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Microplastics (MPs) and organophosphate flame retardants (OPFRs) have recently become ubiquitous and cumulative pollutants in the oceans. Since OPFRs are added to or adsorbed onto MPs as additives, it is necessary to study the composite contamination of OPFRs and MPs, with less focus on bio-based PLA. Therefore, this study focused on the ecotoxicity of the biodegradable MP polylactic acid (PLA) (5 μm, irregular fragments, 10 2 and 10 6 particles/L), and a representative OPFRs tris(1-chloro-2-propyl) phosphate (TCPP, 0.5 and 50 μg/L) at environmental and high concentrations. The mussel Mytilus coruscus was used as a standardised bioindicator for exposure experiments. The focus was on examining oxidative stress (catalase, CAT, superoxide dismutase, SOD, malondialdehyde, MDA), immune responses acid (phosphatase, ACP, alkaline phosphatase, AKP, lysozyme, LZM), neurotoxicity (acetylcholinesterase, AChE), energy metabolism (lactate dehydrogenase, LDH, succinate dehydrogenase, SDH, hexokinase, HK), and physiological indices (absorption efficiency, AE, excretion rate, ER, respiration rate, RR, condition index, CI) after 14 days exposure. The results of significantly increased oxidative stress and immune responses, and significantly disturbed energy metabolism and physiological activities, together with an integrated biomarker response (IBR) analysis, indicate that bio-based PLA MPs and TCPP could cause adverse effects on mussels. Meanwhile, TCPP interacted significantly with PLA, especially at environmental concentrations, resulting in more severe negative impacts on oxidative and immune stress, and neurotoxicity. The more severe adverse effects at environmental concentrations indicate higher ecological risks of PLA, TCPP and their combination in the real marine environment. Our study presents reliable data on the complex effects of bio-based MP PLA, TCPP and their combination on marine organisms and the environment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Global climate change is a major trigger of unexpected temperature fluctuations. The impacts of marine heatwaves (MHWs) and nano-titanium dioxide (nano-TiO 2 ) 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-TiO 2 (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-TiO 2 exposure. However, even at ambient concentration (25 μg/L), prolonged nano-TiO 2 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-TiO 2 particles but increased the hydrodynamic diameter. Additionally, exposure to nano-TiO 2 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-TiO 2 . 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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

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 (nTiO 2 ) 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 nTiO 2 , and a group exposed to a combination of TCPP and 1 mg/L nTiO 2 . 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 nTiO 2 , 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 nTiO 2 , 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 nTiO 2 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 nTiO 2 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 nTiO 2 and TCPP, as well as the influence of nTiO 2 concentration on mitigating TCPP toxicity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

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 10 11 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 10 11 CFU/L V. parahaemolyticus; and a co-exposure group with both 50 particles/L PA and 10 11 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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Rayon microfibers, micro-sized semi-synthetic polymers derived from cellulose, have been frequently detected and reported as "micropollutants" in marine environments. However, there has been limited research on their ecotoxicity and combined effects with persistent organic pollutants (POPs). To address these knowledge gaps, thick-shell mussels (Mytilus coruscus) were exposed to rayon microfibers at 1000 pieces/L, along with polychlorinated biphenyls (PCBs) at 100 and 1000 ng/L for 14 days, followed by a 7-day recovery period. We found that rayon microfibers at the environmentally relevant concentration exacerbated the irreversible effects of PCBs on the immune and digestive systems of mussels, indicating chronic and sublethal impacts. Furthermore, the results of 16 s rRNA sequencing demonstrated significant effects on the community structure, species richness, and diversity of the mussels' intestinal microbiota. The branching map analysis identified the responsive bacteria to rayon microfibers and PCBs belonging to the Proteobacteria, Actinobacteriota, and Bacteroidota phyla. Despite not being considered a conventional plastic, the extensive and increasing use of rayon fibers, their direct toxicological effects, and their interaction with POPs highlight the need for urgent attention, investigation, and regulation to address their contribution to "micropollution"., Competing Interests: Declaration of Competing Interest The authors state that they have no known competing financial interests or personal relationships that could affect the work reported in this article., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Perfluorooctanoate (PFOA) is widely used as a surfactant and has metabolic, immunologic, developmental, and genetic toxicity on marine organisms. However, the effects of PFOA on individual defense functions in mussels in the presence of titanium dioxide nanoparticles (nano-TiO 2 ) are poorly understood. To investigate the defense strategies and regulatory mechanisms of mussels under combined stressors, the thick-shell mussels Mytilus coruscus were exposed to different PFOA concentrations (0, 2 and 200 μg/L) and nano-TiO 2 (0 and 0.1 mg /L, size: 25 nm) for 14 days. The results showed that, compared to the control group, PFOA and nano-TiO 2 significantly reduced the number of byssal threads (NBT), byssal threads length (BTL), diameter of proximal threads (DPB), diameter of middle threads (DMB), diameter of distal byssal threads (DDB), adhesive plaque area (BPA), and breaking force of byssal threads (N). Under the influence of PFOA and nano-TiO 2 , the morphological surface smoothness of the fractured byssal threads surface increased, concurrently inducing an increased surface roughness in the adhesive plaques. Additionally, under the presence of PFOA and nano-TiO 2 , the foot displayed dispersed tissue organization and damaged villi, accompanied by an increased incidence of cellular apoptosis and an upregulation of the apoptosis gene caspase-8. Expression of the adhesion gene mfp-3 and byssal threads strength genes (preCOL-D, preCOL-NG) was upregulated. An interactive effect on the performance of byssal threads is observed under the combined influence of PFOA and nano-TiO 2 . Under co-exposure to PFOA and nano-TiO 2 , the performance of the byssal threads deteriorates, the foot structure is impaired, and the genes mRNA expression of byssal thread secretory proteins have compensated for the adhesion and byssal threads strength by up-regulation. Within marine ecosystems, organic and particulate contaminants exert a pronounced effect on the essential life processes of individual organisms, thereby jeopardizing their ecological niche within community assemblages and perturbing the dynamic equilibrium of the overarching ecosystem. ENVIRONMENTAL IMPLICATION: Perfluorooctanoic acid (PFOA) is prone to accumulate in marine organisms. TiO 2 nanoparticles (nano-TiO 2 ) are emerging environmental pollutants frequently found in marine environment. The effects of PFOA and nano-TiO 2 on marine mussels are not well understood, and their toxic mechanisms remain largely unknown. We investigated the impacts of PFOA and nano-TiO 2 on mussel byssus defense mechanisms. By assessing byssus performance indicators, morphological structures of the byssus, subcellular localization, and changes in byssal secretion-related genes, we revealed the combined effects and mechanisms through which these two types of pollutants may affect the functional capabilities and survival of mussels in the complex marine ecosystem., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

With the wide use of nanomaterials in daily life, nano-titanium dioxide (nano-TiO 2 ) presents potential ecological risks to marine ecosystems, which can be exacerbated by ocean warming (OW). However, most previous studies have only centered around waterborne exposure, while there is a scarcity of studies concentrating on the impact of trophic transfer exposure on organisms. We investigated the differences in toxic effects of 100 μg/L nano-TiO 2 on mussels via two pathways (waterborne and foodborne) under normal (24 °C) and warming (28 °C) conditions. Single nano-TiO 2 exposure (waterborne and foodborne) elevated the superoxide dismutase (SOD) and catalase (CAT) activities as well as the content of glutathione (GSH), indicating activated antioxidatant response in the intestine. However, depressed antioxidant enzymes and accumulated peroxide products (LPO and protein carbonyl content, PCC) demonstrated that warming in combination with nano-TiO 2 broke the prooxidant-antioxidant homeostasis of mussels. Our findings also indicated that nano-TiO 2 and high temperature exhibited adverse impacts on amylase (AMS), trypsin (PS), and trehalase (THL). Additionally, activated immune function (lysozyme) comes at the cost of energy expenditure of protein (decreased protein concentration). The hydrodynamic diameter of nano-TiO 2 at 24 °C (1693-2261 nm) was lower than that at 28 °C (2666-3086 nm). Bioaccumulation results (range from 0.022 to 0.432 μg/g) suggested that foodborne induced higher Ti contents in intestine than waterborne. In general, the combined effects of nano-TiO 2 and warming demonstrated a more pronounced extent of interactive effects and severe damage to antioxidant, digestive, and immune parameters in mussel intestine. The toxicological impact of nano-TiO 2 was intensified through trophic transfer. The toxic effects of nano-TiO 2 are non-negligible and can be exerted together through both water- and foodborne exposure routes, which deserves further investigation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Micro-/nano-plastic particles (MNPs) are present in the ocean with potential detrimental impacts on marine ecosystems. Bivalves are often used as marine bioindicators and are ideal to evaluate the threat posed by various-sized MNPs. We exposed the mussel Mytilus coruscus to MNPs with different particle sizes (70 and 500 nm, 5, 10 and 100 μm) for 3, 72 h and 30 days. The antioxidant responses in digestive gland and the hemolymph were then evaluated. The time of exposure played a strong modulating role in the biological response. A 3-hour exposure had no significant impact on the digestive gland. After 72 h, an increase in oxidative stress was observed in the digestive gland, including increased hydrogen peroxide (H 2 O 2 ) level, catalase (CAT), glutathione peroxidase (GPx) activities and malondialdehyde (MDA) production. After a 30-day exposure, the oxidative stress decreased while lipid peroxidation increased. A 30-day exposure increased hemocyte mortality (HM) and reactive oxygen species (ROS) levels in the hemolymph, while phagocytosis (PA), lysosome content (LC), mitochondrial number (MN) and mitochondrial membrane potential (MMP) significantly decreased. Longer-term exposure to MNPs caused oxidative stress in the digestive gland as well as impaired viability and immunity of hemocytes. Particle size also influenced the response with smaller particles having more severe effects. A depuration for 7 days was enough to reverse the negative effects observed on the digestive gland and hemolymph. This study provides new insights on the effects of small-sized MNPs, especially nanoplastic particles (NPs), on aquatic organisms, and provides a solid theoretical knowledge background for future studies on toxic effects of MNPs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Organic and nanoparticle pollutants are the main environmental problems affecting marine species, which have received great attention. However, the combined effect of pollutants on marine life in the presence of predators needs to be clarified. In this study, the effects of pentachlorophenol (PCP) and titanium dioxide nanoparticles (nano-TiO 2 ) on the energy metabolism of mussels (Mytilus coruscus) in the presence of predators were assessed through cellular energy allocation (CEA) approach. Mussels were exposed to PCP (0, 1, and 10 μg/L), nano-TiO 2 (1 mg/L, 25 and 100 nm), and predators (Portunus trituberculatus presence/absence) for 14 days. Exposure to high concentrations of PCP (10 μg/L) with small particle size nano-TiO 2 (25 nm) decreased cellular energy stores (carbohydrates, lipids, and proteins) and increased cellular energy demand (measured as the activity of the mitochondrial electron transport system, ETS). During the first 7 days, energy was supplied mainly through the consumption of carbohydrates, while lipids are mobilized to participate after 7 days. The presence of predators caused a further decrease in energy stores. These findings demonstrate that PCP, nano-TiO 2 and predators have a negative impact on energy metabolism at the cellular level. Carbohydrates are not able to meet the metabolic demand, lipids need to be consumed, and energy metabolism was also mediated by the involvement of proteins. Overall, our results suggest that PCP, nano-TiO 2 and predators disrupt the cellular energy metabolism of mussels through reduced cellular energy allocation, small particles and predators drive mussels to exert energetic metabolic adjustments for detoxification reactions when toxic contaminants are present., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Coastal habitats are exposed to increasing pressure of nanopollutants commonly combined with warming due to the seasonal temperature cycles and global climate change. To investigate the toxicological effects of TiO 2 nanoparticles (TiO 2 NPs) and elevated temperature on the intestinal health of the mussels (Mytilus coruscus), the mussels were exposed to 0.1 mg/L TiO 2 NPs with different crystal structures for 14 days at 20 °C and 28 °C, respectively. Compared to 20 °C, the agglomeration of TiO 2 NPs was more serious at 28 °C. Exposure to TiO 2 NPs led to elevated mortality of M. coruscus and modified the intestinal microbial community as shown by 16S rRNA sequence analysis. Exposure to TiO 2 NPs changed the relative abundance of Bacteroidetes, Proteobacteria and Firmicutes. The relative abundances of putative mutualistic symbionts Tenericutes and Fusobacteria increased in the gut of M. coruscus exposed to anatase, which have contributed to the lower mortality in this group. LEfSe showed the combined stress of warming and TiO 2 NPs increased the risk of M. coruscus being infected with potential pathogenic bacteria. This study emphasizes the toxicity differences between crystal structures of TiO 2 NPs, and will provides an important reference for analyzing the physiological and ecological effects of nanomaterial pollution on bivalves under the background of global climate change., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Pollution with complex mixtures of contaminants including micro- and nano-plastics (MNPs) and organic pollutants like polycyclic aromatic hydrocarbons (PAH) poses a major threat to coastal marine ecosystems. Toxic mechanisms of contaminant mixtures are not well understood in marine organisms. We studied the effects of single and combined exposures to polycyclic aromatic hydrocarbon phenanthrene (Phe) and MNPs mixture with sizes of 70 nm, 5 μm and 100 μm on the immune health and oxidative stress parameters in the thick-shell mussel Mytilus coruscus. Immune cells (hemocytes) were more sensitive to the pollutant-induced oxidative stress than the gills. In hemocytes of co-exposed mussels, elevated mortality, lower lysosomal content, high production of reactive oxygen species (ROS) and decrease mitochondrial were found. Disparate responses of antioxidant enzymes in the hemolymph (e.g. increased superoxide dismutase (SOD) activity without a corresponding increase in catalase (CAT) in Phe exposures and an increase in CAT without a change in SOD in MNPs exposures) suggests misbalance of the antioxidant defense in the pollutant-exposed mussels. Gill lacked pronounced oxidative stress response showing a decline in ROS and antioxidant levels. Tissue-specific single and combined effects of Phe and MNPs suggest variation in bioavailability and/or different sensitivity to these pollutants in the studied tissues. Notably, the combined effects of MNPs and Phe were additive or antagonistic, showing that MNPs do not enhance and occasionally mitigate the toxic effects of Phe on the hemocytes and the gills of the mussels. Overall, our study sheds light on the impact of long-term exposure to MNPs and Phe mixtures on mussels, showing high sensitivity of the immune system and modulation of the Phe toxicity by MNPs co-exposure. These findings that may have implications for understanding the impacts of combined PAH and MNPs pollution on the health of mussel populations from polluted coastal habitats., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Nanoplastics (NPs) and antibiotics (ABs) are two of the emerging marine contaminants that have drawn the most attention in recent years. Given the necessity of figuring out the effects of plastic and antibiotic contamination on marine organism life and population in the natural environment, it is essential to apply rapid and effective biological indicators to evaluate their comprehensive toxic effects. In this study, using mussel (Mytilus coruscus) as a model, we investigated the combined toxic effects of NP (80 nm polystyrene beads) and AB (Norfloxacin, NOR) at environmental-relevant concentrations on antioxidant and immune genes. In terms of the antioxidant genes, NPs significantly increased the relative expression of Cytochrome P450 3A-1 (CYP3A-1) under various concentrations of NOR conditions, but they only significantly increased the relative expression of CYP3A-2 in the high concentration (500 μg L -1 NOR) co-exposure group. In the NP-exposure group which exposed to no or low concentrations of NOR, nuclear factor erythroid 2-related factor 2 (Nrf2) was upregulated. In terms of the immune genes, interleukin-1 receptor-associated kinase (IRAK) -1 showed a significant increase in the low-concentration NOR group while a significant inhibition in the high-concentration NOR group. Due to the presence of NPs, exposure to NOR resulted in a significant increase in both IRAK-4 and heat shock protein (HSP) 70. Our findings indicate that polystyrene NPs can exacerbate the effects of NOR on the anti-oxidant and immune defense performance of mussels. This study delves into the toxic effects of NPs and ABs from a molecular perspective. Given the expected increase in environmental pollution due to NPs and ABs, future research is needed to investigate the potential synergistic effect of NPs and ABs on other organisms., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Youji Wang reports financial support was provided by State Key Laboratory of Estuarine and Coastal Research. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Numerous studies have shown that microplastics can interact with other pollutants in the environment to produce synergistic effects, leading to more serious impacts. To date, there is little consensus on the combined effects of microfibers (MFs) and polychlorinated biphenyls (PCBs, Aroclor 1254), two legacy and alarming environmental pollutants. There is an urgent need to assess the impact of combined exposures on bivalve immune defences. In this study, we assessed the immune response of the mussels (Mytilus coruscus) hemocyte to MFs and PCBs alone and in combination by using flow cytometry. M. coruscus were exposed to MFs (1000 pieces/L) and PCBs (PCBs) (100 ng/L and 1000 ng/L) alone or in combination for 14 consecutive days and recovered for 7 days. The hemocyte of M. coruscus was collected on day 7, 14 and 21. MF exposure alone had no effect on the hemocyte. The total hemocyte count (THC), esterase (EA), lysosomal contents (LC), mitochondrial number (MN) and mitochondrial membrane potential (MMP) of mussels showed a decreasing trend with increasing PCB concentrations, both individually and in combination; The decreases in EA, MN and MMP were associated with the induction of reactive oxygen species (ROS). Hemocyte mortality (HM) was associated with a decrease in THC. Combined exposure to MFs and PCBs would exacerbate the effects on hemocyte immunity. These new findings improve our understanding of the toxic effects of MFs and organic chemical pollutants, and demonstrate the potential mechanism of PCBs to bivalves through changes in hemolymph immunity-related indicators., Competing Interests: Declaration of competing interest The authors state that they have no known competing financial interests or personal relationships that could affect the work reported in this article., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Nano-TiO 2 can act as a vector to organic compounds, such as pentachlorophenol (PCP) posing a potential threat to the marine ecosystems. Studies showed that nano pollutant toxicity can be modulated by abiotic factors, but little is known about the potential influence of biotic stressors (such as predators) on the physiological responses to pollutants in marine organisms. We explored the effects of n-TiO 2 and PCP on the mussel Mytilus coruscus in the presence of its natural predator, the swimming crab Portunus trituberculatus. Exposure to n-TiO 2 , PCP, and predation risk showed interactive effects on antioxidant and immune parameters of the mussels. Elevated activities of catalase (CAT), glutathione peroxidase (GPX), acid phosphatase (ACP) and alkaline phosphatase (AKP), suppressed activity of superoxide dismutase (SOD), lower levels of glutathione (GSH) and increased malondialdehyde (MDA) levels indicated dysregulation of the antioxidant system and immune stress induced by single PCP or n-TiO 2 exposure. Integrated biomarker (IBR) response values showed the effect of PCP was concentration dependent. Of the two used n-TiO 2 sizes (25 and 100 nm), larger particles induced higher antioxidant and immune disturbances indicating higher toxicity possibly due to higher bioavailability. Compared to single PCP exposure, the combination of n-TiO 2 and PCP enhanced the imbalance of SOD/CAT and GSH/GPX and led to elevated oxidative lesions and activation of immune-related enzymes. Overall, the combined impacts of pollutants and biotic stress exhibited a greater magnitude of adverse effects on antioxidant defense and immune parameters in mussels. The toxicological effects of PCP were exacerbated in the presence of n-TiO 2 , and the deleterious impact of these stressors was further amplified under predator-induced risk after prolonged (28 days) exposure. However, the underlying physiological regulatory mechanisms governing the interplay of these stressors and predatory cues on mussels remain elusive, warranting further investigation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Polybrominated diphenyl ethers (PBDEs) are one of the most used halogenated flame retardants worldwide, and exert neurotoxicity, reproductive toxicity, endocrine interference, and carcinogenic effects on organisms. However, there are insufficient studies on the physical and immune defense at the individual level of mussels under different food conditions. To explore the defense strategy and individual health status, the thick-shelled mussels Mytilus coruscus were exposed to different BDE-47 concentrations (0, 0.1 and 10 μg/L) and nutritional conditions (feeding and starvation) for 21 days. The results showed that BDE-47 exposure and starvation significantly decreased the number of byssus threads (NBT), adhesion, and condition index (CI) of mussels, whereas increased the reactive oxygen species (ROS) production and the combined stress further declined the CI. BDE-47 exposure and starvation induced decreased adhesive capability and healthy state along with oxidative lesions in mussels. The downregulation gene expression of foot adhesion proteins (mfp-2/3/4/5/6) under starvation or combined exposure also proved the reduced adhesion of mussels. However, up-regulated mfp-1 and pre-collagens proteins (preCOL-D/P/NG) indicated mussels would adjust energy allocation to enhance the strength and extensibility of byssal threads for compensating reduced adhesion and CI. As global climate change and organic pollution have dramatically impacted the ocean, hazardous substances and the fluctuated primary productivity have frequently co-occurred, which will affect the structure of coastal biomes and fishery production., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Intestinal microbes are essential participants in host vital activities. The composition of the microbiota is closely related to the environmental factors. Predator presence may impact on intestinal microbiota of prey. In the present study, stone crab Charybdis japonica was used as potential predator, an external stress on mussel Mytilus coruscus, to investigate the intestinal microbiota alteration in M. coruscus. We set up two forms of predator presence including free crab and trapped crab, with a blank treatment without crab. The composition of intestinal microbiota in mussels among different treatments showed significant differences by 16S rRNA techniques. The biodiversity increased with trapped crab presence, but decreased with free crab presence. Neisseria, the most abundant genus, fell with the presence of crabs. Besides, the Arcobacter, a kind of pathogenic bacteria, increased with free crab presence. Regarding PICRUTs analysis, Environmental Information Processing, Genetic Information Processing and Metabolism showed differences in crab presence treatments compared with the blank, with a bit higher in the presence of free crab than trapped crab. In conclusion, trapped crab effects activated the metabolism and immunity of the intestinal flora, but free crabs made mussels more susceptible to disease and mortality, corresponding to the decreased biodiversity and the increased Arcobacter in their intestine., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Nano‑titanium dioxide (nTiO 2 ) is a widely used nanomaterial posing potential ecological risk for marine ecosystems that might be enhanced by elevated temperatures such as expected during climate change. nTiO 2 may affect benthic filter feeders like mussels through waterborne exposures and via food chain due to the adsorption on/in algae. Mussel byssus are proteinaceous fibers secreted by byssal glands of the mussels for attachment. Byssus production and mechanical properties are sensitive to environmental stressors but the combined effects of warming and nTiO 2 on byssus performance of mussels are unclear hampering our understanding of the predation and dislodgement risk of mussels under the multiple stressor scenarios. We explored the effects of a short-term (14-day) single and combined exposures to warming (28 °C) and 100 μg L -1 nTiO 2 (including food co-exposure) on the byssus performance of the thick shell mussel Mytilus coruscus. The mechanical strength (measured as the breaking force) of the byssal threads was impaired by warming and nTiO 2 (including food co-exposure), but the number and length of the byssal threads were increased. The mRNA expression levels of mussel foot proteins (mfp-3, mfp-5) and pre-collagens (preCOL-D, preCOL-P, preCOL-NG) were up-regulated to varying degrees, with the strongest effects induced by warming. This indicates that the physiological and molecular mechanisms of byssus secretion are plastic. However, downregulation of the mRNA expression of preCOL-D and preCOL-P under the combined warming and nTiO 2 exposures indicate the limits of these plasticity mechanisms and suggest that the attachment ability and survival of the mussels may be impaired if the pollution or temperature conditions further deteriorate., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Antibiotics and nanoplastics (NPs) are among the two most concerned and studied marine emerging contaminants in recent years. Given the large number of different types of antibiotics and NPs, there is a need to apply efficient tools to evaluate their combined toxic effects. Using the thick-shelled mussel (Mytilus coruscus) as a marine ecotoxicological model, we applied a battery of fast enzymatic activity assays and 16S rRNA sequencing to investigate the biochemical and gut microbial response of mussels exposed to antibiotic norfloxacin (NOR) and NPs (80 nm polystyrene beads) alone and in combination at environmentally relevant concentrations. After 15 days of exposure, NPs alone significantly inhibited superoxide dismutase (SOD) and amylase (AMS) activities, while catalase (CAT) was affected by both NOR and NPs. The changes in lysozyme (LZM) and lipase (LPS) were increased over time during the treatments. Co-exposure to NPs and NOR significantly affected glutathione (GSH) and trypsin (Typ), which might be explained by the increased bioavailable NOR carried by NPs. The richness and diversity of the gut microbiota of mussels were both decreased by exposures to NOR and NPs, and the top functions of gut microbiota that were affected by the exposures were predicted. The data fast generated by enzymatic test and 16S sequencing allowed further variance and correlation analysis to understand the plausible driving factors and toxicity mechanisms. Despite the toxic effects of only one type of antibiotics and NPs being evaluated, the validated assays on mussels are readily applicable to other antibiotics, NPs, and their mixture., Competing Interests: Declaration of competing interest The authors state that they have no known competing financial interests or personal relationships that could affect the work reported in this article., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

With the development of industry, agriculture and intensification of human activities, a large amount of nano-TiO 2 dioxide and pentachlorophenol have entered aquatic environment, causing potential impacts on the health of aquatic animals and ecosystems. We investigated the effects of predators, pentachlorophenol (PCP) and nano titanium dioxide (nano-TiO 2 ) on the gut health (microbiota and digestive enzymes) of the thick-shelled mussel Mytilus coruscus. Nano-TiO 2 , as the photocatalyst for PCP, enhanced to toxic effects of PCP on the intestinal health of mussels, and they made the mussels more vulnerable to the stress from predators. Nano-TiO 2 particles with smaller size exerted a larger negative effect on digestive enzymes, whereas the size effect on gut bacteria was insignificant. The presence of every two of the three factors significantly affected the population richness and diversity of gut microbiota. Our findings revealed that the presence of predators, PCP, and nano-TiO 2 promoted the proliferation of pathogenic bacteria and inhibited digestive enzyme activity. This research investigated the combined stress on marine mussels caused by nanoparticles and pesticides in the presence of predators and established a theoretical framework for explaining the adaptive mechanisms in gut microbes and the link between digestive enzymes and gut microbiota., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

As a good carrier of contaminants, nanotitanium dioxide (nTiO 2 ) can absorb organic pollutants, producing toxicological effects on organisms. However, the complex effects of nTiO 2 with contaminants on marine mussels are still unclear. In this study, we exposed mussels to tris (2-chloropropyl) phosphate (TCPP) 100 μg/L (T1), 0.5 mg/L nTiO 2 + 100 μg/L TCPP (T2), 1.0 mg/L nTiO 2 + 100 μg/L TCPP (T3) and control (0 nTiO 2 + 0 μg/L TCPP) treatments, and assessed the combined effects of TCPP with nTiO 2 on the thick-shelled mussel Mytilus coruscus by detecting the activities of gill pyruvate kinase (PK), hexokinase (HK), lactate dehydrogenase (LDH) and succinate dehydrogenase (SDH), also gill acetylcholine (Ach) and muscle lactic acid (LD) contents and gut microbiota after 14-d exposure. Compared with the control group, PK activity was increased significantly, but SDH, LDH activities and LD content were decreased significantly in T1, with the addition of nTiO 2 , there were not significantly different in T3. However, Ach content in T3 was significantly higher than the control and T1. Moreover, KEGG of the gut microbiota via 16 s rRNA sequencing showed that most pathways returned to the control level in T3. The results showed that TCPP affected the respiratory metabolism of mussels, changed the community structure of intestinal microflora in mussels, and nTiO 2 alleviated the toxicity of TCPP. Our study provides new insights for ecological risk assessment of TCPP in bivalves in the complex aquatic environment and the novel role of nTiO 2 in regulating the toxicity of TCPP., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Ocean acidification may increase the risk of disease outbreaks that would challenge the future persistence of marine organisms if their immune system and capacity to produce vital structures for survival (e.g., byssus threads produced by bivalves) are compromised by acidified seawater. These potential adverse effects may be exacerbated by microplastic pollution, which is forecast to co-occur with ocean acidification in the future. Thus, we evaluated the impact of ocean acidification and microplastics on the health of a mussel species (Mytilus coruscus) by assessing its physiological performance, immunity and byssus properties. We found that ocean acidification and microplastics not only reduced hemocyte concentration and viability due to elevated oxidative stress, but also undermined phagocytic activity of hemocytes due to lowered energy budget of mussels, which was in turn caused by the reduced feeding performance and energy assimilation. Byssus quality (strength and extensibility) and production were also reduced by ocean acidification and microplastics. To increase the chance of survival with these stressors, the mussels prioritized the synthesis of some byssus proteins (Mfp-4 and Mfp-5) to help maintain adhesion to substrata. Nevertheless, our findings suggest that co-occurrence of ocean acidification and microplastic pollution would increase the susceptibility of bivalves to infectious diseases and dislodgement risk, thereby threatening their survival and undermining their ecological contributions to the community., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Ocean acidification and microplastic pollution is a global environmental threat, this research evaluated the effects of ocean acidification and microplastics on mussel digestive tract microbial community. The 16S rRNA gene was sequenced to characterize the flora. Species diversity in the samples was assessed by clustering valid tags on 97% similarity. Bacteroidetes, Firmicutes and Proteobacteria were the three most abundant genera in the four groups, with Bacteroidetes showing the highest diversity. However, no differences in flora structure were evident under various treatments. Phylogenetic relationship analysis revealed Bacteroidetes and Firmicutes had the highest OTU diversity. The weighted UniFrac distance, principal coordinate analysis (PCoA), unweighted pair group method with arithmetic mean (UPGMA) cluster tree and analysis of molecular variance (AMOVA) evaluation results for all samples also showed that changes in pH and microplastics concentration did not significantly affect the microbial community structure in the mussel digestive tract. The results presented the no significant effects of ocean acidification and microplastics intake on mussel intestinal diversity., (© 2020. Springer Science+Business Media, LLC, part of Springer Nature.)

This study investigated the combined effects of seawater acidification and hypoxia on the antioxidant response in gonads of the thick shell mussel Mytilus coruscus. Mussels were collected along the Shengsi Island, East China Sea, where oxygen and pH fluctuations frequently occur in summer. Mussels were exposed to three pH (8.1, 7.7, and 7.3) and two dissolved oxygen (DO) levels (6 and 2 mg L -1 ) for 21 days followed by a 10-day recovery period (pH 8.1 and DO 6 mg L -1 ). Gonad surface area (GSA) and activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), glutathione (GSH), glutathione S-transferase (GST), and malondialdehyde (MDA) in gonad were measured at days 21 and 31. Complex and enzyme-specific responses were observed after the 21-day exposure period. Overall, PCA analysis revealed a stronger effect of pH than DO. Integrated biomarker response (IBR) analysis demonstrated that low pH and DO decreased mussel's antioxidant system and increased oxidative damage with potential consequences for gonad development. Mussels exposed to low pH and DO were only partly able to recover a normal enzymatic activity after 10-day recovery period. This suggests that mussels exposed to short-term pH and DO fluctuations event in the field may suffer lasting negative impacts., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Microplastics (MPs) have become a ubiquitous emerging pollutant in the global marine environment. The potential toxic effects of MPs and interactions of MP pollution with other stressors such as food limitation on marine organisms' health are not yet well understood. This study investigated the effects of three-week exposure to different MPs and food shortage on the physical defense mechanisms (byssus production and properties) of Mytilus coruscus. Starvation significantly reduced the number of byssus threads, and combined exposure to MPs and food shortage suppressed the adhesion ability and condition index of mussels. The length of the byssus threads was not affected by all experimental exposures. Transcript levels of genes encoding key proteins involved in byssus formation (the mussel foot proteins mfp-1, -2, -3, -4, -5 and -6, and prepolymerized collagen proteins preCOL-D, -P and -NG) were altered by interactions between the MPs and food shortage. These findings show that insufficient food supply can exacerbate the adverse effects of MPs on mussel defense which might have implications for survival and fitness of mussels under food limited conditions (e.g. in winter) in polluted coastal habitats., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Marine microplastic has become an important environmental issue of global concern due to its wide distribution and harmful impacts. However, there is still insufficient information on the toxicity mechanism of microplastics to marine organisms. In this study, we developed and applied a high-coverage quantitative metabolomics technique to investigate the toxicity mechanisms of the polystyrene microspheres (micro-PS) on marine mussels (Mytilus coruscus). A total of 3599 metabolites were quantified, including 163 positively identified metabolites, 318 high-confident putatively identified metabolites, and 2602 mass-matched metabolites from the hemolymph of mussels. Metabolomics analysis indicated that micro-PS disrupted the amino acid metabolism, particularly phenylalanine metabolism, which may lead to oxidative stress and neurotoxicity. Micro-PS at environmentally relevant concentrations induced oxidative stress and immunotoxicity in mussels. After 7 days of recovery, along with the significant clearance of micro-PS by mussels, both metabolite levels and biochemical indicators generally returned to the same level as the control group. Overall, the results showed that microplastics at environmentally-relevant concentrations can cause toxic effects on mussels but these influences are reversible. We envisage the usages of high-coverage metabolomics for investigating the toxicity of various types of microplastics under many different conditions, including those relevant to the marine environment., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Ocean acidification and hypoxia have become increasingly severe in coastal areas, and their co-occurrence poses emerging threats to coastal ecosystems. Here, we investigated the combined effects of ocean acidification and hypoxia on the reproductive capacity of the thick-shelled mussel Mytilus coruscus. Our results demonstrated low pH but not low oxygen induced decreased gonadosomatic index (GSI) in mussels. Male mussels had a lower level of sex steroids (estradiol, testosterone, and progesterone) when kept at low pH. Expression of genes related to reproduction were also impacted by low pH with a downregulation of genes involved in gonad development in males (β-catenin and Wnt-7b involved in males) and an upregulation of testosterone synthesis inhibition-related gene (Wnt-4) in females. Overall, our results suggest that ocean acidification has an impact on the gonadal development through an alternation of gene expression and level of steroids while hypoxia had no significant effect., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Plastic particles cause toxic effects on marine organisms but whether food particles can affect the toxic effects of plastic particles on filter feeding animals remains unknown. To evaluate the intake and physiological effects of different size particles and their exposure ways, the thick shell mussels Mytilus coruscus were exposed to polystyrene (PS) nanoplastics (NPs, 70 nm) and microplastics (MPs, 10 µm) respectively for two weeks by mixing NPs/MPs with microalgae or exposed to MNPs individually after feeding. Intake of particles and their physiological effects including energy budget, digestive enzymes and oxidative responses were assessed after exposure. Results indicated food presence mediate the effects while MPs decrease the energy budget and increase the catalase activity and malondialdehyde levels. Moreover, exposure way significantly affected energy budget and size of particle had a significant impact on enzyme activities. Our results showed MPs induce more significant effects than NPs on mussels, emphasized the importance of particle exposure way and suggested that mixture exposure with microalgae alleviate the influences on mussels caused by plastic particles alone. This study emphasized that we need to take the food particles into account for evaluating the toxic effects of plastic particles on filter feeding animals in the natural environment., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Plastic particles are thought to accumulate in aquatic organisms and cause potential physiological effects. The uneven sizes of plastic particles may affect the ingestion by marine filter feeding bivalves and may lead to differential further physiological effects. To tackle this scientific question, we investigated the size dependent ingestion and dynamic accumulation of nano/micro plastic particles with different diameters (0.07, 0.5, 5, 10 and 100 μm) in the thick shell mussel Mytilus coruscus. The accumulation of particles in gill, digestive tract and mantle of mussels was measured after 3, 15, 87 h exposure and following 87 h depuration. The results showed that particle ingestion was negatively size dependant and positively related to time in digestive tract. In mantle, particles accumulated over the depuration time with a delay, indicating the translocation of particles. Moreover, our results showed that gill was not a target tissue for steady particle accumulation but the digestive tract was. This study showed size dependent and dynamic ingestion of nano/micro particles in mussels which are one of the main marine organisms for accumulating microplastics., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

In the present study, the combined effects of pH, dissolved oxygen (DO) and temperature levels on the antioxidant responses of the mussel Mytilus coruscus were evaluated. Mussels were exposed to two pH (8.1, 7.7-acidification), two DO (6 mg L -1 , 2 mg L -1 -hypoxia) and two temperature levels (20 °C, 30 °C-warming) for 30 days. SOD, CAT, MDA, GPx, GSH, GST, TAOC, AKP, ACP, GPT, AST levels were measured in the gills of mussels. All tested biochemical parameters were altered by these three environmental stressors. Values for all the test parameters except GSH first increased and then decreased at various experimental treatments during days 15 and 30 as a result of acidification, hypoxia and warming. GSH content always increased with decreased pH, decreased DO and increased temperature. PCA showed a positive correlation among all the measured biochemical indexes. IBR results showed that M. coruscus were adversely affected by reduced pH, low DO and elevated temperature., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The highly hydrophobic surfaces make microplastics a potential carrier of organic pollutants in the marine environment. In order to explore the toxic effects of polybrominated diphenyl ethers (BDE-47) combined with microplastics on marine organisms, we exposed the marine mussel Mytilus coruscus to micro-PS combined with BDE-47 for 21 days to determine the immune defense, oxidative stress and energy metabolism of the mussels. The results showed that the clearance rate (CR) of mussels exposed to single micro-PS, single BDE-47 or both was lower than control group. In general, compared to single BDE-47 exposure, the combination of micro-PS and BDE-47 significantly increased respiration rate (RR), activities of acid phosphatase (ACP) and alkaline phosphatase (ALP), reactive oxygen species (ROS) production and malondialdehyde (MDA) concentrations, but significantly decreased lactate dehydrogenase (LDH) activity and the relative expression of heat shock protein (Hsp70 and 90). Overall, combined stress has more adverse effects on defense performance and energy metabolism in mussels and micro-PS seem to exacerbate the toxicological effects of BDE-47. As microplastics pollution may deteriorate in the future, the health of mussels may be threatened in organically polluted environment, which eventually change the stability of the structure and function of intertidal ecosystem., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Global change and anthropogenic activities have driven marine environment changes dramatically during the past century, and hypoxia, acidification and warming have received much attention recently. Yet, the interactive effects among these stressors on marine organisms are extremely complex and not accurately clarified. Here, we evaluated the combined effects of low dissolved oxygen (DO), low pH and warming on the digestive enzyme activities of the mussel Mytilus coruscus. In this experiment, mussels were exposed to eight treatments, including two degrees of pH (8.1, 7.7), DO (6, 2 mg/l) and temperature (30 °C and 20 °C) for 30 days. Amylase (AMS), lipase (LPS), trypsin (TRY), trehalase (TREH) and lysozyme (LZM) activities were measured in the digestive glands of mussels. All the tested stress conditions showed significant effects on the enzymatic activities. AMS, LPS, TRY, TREH showed throughout decreased trend in their activities due to low pH, low DO, increased temperature and different combinations of these three stressors with time but LZM showed increased and then decreased trend in their activities. Hypoxia and warming showed almost similar effects on the enzymatic activities. PCA showed a positive correlation among all measured biochemical parameters. Therefore, the fitness of mussel is likely impaired by such marine environmental changes and their population may be affected under the global change scenarios., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Ocean acidification and hypoxia are concurrent in some coastal waters due to anthropogenic activities in the past decades. In the natural environment, pH and dissolved oxygen (DO) may fluctuate and follow diel-cycling patterns, but such effects on marine animals have not been comprehensively studied compared to their constant effects. In order to study the effects of diel-cycling seawater acidification and hypoxia on the fitness of marine bivalves, the thick shell mussels Mytilus coruscus were exposed to two constant levels of dissolved oxygen (2 mg/L, 8 mg/L) under two pH treatments (7.3, 8.1), as well as single diel fluctuating pH or DO, and the combined diel fluctuating of pH and DO for three weeks. The experimental results showed that constant acidification and hypoxia significantly reduced the extracellular pH (pH e ) and condition index (CI) of mussels, and significantly increased HCO 3 - , pCO 2 and standard metabolic rate (SMR). Diel fluctuating hypoxia and acidification also significantly reduced the pH e and CI, and significantly increased pCO 2 and SMR, but had no significant effects on HCO 3 - . However, the diel-cycling acidification and hypoxia resulted in a higher CI compared to continuous exposure. In general, continuous and intermittent stress negatively impact the hemolymph and growth performance of mussels. However, mussels possess a little stronger resistance to diel-cycling seawater acidification and hypoxia than sustained stress., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

In the marine environment, microplastic contamination and acidification may occur simultaneously, this study evaluated the effects of ocean acidification and microplastics on oxidative stress responses and digestive enzymes in mussels. The thick shell mussels Mytilus coruscus were exposed to four concentrations of polystyrene microspheres (diameter 2 μm, 0, 10, 10 4 and 10 6 particles/L) under two pH levels (7.7 and 8.1) for 14 days followed by a 7-day recovery acclimation. Throughout the experiment, we found that microplastics and ocean acidification exerted little oxidative stress to the digestive gland. Only catalase (CAT) and glutathione (GSH) showed a significant increase along with increased microplastics during the experiment, but recovered to the control levels once these stressors were removed. No significant effects of pH and microplastics on glutathione peroxidase (GPx) and superoxide dismutase (SOD) were observed. The responses of digestive enzymes to both stressors were more pronounced than antioxidant enzymes. During the experiment, pepsin (PES), trypsin (TRS), alpha-amylase (AMS) and lipase (LPS) were significantly inhibited under microplastics exposure and this inhibition was aggravated by acidification conditions. Only PES and AMS tended to recover during the recovery period. Lysozyme (LZM) increased significantly under microplastic exposure conditions, but acidification did not exacerbate this effect. Therefore, combined stress of microplastics and ocean acidification slightly impacts oxidative responses but significantly inhibits digestive enzymes in mussels., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Both nanoparticles (NPs) and ocean acidification (OA) pose threats to marine animals as well as marine ecosystems. The present study aims to evaluate the combined effects of NPs and OA on specific dynamic action (SDA) of mussels. The thick shell mussels Mytilus coruscus were exposed to two levels of pH (7.3 and 8.1) and three concentrations of TiO 2 NPs (0, 2.5, and 10 mg L -1 ) for 14 days followed by a 7-day recovery period. The SDA parameters, including standard metabolic rate, peak metabolic rate, aerobic metabolic scope, SDA slope, time to peak, SDA duration and SDA, were measured. The results showed that TiO 2 NPs and low pH significantly affected all parameters throughout the experiment. When the mussels were exposed to seawater acidification or TiO 2 NPs conditions, standard metabolic rate, aerobic metabolic scope, SDA slope and SDA significantly decreased, whereas peak metabolic rate, time to peak and SDA duration significantly increased. In addition, interactive effects between TiO 2 NPs and pH were observed in SDA parameters except time to peak and SDA. Therefore, the synergistic effect of TiO 2 NPs and low pH can adversely affect the feeding metabolism of mussels., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

With the development of nanotechnology and increased nanomaterial application, TiO 2 nanoparticles have been released into the aquatic environment, causing potential ecotoxicological effects on aquatic organisms. Ocean acidification caused by anthropogenic CO 2 is one of the most common environmental stressors, occurring simultaneously with marine contaminants, e.g., nanoparticles. Marine bivalves can accumulate nanoparticles and their digestive functions may be affected. In this study, we investigated the potential influences of TiO 2 nanoparticles on the digestive enzyme activities of marine mussels Mytilus coruscus under ocean acidification. Mussels were exposed to combined treatments with three concentrations of nano-TiO 2 (0, 2.5, 10 mg/L) and two pH values (8.1, 7.3) for 14 days, and then recovered under ambient condition (pH 8.1 and no nanoparticle) for 7 days. Samples were taken on the 1st, 3rd, 7th, 14th, and 21st day, the digestive enzymes, including amylase, pepsin, trypsin, lipase, and lysozyme, were investigated. Our results showed that nano-TiO 2 and low pH had negative effects on amylase, pepsin, trypsin, and lipase, while both of them led an increase in lysozyme activity. Nano-TiO 2 showed greater effects on the digestive capacity of M. coruscus rather than low pH. Moreover, a recovery period of 7 days was not sufficient for these enzymes to fully recover., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The effects of short-term (7 days) experimental ocean acidification (-0.4 pH units) and warming (+5 °C) on anti-predator defenses of two sympatric Mytilus species from China, M. coruscus and M. edulis, in the presence and absence of predator cues were investigated. Results suggested species-specific independent negative effects of acidification and warming on the number and weight of byssal threads, the force of thread attachment, and total thread plaque area. Similar negative effects were observed for clustering behaviour, with acidification and warming independently increasing the number of solitary individuals and decreasing the percentage of mussels in clusters. Acidification effects on byssus were strongly exacerbated when predators were present. Ultimately, this study suggests that short-term exposure to experimental warming and acidification can negatively impact anti-predator defense strategies in mussels with potential ramifications for predator-prey interactions and ecological functioning in systems where mussel beds play a key ecological role., (Copyright © 2019. Published by Elsevier Ltd.)

Titanium dioxide nanoparticles (TiO 2 NPs) are widely used in various products and inevitably released with different sizes and forms into aquatic environment. The purpose of this study was to assess the differential immune toxicity of TiO 2 NPs with size difference on mussel hemocytes using flow cytometry (FCM) assays. Hemocyte parameters, including total hemocyte count (THC), hemocyte mortality (HM), phagocytosis activity (PA), lysosomal content (LC), esterase activity (EA), mitochondrial number (MN), mitochondrial membrane potential (MMP) and reactive oxygen species content (ROS) were evaluated in the mussels Mytilus coruscus exposed to two types of TiO 2 NPs (25nm & 100nm: 0.1, 1, 10 mg/L, respectively). In general, size- and concentration-dependent toxicity was pronounced with 25nm-NP and highest concentration (10mg/L) being the most toxic. Alhough a slight recovery from the TiO 2 exposure was observed, significant carry-over effects were still detected. These results highlight the importance of differential size effects of metal oxide NPs on toxicity mechanisms in aquatic animals., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Biochemical responses of the mussel Mytilus coruscus exposed to different concentrations of titanium dioxide nanoparticles (nano-TiO 2 ) (0, 2.5, 10 mg L -1 ) and two pH levels (pH 8.1 and pH 7.3) for 14 days. Mussel responses were also investigated after a 7 days recovery period (pH 8.1 and no nanoparticle). Exposure to nano-TiO 2 led changes in antioxidant indexes (superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione (GSH)), biotransformation enzyme activity (GST) and malondialdehyde level (MDA) in gills and digestive glands. An increase in MDA level and a decrease in SOD and GSH activities were observed in gill of mussels exposed to 10 mg L -1 nano-TiO 2 . This effect was more severe in mussels kept at pH 7.3 as compared to pH 8.1. A different response was observed in the digestive gland as SOD, CAT and GSH levels increased in mussels exposed to nano-TiO 2 . These contrasting results in digestive glands and gills were only evident at high concentration of nano-TiO 2 and low pH. A 7 days recovery period was not sufficient to fully restore SOD, GPx, GST, GSH and MDA levels to levels before exposure to nano-TiO 2 and low pH. Overall, our results confirmed that seawater acidification modulates effects of nanoparticles in mussels, and that gills are more sensitive to these stressors as compared with digestive glands., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Flow cytometry was used to investigate the immune parameters of haemocytes in thick-shell mussel Mytilus coruscus exposed to different concentrations of ZnO nanoparticles (NPs) (0, 2.5, and 10mgl -1 ) at two pH levels (7.3 and 8.1) for 14days following a recovery period of 7days. ZnO NPs significantly affected all of the immune parameters throughout the experiment. At high ZnO NPs concentrations, total haemocyte counting, phagocytosis, esterase, and lysosomal content were significantly decreased whereas haemocyte mortality and reactive oxygen species (ROS) were increased. Although low pH also significantly influenced all of the immune parameters of the mussels, its effect was not as strong as that of ZnO NPs. Interactive effects were observed between pH and ZnO NPs in most haemocyte parameters during the exposure period. Although a slight recovery from the stress of ZnO NPs and pH was observed for all immune parameters, significant carry-over effects of low pH and ZnO NPs were still detected. This study revealed that high concentration of ZnO NPs and low pH exert negative and synergistic effects on mussels, and these effects remain even after the mussels are no longer exposed to such stressors., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Increased production of engineered nanoparticles (NPs) has raised extensive concerns about the potential toxic effects on marine organisms. Extensive evidences documented the impact of ocean acidification (OA) on the physiology and fitness of bivalves. In the present study, we investigated the biochemical responses of the mussel Mytilus coruscus exposed to both nano-ZnO and low pH relevant for ocean acidification conditions for 14 d followed by a 7-d recovery period. Most biochemical indexes (superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), acid phosphatase (ACP) and alkaline phosphatase (ALP)) measured in gills and hemocytes were increased when the mussels were subject to low pH or high concentration of nano-ZnO, suggesting oxidative stress responses. No significant interactions between the two stressors were observed for most measured parameters. After a 1 week recovery period, low pH and nano-ZnO had less marked impact for SOD, GPx, ACP and ALP in hemocytes as compared to the end of the 14 d exposure. However, no recovery was observed in gills. Overall, our results suggest that both low pH and nano-ZnO induce an anti-oxidative response in Mytilus coruscus with gills being more sensitive than hemocytes., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Ocean acidification (OA) and hypoxic events are increasing worldwide problems, their interactive effects have not been well clarified, although their co-occurrence is prevalent. The East China Sea (the Yangtze River estuary area) suffers from not only coastal hypoxia but also pH fluctuation, representing an ideal study site to explore the combined effect of OA and hypoxia on marine bivalves. We experimentally evaluated the antioxidant response of the mussel Mytilus coruscus exposed to three pH levels (8.1, 7.7 and 7.3) at two dissolved oxygen (DO) levels (2.0mgL -1 and 6.0mgL -1 ) for 72h. Activities of superoxide dismutase, catalase, glutathione peroxidase, acid phosphatase, and alkaline phosphatase and levels of malondialdehyde were measured in gills and hemolymph. All enzymatic activities in hemolymph and gills followed a similar pattern throughout the experiment duration. Generally, low DO showed greater effects on enzyme activities than elevated CO 2 . Significant interactions between DO, pH and time were only observed at superoxide dismutase and catalase in both tissues. PCA revealed positive relationships between most enzyme activities in both gills and hemolymph with the exception of alkaline phosphatase activity and the level of malondialdehyde in the hemolymph. Overall, our results suggested that decreased pH and low DO induced similar antioxidant responses in the hard shelled mussel, and showed an additive effect on most enzyme activities. The evaluation of multiple environmental stressors, a more realistic scenario than single ones, is crucial to predict the effect of future global changes on coastal species and our results supply some insights on the potential combined effects of reduced pH and DO on marine bivalves., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The increasing usage of nanoparticles has caused their considerable release into the aquatic environment. Meanwhile, anthropogenic CO 2 emissions have caused a reduction of seawater pH. However, their combined effects on marine species have not been experimentally evaluated. This study estimated the physiological toxicity of nano-TiO 2 in the mussel Mytilus coruscus under high pCO 2 (2500-2600 μatm). We found that respiration rate (RR), food absorption efficiency (AE), clearance rate (CR), scope for growth (SFG) and O:N ratio were significantly reduced by nano-TiO 2 , whereas faecal organic weight rate and ammonia excretion rate (ER) were increased under nano-TiO 2 conditions. High pCO 2 exerted lower effects on CR, RR, ER and O:N ratio than nano-TiO 2 . Despite this, significant interactions of CO 2 -induced pH change and nano-TiO 2 were found in RR, ER and O:N ratio. PCA showed close relationships among most test parameters, i.e., RR, CR, AE, SFG and O:N ratio. The normal physiological responses were strongly correlated to a positive SFG with normal pH and no/low nano-TiO 2 conditions. Our results indicate that physiological functions of M. coruscus are more severely impaired by the combination of nano-TiO 2 and high pCO 2 .

With increasing production from nanotechnology industries, nanomaterials are inevitably released into the aquatic environment, thereby posing a potential risk to aquatic organisms. Thus, concerns have been raised on the potential ecotoxicological effect of nanoparticle. Furthermore, the ecotoxicological consequences caused by the interaction of nanoparticles with other environmental stresses, such as seawater acidification on marine animals, have not been evaluated. In particular, whether acidification enhances the susceptibility to nanoparticles in bivalves needs to be evaluated. In the present study, we investigated the combined effects of low pH and nanoscale titanium dioxide (nano-TiO 2 ) on some immune parameters of hemocytes in the mussel Mytilus coruscus by flow cytometry under six combinations of two pH values (7.3 and 8.1) and three nano-TiO 2 concentrations (0, 2.5, and 10mgL -1 ) for 14 d. Afterward, the mussels were shifted to normal conditions without nano-TiO 2 at pH 8.1 for 7 d further to test their recovery from the multiple stresses. Total hemocyte count (THC), phagocytosis (Pha), esterase (Est), and lysosomal content (Lyso) decreased under low pH and high nano-TiO 2 concentration conditions, whereas hemocyte mortality (HM) and reactive oxygen species (ROS) increased with nano-TiO 2 concentrations under low pH conditions. The interactive effects between pH and nano-TiO 2 were observed at the latter part of the exposure experiment (7 and 14 d) in most hemocyte parameters. Nano-TiO 2 influenced the immune functions of mussel more severely than low pH. Slight recovery from the combined stresses was observed for HM, THC, Pha, and Lyso, but significant carry-over effects of nano-TiO 2 and low pH were still observed. This study demonstrated that both low pH and high concentration of nano-TiO 2 had negative effects on mussels, and these effects still acted for some time even though the mussels were already out of such stressors., (Copyright © 2016 Elsevier B.V. All rights reserved.)

In this work, flow cytometry was used to examine the immune responses of hemocytes in the thick shell mussel Mytilus coruscus exposed to six combinations of pH (7.3, 7.7, and 8.1) and temperature (25 °C and 30 °C) for 14 days. Temperature showed significant effects on all immune parameters throughout the experiment. Generally, the total hemocyte count (THC), phagocytosis (Pha), esterase (Est), and lysosomal content (Lyso) significantly decreased at high temperature. By contrast, the hemocyte mortality (Hm) and reactive oxygen species (ROS) levels increased at high temperature. Moreover, pH significantly influenced all the immune parameters, but its effects are not as strong as those of temperature; only Hm, Est, and THC were negatively affected by pH throughout the experiment. After 7 days, low pH resulted in decreased Lyso and increased Hm and ROS levels. Significant interactions between temperature and pH in most measured parameters from 7 days suggested that long-term combined stress, i.e., low pH and high temperature, would cause more severe effects on mussel health than an individual stressor in the field., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Hypoxia often intensifies with rising dissolved CO2, but the concurrent effects of hypoxia and acidification on bivalves are largely unknown. In this study, immune responses of hemocytes in the mussel Mytilus coruscus were examined under six combinations of pH (7.3, 7.7 and 8.1) and dissolved oxygen (DO) concentrations (2mgL(-1), 6mgL(-1)) for 72h. Generally, total hemocyte account, phagocytosis, esterase and lysosomal content were reduced under low DO and pH conditions, whereas hemocyte mortality and reactive oxygen species production increased under low DO and pH. Both hypoxia and low pH have negative effects on mussels, but the effects of pH are not as strong as DO. Moreover, significant interactions between DO and pH occurred. However, acidification generally doesn't aggravate the effects induced by hypoxia. Acidification and hypoxia may increase disease risk and impact the aquaculture of this species., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Hypoxia and ocean acidification are two consequences of anthropogenic activities. These global trends occur on top of natural variability. In environments such as estuarine areas, short-term acute pH and O2 fluctuations are occurring simultaneously. The present study tested the combined effects of short-term seawater acidification and hypoxia on the physiology and energy budget of the thick shell mussel Mytilus coruscus. Mussels were exposed for 72 h to six combined treatments with three pH levels (8.1, 7.7 and 7.3) and two dissolved oxygen (DO) levels (2 mg L(-1), 6 mg L(-1)). Clearance rate (CR), food absorption efficiency (AE), respiration rate (RR), ammonium excretion rate (ER), O:N ratio and scope for growth (SFG) were significantly reduced, and faecal organic dry weight ratio (E) was significantly increased at low DO. Low pH did not lead to a reduced SFG. Interactive effects of pH and DO were observed for CR, E and RR. Principal component analysis (PCA) revealed positive relationships among most physiological indicators, especially between SFG and CR under normal DO conditions. These results demonstrate that Mytilus coruscus was sensitive to short-term (72 h) exposure to decreased O2 especially if combined with decreased pH levels. In conclusion, the short-term oxygen and pH variation significantly induced physiological changes of mussels with some interactive effects., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

This study evaluated the combined effects of seawater pH decrease and temperature increase on the activity of antioxidant enzymes in the thick shell mussel Mytilus coruscus, an ecological and economic bivalve species widely distributed along the East China Sea. Mussels were exposed to three pH levels (8.1, 7.7 and 7.3) and two temperatures (25 °C and 30 °C) for 14 days. Activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), glutathione (GSH), acid phosphatase (ACP), alkaline phosphatase (AKP) and glutamic-pyruvic transaminase (GPT) were measured in gills and digestive glands after 1, 3, 7 and 14 days of exposure. All enzymatic activities were significantly impacted by pH, temperature. Enzymatic activities at the high temperature were significantly higher than those at the low temperature, and the mussels exposed to pH 7.3 showed significantly higher activities than those under higher pH condition for all enzymes except ACP. There was no interaction between temperature and pH in two third of the measured activities suggesting similar mode of action for both drivers. Interaction was only consistently significant for GPX. PCA revealed positive relationships between the measured biochemical indicators in both gills and digestive glands. Overall, our results suggest that decreased pH and increased temperature induce a similar anti-oxidative response in the thick shell mussel., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Ocean acidification and hypoxia, both caused by anthropogenic activities, have showed deleterious impacts on marine animals. However, their combined effect on the mussel's defence to its predator has been poorly understood, which hinders us to understand the prey-predator interaction in marine environment. The thick shell mussel Mytilus coruscus and its predator, the Asian paddle crab Charybdis japonica were exposed to three pH levels (7.3, 7.7, 8.1) at two concentrations of dissolved oxygen (2.0 mg L(-1), 6.0 mg L(-1)) seawater. The anti-predatory responses of mussels, in terms of byssus thread production were analyzed after 72 h exposure. During the experiment, frequency of shedding stalks (mussels shed their byssal stalks to release themselves from attachment and allow locomotion) and number of byssus threads increased with time, were significantly reduced by hypoxia and low pH levels, and some interactions among time, predator, DO and pH were observed. As expected, the presence of the crab induced an anti-predator response in M. coruscus (significant increases in most tested parameters except the byssus thread length). Acidification and hypoxia significantly reduced byssus thread diamter at the end of the experiment, but not the byssus thread length. Cumulative byssus thread length and volume were significantly impaired by hypoxia and acidification. Our results highlight the significance of anti-predatory responses for adult mussel M. coruscus even under a stressful environment in which stress occurs through ocean acidification and hypoxia. By decreasing the strength of byssus attachment, the chance of being dislodged and consumed by crabs is likely increased. Our data suggest that there are changes in byssus production induced by hypoxia and acidification, which may affect predation rates on M. coruscus in the field., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Anthropogenic CO₂ emissions have caused seawater temperature elevation and ocean acidification. In view of both phenomena are occurring simultaneously, their combined effects on marine species must be experimentally evaluated. The purpose of this study was to estimate the combined effects of seawater acidification and temperature increase on the energy budget of the thick shell mussel Mytilus coruscus. Juvenile mussels were exposed to six combined treatments with three pH levels (8.1, 7.7 and 7.3)×two temperatures (25 °C and 30 °C) for 14 d. We found that clearance rates (CRs), food absorption efficiencies (AEs), respiration rates (RRs), ammonium excretion rates (ER), scope for growth (SFG) and O:N ratios were significantly reduced by elevated temperature sometimes during the whole experiments. Low pH showed significant negative effects on RR and ER, and significantly increased O:N ratios, but showed almost no effects on CR, AE and SFG of M. coruscus. Nevertheless, their interactive effects were observed in RR, ER and O:N ratios. PCA revealed positive relationships among most physiological indicators, especially between SFG and CR under normal temperatures compared to high temperatures. PCA also showed that the high RR was closely correlated to an increasing ER with increasing pH levels. These results suggest that physiological energetics of juvenile M. coruscus are able to acclimate to CO2 acidification with a little physiological effect, but not increased temperatures. Therefore, the negative effects of a temperature increase could potentially impact the ecophysiological responses of M. coruscus and have significant ecological consequences, mainly in those habitats where this species is dominant in terms of abundance and biomass., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Coastal habitats are exposed to increasing pressure of nanopollutants commonly combined with warming due to the seasonal temperature cycles and global climate change. To investigate the toxicological effects of TiO 2 nanoparticles (TiO 2 NPs) and elevated temperature on the intestinal health of the mussels (Mytilus coruscus), the mussels were exposed to 0.1 mg/L TiO 2 NPs with different crystal structures for 14 days at 20 °C and 28 °C, respectively. Compared to 20 °C, the agglomeration of TiO 2 NPs was more serious at 28 °C. Exposure to TiO 2 NPs led to elevated mortality of M. coruscus and modified the intestinal microbial community as shown by 16S rRNA sequence analysis. Exposure to TiO 2 NPs changed the relative abundance of Bacteroidetes, Proteobacteria and Firmicutes. The relative abundances of putative mutualistic symbionts Tenericutes and Fusobacteria increased in the gut of M. coruscus exposed to anatase, which have contributed to the lower mortality in this group. LEfSe showed the combined stress of warming and TiO 2 NPs increased the risk of M. coruscus being infected with potential pathogenic bacteria. This study emphasizes the toxicity differences between crystal structures of TiO 2 NPs, and will provides an important reference for analyzing the physiological and ecological effects of nanomaterial pollution on bivalves under the background of global climate change. [Display omitted] • Exposure to TiO 2 NPs led to elevated mortality of M. coruscus and modified the gut microbial community. • The response mechanisms of gut microbiota to TiO 2 NPs with different crystal structure were different. • TiO 2 -induced changes of gut microbiota were correlated with community function and host metabolism alterations. • The agglomeration behaviour of TiO 2 NPs and gut microbiota was modified by temperature. • An interaction between TiO 2 NPs and temperature on the gut microbiota of M. coruscus was found. [ABSTRACT FROM AUTHOR]