The anthropogenic rise in atmospheric CO₂ leads to its absorption by the oceans resulting in the decreasing of seawater pH, known as ocean acidification (OA). Under current emissions, a drop in oceanic pH by 0.4 units is projected by the end of the 21st century compared to the current values (Allan et al., 2021). OA severely threatens marine ecosystems (Dupont and Pörtner, 2013), due to the reduction in the availability of carbonate ions, which affects marine calcifiers who use these minerals to synthesize carbonate biostructures. This vulnerability encompasses a broad range of taxa, including echinoderms (Byrne and Hernández, 2020). The consequences of vulnerable systems are disruptions to food webs, alteration of biodiversity on marine invertebrates, and impacts on the overall health of the oceans (Stumpp et al., 2013).

The sea urchins Paracentrotus lividus (Lamarck, 1816) and Arbacia lixula (Linnaeus, 1758) are calcifying species and key components in benthic communities of the Mediterranean Sea and northeast Atlantic due to their role in algae population regulation (Palacı́n et al., 1998). Within the Mediterranean, the combined grazing action at high densities promotes the formation of barren ground areas (Bonaviri et al., 2011; Agnetta et al., 2015), making changes in their abundance a concern for coastal conservation (Vergés et al., 2014). Studying their adaptability to overcome OA is essential to predict potential impacts on shallow Atlantic-Mediterranean waters.

Seawater pH affects the metabolism of the organisms by altering biochemical pathways (Dupont et al., 2010). External rising CO₂ concentration can diffuse into body tissues causing hypercapnia and acidosis (a decreased internal pH) (Collard et al., 2013) which disrupt physiological processes such as calcification, nutrition, and metabolism (Melzner et al., 2009; Byrne and Fitzer, 2019; Pörtner, 2008). Understanding metabolic responses is necessary for assessing the species survival, health, and fitness under environmental challenges (Brown et al., 2004; Calosi et al., 2013). Stabilized metabolic rates can occur via adaptation (selecting genotypes), or acclimatization (phenotypic plasticity) (Norin and Metcalfe, 2019). However, physiological plasticity and adaptation to low pH in marine organisms remain poorly understood (Calosi et al., 2013; Di Giglio et al., 2020). Both P. lividus and A. lixula have a likely efficient pH compensation system, essential for survival in acidified areas. However, P. lividus is less effective in regulating extracellular acid-base balance than A. lixula (Calosi et al., 2013). Nevertheless, this buffering ability may involve an energy reallocation (Catarino et al., 2012), making it essential to study these hidden physiological responses for predicting their long-term viability and ecosystem dynamics.

Much of our knowledge of OA effects mainly comes from short to medium-term laboratory experiments (e.g. Stumpp et al., 2012; Spicer et al., 2011; Wood et al., 2008), but long-term exposure often reveals more complex responses. Long-term studies on tropical sea urchins, particularly those in the Echinometra genus, showed varied OA responses, depending on life stage and environmental context (Uthicke et al., 2020, 2021; Karelitz et al., 2019). Moreover, studies on natural CO₂ emission points, also known as CO₂ vents, such as those at Ischia, Papua New Guinea and Canary Island, show that some populations of sea urchins exhibit adaptations to high CO₂ environments, with adults demonstrating resilience (Foo et al., 2022; Di Giglio et al., 2020; Uthicke et al., 2016, 2019; González-Delgado et al., 2024). In this sense, marine CO₂ vents, which create natural pH gradients, offer valuable scenarios to study the long-term effects of OA on marine ecosystems (González-Delgado and Hernández, 2018; González-Delgado et al., 2023), allowing scientists to investigate the biological responses of organisms, as well as their adaptive potential and resilience (Hofmann et al., 2014).

The Canary Islands, situated in the northeastern Atlantic Ocean, are home to the only subtropical marine CO₂ vent under investigation (Hernández et al., 2024). La Palma Island presents a continuous degassing of CO₂ in the south, in Fuencaliente ( Figure 1 ) (Carracedo et al., 2001; Padrón et al., 2015). The features of the natural CO₂ vent include a pH gradient that decreases from 8.1 at open sea (150m from the vent center) to less than 7.4 at the source during low tide (González-Delgado et al., 2021), allowing us to test the effect of decreasing pH on marine organisms in a situation analogous to future oceans.

This study investigates how the metabolic rates vary in two calcifying echinoderms, the sea urchins P. lividus and A. lixula, under different pH conditions during experiments in a laboratory and along a naturally acidified area. The main goal is to better understand their physiological responses to OA conditions predicted for the near future and whether those responses are a result of large phenotypic plasticity or local adaptation to long-term exposure to low pH. Specifically, we measured metabolic responses by analyzing respiration rates (VO₂ consumption) of organisms under experimental pH changes to assess their innate plasticity to pH shifts. Additionally, we compared organisms from natural CO₂ vent systems (pH ~7.5) and ambient sites (pH ~8.1) to identify potential adaptive differences between them. We expected organisms from more stable environments to be more affected by environmental changes, while those accustomed to greater environmental variability may respond more effectively, having adapted to wider fluctuations in abiotic factors (Asnicar et al., 2021). It is critical to have a clear understanding of when plastic responses occur instead of genetic adaptations and vice versa, as this knowledge is essential for predicting how OA affects the distribution and abundance of species and thus predicting the responses of marine organisms to ongoing climate change.

Our study is divided into two different parts. For investigating the plasticity response over time experiments were performed with individuals collected from ambient sites at pH 8.1 to measure their plasticity responses to low pH conditions over seven–days. Adaptation and plasticity under changing pH was analyzed using individuals living along a pH gradient from a natural CO₂ vent system. Each part of the study was performed in a different laboratory and locality due to infrastructure and field requirements.

Echinoderms, as marine calcifiers, are among the most sensitive organisms to future OA conditions (Byrne and Hernández, 2020; Byrne and Fitzer, 2019). Studies that combine laboratory experiments and OA natural analogues are useful for assessing the stress response and long-term effects of OA (Hernández et al., 2024). In the present study, we combined the study of short, medium, and long-term metabolic responses to OA conditions in two keystone species of sea urchins.

The ability of sea urchins such as P. lividus and A. lixula to cope with pH fluctuations stems from their coelomic fluid’s buffering capacity (Calosi et al., 2013; Di Giglio et al., 2020). Moreover, other physiological processes such as osmoregulation, and excretion, are also interrelated for maintaining metabolic activities (Catarino et al., 2012). After seven days of experiments at acidified conditions (pH=7.5), respiration rates of both species reached similar VO₂ levels to Day 0 (pH=8.1) before the low pH exposure, indicating a potential recovery of the basal respiration rates. However, A. lixula showed significant differences when compared to the respiration rates found in the controls, possibly related to the sampling season. The specimens for the control conditions were collected at the end of December of 2023 while the specimens used for the experimental conditions were collected at the beginning of May of 2024. Although similar seawater temperature (16°C ± 1°C), other internal physiological (such as reproductive season) and external ecological (such as food supply) processes might be influencing the respiration rate of collected specimens (Burton et al., 2011).

During the seven days of low pH exposure, VO₂ consumption alternate between increases and decreases in both species, following opposite patterns. In P. lividus, elevated respiration rates may be explained through metabolic upregulation to compensate for tissue acidosis (Stumpp et al., 2012). This finding aligns with Calosi et al. (2013), who reported that P. lividus adjusts to low pH by accumulating bicarbonates, a buffering mechanism likely requiring increased metabolic activity. In contrast, A. lixula exhibited an initial decrease in VO₂ consumption, likely due to its non-bicarbonate buffering mechanisms requiring less metabolic adjustment (Calosi et al., 2013). These distinct physiological responses underline the species-specific strategies for coping with low pH.

Both species show potential for adaptation and/or long-term acclimatization in physiological responses since the basal respiration rates showed no differences between populations from the ambient sites (pH=8.1) and the CO₂ vent sites (pH=7.5 and 7.8) at La Palma. Migliaccio et al. (2019) also found a lack of differences in P. lividus respiration rates between specimens from a CO₂ vent and ambient sites, suggesting physiological mechanisms to survive under OA. Acclimatization to OA over time is not rare in sea urchins since several studies have already found no differences in respiration rates between pH treatments in the long-term, for instance, P. lividus (Catarino et al., 2012; Cohen-Rengifo et al., 2019), Echinometra sp (Asnicar et al., 2021; Moulin et al., 2014; Uthicke et al., 2020, 2021), Hemicentrotus pulcherrimus (Kurihara et al., 2013), Strongylocentrotus droebachiensis (Stumpp et al., 2012), and Sterechinus neumayeri (Suckling et al., 2015). However, studies such as Holtmann (2013) and Marčeta et al. (2020), indicate that maintaining coelomic fluid pH may involve metabolic trade-offs impacting growth, reproduction and immune functions. In this study, the sex of the individuals was not recorded to avoid sacrificing them. Marčeta et al. (2020) found that females exposed to acidified conditions showed reduced gonad quality and impaired oocyte development. These findings suggests that sex-specific differences could exist in the responses of P. lividus and A. lixula to OA, with females potentially being more sensitive due to the energetic demands of reproduction. While maintaining acid-base homeostasis is energetically demanding and can reduce the energy available for other essential processes (Catarino et al., 2012; Collard et al., 2013) it may not fully explain the shifts in energy budgets observed in OA environments. Indeed, other cofounding factors could contribute to a shift in energy budgets and VO₂ consumption rates, such as biomineralization (Kurihara et al., 2012). These trade-offs further highlight the metabolic challenges associated with ocean acidification (Dupont and Thorndyke, 2012; Stumpp et al., 2012; Hernroth et al., 2011).

To assess the phenotypic plasticity, individuals from ambient sites in La Palma of both species were placed under short-term exposure to low pH conditions (pH=7.5). VO₂ consumption increased significantly, especially in A. lixula, indicating a stress response to the sudden change. Interestingly, P. lividus showed a higher increase in VO₂ consumption when ambient individuals were exposed to low pH conditions (A-V) compared to vent specimens exposed to ambient pH conditions (V-A). This might be related to the natural exposure to fluctuating pH of individuals living at vent sites, ranging from pH=8.1 to pH=7.4 during tides (González-Delgado et al., 2021) while organisms from ambient sites live under constant pH values (pH=8.1). Differences in food consumption at varying pH levels may have influenced the observed VO₂ patterns, as sea urchins might adjust feeding to cope with acidification stress, aligning with other studies (e.g., Dupont et al., 2010). However, food consumption was not directly measured in this study. Populations exposed to high environmental variability may develop tolerance to environmental stressors (Asnicar et al., 2021; Teixidó et al., 2020) as well as maternal anticipatory effects (Palombo et al., 2023; Karelitz et al., 2019; Thor and Dupont, 2015). Studies have shown that echinoderms exhibit reduced sensitivity to OA when exposed to a highly variable environmental history (Asnicar et al., 2022; Kapsenberg and Cyronak, 2019) indicating higher phenotypic plasticity in specimens from more fluctuating environments, such as the CO₂ vent sites studied here. In those lines, high pH variability areas have been proposed as OA refugia, offering adaptive advantages for marine organisms (Bednaršek et al., 2022; García et al., 2018; Kapsenberg and Cyronak, 2019). Thus, CO₂ vent systems in particular, may serve as significant OA refugia to consider.

While P. lividus and A. lixula may acclimate their respiration rates to low pH over time, understanding their long-term physiological responses is key to predicting viability and ecosystem dynamics. This study suggests that adult sea urchins of both species can potentially acclimate to low pH conditions over the long-term, supporting previous findings (Calosi et al., 2013; Collard et al., 2013; Di Giglio et al., 2020; and Migliaccio et al., 2019). Incorporating sex identification and measuring feeding rates could reveal sex-specific differences and clarify dietary roles in metabolic homeostasis. Future research combining physiological data with population genomics and transcriptomics could uncover adaptive mechanisms and their inheritance.