The experiment was carried out for 66 days in Potter Cove (PC) (62^°14′S., 58^°40′W; King George/25 de Mayo Island - South Shetlands - Antarctica) during the summer campaign of 2015-2016 (from December to March) (Figure 1). N. eatoni was collected by scuba diving at approximately 15 m depth, and they were immediately placed in seawater containers and transported to the experimental aquarium at the Argentinian research station Carlini. Prior to altering the pCO₂, the snails were placed for seven days in an individual acclimation tank [with a continuous flow of seawater, maintained at in situ natural conditions (approximately 8.03)].

We implemented an experimental CO₂-manipulation system following the same experimental design utilized in previous studies on zoo-benthic Antarctic species exposed to OA (de Aranzamendi et al., 2021; Servetto et al., 2021, 2023, 2025) (Figure 2). Seawater was continuously supplied from the field to two main header tanks (150 L each), designated as the acidified treatment (hereafter referred to as low pH, LpH) and the control. pH levels were continuously monitored using glass electrodes (LL Ecotrodeplus, Metrohm) connected to a pH controller (Consort R3610, Turnhout, Belgium). The LpH tank was gradually acidified by bubbling CO₂ gas (99.9% purity) until reaching a target pH of 7.68 ± 0.17, representing ~1000 µatm pCO₂, a reduction of approximately 0.3–0.4 pH units relative to the control. The control tank was maintained to reflect the natural pH variability recorded at the snails’ collection depth (15 m). It continuously received unaltered seawater directly supplied from the PC. Individual snails (N = 6; two individuals per tank) were placed in separate subsidiary smaller aquaria (~6 L), each connected to a continuous flow of seawater supplied from either the LpH or control head tank (Figure 2). The experimental sample size (5–6 cm) was carefully selected to minimize potential impacts on the individuals. As these snails are Antarctic organisms and little is known about their population in PC (Sahade et al., 2015; personal observation), a precautionary approach was taken by limiting the number of specimens collected. To minimize evaporation and reduce gas exchange with the air, each tank was covered with a methacrylate lid. Electrodes were calibrated daily using Tris buffers of known pH values, following standard procedures (SOP6a of Dickson et al., 2007). Weekly, 50 mL water samples were collected from the head tanks and fixed with HgCl₂ for subsequent total alkalinity (TA) analysis. TA was quantified via Gran titration (Smith and Kinsey, 1978) employing a sample exchanger coupled to a TitroLine alpha plus titration system (SI Analytics, Mainz, Germany) equipped with an A157–1 M-DIN-ID pH electrode, and conducted in accordance with standard operating procedure SOP 3a (Dickson et al., 2007). Carbonate chemistry parameters were calculated with the CO2SYS spreadsheet (Pierrot et al., 2006), using dissociation constants for carbonate determined by Mehrbach et al. (1973) and refitted by Dickson and Millero (1987). A summary of the physicochemical parameters of seawater is provided in Table 1. Temperature and salinity were not experimentally manipulated; instead, natural conditions from the cove were maintained throughout the study. Nevertheless, both parameters were continuously monitored during the experimental period to account for natural oscillation and support data interpretation (Table 1).

After two months of incubation under experimental conditions, the animals were dissected. Different tissues were selected for the FA analysis based on their functional and metabolic roles. Samples of mantle, gill, gonads, and foot were separated, stored at –80°C, and subsequently transported to Ghent University (Ghent, Belgium) for FA analysis. Results are expressed as both percentages and in μg·mg⁻¹ dry weight (DW).

FA methyl esters (FAME) were prepared via a direct transesterification procedure with 2.5% (v:v) sulfuric acid in methanol as described by De Troch et al. (2012) to achieve total FA analysis. An internal standard (FA 19:0 5 µg) was added to the freeze-dried tissue samples (~ 10 mg). FAME was extracted twice with hexane. FA composition was carried out using a gas chromatograph (GC) (HP 7890B. Agilent Technologies, Diegem. Belgium) equipped with a flame ionization detector (FID) and connected to an Agilent 5977A Mass Selective (MS) Detector (Agilent Technologies). The GC was equipped with a PTV injector (CIS-4. Gerstel. Mülheim an der Ruhr. Germany). A 60 m×0.25 mm×0.20 μm film thickness HP88 fused silica capillary column (Agilent Technologies) was used for the GC analysis at a constant Helium flow rate (2 mL min^-1). The injection sample volume was 2 μL, and the oven temperature program was set as Boyen et al. (2020) described. The signal obtained with the FID detector was used to generate quantitative data on all compounds (MassHunter Quantitative Analysis Software, Agilent Technologies). Chromatogram peaks were identified based on their retention times. Quantification was done through the external standards (Supelco 37 Component FAME Mix, Sigma-Aldrich). Mean FA chain length (MCL) was calculated using the equation from Guerzoni et al. (2001):

where FAP is the percentage of fatty acid; C number of carbon atoms.

QC/QA procedures included repeated measurements of water chemistry parameters using calibrated instruments, verification of FA identification against reference standards, and cross-validation of statistical outputs. Residuals and diagnostic plots were systematically examined to ensure model assumptions were met, and all analyses were independently reproduced to confirm consistency.

To verify the consistency of experimental conditions, a t-test was performed on the measured water parameters (pH, CaCO₃, total alkalinity (TA), and temperature) to detect any significant differences between control and LpH treatments. FA profiles of different tissues were analyzed using Principal Component Analysis (PCA), while Non-Metric Multidimensional Scaling (nMDS) was conducted separately for each tissue type. The homogeneity of variances was assessed using Bartlett’s test, and homoscedasticity and normality were further evaluated through residual analysis. Differences in FA composition among treatments were tested using an ANOVA (with tanks nested within treatments to account for potential tank effects). Additionally, a t-test was performed to compare the LpH treatment and control groups within each tissue after 66 days of the experimental condition. All statistical analyses were conducted using R version 3.6 for Microsoft Windows (R Core Team, 2020), with significance set at p < 0.05.

The experimental setup successfully verified the targeted pH conditions (7.70 ± 0.09) despite variability in the incoming natural seawater throughout the experiment. The treatment was consistently maintained at a lower pH (7.70) compared to the control (8.00) (t-test, T = 4.94, p < 0.01) (Table 1). Additionally, the average bicarbonate ion concentration (HCO₃⁻) and aragonite saturation state (Ω_Ar) differed significantly between the two conditions over the 66-day exposure period (T = -7.95, p < 0.01 and T = 2.60, p = 0.0266, respectively). In contrast (Table 1). Similarly, pCO₂ (µatm) was significantly higher in the LpH treatment compared to ambient conditions (mean ± SD: 975.74 ± 61.25 vs. 473.43 ± 40.24; T = -9.45, p < 0.0001), and aqueous CO₂ concentration ([CO₂]aq, µmol kg⁻¹) also increased significantly under elevated pCO₂ (62.26 ± 7.49 vs. 30.06 ± 3.99; T = -9.65, p < 0.0001). TA did not vary significantly between treatments for either variable (T = -1.53, p > 0.1407).

A total of 20 FAs were identified in N. eatoni, grouped as saturated (SFA), monounsaturated (MUFA), and polyunsaturated (PUFA) fatty acids (Table 2). PUFA was the predominant group across all tissues (Supplementary Figure S1). FA composition varied among tissues and treatments, with the foot and mantle showing similar ranges, while the gonads exhibited the highest MUFA content (19.43–40.40%).

Multivariate analyses supported these tissue-specific patterns. Principal Component Analysis (PCA) revealed a clear separation along PC1, differentiating the gonad’s FA composition from that of gill, foot, and mantle tissues (Figure 3). Within this framework, differences between control and LpH conditions were most evident in the gills. Similarly, non-metric Multidimensional Scaling (nMDS) indicated a marked distinction between control and LpH treatments in gill and mantle tissues, while this separation was less pronounced in the other tissues (Figure 4).

Under LpH conditions, opposite patterns were observed in gill and mantle tissues. In the gills, total SFA, MUFA, PUFA, and LC-PUFA contents were significantly higher in the control group than in LpH-exposed snails (Figure 5; Supplementary Table S1). The PUFA/SFA and MCL ratios followed the same trend, whereas the 20:5n-3/20:4n-6 and n-3/n-6 ratios were elevated under LpH. EPA was the most abundant FA in the gills, although no significant difference was detected between treatments. Several other FAs, including 20:2n-6, ARA, DHA, 22:5n-3, and MUFAs such as 18:1n-7, 20:1n-11, and 20:1n-9, were significantly higher in control samples (Table 2).

Conversely, in the mantle, SFA, MUFA, PUFA, and LC-PUFA levels were significantly higher under LpH exposure. The PUFA/SFA ratio and MCL indicator also increased in LpH samples compared to controls (Figure 5; Supplementary Table S1). Most mantle FAs were significantly altered by acidification, with EPA, DHA, 22:5n-3, 22:6n-3, and 24:5n-6 showing particularly elevated levels under LpH conditions (Table 2; Supplementary Table S2).

FA composition varied considerably among tissues, with PUFAs predominating across all samples. The gonads displayed the highest MUFA content, likely reflecting their reproductive function and energy storage role. In contrast, the gills and mantle were more responsive to OA exposure. In gills, total SFA, MUFA, PUFA, and LC-PUFA levels were significantly reduced under LpH conditions, indicating a reorganization of FA composition in response to environmental stress. Notably, ARA (20:4 n-6) levels also decreased, which could reduce the production of eicosanoids, potentially compromising inflammatory signaling and immune plasticity (Supplementary Table S3). Conversely, in the mantle, these same FA classes were elevated under LpH. The increased LC-PUFA content in the mantles may represent an adaptive response to mitigate oxidative damage and maintain membrane integrity under environmental stress. LC-PUFAs, particularly DHA and EPA, play a crucial role in modulating oxidative stress, as they are involved in the regulation of antioxidant defense mechanisms and inflammatory responses (Monroig et al., 2013). While this mechanism has been previously documented in marine microorganisms (Okuyama et al., 2008), studies have also shown the involvement of LC-PUFAs in oxidative stress responses in marine invertebrates, such as mollusks and crustaceans (Monroig et al., 2013; Lv et al., 2021).

The observed tissue-specific variations in DHA levels between the mantle and gill tissues of N. eatoni under LpH conditions may be attributed to the distinct physiological roles and environmental exposures of these tissues. The mantle, primarily responsible for shell formation and protection, exhibited elevated DHA levels under LpH conditions, suggesting an adaptive mechanism to maintain membrane fluidity and integrity in response to environmental stressors. This lipid remodeling could enhance the mantle’s resilience to oxidative stress induced by ocean acidification. However, this strategy may involve a metabolic cost, diverting resources from other physiological processes such as growth or reproduction.

In contrast, the gills, which are directly exposed to the external environment and are the main site for respiration and ion regulation, contained higher DHA concentrations in the control samples. Under LpH conditions, the reduced DHA levels in the gills likely reflect increased lipid peroxidation caused by elevated reactive oxygen species (ROS) (Fadhlaoui and Lavoie, 2021; Lushchak, 2021). Gills are known to be particularly susceptible to environmental stressors, including changes in pH, which can lead to oxidative damage and lipid degradation (Harms et al., 2014; Zutshi et al., 2019). This susceptibility necessitates efficient antioxidant defenses to mitigate oxidative damage and maintain cellular function (Mathieu-Resuge et al., 2020). Moreover, the loss of PUFA and increased SFA content in gills could reduce membrane fluidity, potentially impairing critical physiological functions such as ion transport, respiration efficiency, and filtration. These findings underscore the importance of DHA in modulating tissue-specific responses to environmental stress, reflecting the organism’s strategic allocation of lipid resources to maintain functionality under varying conditions.

In mantle tissue, EPA levels were elevated in individuals exposed to LpH conditions. These FAs are well known for their anti-inflammatory properties and their ability to modulate membrane-associated proteins, such as ion channels and transporters (Banaszak et al., 2024). Additionally, they play a crucial role in defining key biophysical properties of biological membranes, including organization, ion permeability, elasticity, and the formation of microdomains (Bruno et al., 2007). A similar pattern was observed by Silva et al. (2017) in Gibbula umbilicalis exposed to metal contamination, further supporting the idea that DHA and EPA serve as reliable biomarkers of environmental stressors, in that particular case, metal pollutants.

Additionally, independent of the OA experiment, we observed differences in FA concentrations between the gonads and the other three analyzed tissues. These differences are primarily driven by the higher abundance of MUFAs in the gonads, where MUFAs represent the predominant FA class. The elevated MUFA suggests a critical role in reproductive processes, likely associated with energy storage and the maintenance of membrane fluidity during gametogenesis. In N. eatoni, this pattern may indicate a physiological adaptation to meet the energetic and structural requirements of gamete development. For example, Kapranova et al. (2019) reported that in the mussel Mytilus galloprovincialis, MUFA concentrations peak in male gonads at the onset of stage 2, whereas SFAs predominate in female gonads throughout stages 1 to 4. This pattern is consistent with our findings in N. eatoni, where the predominance of MUFAs in gonads suggests a comparable role of these fatty acids in supporting reproductive processes. These findings highlight the necessity for further research to elucidate the specific FA composition patterns associated with the reproductive cycle of N. eatoni and their potential ecological implications. Overall, the comparison underscores that differences in gonadal FA profiles across species can inform interpretations of reproductive status and resilience in N. eatoni under changing environmental conditions.

In addition to their structural and energetic roles, fatty acids contribute to immune regulation. In particular, the balance between n-3 and n-6 PUFAs influences the production of pro- and anti-inflammatory eicosanoids, as both groups compete for the same desaturation and elongation pathways (Monroig and Kabeya, 2018). In our study, the n-3/n-6 ratio remained largely unchanged in mantle, foot, and gonadal tissues, suggesting a stable immune–lipid balance under OA conditions. However, in gill tissues, this ratio was significantly elevated in the LpH group, indicative of a potential shift toward an anti-inflammatory lipid profile under acidified conditions.

Similar findings have been reported in Crassostrea gigas, where OA exposure increased n-3 PUFA content in gills, influencing immune performance and disease susceptibility (Wang et al., 2016). In N. eatoni, this tissue-specific shift may serve as a compensatory immune response to the heightened oxidative and ionic challenges posed by OA. However, whether this adjustment enhances or compromises immune competency remains to be further investigated.

Homeoviscous adaptation (HVA) refers to modifications in the chemical and mechanical properties of the lipid bilayer that help preserve membrane fluidity under changing stress conditions (Parrish, 2013). In this study, HVA biomarkers—specifically PUFA/SFA ratios and mean chain length (MCL)—revealed effects of elevated CO₂ exposure in both mantle and gill tissues. In the gills, a higher PUFA/SFA index under control conditions suggests a decrease in membrane fluidity in response to acidification, potentially indicating an adaptive mechanism for maintaining membrane function. This aligns with known functions of PUFAs enhancing membrane fluidity, and SFAs contributing to membrane stability (Hąc-Wydro and Wydro, 2007). Elevated seawater pCO₂ in treatment also influenced MCL in both tissues. The mantle exhibited higher MCL under LpH conditions, suggesting a reduced membrane fluidity as a compensatory response to acidification (Guerzoni et al., 2001). These findings are evidence that OA can modulate HVA pathways in polar invertebrates. In addition, under stress, HVA, through the regulation of lipid class ratios (e.g., ST/PL ratio), may be more energy-efficient than modifications in PUFA/SFA ratios and MCL. HVA pathways have been previously reported as adaptive responses to abiotic stressors in other marine organisms (Somero, 2022). For example, the impact of increasing seawater CO₂ and temperature on HVA has been studied in marine sponges (Bennett et al., 2018). They found that certain species used the additional carbon to enhance sterol biosynthesis, which likely reflects an HVA mechanism, providing a potential pathway where elevated CO₂ helps to mitigate thermal stress (Bennett et al., 2018).

While our results support the use of FA profiles as tissue-specific biomarkers of physiological stress in N. eatoni, a more mechanistic understanding of the biochemical underpinnings driving these shifts under pCO₂/pH variability is essential. FA composition in marine invertebrates is not static, but rather dynamically regulated through enzymatic pathways such as desaturation and elongation, mediated by desaturases and elongases whose activities are often sensitive to environmental stressors (Monroig et al., 2013; Bell et al., 2016). Changes in pH and CO₂ availability can alter cellular acid-base balance, indirectly affecting enzymatic efficiency and gene expression linked to lipid metabolism (Tocher, 2010). Moreover, the remodeling of membrane lipid composition, particularly increases in LC-PUFAs like EPA and DHA, may reflect adaptive strategies to maintain membrane fluidity and functionality under acidified conditions, as observed in both invertebrate and vertebrate systems (Mourente et al., 2022). In gill tissues, reductions in PUFAs under LpH (7.70 ± 0.09) may be associated with impaired membrane-bound processes such as ion transport or respiration, whereas the mantle’s elevated LC-PUFA content could suggest compensatory regulation aimed at preserving cellular performance. Collectively, these tissue-specific FA shifts likely represent a balance between maintaining membrane integrity, oxidative defense, and energetic allocation, highlighting the complex trade-offs faced by N. eatoni under OA stress. Such tissue-specific responses align with broader findings across phyla, including mollusks and crustaceans, where FA remodeling serves as a plastic response to environmental change (Arts and Kohler, 2009; Soudant et al., 2020). Thus, interpreting FA shifts not solely as stress indicators, but as reflections of underlying biochemical and physiological strategies, provides a richer framework for understanding organismal resilience to ocean acidification.