Effect of the High Temperature on Growth, Metabolism, and Fatty Acid Profile of the Clam Ruditapes decussatus Culture with and Without Substrate

TL;DR

High temperatures harm clam growth and metabolism, altering fatty acid profiles, which could threaten clam aquaculture sustainability.

Contribution

This study reveals how chronic high temperatures affect clam physiology and lipid composition, emphasizing thermal stress in aquaculture management.

Findings

High temperatures reduced growth, biomass, and meat yield in clams.

Thermal stress shifted metabolism toward anaerobic pathways and increased lipid mobilization.

Fatty acid profiles changed significantly, with decreases in ARA and EPA but stable DHA levels.

Abstract

Increasing seawater temperatures could seriously affect bivalve aquaculture, chiefly in estuarine areas, which are highly sensitive to environmental changes. The grooved carpet clam (Ruditapes decussatus), an ecologically and economically important species in southern Europe, is especially vulnerable to abiotic factors. In this study, clams reared with or without substrate were exposed to elevated (28 °C) and control (18 °C) temperatures for 21 days, and their growth, metabolism, and fatty acid composition were analyzed. High temperature significantly reduced growth, tissue biomass, and meat yield, while inducing metabolic shifts toward anaerobic pathways and increased lipid mobilization. Fatty acid profiles were affected by temperature, observing important variations in total lipids and key unsaturated fatty acids such as arachidonic acid (ARA) and eicosapentaenoic acid (EPA), while docosahexaenoic acid (DHA) remained stable. Sediment presence had only minor effects. These findings indicate that sustained warming could compromise clam health and growth, highlighting the importance of considering thermal stress in the management and sustainability of clam aquaculture.

Rising seawater temperatures associated with climate change are expected to increasingly challenge the sustainability of bivalve aquaculture, particularly in estuarine environments where thermal variability is naturally high. The grooved carpet clam (Ruditapes decussatus), a species of high ecological and economic value in southern Europe, is strongly influenced by weather conditions. This study aimed to investigate the effects of a chronic thermal challenge (21 days at 28 °C) on the growth performance, intermediary metabolism, and fatty acid composition of R. decussatus raised with and without substrate. Clams were acclimated to either control (18 °C) or high-temperature (28 °C) conditions, and biometric, biochemical parameters (glucose, glycogen, lactate, triglycerides, and cholesterol) and fatty acid profiles were analyzed. Our results denote that exposure to elevated temperature significantly reduced total weight, tissue biomass, meat yield, and condition index in both clams reared with and without substrate. Thermal stress induced marked metabolic alterations, characterized by increased lactate accumulation and depletion of triglyceride reserves, indicating a shift toward anaerobic metabolism and enhanced lipid mobilization. However, glycogen and cholesterol levels remained largely unchanged. Fatty acid analysis revealed a strong temperature-driven remodeling of lipid composition, characterized by significant reductions in total lipids and unsaturated fatty acids, which highlighted changes in key fatty acids, such as arachidonic acid (ARA; 20:4n-6) and eicosapentaenoic acid (EPA; 20:5n-3). In contrast, docosahexaenoic acid (DHA; 22:6n-3) levels remained unchanged under high-temperature conditions. Principal component analysis confirmed temperature as the main factor structuring fatty acid profiles, while substrate exerted only minor effects. Overall, these findings demonstrate that sustained exposure to sublethal high temperature profoundly affects growth performance, metabolic balance, and lipid homeostasis in R. decussatus, overriding the possible physiological benefits associated with substrate presence. The results highlight the vulnerability of this species to future warming scenarios and underscore the importance of incorporating thermal stress considerations into sustainable clam aquaculture management strategies in estuarine environments.

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1. Introduction

The diversification of aquaculture species represents a promising strategy to strengthen the economic resilience and long-term sustainability of the aquaculture sector. Bivalves, in particular, are key components of coastal ecosystems due to their ecological functions, and many species also constitute an important source of high-quality protein for human consumption [1]. Within the European Union, this approach has highlighted the relevance of certain species, such as the grooved carpet clam (Ruditapes decussatus), owing to its high biological, economic, and gastronomic value [1,2]. This species is widely distributed along European, Mediterranean, and North African coasts and estuaries, and it is extensively cultured in Portugal and Spain [2,3,4].

Ensuring the sustainable aquaculture of R. decussatus is increasingly important, as natural populations have declined due to overexploitation [5], disease outbreaks [6], and competition with non-native bivalves [7,8]. These pressures are expected to intensify under projected climate warming scenarios [9,10], with heatwaves predicted to become more frequent and severe in the coming decades [11]. Since growth, physiology, and metabolism in marine invertebrates are tightly regulated by environmental temperature [9,10,12], understanding the effects of thermal stress is essential for improving aquaculture resilience. This is particularly relevant in estuarine and salt marsh ecosystems, typical habitats for R. decussatus, where temperature naturally fluctuates due to seasonal variations, tidal cycles, and rainfall patterns [13,14,15]. Such fluctuations are predicted to intensify under climate change [11,14], potentially generating physiological stress in both wild and cultured bivalve populations, altering metabolic performance, and, in severe cases, increasing mortality [10,16]. Temperature variability can also influence a wide range of bivalve traits, including spatial distribution, growth, reproduction, feeding activity, behavior, and respiration [17,18,19]. Given the ecological significance of bivalves and the economic relevance of R. decussatus aquaculture in Europe, comprehensive assessments of heat stress impacts and potential mitigation strategies are urgently needed.

Among abiotic factors, substrate type is a critical determinant for burrowing bivalves. For species such as R. decussatus, the substrate provides physical protection while also modulating physiological and metabolic responses [20]. As a result, several studies have evaluated how different substrates affect aquaculture performance, particularly during early developmental stages, focusing on growth and mortality patterns [20,21,22,23]. Although substrate use in bivalve production offers environmental benefits [24], its application also entails challenges. Substrates may alter water quality and provide surfaces conducive to pathogenic microbial growth, raising concerns about their suitability [25,26]. Practical issues such as handling, cleaning, and the limited availability of high-quality substrate further complicate its use in bivalve aquaculture. Moreover, the current lack of a structured breeding program for R. decussatus, a key requirement for ensuring stable aquaculture production in its native regions [27], emphasizes the need to optimize rearing protocols, including substrate management. Despite the recognized role of substrate in facilitating thermoregulatory behaviors in marine mollusks [28], little is known about how temperature fluctuations interact with the presence or absence of substrate to influence physiological and metabolic responses in R. decussatus, particularly regarding lipid and fatty acid regulation.

Fatty acids (FAs) are considered essential dietary components for bivalves, supporting a broad array of physiological functions [16]. Among them, polyunsaturated fatty acids (PUFAs), including arachidonic acid (ARA; 20:4n-6), eicosapentaenoic acid (EPA; 20:5n-3), and docosahexaenoic acid (DHA; 22:6n-3), are particularly relevant due to their key role in maintaining membrane structure and, ultimately, the overall physiological condition of the aquatic organism [29,30,31,32]. In poikilothermic animals such as bivalves, the ability to cope with environmental temperature shifts is closely linked to the PUFA composition of membrane phospholipids and cholesterol, as these lipids modulate membrane fluidity and influence the activity of associated proteins [33,34]. For sessile species like R. decussatus, the regulation of lipid metabolism and the ability to accumulate FAs in storage pools, mainly as triglycerides, are key mechanisms for coping with fluctuating environmental conditions [16,29]. Temperature is one of the most influential environmental variables because it impacts a wide array of biochemical and physiological processes, including reaction kinetics, diffusion rates, membrane biophysics, and protein stability [16,35]. Consequently, poikilothermic organisms routinely adjust the lipid composition of their cellular membranes during thermal acclimation to safeguard membrane integrity, sustain cellular functionality, and maintain homeostasis [35,36,37]. Achieving such stability requires an adequate supply of metabolic energy, which in aquaculture species depends on the effective digestion and assimilation of dietary nutrients. This process breaks down macronutrients into absorbable molecules, including amino acids, FAs, and glucose, ensuring the availability of essential bioenergetic substrates for physiological maintenance.

To improve our understanding of how climate-driven temperature increases affect life-cycle processes and aquaculture performance, and to elucidate the metabolic mechanisms underlying thermal adaptation in bivalves, this study examined the effects of elevated environmental temperature (28 °C) on the metabolic condition of R. decussatus reared with and without substrate. Beyond assessing growth, temperature-induced changes in key metabolic parameters (glucose, glycogen, lactate, triglycerides, and cholesterol) were evaluated. In addition, the combined effects of substrate and temperature on the FA composition of R. decussatus were analyzed, providing a comprehensive assessment of their combined effects on the species’ intermediary metabolism.

2. Materials and Methods

2.1. Clam Maintenance and Experimental Design

R. decussatus clams, obtained from thermal shock-induced spawning in an experimental hatchery located at the IFAPA-Agua del Pino facilities (Cartaya, Huelva, Spain), were reared for 15 months under two distinct conditions: (i) with substrate and (ii) without substrate, both under identical environmental and zootechnical conditions. The substrate depth was 10 cm and consisted of silica sand (95%) and aragonite (CaCO_3_) (5%), with a grain size of 3–4 mm.

After this rearing period, a total of 240 clams were individually measured, weighed, and randomly allocated into 12 perforated (2 mm mesh) rectangular baskets (44 × 33 × 10 cm^3^; 20 clams per basket), maintaining the respective substrate conditions: 120 clams in six baskets with substrate and 120 clams in six baskets without substrate.

Baskets were assigned to four experimental groups (in triplicate): with and without substrate, each maintained at either control temperature (Ctrl: 18 ± 0.5 °C) or elevated temperature (High: 28 ± 0.5 °C) for 21 days (14 March–4 April 2025). Both temperatures (Ctrl and High) were established in accordance with Rato et al., 2022 [2], da Costa et al., 2020 [27], and Macho et al., 2026 [38]. Before the thermal challenge, clams were gradually acclimated to the experimental temperature over one week.

Natural seawater was used under open flow-through conditions with a renewal rate of 100–150 mL min^−1^ (i.e., 6–9 L h^−1^). Throughout the experiment, water quality parameters (pH, nitrite, nitrate, and ammonia), dissolved oxygen, salinity (36.5 ± 0.5 g L^−1^), and photoperiod (12 h light: 12 h dark) were kept constant. Clams were fed daily with a mixed microalgae diet (~0.5 × 10^6^ cells mL^−1^) composed of Skeletonema costatum (70–90%), Chaetoceros gracilis (10–20%), and Isochrysis galbana (≈5%). The fatty acid composition of the diet is presented in Table 1.

2.2. Sampling Protocols and Biometric Parameters

Regarding biometric parameters, total weight (g) and shell length (mm) were recorded for all live clams (240 individuals, i.e., 120 clams reared with and without substrate, respectively) at the beginning of the trial (T_0_) and re-measured after 21 days for all experimental groups. To determine individual shell weight and meat wet weight, clams were sacrificed and dissected. Shells were cleaned, dried, and weighed, whereas soft tissues were first weighed (60 clams per group) to obtain the meat wet weight (g). Subsequently, 15 clams per experimental group were oven-dried at 80 °C for 24 h and reweighed to determine the dry tissue mass (g).

The meat yield (%), shell component index (%), and condition index (%) were calculated according to the following equations [39,40,41]:

• -Meat Yield = Wet meat weight (g)/Total weight (g) × 100
• -Shell Component Index = Shell weight (g)/(Shell + Wet meat weight (g)) × 100
• -Condition Index = Meat dry weight (g)/Shell dry weight (g) × 100

Finally, soft tissue samples (whole meat) from each experimental group were collected and stored at −80 °C for subsequent biochemical analyses.

2.3. Biochemical Parameters

To evaluate metabolic parameters, whole clam tissues (n = 12) were processed following the procedure described by Torres-Rodríguez et al., 2025 [42]. Briefly, individual wet meat samples were finely minced and homogenized in 3.5 volumes (w/v) of ice-cold 0.6 N perchloric acid. The homogenates were subsequently neutralized with an equal volume of 1 M KHCO_3_ and centrifuged at 3500× g for 30 min at 4 °C. The resulting supernatants were transferred to 0.5 mL Eppendorf tubes. For triglyceride and cholesterol analyses, aliquots were collected before centrifugation. All extracts were stored at −80 °C until further biochemical assays.

Spectrophotometric determinations were carried out in duplicate using a Varioskan LUX 3020 microplate reader (Thermo Scientific, Alcobendas, Madrid, Spain) controlled with Thermo Scientific SkanIt^TM^ software v6.0 (Thermo Scientific). Metabolite concentrations were assessed with commercial enzymatic kits (SpinReact SA, St. Esteve d’en Bas, Girona, Spain) adapted to 96-well microplates. The metabolites quantified included glucose (Ref. 1001200), lactate (Ref. 1001330), cholesterol (Ref. 41021), and triglycerides (Ref. 1001311). Glycogen content was determined according to the procedure of Keppler and Decker [43], in which glucose released after glycogen hydrolysis with amyloglucosidase (Sigma-Aldrich^®^, Ref. A7420, Madrid, Spain) was quantified using the aforementioned glucose kit (SpinReact SA, Ref. 1001200, Girona, Spain).

2.4. Total Lipids Extraction and Fatty Acid Analysis

Lipid and fatty acid analyses were carried out in clams as described by Fernández-Cabanás and Cruzado [44]. Briefly, lipids were extracted from approximately 200 mg of tissue (previously freeze-dried at −80 °C for 48 h) using an ice-cold chloroform:methanol mixture (2:1, v/v). Samples (n = 6) were homogenized with a MiniG Tissue Homogenizer (SPEX CertiPrep, Metuchen, NJ, USA). Phase separation of lipid and non-lipid components was achieved by adding 0.88% (w/v) KCl. The upper aqueous phase was removed and discarded, and the lower organic phase was evaporated to dryness under oxygen-free nitrogen. Total lipid content was determined gravimetrically after overnight drying in a vacuum desiccator.

Fatty acid methyl esters (FAMEs) were prepared from total lipids by acid-catalyzed transesterification at 50 °C for 16 h, following the method described by Christie (2003) [45]. FAMEs were separated and quantified using a Shimadzu GC-2010 gas chromatograph equipped with a flame ionization detector (280 °C) and a fused silica capillary column (SU-PRAWAX-280, 15 m × 0.1 mm i.d.; Teknokroma, Sant Cugat del Vallès, Spain). Hydrogen was used as the carrier gas. The oven temperature was initially set at 150 °C for 1 min, then increased at a rate of 8 °C min^−1^ to a final temperature of 250 °C, which was held for 3 min. FAMEs were identified by comparison with a standard mixture (Supelco 37 Component FAME Mix, CRM47885) and expressed as the percentage of total fatty acids.

2.5. Statistical Analysis

All data were tested for normality and homogeneity of variances using the Kolmogorov–Smirnov and Levene’s tests, respectively. Outliers were identified using the ROUT method with a Q value of 1%. Results are expressed as means ± SEM (standard error of the mean). Differences in biometric parameters were analyzed using one-way analysis of variance (ANOVA; p ≤ 0.05), followed by Tukey’s HSD post hoc test. The effects of environmental temperature on biochemical parameters and fatty acid profiles were assessed using independent-samples Student’s t-tests. To account for multiple comparisons, p-values were adjusted using the Bonferroni correction, and statistical significance was accepted at an adjusted α level of 0.05. Principal component analysis (PCA) was performed to explore differences in fatty acid composition among clams according to substrate (presence/absence) and temperature conditions. All statistical analyses and graphical representations were conducted using GraphPad Prism v8.0 (GraphPad Software Inc., San Diego, CA, USA).

3. Results

3.1. Growth Parameters and Somatic Indices

No clam mortality was observed during the experimental period. Growth parameters and somatic indices are presented in Table 2. Environmental temperature significantly affected growth performance, regardless of whether substrate was present or not. Under both substrate and no-substrate conditions, clams acclimated to high temperature (28 °C) exhibited lower total weight, meat wet weight, meat dry weight, meat yield, and condition index than those maintained at the control temperature (18 °C). No significant differences in shell weight were observed between temperatures under either substrate condition. For the shell component index, significant differences were detected only in clams cultured without substrate, showing the lowest values in individuals reared at the control temperature.

3.2. Biochemistry Results

Biochemical results for whole clam tissue are presented in Table 3. In both substrate and no-substrate groups, significant differences were observed in lactate and triglyceride levels. In both cases, clams maintained at high temperature (28 °C) exhibited the highest lactate concentrations. In contrast, clams exposed to high temperature showed the lowest triglyceride levels. Glucose levels varied significantly only in the no-substrate group, reaching their highest values at the control temperature (18 °C). Environmental temperature had no significant effect on glycogen or cholesterol levels in either the substrate or the no-substrate groups.

3.3. Fatty Acid Composition

The fatty acid composition of R. decussatus clams is presented in Table 4. Overall, temperature exerted a strong influence on the unsaturated fatty acid profile and total lipid content, regardless of the presence or absence of substrate.

Under both substrate and no-substrate conditions, clams maintained at high temperature (28 °C) exhibited significantly lower levels of several polyunsaturated fatty acids (PUFAs), including the n-3 fatty acids 18:3n-3, 18:4n-3, 20:3n-3, 20:4n-3, and 20:5n-3 (EPA), as well as the n-6 fatty acids 18:2n-6, 18:3n-6, and 20:3n-6 (in the substrate group only). Conversely, the levels of 20:4n-6 (ARA) and 22:4n-6 increased at high temperature.

Monounsaturated fatty acids (MUFAs), such as 18:1n-7, were significantly reduced at high temperature in both culture conditions, whereas total MUFA levels decreased significantly only in clams reared without substrate. Total unsaturated fatty acids (UFAs) and total lipid content were significantly lower in clams exposed to high temperature, irrespective of substrate availability.

Principal component analysis (PCA) of the fatty acid profiles of clams revealed a clear separation among groups (Figure 1). The first two principal components explained 53.5% (PC1) and 26.8% (PC2) of the total variance, respectively. Along PC1, samples clustered primarily according to temperature, with clams maintained at 18 °C (both substrate and no substrate) positioned on the right side of the plot and those at 28 °C on the left. Within each temperature group, a secondary separation along PC2 was observed between substrate and no-substrate treatments. The distinct clustering patterns and non-overlapping confidence ellipses support the existence of clear differentiation in the FA profile depending on the temperature.

4. Discussion

Global warming predictions indicate that the rise in seawater temperature, together with the increased intensity and frequency of extreme climatic events such as heavy rainfall, heatwaves, and droughts, will intensify in the coming decades [2,10], potentially affecting food production systems, including aquaculture [13]. Temperature fluctuations associated with these events may impact the life cycle of aquatic organisms, particularly those inhabiting or cultured in intertidal environments such as estuaries and salt marshes [14]. This is especially relevant for bivalves, whose populations and ecological dynamics are strongly influenced by weather conditions [10,12].

In this context, the present work provides new insights into the physiological and metabolic responses of the clam R. decussatus to a chronic (21 days) yet ecologically realistic thermal challenge, demonstrating that sublethal high water temperature (28 °C) [38] could exert a strong effect on growth, intermediary metabolism, and fatty acid composition regardless of these having been raised in culture conditions with or without substrate.

Under natural conditions, substrate is a fundamental abiotic element for burrowing bivalves, providing not only physical support but also exerting a notable influence on their physiological functioning [20,22,23,46,47]. In our assay, this beneficial role is already evident at the onset of the thermal challenge (T_0_), where clams raised with substrate displayed superior growth and body condition relative to individuals kept without it. Such differences likely reflect more favorable feeding dynamics, as the presence of sediment enables appropriate shell positioning that facilitates effective microalgae filtration and ingestion [20]. When clams can settle their valves against the surrounding substrate, the adductor muscles can relax during filtration activity [48,49]. In contrast, animals deprived of substrate must sustain adductor tension to maintain valve alignment, a condition that may compromise filtration efficiency and, consequently, the bioassimilation of nutrients. Moreover, substrate can contribute to the thermoregulatory behavior of marine mollusks by enabling stable and functional shell positioning [28,50,51]. Its presence supports behavioral strategies such as shell-lifting, shell-standing, and towering, all of which act as adaptive responses to thermal elevation [28,50]. Despite these behavioral advantages, our results denoted that exposure to high temperatures markedly compromises growth and body condition in R. decussatus raised both with and without substrate. Thus, either in the presence or absence of substrate, clams exposed to a higher temperature (28 °C) showed reduced growth performance, including lower total weight, meat weights (wet and dry), meat yield, and condition index. These responses are consistent with thermal inhibition of physiological processes in bivalves when temperatures exceed their optimal thermal window [52,53]. Previous studies have reported that R. decussatus experiences feeding reduction, metabolic imbalance, and increased vulnerability above 25–27 °C [2,9,54]. Similar declines in somatic performance under heat exposure have been described in other venerids, such as Ruditapes philippinarum [38] and Paphia undulata [20], supporting the idea that prolonged exposure to temperatures near the upper tolerance limit diverts energy away from growth toward stress mitigation and maintenance of homeostasis in bivalves [10,55,56,57].

Thermal stress induced clear changes in intermediary metabolism. The significant increase in lactate levels at 28 °C in both rearing conditions, with and without substrate, indicates a metabolic shift toward anaerobic pathways, likely reflecting a reduced aerobic capacity at elevated temperatures [58]. Accumulation of lactate has similarly been documented in bivalves subjected to stressful conditions such as hypoxia, acidification, and thermal extremes [59,60,61]. Consistent with responses described in other marine ectotherms [57,62,63], this pattern suggests the activation of anaerobic metabolism during sustained thermal exposure, whereby increased energetic demands are met predominantly through ATP synthesis via anaerobic glycolysis [58,64]. This response appears particularly evident in clams maintained without substrate, which showed a significant decline in glucose levels at high temperature, further supporting the prevalence of anaerobic glycolysis under heat stress. Concomitantly, triglyceride reserves decreased markedly in heated clams. Mobilization of lipid reserves is a common compensatory mechanism when energy demand rises and feeding efficiency declines, as lipids represent a major long-term energy store in many bivalves [29,57,58,65,66]. The reduction in triglycerides, combined with conserved glycogen levels, suggests that clams prioritized lipid mobilization rather than carbohydrate use during thermal stress [57,65]. This observation aligns with evidence that bivalves often spare glycogen during the adaptation stress process to sustain essential cellular functions [67,68,69,70]. Overall, these metabolic adjustments under high temperature reflect a greater dependence on anaerobic pathways coupled with enhanced lipid catabolism.

Consistent with observations reported for other bivalve species [16,66,71,72], temperature exerted a pronounced and systematic influence on the fatty acid composition of R. decussatus. Exposure to elevated temperature resulted in marked decreases in several n-3 and n-6 polyunsaturated fatty acids, notably 18:3n-3, 18:4n-3, 20:4n-3, 20:5n-3 (EPA), and 18:3n-6. These changes likely reflect a homeoviscous adjustment mechanism, through which ectothermic organisms modulate membrane lipid composition by lowering PUFA levels to compensate for the increased membrane fluidity associated with warming [35,37,73,74]. This interpretation is further supported by the absence of significant variations in cholesterol content in clams, as cholesterol plays a central role in stabilizing animal cell membranes and may buffer temperature-driven alterations in membrane organization across fluctuating thermal environments [75].

In contrast, the relative increase in ARA (20:4n-6) and adrenic acid (22:4n-6) at 28 °C suggests selective retention and/or biosynthesis of long-chain n-6 PUFAs. These fatty acids act as key substrates for eicosanoid production, mediating inflammatory and stress-related pathways [76,77,78], and may therefore contribute to limiting oxidative damage and maintaining cellular integrity during prolonged exposure to elevated temperatures. Although DHA levels remained relatively stable, EPA showed a pronounced decrease with increasing temperature, consistent with its high susceptibility to oxidative degradation and rapid utilization under stress conditions [36,79]. As observed in aquatic vertebrates [30,42], this pattern may reflect the critical role of DHA in correct cellular development and the functionality of essential tissues, including the nervous system, as well as its contribution to physiological resilience under environmental stress in marine invertebrates [79,80,81,82], suggesting a potential tissue-level conservation of DHA under environmental stress. Furthermore, exposure of R. decussatus to elevated temperatures could stimulate the biosynthesis of DHA from EPA, maintaining sufficient DHA reserves for incorporation into tissues with high requirements of this compound [83,84]. This metabolic adaptation process likely supports proper cell signaling, energy metabolism, and other essential physiological functions necessary to cope with environmental stressors [79,80,81,82,83,84,85]. Together, these alterations indicate that rising temperature drives membrane restructuring in bivalves toward a more ordered lipid organization, achieved through increased fatty acid saturation and/or elongation as a compensatory response to thermally induced membrane fluidity [79,80,86]. In addition, the observed reductions in total UFAs and total lipids provide additional evidence that high temperatures accelerate bivalves’ lipid metabolism, likely to meet the elevated metabolic and maintenance demands associated with thermal stress [29,37,68,69]. This observation aligns with previous findings indicating that modifications in membrane fatty acid composition generally emerge following a thermal acclimation phase, the duration of which may range from approximately one week under warm conditions to several weeks during cold exposure [29,73,74].

Although temperature emerged as the principal driving lipid-profile reorganization in R. decussatus, PCA revealed partial segregation between substrate conditions along PC2, suggesting that substrate had only minor effects on FA composition. These may arise from behavioral differences, since substrate allows natural burrowing, shell-lifting, and shell-standing behaviors that modify exposure to thermal and hydrodynamic conditions [20,28]. Such behavioral thermoregulation could slightly influence metabolic rate or feeding selectivity [87], thereby affecting FA biosynthesis and assimilation. However, these substrate-mediated effects were minor compared to the dominant influence of water temperature, indicating that warming counteracts the substrate-related physiological buffering.

5. Conclusions

This study demonstrates that prolonged exposure to elevated temperature (28 °C) has a pronounced impact on the growth performance, metabolic balance, and FA composition of R. decussatus, regardless of substrate availability. Thermal stress significantly reduced biomass, meat yield, and condition index, while promoting a metabolic shift toward anaerobic pathways, evidenced by increased lactate accumulation and depletion of triglyceride reserves. In parallel, high temperature induced marked remodeling of fatty acid profiles, characterized by reductions in total lipids and several key polyunsaturated fatty acids, particularly EPA, alongside increases in ARA and adrenic acid, reflecting adaptive membrane restructuring under heat stress. Although substrate presence conferred initial growth advantages, it did not mitigate the detrimental effects of sustained warming. Overall, these findings highlight the high vulnerability of R. decussatus to projected climate warming and underscore the need to incorporate thermal stress considerations into sustainable management and aquaculture strategies for this species, especially in estuarine environments.