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Ectotherms are exposed to a range of environmental temperatures and may face extremes beyond their upper thermal limits. Such temperature extremes can stimulate aerobic metabolism toward its maximum, a decline in aerobic substrate oxidation, and a parallel increase of anaerobic metabolism, combined with ROS generation and oxidative stress. Under these stressful conditions, marine organisms recruit several defensive strategies for their maintenance and survival. However, thermal tolerance of ectothermic organisms may be increased after a brief exposure to sub-lethal temperatures, a process known as \"hardening\". In our study, we examined the ability of M. galloprovincialis to increase its thermal tolerance under the effect of elevated temperatures (24, 26 and 28 °C) through the \"hardening\" process. Our results demonstrate that this process can increase the heat tolerance and antioxidant defense of heat hardened mussels through more efficient ETS activity when exposed to temperatures beyond 24 °C, compared to non-hardened individuals. Enhanced cell protection is reflected in better adaptive strategies of heat hardened mussels, and thus decreased mortality. Although hardening seems a promising process for the maintenance of aquacultured populations under increased seasonal temperatures, further investigation of the molecular and cellular mechanisms regulating mussels’ heat resistance is required.

The global seawater temperature is expected to further rise in the following years. While species have historically adapted to climatic variations, the current pace of climate change may exceed their ability to adapt. The abnormally increased seawater temperatures occasionally lead to high mortalities of marine bivalve mollusks, threatening the productivity of aquaculture and the sustainability of wild populations. This study investigates the antioxidant and cell death mechanisms of the Mediterranean mussel Mytilus galloprovincialis during a 25-day exposure to temperatures of 24°C, 26°C, and 28°C, by analyzing the transcription of key genes and assessing the oxidative damage on days 1, 3, 12, and 25. In addition, individuals resilient (survived at 28°C until day 30) and susceptible (died early at 26°C and 28°C) to thermal stress were collected to investigate potential polymorphisms in associated genes. The results showed increased transcription of antioxidant genes at higher temperatures. Elevated pro-apoptotic indices were initially observed at 26°C and a higher mortality than at 28°C. However, final mortality was much higher at 28°C. At 26°C, mussels exhibited the highest oxidative damage and pro-apoptotic indices after 25 days. At 28°C, although oxidative damage occurred after 24 hours, survivors maintained a prolonged activated antioxidant defense and increased lc3b transcription, which likely contributed to the observed reduction of pro-apoptotic and oxidative damage metrics on day 25, compared to 26°C. Further, the coding sequences of catalase , intracellular Cu-Zn superoxide dismutase ( Cu-Zn sod ), and fas-associated protein with death domain ( fadd ) from heat-resilient and heat-susceptible mussels were analyzed. Based on statistical correlation of nucleotide and genotype frequencies with resilience phenotypes, two novel single nucleotide polymorphisms (SNPs) in Cu-Zn sod and one in fadd were detected, potentially correlating with thermal stress resilience. These findings offer valuable insights into the physiological and genetic adaptations of M. galloprovincialis to rising temperatures and highlight loci potentially linking to thermal resilience.

Freshwater crayfish Pontastacus leptodactylus is an indigenous European species of considerable ecological and commercial significance. However, it is highly susceptible to the oomycete Aphanomyces astaci , the causative agent of crayfish plague. Although its importance is widely recognized, little information exists on how seasonality affects the physiology and welfare of this species. The present study was designed to (i) investigate seasonal effects on the physiological responses of P. leptodactylus in relation to abiotic factors and crayfish plague outbreak, and (ii) identify the most suitable period for restocking in temperate regions. To achieve this, we characterized for the first time the mRNA expression of hsp70, hsp90, hif-1α, ubiquitin, and gapdh (as endogenous control gene), alongside protein levels of Hsp70, Hsp90, hydroxylated Hif-1α, ubiquitin conjugates, cleaved caspases, Bax/Bcl-2 ratio, and interleukin-6 (IL-6). Our findings revealed that cold stress triggered both the heat shock response and apoptosis. Interestingly, despite the activation of apoptosis, which generally suppresses inflammation, IL-6 levels increased, suggesting a possible association with A. astaci infection. Moreover, elevated Hif-1α levels indicated hypoxic stress during warmer months, likely linked to reduced oxygen availability and turbidity. Overall results suggest that crayfish plague epidemics may intensify with rising temperatures, whereas winter appears to be the optimal period for restocking. Interestingly, in addition, freshwater crayfish in temperate latitudes are organisms that face both cold and heat stress seasonally in the same ecosystem.

For a comprehensive understanding of fish responses to increasing thermal stress in marine environments, we investigated tissue energetics, antioxidant levels, inflammatory and cell death responses in Sparus aurata (gilthead seabream) red muscle during exposure to elevated temperatures (24 °C, 26 °C, 30 °C) compared to the control temperature of 18 °C. Energetic aspects were assessed by determining lactate, glucose and lipids levels in blood plasma, ATP, ADP and AMP levels, and AMPK phosphorylation as an indicator of regulatory changes in energy metabolism, in tissue extracts. Oxidative defence was assessed by determining superoxide dismutase, catalase and glutathione reductase maximum activities. Moreover, xanthine levels were determined as an indicator of purine conversion to xanthine and associated ROS production. In the context of inflammatory response and cell death due to oxidative stress, pro-inflammatory cytokines (IkBα phosphorylation, IL-6 and TNFα) levels, and LC3 II/I ratio and SQSTM1/p62 as indicators of autophagic-lysosomal pathway were also determined. A recovery in the efficacy of ATP production after a marked decrease during the 1st day of exposure to 24 °C is observed. This biphasic pattern is paralleled by antioxidant enzymes’ activities and inflammatory and autophagy responses, indicating a close correlation between ATP turnover and stress responses, which may benefit tissue function and survival. However, exposure beyond 24 °C caused tissue’s antioxidant capacity loss, triggering the inflammatory and cell death response, leading to increased fish mortality. The results of the present study set the thermal limits of the gilthead seabream at 22–24 °C and establish the used cellular and metabolic indicators as tools for the definition of the extreme thermal limits in marine organisms.

Heat hardening induces complex biochemical reprogramming that enhances thermal resilience in marine bivalves. Despite this technique's promising results in marine animals, the molecular basis of heat hardening is far from understood. This study elucidates the molecular mechanisms underlying the hardening process in exposed to a 4-day sublethal heat treatment. Induction of , , and genes revealed the activation of the heat shock response and proteostasis machinery, ensuring proper protein folding and preventing oxidative and proteotoxic stress. Simultaneous upregulation of mitochondrial ( , , ) and glycolytic ( , ) genes reflects enhanced oxidative phosphorylation and glycolytic flux, maintaining ATP supply and metabolic flexibility under elevated temperatures. Increased expression suggests transient hypoxia signaling, coordinating oxygen utilization with redox control. Reinforcement of antioxidant defenses, together with elevated autophagy-related transcription, denotes a shift toward oxidative stress mitigation and damaged organelle clearance. Balanced expression of pro- ( ) and anti-apoptotic ( ) factors, along with modulation, supports tight regulation of cell survival and inflammatory responses. These findings underscore a highly integrated biochemical network linking proteostasis, intermediary metabolism, redox balance, and antioxidant defense with cellular quality control, which together underpin the physiological plasticity of heat-hardened , enhancing survival under transient thermal stress.

Global warming significantly impacts coastal zones, particularly affecting ectothermic inhabitants such as bivalve mollusks. This study evaluates the response of the grooved carpet shell clam Ruditapes decussatus (Linnaeus, 1758) to increasing temperatures (22.5°C, 24.5°C, 26.5°C) over 25 days through the transcription of key genes involved in antioxidant defense [Cu‐Zn superoxide dismutase (Cu‐Zn sod), catalase, metallothionein], anti‐apoptotic procedures [b‐cell lymphoma 2 (bcl2)], and energy metabolism [pyruvate kinase (pk), phosphoenolpyruvate carboxykinase (pepck)]. Additionally, the genes catalase and Cu‐Zn sod were characterized for the first time, and along with the metallothionein gene, were sequenced in heat‐resilient and heat‐susceptible individuals to identify polymorphisms potentially associated with thermal tolerance. At 22.5°C, clams showed a delayed increase in glycolytic flux and a gradual up‐regulation of antioxidant and anti‐apoptotic mechanisms. At 24.5°C and 26.5°C, a strong initial stress response resulted in equally high mortality during the early days of exposure. Subsequently, clams appeared to shift toward a reduced energy metabolism, with mildly upregulated antioxidant defenses and anti‐apoptotic activity. With prolonged exposure, there was evidence of enhanced aerobic glycolysis, antioxidant, and anti‐apoptotic responses at these temperatures. However, increased pepck transcription on days 12 and 25 suggests that metabolic demands may have exceeded aerobic capacity, potentially triggering apoptotic processes at 26.5°C. Clams at 24.5°C maintained aerobic capacity upon the final day, also engaging anaerobic pathways to meet energy demands. Eventually, three SNPs were statistically correlated with heat resilience. These included one non‐synonymous SNP in catalase, one SNP in the 3′ untranslated region (3′UTR) of metallothionein, and one synonymous SNP in Cu‐Zn sod. These findings underscore the sensitivity of R. decussatus populations from the northeastern Mediterranean to persistent thermal stress and reveal several polymorphisms in antioxidant genes with potential adaptive significance. However, the limited sample size and the weak correlations observed in some cases highlight the need for further research to clarify the relationship between these polymorphisms and thermal resilience. Climate change is known to affect all levels of biology, setting at risk ecological balance. Among other organisms' groups, aquatic inhabitants, and particularly bivalves, are expected to be influenced to a greater extent owing to their inability to move to more mild temperatures. Therefore, these organisms represent excellent models to study the adaptation to global warming. Keeping this in mind, in the present study, we molecularly characterized the evolutionary response of the marine bivalve Ruditapes decussatus to increased seawater temperature by means of both antioxidant gene expression and resilience‐associated genetic loci. Several genes were characterized for the first time, and novel SNPs were detected that are linked with tolerance to climate change.

Aquaculture is affected by numerous factors that may cause various health threats that have to be controlled by the most environmentally friendly approaches. In this context, prebiotics, probiotics, and synbiotics are frequently incorporated into organisms’ feeding rations to ameliorate the health status of the host’s intestine, enhancing its functionality and physiological performance, and to confront increasing antimicrobial resistance. The first step in this direction is the understanding of the complex microbiome system of the organism in order to administer the optimal supplement, in the best concentration, and in the correct way. In the present review, pre-, pro-, and synbiotics as aquaculture additives, together with the factors affecting gut microbiome in crayfish, are discussed, combined with their future prospective outcomes. Probiotics constitute non-pathogenic bacteria, mainly focused on organisms’ energy production and efficient immune response; prebiotics constitute fiber indigestible by the host organism, which promote the preferred gastrointestinal tract microorganisms’ growth and activity towards the optimum balance between the gastrointestinal and immune system’s microbiota; whereas synbiotics constitute their combination as a blend. Among pro-, pre-, and synbiotics’ multiple benefits are boosted immunity, increased resistance towards pathogens, and overall welfare promotion. Furthermore, we reviewed the intestinal microbiota abundance and composition, which are found to be influenced by a plethora of factors, including the organism’s developmental stage, infection by pathogens, diet, environmental conditions, culture methods, and exposure to toxins. Intestinal microbial communities in crayfish exhibit high plasticity, with infections leading to reduced diversity and abundance. The addition of synbiotic supplementation seems to provide better results than probiotics and prebiotics separately; however, there are still conflicting results regarding the optimal concentration.

Pinna nobilis populations, constituting the largest bivalve mollusk endemic to the Mediterranean, is characterized as critically endangered, threatened by extinction. Among the various factors proposed as etiological agents are the Haplosporidium pinnae and Mycobacterium sp. parasites. Nevertheless, devastation of the fan mussel populations is still far from clear. The current work is undertaken under a broader study aiming to evaluate the health status of Pinna nobilis population in Aegean Sea, after the mass mortalities that occurred in 2019. A significant objective was also (a) the investigation of the etiological agents of small-scale winter mortalities in the remaining populations after the devastating results of Haplosporidium pinnae and Mycobacterium sp. infections, as well as (b) the examination of the susceptibility of the identified bacterial strains in antibiotics for future laboratory experiments. Microbiological assays were used in order to detect the presence of potential bacterial pathogens in moribund animals in combination with molecular tools for their identification. Our results provide evidence that Vibrio bacterial species are directly implicated in the winter mortalities, particularly in cases where the haplosporidian parasite was absent. Additionally, this is the first report of Vibrio mediterranei and V. splendidus hosted by any bivalve on the Greek coastline.

The impact of pathogenic Vibrio on bivalves is expected to be aggravated by global warming, posing a growing threat to aquaculture. Clam production has been particularly vulnerable, with significant losses attributed to the lack of pathogen-resistant strains. In this study, the mantle cavity of the grooved carpet shell clam Ruditapes decussatus (Linnaeus, 1758) was injected with Vibrio splendidus at 18 °C, 22 °C, and 24 °C and the transcription of the phagelysozyme, Cu-Zn superoxide dismutase (Cu-Zn sod), tumor necrosis factor receptor associated factor 6 (traf6), inhibitor of NF-κB (IκB), and Fas-associated protein with death domain (fadd) genes were assessed during a 20-day period. Additionally, the coding sequences of lysozyme, fadd, and IκB in Ruditapes decussatus were characterized for the first time, and SNPs were identified. Some SNPs showed significantly different distributions between infection-resistant and infection-susceptible individuals. Infected clams experienced increased mortality at elevated temperatures. Lysozyme mRNA was upregulated in infected groups across all temperatures. The sustained increase on day 20, coinciding with elevated traf6 mRNA, suggests a prolonged activation of the immune response. Cu-Zn sod transcription at 18 °C and 22 °C peaked on day 7 and returned to control levels by day 20, indicating an effective immune response, while at 24 °C, infected animals showed a continuously increased transcription. IκΒ and traf6 transcription, reflecting NF-κB pathway activity, varied with temperature and showed transient stimulation at higher temperatures. The pattern of fadd expression indicated a late induction of apoptosis, particularly at 18 °C and 24 °C. Overall, this study illustrates the involvement of five key genes in host–pathogen interactions and identifies potential markers for selection for Vibrio resistance in Ruditapes decussatus. However, given the weak correlation observed, further research is needed on the link between these polymorphisms and pathogen resilience.

Biofouling constitutes one of the main obstacles in the sector of shellfish farming. Under this perspective, it is of highly importance to critically combine the updated information regarding the invasive potential of ascidian species, together with the factors contributing toward these events. The biological features of each species in relation to the gathering of the main non-indigenous species in the Mediterranean basin represent the first step toward mitigation of negative effects of the phenomenon. Further, there are limited studies investigating the physiological changes of bivalves caused by biofouling while leading to an increase in stress biomarkers. In the present review, the major ascidian species negatively affecting bivalve culture in the Mediterranean Sea are presented, alongside monitoring of ascidians from four Greek mussel farming locations as typical mussel culture cases. Among the main ascidian species, , , , sp., sp., , and are included, with the last three being the most harmful for this aquaculture sector. Based on the existing literature and research conducted so far, future research directions are proposed, in an effort to effectively control or efficiently manage ascidian biofouling organisms. Overall, perspectives toward the way we manage the biofouling phenomenon, such as the use of ascidian’s by-products in feedstuffs, chemical and pharmaceutical industry, or their incorporation in bivalve co-culture and integrated multi-trophic aquaculture systems represent promising alternative approaches.