and opportunities for education, holding significant existence values (e.g., [START_REF] De Oliveira | What value São Pedro's procession? Ecosystem services from local people's perceptions[END_REF][START_REF] Fletcher | Revealing marine cultural ecosystem services in the Black Sea[END_REF][START_REF] Hashimoto | Mapping and characterizing ecosystem services of social-ecological production landscapes: case study of Noto, Japan[END_REF][START_REF] Martínez-Pastur | Spatial patterns of cultural ecosystem services provision in Southern Patagonia[END_REF][START_REF] Outeiro | Framing local ecological knowledge to value marine ecosystem services for the customary sea tenure of aboriginal communities in southern Chile[END_REF]). Furthermore, De Groot et al. (2002) refers to 'aesthetic information' as an ecosystem service category, provide 'scenery' as an example of an aesthetic service, and a feeling of 'enjoyment' as the benefit of this service. [START_REF] Boyd | What are ecosystem services? The need for standardized environmental accounting units[END_REF] suggest that 'amenity and fulfilment' are examples of aesthetic benefits and 'natural land cover in viewsheds' is the ecosystem service providing the benefits. They argue this differentiation is essential in defining a workable accounting system to facilitate land-use decision-making and management.

For the purpose of this paper, the term aesthetic value(s) has been taken as defined by Context Pty Ltd (2013 p. 33), being: ".. the response (the aesthetic response) derived from the experience of an environment or parts of an environment. Human sensessight, touch, smell, sound, movement -are important in how humans experience an environment. And culture, knowledge, expectations and experience mediate sensory perceptions. Aesthetic response can therefore be said to be linked to:

 the characteristics of an environment  culturally or personally derived preferences." Furthermore, we have summarised the by the many contributing environmental and experiential characteristics that contribute to aesthetic response, satisfaction and derived value in a natural setting (Fig. 1). The natural beauty and aesthetic appreciation of landscapes has previously been studied in various terrestrial ecosystems around the world [START_REF] Arriaza | Assessing the visual quality of rural landscapes[END_REF][START_REF] Beza | The aesthetic value of a mountain landscape: a study of the Mt. Everest Trek[END_REF][START_REF] De Pinho | Influence of aesthetic appreciation of wildlife species on attitudes towards their conservation in Kenyan agropastoralist communities[END_REF][START_REF] Frank | Assessment of landscape aesthetics-Validation of a landscape metrics-based assessment by visual estimation of the scenic beauty[END_REF][START_REF] Gobster | The shared landscape: what does aesthetics have to do with ecology?[END_REF][START_REF] Hoffman | Silviculture and forest aesthetics within stands, The New York center for forestry research and development[END_REF][START_REF] Howley | Landscape aesthetics: Assessing the general publics' preferences towards rural landscapes[END_REF][START_REF] Rosley | Perceiving the Aesthetic Value of the Rural Landscape Through Valid Indicators[END_REF][START_REF] Schirpke | Predicting scenic beauty of mountain regions[END_REF][START_REF] Sheppard | Visual analysis of forest landscapes[END_REF][START_REF] Van Den Berg | Group differences in the aesthetic evaluation of nature development plans: a multilevel approach[END_REF][START_REF] Van Der Jagt | Unearthing the picturesque: The validity of the preference matrix as a measure of landscape aesthetics[END_REF][START_REF] Van Zanten | Continental-scale quantification of landscape values using social media data[END_REF]Wet Tropics Management Authority, 2017;[START_REF] Xu | Valuing Biodiversity, Aesethetics, and Job Losses Associated with Ecosystem Management Using Stated Preferences[END_REF], however very few studies have occurred in marine ecosystems [START_REF] Fenton | Perception and evaluation of the coastal zone: implications for coastal zone planning[END_REF] or more specifically within coral reefs [START_REF] Dinsdale | Assessing coral reef condition: eliciting community meanings[END_REF][START_REF] Fenton | Re-presenting the great barrier reef to tourists: Implications for tourist experience and evaluation of coral reef environments[END_REF]. Coral reef ecosystems worldwide are significant sources of consumptive and non-consumptive economic values [START_REF] Wielgus | Dose-Response Modeling of Recreationally Important Coral Reef Attributes: A Review and Potential Application to the Economic Valuation of Damage[END_REF] and supply vast numbers of people with goods and services such as seafood, tourism and recreational opportunities, coastal protection, as well as aesthetic and cultural benefits [START_REF] Martin | A systematic quantitative review of coastal and marine cultural ecosystem services: Current status and future research[END_REF][START_REF] Moberg | Ecological goods and services of coral reef ecosystems[END_REF][START_REF] Werner | Rapid prioritization of marine ecosystem services and ecosystem indicators[END_REF]. Many attributes have been described as to what contributes to these non-consumptive aesthetic values including the abundance of fish and corals [START_REF] Dixon | Meeting Ecological and Economic Goals: Marine Parks in the Caribbean[END_REF][START_REF] Wilkinson | Global Change and Coral Reefs: Impacts on Reefs, Economies and Human Cultures[END_REF][START_REF] Williams | Differences between Protected and Unprotected Reefs of the Western Caribbean in Attributes Preferred by Dive Tourists[END_REF], existence of iconic species/habitats [START_REF] Jobstvogt | Looking below the surface: The cultural ecosystem service values of UK marine protected areas (MPAs)[END_REF][START_REF] Levin | Developing meaningful marine ecosystem indicators in the face of a changing climate[END_REF][START_REF] Werner | Rapid prioritization of marine ecosystem services and ecosystem indicators[END_REF], and water clarity [START_REF] Dixon | Meeting Ecological and Economic Goals: Marine Parks in the Caribbean[END_REF][START_REF] Hatton Macdonald | Valuing coastal water quality: Adelaide, South Australia metropolitan area[END_REF][START_REF] Lee | Appearance's Aesthetic Appreciation to Inform Water Quality Management of Waterscapes[END_REF][START_REF] Tosic | Selection of parameters in the design of beach coastal water quality monitoring programs[END_REF][START_REF] Wilkinson | Global Change and Coral Reefs: Impacts on Reefs, Economies and Human Cultures[END_REF].

The development of indicators and quantifying the aesthetic value that people place on different landscapes, seascapes, and ecosystems has been approached in a number of ways by researchers over time. Primarily these have focused on using landscape assessment methods which either focus on understanding experiential preferences, the physical attributes/attractiveness of a place [START_REF] Tardieu | From supply to demand factors: What are the determinants of attractiveness for outdoor recreation?[END_REF], landscape preferences, [START_REF] Atauri | Visitors' landscape preferences as a tool for management of recreational use in natural areas: a case study in Sierra de Guadarrama (Madrid, Spain)[END_REF][START_REF] De La Fuente De Val | Relationship between landscape visual attributes and spatial pattern indices: a test study in Mediterranean-climate landscapes[END_REF][START_REF] De Lucio | Landscape preferences and behavior of visitors to Spanish National Parks[END_REF][START_REF] Huang | Visitor responses to the changing character of the visual landscape as an agrarian area becomes a tourist destination: Yilan County, Taiwan[END_REF][START_REF] Múgica | The role of on-site experience on landscape preferences. A case study at Doñana National Park (Spain)[END_REF], scenic quality, or the experience of the place. Two different methodological approaches that assess landscape aesthetics either from an objective or a subjective point of view have been established in the past [START_REF] Frank | Assessment of landscape aesthetics-Validation of a landscape metrics-based assessment by visual estimation of the scenic beauty[END_REF]. More recent approaches such as those by [START_REF] Casalegno | Spatial covariance between aesthetic value & other ecosystem services[END_REF] and [START_REF] Van Zanten | Continental-scale quantification of landscape values using social media data[END_REF] have used crowd-source datasets, machine learning, [START_REF] Haas | Can we measure beauty? Computational evaluation of coral reef aesthetics[END_REF][START_REF] Seresinhe | Quantifying the Impact of Scenic Environments on Health[END_REF][START_REF] Spalding | Mapping the global value and distribution of coral reef tourism[END_REF] and geo-tagged digital photographs uploaded to social media platforms to quantify and objectively measure the perceived aesthetic value of ecosystems.

The emergence of aesthetic values methods in the Australian heritage practice although only new, is important as there is no established World Heritage methodology for assessing natural beauty and aesthetic values under Criterion vii (i.e. contain superlative natural phenomena or areas of exceptional natural beauty and aesthetic importance). In the Great Barrier Reef and Wet Tropics World Heritage Areas in Australia attempts have been made to define and measure aesthetic values (Context Pty Ltd, 2013;Wet Tropics Management Authority, 2017). More recently new methods have been developed utilising non-expert visual assessments [START_REF] Marshall | Identifying indicators of aesthetics in the Great Barrier Reef for the purposes of management[END_REF] and innovative technologies using artificial intelligence [START_REF] Becken | Monitoring aesethetic value of the Great Barrier Reef by using innovative technologies and artificial intelligence[END_REF]. Given the importance of the tourism industry in these areas, those features and attributes that currently draw visitors to these areas are important, particularly to tourism operators in the areas. Furthermore, a number of values for which the Great Barrier Reef was originally inscribed for are on the decline after back to back coral bleaching events recently in 2016 and 2017, terrestrial run-off of sediments and nutrients and associated Crown-of-Thorns Starfish (COTS) (Acanthaster planci) outbreaks, acidification associated with climate change, impacts from severe cyclones, and coral diseases [START_REF] Brodie | A critical review of environmental management of the 'not so Great' Barrier Reef[END_REF][START_REF] De'ath | The 27-year decline of coral cover on the Great Barrier Reef and its causes[END_REF][START_REF] Hughes | Securing the future of the Great Barrier Reef[END_REF][START_REF] Hughes | Global warming and recurrent mass bleaching of corals[END_REF], which have all affected the aesthetics and natural beauty of some parts of the Region above and below the water (Great Barrier Reef Marine Park Authority, 2019).

Appealing features of methods which use non-expert rating scores of aesthetic beauty include simplicity and scalability, and as [START_REF] Marshall | Identifying indicators of aesthetics in the Great Barrier Reef for the purposes of management[END_REF] and [START_REF] Haas | Can we measure beauty? Computational evaluation of coral reef aesthetics[END_REF] have shown, relevance/correlation to environmental attributes associated with Reef health. However, before such a method can be implemented and used in a management context, managers need to understand the method's sensitivity, extrapolative power, and any potential biases associated with non-expert visual ratings of scenes. In this study we evaluate (a) the sensitivity of rating scores used in [START_REF] Marshall | Identifying indicators of aesthetics in the Great Barrier Reef for the purposes of management[END_REF] to compare underwater coral reef scenes, (b) the sampling design requirements to establish appropriate confidence levels, and (c) biases associated with demography and knowledge levels/expertise.

Methods

Study site

The Great Barrier Reef (GBR) of Australia is one of the world's largest coral reef systems. It houses globally-significant biodiversity, a rich diversity of underwater habitats, and a scenic appeal that is universally recognised [START_REF] Hughes | Climate change, human impacts, and the resilience of coral reefs[END_REF]. These values make it an international tourism attraction: in 2013, the Great Barrier Reef received an estimated 53.3M days of use, with 98% of all days comprising tourism visitation [START_REF] Marshall | Advances in monitoring the human dimension of natural resource systems: an example from the Great Barrier Reef[END_REF]. Tourism operators and commercial fishers are particularly appreciative of the natural beauty of the region, with the tourism industry alone worth over $5.2 billion each year [START_REF] Marshall | Advances in monitoring the human dimension of natural resource systems: an example from the Great Barrier Reef[END_REF].

It also holds immense value for local communities, where 'beautiful' is one of the first words that come to local residents' minds when thinking of the GBR [START_REF] Marshall | Advances in monitoring the human dimension of natural resource systems: an example from the Great Barrier Reef[END_REF]. Residents and tourists seek opportunities to appreciate the natural aesthetics of the GBR through experiences such as diving and snorkelling, visiting beaches, boating and scenic flights [START_REF] Marshall | Advances in monitoring the human dimension of natural resource systems: an example from the Great Barrier Reef[END_REF].

Data analysis

We developed a linear mixed effect model to generate expectations of how image scores were related to eight covariables expected to influence the aesthetic score. These included coral size, fish size, fish diversity, presence of algae, coral cover, topography, fish density, and visibility [START_REF] Marshall | Identifying indicators of aesthetics in the Great Barrier Reef for the purposes of management[END_REF]. Respondents and images were used as random effects. Variance inflation factor (VIF) scores 3.28, indicating low collinearity among covariates. We then identified XXXX and YYYY, defined as respondents scoring more than two standard deviations higher or lower than expectations from our model, respectively. Our approach thus reveals the respondents that tend to provide more extreme scores given the general characteristics of the images they evaluate. Finally, we assessed the link between socioeconomic characteristics and whether a respondent was XXXX, average, or YYYY using a Fisher's exact test.

In order to help practitioners find a balance between low cost and high accuracy, we examined how error in the image scores varied according to the number of respondents (i.e. sample size). Specifically, we calculated the error (i.e. score's 95% confidence interval) at varying sample sizes (N = 3 to 380 respondents in increments of 2, taken randomly without replacement from the original pool of respondents). This process was repeated 100 times for each of the 181 images in order to calculate the median error each image add at various sample size.

Five indicators were selected that reflected distinct attributes of coral reefs: (i) coral cover; (ii) coral pattern; (iii) coral topography; (iv) fish abundance; and (v) visibility [START_REF] Marshall | Identifying indicators of aesthetics in the Great Barrier Reef for the purposes of management[END_REF].

All analyses were implemented in R statistical analysis software v3.4.0.

Results

The average aesthetic score ranged between X and X. Four images (Fig. 2) indicate the frequency and average aesthetic score.

Discussion

• CHALLENGES Reporting through monitoring, and actively managing, aesthetic quality is potentially a critically useful strategy for natural resource managers that aim to effectively deliver both conservation and social outcomes. The advantages of monitoring aesthetic values lie essentially around the opportunity to better engage with the public, correspondingly achieving conservation [START_REF] Frank | Assessment of landscape aesthetics-Validation of a landscape metrics-based assessment by visual estimation of the scenic beauty[END_REF]. People appreciate and relate to aesthetics more than any other aspect of the natural environment [START_REF] Marshall | Measuring What Matters in the Great Barrier Reef[END_REF][START_REF] Marshall | Advances in monitoring the human dimension of natural resource systems: an example from the Great Barrier Reef[END_REF][START_REF] Marshall | The Dependency of People on the Great Barrier Reef, Australia[END_REF]. Aesthetics is also a unifying value associated with natural places, whilst biodiversity is often a divisive factor [START_REF] Hill | Reconciling Biodiversity Indicators to Guide Understanding and Action[END_REF][START_REF] Lecuyer | Building on common ground to address biodiversity conflicts and foster collaboration in environmental management[END_REF][START_REF] Schmidt | Biodiversity Conservation and Indigenous Land Management in the Era of Self-Determination[END_REF][START_REF] Young | The emergence of biodiversity conflicts from biodiversity impacts: characteristics and management strategies[END_REF]. In the case of iconic places such as the Great Barrier Reef, aesthetic values were a major factor in the establishment of World Heritage Area status, and the reporting of aesthetic quality and its maintenance is likely to become mandatory. For these reasons, efforts are currently underway to explore how aesthetic quality might be recorded and reported on within the Great Barrier Reef (NESP reference).

Perhaps the reason that aesthetic values have not been explicitly used by natural resource managers to further conservation goals and support is because aesthetics are seen as subjective and essentially unmanageable (Context Pty Ltd, 2013;[START_REF] Dramstad | Relationships between visual landscape preferences and map-based indicators of landscape structure[END_REF]. However, preliminary work by the National Environment and Science Program [START_REF] Becken | Monitoring aesethetic value of the Great Barrier Reef by using innovative technologies and artificial intelligence[END_REF][START_REF] Marshall | Identifying indicators of aesthetics in the Great Barrier Reef for the purposes of management[END_REF] has strongly suggested that developing indicators to rate aesthetic quality is possible [START_REF] Marshall | Identifying indicators of aesthetics in the Great Barrier Reef for the purposes of management[END_REF]. In sum, the development of aesthetic indicators for use in specific environments such as marine environments are still very much in their infancy, but the momentum supporting the development of aesthetic metrics, or indicators, is growing [START_REF] Belhassen | Evaluating the attractiveness and effectiveness of artificial coral reefs as a recreational ecosystem service[END_REF][START_REF] Haas | Can we measure beauty? Computational evaluation of coral reef aesthetics[END_REF][START_REF] Hein | Coral restoration: Socio-ecological perspectives of benefits and limitations[END_REF][START_REF] Jaap | Coral reef restoration[END_REF][START_REF] Tamayo | Estimates of Values of Philippine Reefs' Ecosystem Services[END_REF][START_REF] Turek | Science and technology needs for marine fishery habitat restoration[END_REF][START_REF] Vercelloni | Using virtual reality to estimate aesthetic values of coral reefs[END_REF][START_REF] Vlami | Cultural landscapes and attributes of "culturalness" in protected areas: An exploratory assessment in Greece[END_REF][START_REF] Wessel | An evaluative tool for rapid assessment of derelict vessel effects on coastal resources[END_REF].

The emerging social-ecological field continues to expand methodologies to improve techniques in monitoring aesthetic heritage values, using potential indicators and computations of aesthetic value. However, ongoing examination of which locations or biophysical elements are the most important to the Reef's spectacular seascapes and scenery, remains an information gap.

Conclusion

There are numerous potential management (and other) uses of aesthetic assessments and monitoring with information needs that vary substantially. The rapid assessment approach is potentially useful for some purpose with statistical design requirements and limitations now better understood. Our findings can help World Heritage site managers better contextualise the results and limitations from such monitoring and ensure that future aesthetic monitoring programs can be designed to meet management and reporting needs.

Funding

This research was funded by the National Environmental Science Program (Australia).

Fig. 1 .

 1 Fig. 1. Characteristics and attributes that contribute to aesthetic response, satisfaction and

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Fig. 4 .

 4 Fig. 4. Sample size describes how many people you would need to assess a photo/coral reef