The continuous increase in anthropogenic carbon dioxide (CO2) emissions into the atmosphere is one of the main factors contributing to ocean acidification (OA). In fact, CO2 is partially absorbed by the oceans, where it alters carbonate chemistry and seawater pH, which is expected to decrease from the current level of 8.1 to 7.7 by 2100. OA exerts harmful impacts primarily on calcifying organisms, as it affects the availability of carbonates, which makes their calcareous structures thinner and more fragile. Moreover, several studies have described the detrimental effects of OA across many marine taxa, affecting important physiological and metabolic mechanisms. On the other hand, research conducted at CO2 vent systems, which are volcanic naturally acidified sites, showed that several organisms can survive under acidified conditions through specific tolerance and/or adaptive strategies. Among these organisms, the gastropod Patella spp. is one of the few calcifiers capable of inhabiting naturally acidified sites, such as the Castello Aragonese vent systems at Ischia Island and the San Giorgio vent systems at Sicily Island. Nonetheless, the complex mechanisms that allow survival and potential adaptation of these organisms to natural OA conditions need to be understood. Therefore, this PhD study aimed at investigating the potential molecular, physiological, metabolic, genetic, and epigenetic mechanisms that enable these organisms to tolerate and survive under OA conditions through a stepwise approach. Specifically, this PhD research attempted to answer the following questions: • Does OA entail a stressful condition in resident populations of Patella spp. living at reduced pH conditions, thereby affecting their overall well-being and health status? • Are there specific physiological, metabolic, and biochemical mechanisms that contribute in defining tolerance to OA? • Are limpets genetically adapted to OA? • Is DNA methylation contributing to promote tolerance to OA in limpets? • What is the role of environmental conditions in shaping the response to OA? The first chapter of this thesis considered three Patella species (P. caerulea, P. rustica, and P. ulyssiponensis) collected from the CO2 vent systems of the Castello Aragonese (Ischia Island). This vent system exhibits a natural acidification gradient ranging from ambient pH (N1: pH = 8.1), to intermediate pH (N2: pH = 7.7), and to extremely low pH (N3: pH < 7.4). Resident populations were collected along the gradient and at San Pietro, an additional ambient pH site (pH = 8.1), located at a distance of 4 km from the Castello vent. In addition, a 30-day in situ transplant experiment was conducted using P. caerulea, in order to evaluate the short-term responses induced by OA. Morphometric traits, such as shell length, height, width, and soft-tissue weight, were measured, and a set of biomarkers related to antioxidant system, energy metabolism, neurotoxicity, and biomineralization was applied. For resident populations, P. caerulea showed increased size and energy reserves at the extremely acidified site, likely related to a shift from erect calcified algae to biofilm, or to reduced competition and/or predatory pressure under acidified conditions. Biochemical endpoints measured in both P. caerulea and P. ulyssiponensis were not modified by OA. Conversely, P. rustica exhibited significant modulation of nearly all biochemical parameters, likely due to its different position on the rocky shore, which makes this species more exposed to tidal fluctuations and therefore to an additional source of disturbance, besides OA. Short-term exposure of P. caerulea to OA resulted in a decrease in protein content and an increase in glycogen content at the extremely acidified site, with the induction of superoxide dismutase and glutathione-S-transferase activities at intermediate pH, suggesting the activation of compensatory mechanisms to cope with reduced pH conditions. Overall, results revealed a distinct response to OA of the three species of Patella. Moreover, the increased size and energy-related endpoints observed in P. caerulea and P. rustica highlighted the need to further investigate energy metabolism aspects, in order to better understand the trade-offs between compensatory mechanisms and the energetic cost underlying tolerance to OA. Based on these evidences, the second chapter focused exclusively on P. caerulea, with the aim of deeply investigating metabolic and physiological stress-responses, comparing resident populations of the Castello Aragonese vent systems and transplanted organisms, similarly to the first chapter. Respiration and ammonia excretion rates were measured four times across the year. Additionally, untargeted metabolomics analyses were performed to investigate metabolic pathways potentially involved in response to OA. Only during summer, OA increased respiration rate in limpets from the most acidified site, while simultaneously reduced excretion rates, likely to allocate more energy resources to face the increasing temperature, besides OA. Furthermore, both resident and transplanted populations up-regulated carnitine metabolism, suggesting that OA induced an increase of energy production through β-oxidation and subsequent Krebs cycles. Moreover, several metabolites involved in osmoregulation, oxidative stress, and nucleic acid mechanisms were increased. Overall, results seem to confirm the presence of negative effects and of an energetic cost underpinning tolerance to OA. The third and final chapter investigated the potential influence of phenotypic plasticity, genetic adaptation, and DNA methylation in tolerance to OA in adult and juvenile populations of P. caerulea collected from two CO2 vent systems of the Mediterranean Sea. Adult and juvenile specimens were sampled along the acidification gradient of the Castello Aragonese vent systems of Ischia Island (San Pietro/N1: pH = 8.1; N2: pH = 7.7; N3: pH < 7.4) and from the San Giorgio vent systems of Sicily Island (Patti: pH = 8.1; San Giorgio: pH = 7.8). Following genomic DNA extractions from foot tissue and individual libraries preparation with the NEB Next® Enzymatic Methyl-seq Kit, samples were sequenced on the Illumina NovaSeq 6000 sequencer. Data processing and analyses were conducted on Euler platform mainly using biscuit tool, which enabled to simultaneously extract genomic and epigenomic information from DNA methylation sequencing. Population genomics and epigenomics analyses revealed divergent patterns between the Ischia and Sicily populations. Populations from the Ischia vent revealed marked signs of genomic differentiation, particularly in adults from the intermediate and extremely low pH sites, while reduced differences in DNA methylation levels were detected, especially in adults. These findings suggest a strong signature of purifying selection acting on standing genetic variation, through a within-generation response, likely driven by the more pronounced pH fluctuations occurring at these sites. Conversely, no genomic differentiation was observed between the Sicily populations, but greater differences in DNA methylation were detected between acidified and non-acidified sites at both adult and juvenile stages. These results revealed that this epigenetic mechanism, rather than genomic changes, may play a key role in the response to the milder pH variations of this vent and potentially enhance organisms’ tolerance to OA. In conclusion, this PhD project investigated tolerance to OA in limpets through a holistic approach that, for the first time, integrated morphological, physiological, metabolic, biochemical, genetic, and epigenetic analyses. Overall, findings revealed that Patella spp. has the ability to survive under acidified conditions even though with a physiological and metabolic cost, which could be partially compensated by more favorable environmental conditions. This study further highlights the importance of conducting research in naturally acidified environments, since it allows to formulate more realistic hypotheses about the ability of marine organisms to persist in future changing oceans.
Signorini S. G., 2026. Investigation of the adaptive mechanisms to ocean acidification in Patella species from CO2 vent systems of the Mediterranean Sea. PhD thesis, University of Milan. Thesis (embargoed).
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