External fertilization is a common mode of reproduction in marine organisms. However, spawning in the water column poses a risk to eggs and sperm, which are directly exposed to environmental variability and anthropogenic stressors. The increase of atmospheric carbon dioxide (CO₂) and other greenhouse gases is influencing the climate of the planet (IPCC, 2019), as well as the marine environment (Sabine et al., 2004; Doney et al., 2009). Ocean acidification (OA) linked to a climate change scenario is changing the seawater chemistry (Caldeira and Wickett, 2003), increasing the concentration of hydrogen ions in the seawater, disrupting the metabolism and intra-/extra-cellular pH compensation of organisms, in particular at early life stages, which are the most vulnerable (Foo and Byrne, 2017).

Among marine organisms, calcifying invertebrates, such as sea urchins, are the most sensitive to OA scenario and a shift in their presence can alter profoundly the community (Hall-Spencer et al., 2008; Fabricius et al., 2011; Kroeker et al., 2011, 2013).

Gametes have limited ability to regulate intracellular pH when facing changes in pH of seawater (Ciapa and Philippe, 2013; Bögner et al., 2014; Nishigaki et al., 2014). Indeed, after spawning, eggs of external fertilizers absorb the water they are in contact with to increase jelly coat size (Podolsky, 2001), thus being directly influenced by changes in seawater physico-chemical features. The egg size and energy content (lipids and fatty acids in particular) are two important factors related to fitness, offspring development and survival (Tocher, 2003; Byrne et al., 2008; Foo et al., 2018), with the quality of eggs probably being a better predictor of offspring quality rather than their size (Byrne et al., 2008). Egg size and composition are the most studied parameters when assessing the effects of environmental changes (Moran and McAlister, 2009; Foo and Byrne, 2017). Parental environmental history and diet are also fundamental factors to determine survival chances for the following generation as they influence egg nutrient content, in particular energetic lipids (López et al., 1998; Lamare and Barker, 1999; Liu et al., 2007; Clark et al., 2009; Braun et al., 2013; Feiner et al., 2016; Gago et al., 2016; Gosselin et al., 2019; Hoshijima and Hofmann, 2019). OA effects on egg quality are still unclear, with neutral (Uthicke et al., 2013; Parker et al., 2017; Karelitz et al., 2020) or negative (Bögner et al., 2014; Suckling et al., 2015) responses. It seems, however, that with prolonged exposure, animals have the potential to acclimate to the experimental condition (Suckling et al., 2015; Asnicar and Marin, 2022). As egg quality defines the success of the following generation, it is a crucial endpoint deserving investigation, especially in studies on the impact of OA where inter- and intra-specific variations in physiological parameters have been previously documented.

Sperm quality (e.g. morphology, velocity and viability) is a crucial determinant of fertilization success (Benzie and Dixon, 1994; Levitan, 2000; Au et al., 2002), and many studies underline the usefulness of these endpoints to assess effects of potential stressors on external fertilizers (Moschino and Marin, 2002; Fitzpatrick et al., 2008; Fabbrocini et al., 2010; Fitzpatrick et al., 2012; Suquet et al., 2012). A large majority of studies reports detrimental effects of OA on sperm quality, including impairment of the polyspermy block (Reuter et al., 2011; Bögner et al., 2014), as well as a reduction of sperm velocity, motility and viability (Havenhand et al., 2008; Schlegel et al., 2012; Campbell et al., 2016). Contrasting species-specific differences in OA impact have also shown both positive and absence of effect on sperm quality (Havenhand and Schlegel, 2009; Caldwell et al., 2011; Marčeta et al., 2022). Under stressful conditions sperm quality can be also highly variable among individuals, with both improving and impairing intra-specific effects of OA (Gallo et al., 2020b).

Most of the studies aimed at predicting the impact of OA on gametes did not consider the exposure of parental generations to OA. However, when adults were exposed to OA during gametogenesis, a generally positive effect on gamete quality and on offspring emerged (Thor and Dupont, 2015; Uthicke et al., 2020). Considering these experimental limitations and mixed results due to intra-specific variability, more in-depth investigation is certainly needed (Gallo et al., 2020a; Parker et al., 2017).

This study aims to experimentally investigate the impact of OA (pH reduction of 0.4 units) on gamete quality in sea urchins, Paracentrotus lividus. To test for intra-specific variability in relation to different ecological histories, adult specimens from two populations (lagoon vs. coastal environment) experienced both mid-term (two months) and a long-term (six months) exposure. Specifically, the effect of reduced pH on the size and biochemical composition of the eggs (carbohydrates, proteins, lipids, and fatty acids), and on the viability and velocity of sperm are evaluated.

In this study, we investigated potential variations in gamete quality of sea urchins P. lividus with different ecological history after long-term exposure to future OA scenario. Parental exposure pH showed little influence on gamete quality, whereas a greater difference emerged in egg quality at different time-points (i.e. eggs spawned after two and six months of parents’ exposure) and in the specific responses of each site of origin (i.e. lagoon and coastal area).

Except for the fatty acid profile (discussed below), the only significant difference due to pH of exposure was found in the diameter of eggs from Site 1 females. Eggs of specimens maintained at lower pH, indeed, were smaller than those from females at natural pH. These findings are different from those obtained in other sea urchin species where egg size was not affected by exposure to lower pH conditions: Echinometra mathaei maintained for seven weeks at pH 7.5 (Uthicke et al., 2013); Strongylocentrotus purpuratus kept at upwelling conditions (-0.3 pH units and -4°C compared to the control) for four months (Hoshijima and Hofmann, 2019); Sterechinus neumayeri specimens, kept for six and seventeen months at pH 7.7 (Suckling et al., 2015). However, in this latter study an effect emerged after seventeen months at pH 7.5, with larger eggs compared to the control (Suckling et al., 2015). In addition to the variability in response to lower pH across sea urchin species, an intraspecific variability in egg size is also observed in natural populations (George et al., 1990; Moran and McAlister, 2009).

In the present study, the number of eggs spawned in 30 minutes was very different between the time-points considered, with the eggs collected after six months being up to four times more abundant than those collected after two months. This is reasonably due to the accumulation of eggs during the 6 months of rearing in captivity during which females continued to produce eggs but did not have the chance to spawn. The pH of exposure, however, did not influence egg production. This is in line with a previous study on Hemicentrotus pulcherrimus exposed for nine months to pH 7.8 (Kurihara et al., 2013).

The eggs from Site 1 females kept at low pH were slightly lighter and with slightly lower carbohydrate content, although this difference was not statistically significant.

The total energetic content did not show a clear influence of the exposure pH, suggesting that embryo development started at the same energetic conditions for all eggs. Also eggs from Site 2 at reduced pH were slightly less abundant, lighter, with lower content of all three biochemical components and with a lower total energy content (although differences between treatments were not statistically significant). However, it is noteworthy that differences in the egg biochemical composition, based on different pH of maternal exposure, decreased with the prolongation of the exposure, suggesting acclimation and better starting condition for the offspring growth.

It has been hypothesized that producing lighter eggs might represent an advantage since lighter eggs can remain longer in the water column, reaching greater distances, and colonizing new areas (Arai et al., 1993; Harii et al., 2007). However, the lower content of energetic components associated to lighter eggs might force the embryo to enter the exotrophic stage faster, possibly resulting in smaller larvae. As a consequence, embryos could be exposed to a higher risk of predation, a reduced possibility to find food and to reach the right substrate for the settlement on time, since all these aspects are related to larval size (Marshall and Keough, 2003; Allen, 2008). Eggs and sperm analyzed in the present study were used for in vitro fertilization trials in a parallel study regarding OA effects on larvae (Asnicar et al., 2022). Larvae obtained from eggs with a lower energetic content were significantly smaller and reached the exotrophic stage (four-armed echinopluteus) faster. Moreover, differences in the larval size also decreased from two to six months of parental exposure (Asnicar et al., 2022). In Heliocidaris erythrogramma, smaller eggs with lower lipid content produce larvae that need more time to develop and metamorphose, have less chance to reach the juvenile stage and result smaller in size (Emlet and Hoegh-Guldberg, 1997). These and our findings emphasize the key role of gamete quality as a good predictor of offspring performance. To the best of our knowledge, the present study investigates for the first time the effect of ocean acidification on Paracentrotus lividus eggs’ biochemical composition and energetic content while standardizing the exposure conditions (seawater parameters, gonadal development).

Although we did not find a significant effect of the pH of exposure on the egg quality, a higher influence of pH in Site 2 specimens than in specimens from Site 1 was highlighted. This can be possibly related to the different ecological features of the two Sites. Site 1, in the lagoon of Venice, shows a highly variable and inhabited environment subject to relevant levels of anthropogenic stress compared to Site 2. Our results suggest that specimens from the lagoon have higher tolerance to OA, possibly reflecting better adaptation to changing environmental conditions.

In both sites, regardless of the pH, females produced fewer but more energy-rich eggs, after two months of exposure and, at the opposite, more eggs but with a lower energy content after six months of exposure. Therefore, maternal plasticity in reproductive strategy can be hypothesized as a tool to optimize fitness gain over time. Indeed, egg energy content is favored over number after two months (when the first eggs are spawned), whereas significantly more eggs are produced after six months, increasing the likelihood that a good number of offspring would survive on the long term. Further investigations are necessary to support this hypothesis.

Our results also confirm that, as suggested by previous studies (Grosjean et al., 1998; Spirlet et al., 2000; Shpigel et al., 2004; Sartori et al., 2016), the maintenance at 18-19°C, 14:10 L:D hours, with a diet based on spinach and corn is optimal to promote sea urchin gametogenesis and produce mature gametes, even after two months of exposure. In fact, in vitro fertilization trials that were carried out with eggs and sperm of the present work (Asnicar et al., 2022) led to a fertilization success always exceeding 90%.

Of course the diet can also influence the lipid metabolism and storage (e.g. Schiopu et al., 2006; Trocino et al., 2012) and in our study the diet was different from a natural one. P. lividus lives in a natural environment, where several food sources, including brown, red, green algae, and seagrass, are available (Boudouresque and Verlaque, 2020). Our unvariegated spinach-corn diet, necessary for the standardization of the procedure, could have influenced the eggs’ lipid profile and could contribute to explain the difference observed in the FA profile between 2 and 6 months of exposure.

However, despite this difference in eggs’ FA composition across the time of exposure, we interestingly observed an effect of the pH of exposure only in animals from Site 1, whereas the effect is very limited in animals from Site 2. The fatty acid profile importance in diet is very well studied, with much evidence that higher MUFA and especially PUFA levels, compared to SAFA, in food intake are beneficial for the health of animals and humans (Dewhurst et al., 2003; Ramsden et al., 2015). It is known that environmental changes and pollution may alter the FA composition of organisms and gametes (Filimonova et al., 2016). Changes in the fatty acid composition of the eggs may be linked to adjustments that the adults make to favor the appropriate growth of embryos or give them extra protection. For example, non-methylene-interrupted (NMI) fatty acids seem to have a protective function from lipid peroxidation processes, although the mechanism of action is not fully understood (Garrido and Medina, 2002). The lack of long chain unsaturated fatty acids, such as linolenic [C18:3(n-3)] and docosahexanoic acid [DHA C22:6(n-3)], may have deleterious effects, as reported for larvae of the sand dollar Dendraster excentricus (Schiopu et al., 2006). Compared to Site 2, in Site 1 eggs we detected a higher content of PUFAs (heneicosapentaenoic acid (HPA), docosahexaenoic acid (DHA), linolenic acid) that play a key role in the health and functioning of animals. After two months of exposure, DHA made up 0.380% of the total FA in the eggs of control animals, while it was three times higher in those from animals kept at reduced pH (1.117%). This difference disappeared after 6 months. Linolein (C18:2) and linolenic (C18:3) acids are precursors of the eicosapentaenoic acid (EPA; C20:5_n3) which also has an important role in animal health (Cohen et al., 1993). In the present study, at two months, linolein acid % was reduced at low pH, while the linolenic acid % was increased. Overall, the EPA % did not change demonstrating compensatory mechanism in the animals.

The larvae from Site 1 urchins, obtained in a further study with these gametes, performed better than those from Site 2 (Asnicar et al., 2022). This suggest that the changes detected at reduced pH may be compensatory changes, with females from Site 1, already accustomed to a more variable environment, providing more nutritious content in the eggs for the future generation. The egg fatty acid profile represents, therefore, a powerful tool in predicting the performance of the following generations in response to environmental stressors.

In the present study, seawater pH affected only slightly the P. lividus sperm endpoints considered. The only significant differences in the sperm viability and velocity were driven by the activation pH, rather than the exposure pH, and they were found in sperm from males maintained at natural pH. For both Site 1 and Site 2, sperm from sea urchins maintained at low pH and activated at the same low pH, showed a similar or even enhanced response if compared to the corresponding one activated at natural pH and also to sperm from adults maintained at natural pH and activated at natural pH. Our results suggest that exposure to reduced pH for six months does not affect the ability of P. lividus to produce gametes of good quality, potentially guaranteeing a successful fertilization irrespective of the pH of parental maintenance.

Previous studies on the effects of OA exposure on sea urchin sperm viability and velocity gave mixed results, possibly due to differences in ecological history of analyzed population or to different methodologies applied. A pH reduction of 0.2-0.3 units enhanced velocity and motility in Psammechinus miliaris sperm (Caldwell et al., 2011); exposure to pH 7.7 significantly reduced velocity and motility in sperm of H. erythrogramma (Havenhand et al., 2008), but in another study on the same species only sperm motility, and not velocity, was affected by reduced seawater pH values (7.8 and 7.6) (Schlegel et al., 2012). Previous results in P. lividus showed that the exposure of sperm to pH reduced of 0.4 units negatively affected the velocity in the first five minutes post-activation (Munari et al., 2022). Although after 15 and 30 minutes from activation treated sperm reached the same performance of controls, the first 5 minutes gap was enough to reduce the fertilization success of treated individuals (Munari et al., 2022). These studies however, did not include exposure of the parents to OA conditions before their gamete analyses. In the only previous study in which P. lividus adults were maintained at pH conditions of 8.0, 7.7, and 7.4 for two months, sperm produced by treated animals did not differ in motility and oxygen consumption from the control (Marčeta et al., 2022). Sperm velocity, instead, was affected by parental pH: sperm from males reared at pH 7.7 show a lower average initial velocity with no changes over time (0-60 minutes). Whereas sperm ATP content was significantly higher in males reared at low pH (both 7.7 and 7.4), ATP consumption was affected by both parental pH and activation pH (Marčeta et al., 2022).

It has been suggested that sperm speed and longevity are negatively correlated in broadcast spawners (Levitan, 2000; Burness et al., 2004) and that sperm longevity, more than velocity, influences the ejaculate fertilization ability (Fitzpatrick et al., 2012). Our data suggests higher sperm viability in Site 1 males, that might be related to a higher reproductive fitness for sea urchins from the site with higher environmental variability and anthropogenic stress, although this deserves further specific analysis to be confirmed.

Overall, our results suggest a negligible effect of seawater acidification on the quality of P. lividus gametes spawned after long-term parental exposure. In particular, OA levels expected for the next century did not cause severe alterations in the quality of eggs and sperm, especially with the prolongation of the adult exposure (six months vs. two months). This limited effect could be related to a general great tolerance to environmental variations in P. lividus sea urchins. In fact, this species has a very wide distribution range, even latitudinally, suggesting a very high adaptation potential. However, our results suggest a different response toward OA in the two sites of origin, in particular in relation to the eggs’ FA composition. An intraspecific variability related to the ecological history of the animals is suggested, with specimens from the coastal area more strongly suffering the effect of OA compared to animals from the lagoon area, and, then, possibly requiring longer time to acclimate to stressful conditions. Our observations concur with recent evidence of robustness of P. lividus to end-of-century acidification conditions. However, here we provide further evidence that local populations subjected to higher levels of environmental stress may play an important role in promoting adaptation and resilience to future reduction in seawater pH. Indeed, considering that the effects of time and exposure pH are more pronounced in animals from Site 1 (lagoon), coupled with the fact that their offspring were of higher quality compared to Site 2 (coastal area), the detected variations might be linked to higher adaptability of the lagoon population rather than to negative effects of ocean acidification. Moreover, our results highlight the importance of integrating the analyses of parameters of gamete quality, in particular eggs’ biochemical and fatty acid composition, to fully understand the transgenerational adaptation ability of sea urchins to ocean acidification.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The manuscript presents research on animals that do not require ethical approval for their study.

DA: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing – original draft, Writing – review & editing. LL: Conceptualization, Formal analysis, Methodology, Supervision, Validation, Writing – review & editing. LZ: Formal analysis, Investigation, Writing – original draft. RM: Formal analysis, Investigation, Writing – original draft. LM: Formal analysis, Investigation, Writing – original draft. MM: Conceptualization, Formal analysis, Investigation, Supervision, Writing – review & editing. MGM: Conceptualization, Project administration, Resources, Supervision, Writing – review & editing.