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Global climate change is a major trigger of unexpected temperature fluctuations. The impacts of marine heatwaves (MHWs) and nano-titanium dioxide (nano-TiO2) on marine organisms have been extensively investigated. However, the potential mechanisms underlying their interactive effects on physiological processes and metabolism remain poorly understood, especially regarding periodic MHWs in real-world conditions. In this study, the effects of nano-TiO2 (at concentrations of 0, 25, and 250 µg/L) and periodic MHWs on the condition index (CI) and underlying metabolic mechanisms were investigated in mussels (Mytilus coruscus). The results showed that mussels try to upregulate their respiration rate (RR) to enhance aerobic metabolism (indicated by elevated succinate dehydrogenase) under short-term nano-TiO2 exposure. However, even at ambient concentration (25 µg/L), prolonged nano-TiO2 exposure inhibited ingestion ability (decreased clearance rate) and glycolysis (inhibited pyruvate kinase, hexokinase, and phosphofructokinase activities), which led to an insufficient energy supply (decreased triglyceride, albumin, and ATP contents). Repeated thermal scenarios caused more severe physiological damage, demonstrating that mussels are fragile to periodic MHWs. MHWs decreased the zeta potential of the nano-TiO2 particles but increased the hydrodynamic diameter. Additionally, exposure to nano-TiO2 and periodic MHWs further affected aerobic respiration (inhibited lactate dehydrogenase and succinate dehydrogenase activities), metabolism (decreased RR, activities of respiratory metabolism-related enzymes, and expressions of PEPCK, PPARγ, and ACO), and overall health condition (decreased ATP and CI). These findings indicate that the combined stress of these two stressors exerts more detrimental impact on the physiological performance and energy metabolism of mussels, and periodic MHWs exacerbate the toxicological effects of ambient concentration nano-TiO2. Given the potential worsening of nanoparticle pollution and the increase in extreme heat events in the future, the well-being of mussels in the marine environment may face further threats.

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Green Chemistry involves applying a set of principles aimed at minimizing the use of hazardous substances in the design, production, and application of chemical products. In recent decades, Ionic Liquids (ILs) have emerged as more environmentally friendly substitutes for traditional organic solvents. This preference is primarily due to their low vapor pressure, which results in minimal atmospheric pollution and enhanced industrial safety. However, existing literature highlights the toxicity of ILs towards aquatic invertebrates. Consequently, this study points to assess the biochemical effects of a selection of ILs through an in vitro approach. Specifically, digestive gland and gill cellular fractions (S9) of the marine bivalve Mytilus galloprovincialis were exposed to varying concentrations (0.05-2 µM) of three ILs featuring identical cations but different anions. The ILs tested were 1-ethyl-3-methylimidazolium octanoate ([EMIM][Oct]), 1-ethyl-3-methylimidazolium acetate ([EMIM][OAc]), and 1-ethyl-3-methylimidazolium ethyl sulfate ([EMIM][EtSO4]). The results indicate that [EMIM][Oct] induces higher toxicity in both S9 tissues, highlighting a strong effect of the anion. Overall, antioxidant and biotransformation defenses were significantly altered for all three ILs assessed. While acetylcholinesterase activity was significantly inhibited of about half of control activity, indicating neurotoxic damage as part of the toxicity mode of action of these ILs, neither lipid peroxidation nor alterations to DNA integrity were observed (≥100 %). This study supports the use of in vitro techniques as important tools capable of generating reliable ecotoxicological data, which can be further considered as a screening before in vivo testing and used for in silico modeling.

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The severe eutrophication of the Baltic Sea requires mussel (Mytilus spp.) farming to remove nutrients, but farming in a low salinity environment results in smaller mussels that require value enhancement to be economically viable. This study evaluates the biomass valorisation of smaller Baltic mussels, focusing on the extraction of oil, protein and glycogen. It analyses the amino acid profiles, oil and fatty acid contents and glycogen levels of the mussels, as well as their prebiotic properties on beneficial gut bacteria. In addition, the study improves the extraction of bioactive compounds through enzymatic hydrolysis. Results indicate significant seasonal differences, with summer mussels having higher meat and lower ash content, and a rich content of essential fatty acids, particularly omega-3, and amino acids, underscoring the mussels' sustainability as a food source. The enzymatically treated biomass exhibited notable prebiotic activity, proposing health-promoting benefits. The study underscores the valorization of Baltic mussel biomass, highlighting its role in health, nutrition, and environmental sustainability.

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Biomineralisation of bivalve shells raises questions at the level of genes to the final calcified product. For the first time, gene expression has been studied in association with growth increment deposition in the mussel Mytilus galloprovincialis. A short-term experiment highlighted that biomineralisation genes exhibit a rhythm of expression consistent with the observed tidal increment formation. Long-term mark-recapture experiments were conducted in three Mediterranean environments and revealed that the mussel shells harbour complex incrementation regimes, consisting of daily, tidal and a mixed periodicity of ∼1.7 growth increment.d-1 formed. The latter is likely related to the local tidal regime, although the mussels were continuously submerged and exposed to a small tidal range. The pattern of growth increments shifted from mixed to daily in Mediterranean lagoon, and to tidal at sea, probably linked to biological clocks. Based on our results and the literature, a hypothetical model for mussel shell increment formation in various habitats is proposed.

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The global crystallographic texture of calcite and aragonite in the shells of the bivalves Bathymodiolus thermophilus, Mytilus galloprovincialis, M. edulis and M. trossulus was studied by means of neutron diffraction. It was revealed that the general appearance of pole figures isolines of both minerals coincides for the studied species. The crystallographic texture sharpness evaluated by means of pole density on the calcite pole figures ((0006), (101¯4)) and aragonite pole figures ((012)/(121), (040)/(221)) coincides or has close values for deep-sea hydrothermal species B. thermophilus and the studied shallow-water species of the genus Mytilus. The calcite pole figures (0006) and (101¯4) of B. thermophilus show a shift in the position of texture maximum values compared to corresponding pole figures of other mussels. The shell microstructure of all studied mollusks is similar, only the shape of the fibers of B. thermophilus differs. Global crystallographic texture is a stable feature of the family Mytilidae. The extreme habitat conditions of the hydrothermal biotope do not significantly affect the crystallographic texture of B. thermophilus.

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Despite increasing concerns regarding the interactions of microplastic and heavy metal pollution, there is limited knowledge on the molecular responses of marine organisms to these stressors. In this study, we used whole-transcriptome sequencing to investigate the molecular responses of the ecologically and economically important bivalve Mytilus galloprovincialis to individual and combined exposures of environmentally relevant concentrations of PVC microplastics and cadmium (Cd). Our results revealed distinct transcriptional changes in M. galloprovincialis, with significant overlap in the differentially expressed genes between the individual and combined exposure groups. Genes involved in cellular senescence, oxidative stress, and galactose metabolism were differentially expressed. Additionally, key signaling pathways related to apoptosis and drug metabolism were significantly modulated. Notably, the interaction of PVC microplastics and Cd resulted in differential expression of genes involved in drug metabolism and longevity regulating compared to single exposures. This suggests that the interaction between these two stressors may have amplified effects on mussel health. Overall, this comprehensive transcriptomic analysis provides valuable insights into the adaptive and detrimental responses of M. galloprovincialis to PVC microplastics and Cd in the environment.

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Agricultural nitrogen (N) contributes a dominant percentage to global N pollution in the coastal zone. Emerging research on N isotopes in bivalve shells has shown value for reconstructing historical increases in estuarine wastewater inputs. However, applications for fertilizer N are understudied. Here, we integrate the study of organic N isotopes, in concert with δ18O and δ13C, in estuarine bivalve shells to investigate spatial and long-term changes in nitrogen inputs and sources. Modern, museum-collected, and subfossil specimens of the genera Mytilus and Ostrea were profiled in a California estuary with an intensely agricultural watershed. Spatial patterns in bivalve isotopic composition reflected gradients in watershed nutrient inputs and productivity parameters. Furthermore, a comparison between modern and historical periods revealed changes in nutrient source or processing over the last 1,000 years. The N isotope values from shells offer perspective on agricultural pollution in estuaries.

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Nano-zinc oxide (ZnO NPs), as widely used nanomaterials, are inevitably released into aquatic environments, posing potential threats to aquatic organisms. Mytilus galloprovincialis is a bivalve species sensitive to changes in marine ecological environments, but there has been limited research on its toxicity response to ZnO NPs. Therefore, we selected M. galloprovincialis as the research subject and exposed them to 50 µg/L ZnO NPs for 96 h and 30 days to determine the dissolution of ZnO NPs in seawater and their distribution in M. galloprovincialis. The toxicity of ZnO NPs in M. galloprovincialis was then evaluated through gene expression, tissue pathology, and cellular immune response. The results showed that ZnO NPs could enrich Zn in various tissues of the mussel, in the order of gills > hepatopancreas > adductor muscle > mantle. Seven immune-related genes including four heat shock protein genes (HSPA12A, sHSP24.1, sHSP22, TCTP) and three apoptotic genes (Ras, p63 and Bcl-2) were altered to varying degrees. There was a downward trend in lysosomal membrane stability of M. galloprovincialis after exposure to ZnO NPs for 96 h and 30 days, while ROS and apoptosis rates increased significantly. Furthermore, the seven genes, apoptosis, LMS, and ROS were dependent on exposure time, treatment, and their interaction. Histopathological damage included disorganisation of hepatopancreas epithelial cells, gill filament swelling, and contraction of blood sinuses. These results indicated that ZnO NPs exerted toxicity in M. galloprovincialis, affecting the immune system, resulting in changes in the expression of immune-related genes and ultimately leading to histopathological changes. Our research findings could contribute to systematically understand the impact of ZnO NPs on bivalves in aquatic environments and provide a theoretical basis for marine pollution assessment.

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The effects of climate change on coastal biodiversity are a major concern because altered community compositions may change associated rates of ecosystem functioning and services. Whilst responses of single species or taxa have been studied extensively, it remains challenging to estimate responses to climate change across different levels of biological organisation. Studies that consider the effects of moderate realistic near-future levels of ocean warming and acidification are needed to identify and quantify the gradual responses of species to change. Also, studies including different levels of biological complexity may reveal opportunities for amelioration or facilitation under changing environmental conditions. To test experimentally for independent and combined effects of predicted near-future warming and acidification on key benthic species, we manipulated three levels of temperature (winter ambient, +0.8 and +2°C) and two levels of pco 2 (ambient at 450 ppm and elevated at 645 ppm) and quantified their effects on mussels and algae growing separately and together (to also test for inter-specific interactions). Warming increased mussel clearance and mortality rates simultaneously, which meant that total biomass peaked at +0.8°C. Surprisingly, however, no effects of elevated pco 2 were identified on mussels or algae. Moreover, when kept together, mussels and algae had mutually positive effects on each other's performance (i.e. mussel survival and condition index, mussel and algal biomass and proxies for algal productivity including relative maximum electron transport rate [rETRmax], saturating light intensity [I k] and maximum quantum yield [F v/F m]), independent of warming and acidification. Our results show that even moderate warming affected the functioning of key benthic species, and we identified a level of resistance to predicted ocean acidification. Importantly, we show that the presence of a second functional group enhanced the functioning of both groups (mussels and algae), independent of changing environmental conditions, which highlights the ecological and potential economic benefits of conserving biodiversity in marine ecosystems.

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Vibrio parahaemolyticus and microplastics are prevalent in the ocean. Bacteria attach onto plastic particles, forming harmful biofilms that collectively threaten bivalve health. This study investigates the interaction between polyamide microplastics (PA: particle size 38 ± 12 µm) and V. parahaemolyticus, as well as their combined impact on thick-shelled mussels (Mytilus coruscus). We introduced 1011 CFU/L of V. parahaemolyticus into varying PA concentrations (0, 5, 50, and 500 particles/L) to observe growth over 14 h and biofilm formation after 48 h. Our findings indicate that microplastics suppress biofilm formation and virulence gene expression. Four treatments were established to monitor mussel responses: a control group without PA or V. parahaemolyticus; a group with 50 particles/L PA; a group with 1011 CFU/L V. parahaemolyticus; and a co-exposure group with both 50 particles/L PA and 1011 CFU/L V. parahaemolyticus, over a 14-day experiment. However, combined stress from microplastics and Vibrio led to immune dysregulation in mussels, resulting in intestinal damage and microbiome disruption. Notably, V. parahaemolyticus had a more severe impact on mussels than microplastics alone, yet their coexistence reduced some harmful effects. This study is the first to explore the interaction between microplastics and V. parahaemolyticus, providing important insights for ecological risk assessments.

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Formalin baths are the most widely used treatment for ectoparasitic fish diseases. Nonetheless, their use in fish cages has been blamed for a number of problems. Although a considerable amount of literature has been produced on the short-term toxic effects of formaldehyde, there is virtually no data on the long-term side effects of the compound on non-target organisms. Therefore, the purpose of this research was to assess the long-term formaldehyde toxicity in Mediterranean mussel, Mytilus galloprovincialis, a common sentinel species that inhabits the area surrounding cage farms. Mussels were kept in a laboratory microenvironment at 20 ± 1 °C for 21 days and exposed to two different formaldehyde concentrations during experimentation: a low dose (L; 40 ppb) based on formaldehyde field measurements in the vicinity of Mediterranean cages, and a high dose (H; 400 ppb) generated by a factor of 10 of the previous dose. A multi-biomarker approach that included antioxidant enzymes such as catalase (CAT) and superoxide dismutase (SOD), lipid peroxidation (MDA), lysosomal stability (NRRT), genotoxicity tests, condition index (CI), and stress on stress (SoS), was used to evaluate the toxicity of formaldehyde on mussels. The results of the selected tests indicate that formaldehyde does not cause chronic toxicity in mussels subjected to commonly measured concentrations in the aquatic environment following formalin bath treatments. Despite being defined as reversible, the stress brought by the high dose used seems to reduce the antioxidant activity of the tested organism. The significance of this research lies in its contribution to understanding the wider ecological effects of formaldehyde exposure. Moreover, the results highlight the need for further research on other non-target marine organisms to fully understand the cumulative effects of formaldehyde on marine ecosystems.

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Accidental oil spills into the ocean can lead to downward transport and settling of oil onto the seafloor as part of marine snow, as seen during the Deepwater Horizon incident in 2010 in the Gulf of Mexico. The arctic and subarctic regions may favor conditions leading to this benthic oil deposition, prompting questions about the potential impacts on benthic communities. This study investigated the effects of oil-contaminated marine snow uptake on the blue mussel (Mytilus sp.). We exposed mussels for four days to 1) oil-contaminated marine snow (MOS treatment), or to 2) chemically-enhanced water-accommodated fraction (CEWAF) of oil plus unaggregated food particles (CEWAF treatment). Both oil treatments received the same nominal concentration of oil and food. Two controls were included: 1) Clean seawater plus unaggregated food (agg-free control) and 2) clean seawater plus marine snow (marine snow control). After the exposure, mussels were allowed to recover for ten days under clean, running seawater. Samples were taken right before and after the exposure period, and after the recovery phase for the following endpoints: distribution (partitioning) of oil compounds between seawater and MOS, and between seawater and mussel tissue; DNA damage (assessed via the comet assay); clearance rate; and condition index [tissue dry weight (g) divided by shell length (mm)]. Some discernable patterns were found in the partitioning of oil compounds between seawater and MOS. However, these patterns did not translate to any significant differences in the partitioning of oil compounds into mussel tissue between the two oil treatments. DNA damage did not exceed background levels (10% tail DNA or less; to be expected in healthy, viable cells) at any sampling time point, but significantly higher DNA damage was observed in CEWAF-T compared to MOS-T mussels after the recovery phase. After the exposure, a significant difference emerged in the clearance rate between the CEWAF treatment and the agg-free control, but not between the MOS treatment and the marine snow control. All mussels except those from the CEWAF treatment exhibited an increased condition index after the exposure time. Together, these results suggest that aggregates could moderate the effects of oil exposure on blue mussels, possibly by providing better, more concentrated nutrition than unaggregated food particles.

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Environmental impacts are a cause for concern when developing and expanding aquaculture and to be sustainable potential negative effects need to be addressed. The intensity and extent of these impacts likely vary among sites and seasons, depending on multiple factors including the physical and biological setting and operational aspects. Using a combination of sampling techniques, we investigated the spatial variability in epibenthic impacts in eleven commercial mussel farms, on the Swedish west coast. We found increased levels of organic content, changes in epibenthic macrofauna and increased cover of Beggiatoa sp., a documented indicator of hypoxia. The extent of these impacts was generally limited to the extent of the farms. Because the cover of Beggiatoa sp. was particularly clear and because oxygen conditions in the sediment is of great importance to the structure and function of these habitats, we analysed spatial patterns using an index of the benthic footprint (BFI) accounting for both intensity and extent of impacts. In the summer, the BFI varied strongly among farm-sites and subsequent analyses showed that it highly correlated with ambient bottom oxygen concentration. Repeated sampling during early spring, however, showed that impacts were quickly reversible also in the most impacted sites. Thus, we conclude that in Swedish coastal waters the benthic footprint calculated on the % cover of Beggiatoa sp. is highly dependent on ambient oxygen concentration. We suggest that knowledge about spatial and temporal patterns of oxygen in the bottom water can be used to predict the severity of impacts and provide an important criterion in a site-selection process aimed at developing a sustainable food industry.

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The homologous E6-AP carboxy-terminal structural domain (HECT) contained in E3 ubiquitin ligases (E3s) is a key factor in protein degradation and maintenance of cellular homeostasis in animals. However, the functional roles and evolutionary aspects of the HECT gene family in bivalve mussels remain unclear and warrant further investigation. In this study, we identified 22 HECT genes within the genome of Mytilus coruscus Gould, all containing a conserved HECT structural domain derived from dispersed repeats, distributed unevenly across 11 chromosomes. Phylogenetic analysis classified M. coruscus HECT genes into six major classes, with amino acid sequences within the same evolutionary clade displaying similar conserved motifs. Homology analysis with HECT genes of four bivalve species revealed that M. coruscus and Mytilus galloprovincialis possessed the largest number of homologous gene pairs, showing a significant correlation between the two in the evolution of the HECT gene family. Homology analysis with HECT genes of four bivalve species revealed that M. coruscus and M. galloprovincialis possessed the largest number of homologous gene pairs, showing a significant correlation between the two in the evolution of the HECT gene family. M. coruscus exhibited pronounced and specific expression in gills and blood tissues. Notably, Mco_UPL3 gene expression was significantly upregulated after 12 h of acute heat stress (33 °C) and 24 h of Vibrio injection (0.4 OD). Gene ontology analysis of the HECT genes in M. coruscus revealed that it is primarily enriched in protein modification and degradation functions. This suggests that HECT genes may play a key role in protein degradation and immunomodulation in M. coruscus. These findings offer valuable insights for the breeding of stress-tolerant traits in M. coruscus. In summary, our data shed light on the potential functions of HECT E3 ligases in response to heat stress and Vibrio infection, providing practical guidance for enhancing resilience through breeding in M. coruscus.

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In late summer and early autumn 2022, an intense bloom of Protoceratium reticulatum-the main yessotoxin (YTX) producer along Chilean coasts and a major threat to artisanal fisheries, the aquaculture industry, and environmental health-was recorded in the Patagonian fjord system. The high YTX levels (>3.75 mg kg-1) resulted in the first ban of shellfish collection in Chile. At Puyuhuapi Fjord, a global "hotspot" of harmful algal bloom events, the cell density of P. reticulatum determined in integrated tube samples (0-10 m) at the end of April 2022 reached 407,000 cells L-1. At the same time, YTX levels well exceeded the regulatory limit by roughly 2.5-fold, with concentrations as high as 9.42 mg kg-1 measured in native populations of the blue mussel Mytilus chilensis. Five different YTX analogues, 45-OH-YTX, COOH-45-keto-YTX, COOH-45-OH-YTX, COOH-YTX, and 45,55-diOH-YTX, were also detected in relevant amounts. While the ban lasted close to 3 months, accumulation and detoxification processes were monitored over a 1-year period. This study assessed the implications of high levels of YTXs and their analogues on the local economy and ecosystem health, given the increase in P. reticulatum blooms predicted for NW Patagonia in the context of a changing climate.

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Nanoparticles (NPs) are widely used in various fields, including antifouling paints for ships and industrial structures submerged in water. The potential impact of NPs on aquatic organisms, particularly their potential toxicity, is a significant concern, as their negative impact has been relatively poorly studied. In this study, we evaluated the effect of different concentrations of bimetallic Ag-TiO2 and ZnTi2O4-TiO2 NPs, which could potentially be used in antifouling coatings, on the hemocytes of the Mediterranean mussel Mytilus galloprovincialis. Hemocytes were exposed to NPs at concentrations of 0.1-1 mg/L for 1 and 2 h, and the production of reactive oxygen species (ROS), levels of DNA damage, and number of dead cells were measured. Exposure to Ag-TiO2 NPs at 1 mg/L concentration for 1 h suppressed ROS production in hemocytes and reduced the relative number of agranulocytes in cell suspensions, without inducing DNA damage or cell death. Exposure to ZnTi2O4-TiO2 NPs did not cause changes in the ratio of granulocytes to agranulocytes in suspensions, nor did it affect other functional parameters of hemocytes. However, after a 2 h exposure period, ZnTi2O4-TiO2 NPs (1 mg/L) significantly reduced the production of ROS by hemocytes. These findings suggest that Ag-TiO2 and ZnTi2O4-TiO2 NPs have low acute toxicity for marine bivalves.

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Ocean acidification (OA) caused by increased atmospheric carbon dioxide is affecting marine systems globally and is more extreme in coastal waters. A wealth of research to determine how species will be affected by OA, now and in the future, is emerging. Most studies are discrete and generally do not include the full life cycle of animals. Studies that include the potential for adaptation responses of animals from areas with different environmental conditions and the most vulnerable life stages are needed. Therefore, we conducted experiments with the widely distributed blue mussel, Mytilus edulis, from populations regularly exposed to different OA conditions. Mussels experienced experimental conditions prior to spawning, through embryonic and larval development, both highly vulnerable stages. Survivorship to metamorphosis of larvae from all populations was negatively affected by extreme OA conditions (pH 7.3, Ωar, 0.39, pCO2 2479.74), but, surprisingly, responses to mid OA (pH 7.6, Ωar 0.77, pCO21167.13) and low OA (pH 7.9, Ωar 1.53, pCO2 514.50) varied among populations. Two populations were robust and showed no effect of OA on survivorship in this range. One population displayed the expected negative effect on survivorship with increased OA. Unexpectedly, survivorship in the fourth population was highest under mid OA conditions. There were also significant differences in development time among populations that were unaffected by OA. These results suggest that adaptation to OA may already be present in some populations and emphasizes the importance of testing animals from different populations to see the potential for adaptation to OA.

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Perkinsus, a parasitic pathogen of marine bivalves, is widely distributed among various mollusks in numerous countries. However, the prevalence and diversity of Perkinsus species in the two economically important mussels, Mytilus coruscus and M. galloprovincialis, in China remain unknown. The presence of the Perkinsus species was identified in the two mussels sampled along the coast of the East China Sea and the Yellow Sea, using both the alternative Ray's fluid thioglycolate medium (ARFTM) and conventional polymerase chain reaction (PCR). The ARFTM test indicated the presence of Perkinsus-like hypnospores in the two mussels. The diameter of the hypnospores in M. coruscus was significantly smaller than that in M. galloprovincialis. The prevalence of Perkinsus in M. galloprovincialis and M. coruscus ranged from 0 to 37.5% and 0 to 25%, respectively. The mean intensity of Perkinsus in M. galloprovincialis and M. coruscus ranged from 0 to 5.14 and 0 to 4.92, respectively. The PCR assay showed that the prevalence of Perkinsus spp. in M. galloprovincialis and M. coruscus was 0 to 25.0% and 0 to 12.5%, respectively. The homology analysis of the newly obtained internal transcribed spacer (ITS) sequences of Perkinsus revealed the highest identity of 100% with P. beihaiensis. The phylogenetic analysis indicated that the Perkinsus isolates from the two mussels were clustered with P. beihaiensis. The results of the molecular biology indicated that only P. beihaiensis was detected in the two mussels. The highest prevalence of P. beihaiensis was observed in Liaoning province (Dalian, 20.83%), followed by Shandong province, Zhejiang province and Fujian province. Consequently, it is recommended that surveillance should be conducted in Dalian, where the prevalence and mean intensity of P. beihaiensis in M. galloprovincialis are the highest.

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In the marine environment, nanoparticles play a role in adsorbing and catalytically degrading organic pollutants, thereby mitigating their toxic effects on aquatic organisms. This study aimed to investigate the impact of nano titanium dioxide (nTiO2) and tris (2-chloropropyl) phosphate (TCPP) on the hemolymph and digestive function of the thick-shell mussel Mytilus coruscus. Mussels were divided into a control group, a group exposed to TCPP alone, a group exposed to a combination of TCPP and 0.5 mg/L nTiO2, and a group exposed to a combination of TCPP and 1 mg/L nTiO2. After 14 days of exposure, oxidative stress responses, including superoxide dismutase (SOD) activity and malondialdehyde (MDA) content, immune defense responses, including acid phosphatase (ACP) and alkaline phosphatase (AKP) activities, and gene expression, including HSP70 expression, were measured in the hemolymph and digestive glands of the mussels. Compared to the control group, mussels solely exposed to 100 µg/L TCPP exhibited a significant reduction in SOD activity in the hemolymph. When TCPP was co-exposed with 0.5 mg/L nTiO2, there were significant increases in MDA content and AKP activity in both the digestive gland and hemolymph compared to the control group. Upon co-exposure of TCPP with 1 mg/L nTiO2, MDA content and AKP activity in the digestive gland significantly decreased, while SOD, ACP, and AKP activity in the hemolymph significantly increased and MDA content significantly decreased, returning to the control group levels. Furthermore, in the combined exposure, HSP70 gene expression significantly decreased as the nTiO2 concentration increased from 0.5 mg/L to 1 mg/L. In summary, TCPP impacted the hemolymph and digestive function of mussels, whereas a concentration of 1 mg/L nTiO2 effectively alleviated the toxic effects of TCPP. This study is crucial for assessing the ecological risks of nanoparticles and emerging organic pollutants in marine environments, and provides new insights into the interaction between nTiO2 and TCPP, as well as the influence of nTiO2 concentration on mitigating TCPP toxicity.

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The effect of water acidification in combination with normoxia or hypoxia on the antioxidant capacity and oxidative stress markers in gills and hemolymph of the Mediterranean mussel (Mytilus galloprovincialis), as well as on gill microstructure, has been evaluated through an in vivo experiment. Mussels were exposed to a low pH (7.3) under normal dissolved oxygen (DO) conditions (8 mg/L), and hypoxia (2 mg/L) for 8 days, and samples were collected on days 1, 3, 6, and 8 to evaluate dynamic changes of physiological responses. Cytoplasmic concentrations of reactive oxygen species (ROS) and levels of DNA damage were measured in hemocytes, while the activity of catalase (CAT) and superoxide dismutase (SOD) and histopathological changes were assessed in gills. The results revealed that while water acidification did not significantly affect the activity of SOD and CAT in gills under normoxic and hypoxic conditions, there was a trend towards suppression of CAT activity at the end of the experimental period (day 8). Similarly, we did not observe increased formation of ROS in hemocytes or changes in the levels of DNA damage during the experimental period. These results strongly suggest that the oxidative stress response system in mussels is relatively stable to experimental conditions of acidification and hypoxia. Experimental acidification under normoxia and hypoxia caused changes to the structure of the gills, leading to various histopathological alterations, including dilation, hemocyte infiltration into the hemal sinuses, intercellular edema, vacuolization of epithelial cells in gill filaments, lipofuscin accumulation, changes in the shape and adjacent gill filaments, hyperplasia, exfoliation of the epithelial layer, necrosis, swelling, and destruction of chitinous layers (chitinous rods). Most of these alterations were reversible, non-specific changes that represent a general inflammatory response and changes in the morphology of the gill filaments. The dynamics of histopathological alterations suggests an active adaptive response of gills to environmental stresses. Taken together, our data indicate that Mediterranean mussels have a relative tolerance to water acidification and hypoxia at tissue and cellular levels.

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