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Anthropogenic elevation of atmospheric carbon dioxide (CO2) drives global-scale ocean acidification (OA), which has aroused widespread concern for marine ecosystem health. The tri-spine horseshoe crab (HSC) Tachypleus tridentatus has been facing the threat of population depletion for decades, and the effects of OA on the physiology and microbiology of its early life stage are unclear. In this study, the 1st instar HSC larvae were exposed to acidified seawater (pH 7.3, pH 8.1 as control) for 28 days to determine the effects of OA on their growth, molting, oxidative stress, and gut microbiota. Results showed that there were no significant differences in growth index and molting rate between OA group and control group, but the chitinase activity, β-NAGase activity, and ecdysone content in OA group were significantly lower than those of the control group. Compared to the control group, reactive oxygen species (ROS) and malondialdehyde (MDA) contents in OA group were significantly increased at the end of the experiment. Superoxide dismutase (SOD), catalase (CAT), and alkaline phosphatase (AKP) activities increased first and then decreased, glutathione peroxidase (GPX) decreased first and then increased, and GST activity changed little during the experiment. According to the result of 16S rRNA sequencing of gut microbiota, microbial-mediated functions predicted by PICRUSt showed that "Hematopoietic cell lineage," "Endocytosis," " Staphylococcus aureus infection," and "Shigellosis" pathways significantly increased in OA group. The above results indicate that OA had no significant effect on growth index and molting rate but interfered with the activity of chitinolytic enzymes and ecdysone expression of juvenile horseshoe crabs, and caused oxidative stress. In addition, OA had adverse effects on the immune defense function and intestinal health. The present study reveals the potential threat of OA to T. tridentatus population and lays a foundation for the further study of the physiological adaptation mechanism of juvenile horseshoe crabs to environmental change. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. Unlabelled Image • Predator, pH and temperature affected the mussels' anti-predator behaviour. • Species specific effects were observed between Mytilus coruscus and Mytilus edulis. • Low pH and high temperature reduced the anti-predation performance of mussels. • Predator induced mussel clustering while low pH and warming had negative effects. [ABSTRACT FROM AUTHOR]

Anthropogenic climate change exposes marine organisms to CO2 induced ocean acidification (OA). Marine animals may make physiological and behavioral adaptations to cope with OA. Elevated pCO2 may affect metabolism, feeding, and energy partition of marine crabs, and thereby affect their predator-prey dynamics with mussels. Therefore, we examined the effects of simulated future elevated pCO2 on feeding behavior and energy metabolism of the brown crab Cancer pagurus. Following 54 days of pre-acclimation to control CO2 levels (360 μatm) at 11°C, crabs were exposed to consecutively increased oceanic CO2 levels (2 weeks for 1200 and 2300 μatm, respectively) and subsequently returned to control CO2 level (390 μatm) for 2 weeks in order to study their potential to acclimate elevated pCO2 and recovery performance. Standard metabolic rate (SMR), specific dynamic action (SDA) and feeding behavior of the crabs were investigated during each experimental period. Compared to the initial control CO2 conditions, the SMRs of CO2 exposed crabs were not significantly increased, but increased significantly when the crabs were returned to normal CO2 levels. Conversely, SDA was significantly reduced under high CO2 and did not return to control levels during recovery. Under high CO2, crabs fed on smaller sized mussels than under control CO2; food consumption rates were reduced; foraging parameters such as searching time, time to break the prey, eating time, and handling time were all significantly longer than under control CO2, and prey profitability was significantly lower than that under control conditions. Again, a two-week recovery period was not sufficient for feeding behavior to return to control values. PCA results revealed a positive relationship between feeding/SDA and pH, but negative relationships between the length of foraging periods and pH. In conclusion, elevated pCO2 caused crab metabolic rate to increase at the expense of SDA. Elevated pCO2 affected feeding performance negatively and prolonged foraging periods. These results are discussed in the context of how elevated pCO2 may impair the competitiveness of brown crabs in benthic communities. [ABSTRACT FROM AUTHOR]

With the release of large amounts of CO2, ocean acidification is intensifying and affecting aquatic organisms. In addition, salinity also plays an important role for marine organisms and fluctuates greatly in estuarine and coastal ecosystem, where ocean acidification frequently occurs. In present study, flow cytometry was used to investigate immune parameters of haemocytes in the thick shellmusselMytilus coruscus exposed to different salinities (15, 25, and 35‰) and two pH levels (7.3 and 8.1). A 7-day in vivo and a 5-h in vitro experiments were performed. In both experiments, low pH had significant effects on all tested immune parameters. When exposed to decreased pH, total haemocyte count (THC), phagocytosis (Pha), esterase (Est), and lysosomal content (Lyso) were significantly decreased, whereas haemocyte mortality (HM) and reactive oxygen species (ROS) were increased. High salinity had no significant effects on the immune parameters of haemocytes as compared with low salinity. However, an interaction between pH and salinity was observed in both experiments for most tested haemocyte parameters. This study showed that high salinity, low salinity and low pH have negative and interactive effects on haemocytes of mussels. As a consequence, it can be expected that the combined effect of low pH and changed salinity will have more severe effects on mussel health than predicted by single exposure. [ABSTRACT FROM AUTHOR]

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 (2.0 mg L −1 and 6.0 mg L −1 ) for 72 h. 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. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • Ocean acidification reduced phagocytic activity and hence immunity of mussels. • The reduced phagocytic activity was associated with lowered energy budget. • Ocean acidification also reduced byssus strength, extensibility and production. • Microplastics can aggravate these negative effects of ocean acidification. • Mussels would be more prone to diseases and dislodgement in future oceans. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • Ocean acidification adversely affects the gonadal performance of mussels. • Hypoxia does not have effects on fecundity of mussels. • Steroids and Wnt/β-catenin gene expressions have weak correlations with fecundity in mussels. [ABSTRACT FROM AUTHOR]

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. Unlabelled Image • Low pH, low DO and high temperature have adverse effects on the antioxidant parameters. • Exposure time of stressors has significant effects on the antioxidant parameters. • Combined stressors exert more severe effects on the antioxidant indicators than single ones. [ABSTRACT FROM AUTHOR]

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. Image 1 • Low pH, low DO and high temperature showed drastic effects on digestive enzyme activities. • Low pH, low DO and high temperature synergistically reduced digestive enzyme activities. • Lysozyme showed increased and then decreased activities with time. • Combinations of low pH, low DO and high temperature showed more severe effects on digestive enzymes than single factors. • Hypoxia and high temperature showed similar effects on digestive enzyme activities. [ABSTRACT FROM AUTHOR]

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, 104 and 106 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. Image 1 • The immunity and digestion of mussels were impaired by MPs and OA. • OA can enhance the toxicity of MPs to mussels. • Digestion was more severely affected by OA and MPs than antioxidant system. • The negative effects on PES and AMS showed a reversible trend. [ABSTRACT FROM AUTHOR]

Apart from ocean acidification, hypoxia is another stressor to marine organisms, especially those in coastal waters. Their interactive effects of elevated CO 2 and hypoxia on the physiological energetics in mussel Mytilus edulis were evaluated. Mussels were exposed to three pH levels (8.1, 7.7, 7.3) at two dissolved oxygen levels (6 and 2 mg L−1) and clearance rate, absorption efficiency, respiration rate, excretion rate, scope for growth and O: N ratio were measured during a14-day exposure. After exposure, all parameters (except excretion rate) were significantly reduced under low pH and hypoxic conditions, whereas excretion rate was significantly increased. Additive effects of low pH and hypoxia were evident for all parameters and low pH appeared to elicit a stronger effect than hypoxia (2.0 mg L−1). Overall, hypoxia can aggravate the effects of acidification on the physiological energetics of mussels, and their populations may be diminished by these stressors. Unlabelled Image • Combined effects of ocean acidification and hypoxia are investigated in mussels. • Physiological activities of mussels are inhibited by low pH and hypoxia. • OA and hypoxia exert additive effects on the physiological metabolism of mussels. [ABSTRACT FROM AUTHOR]

Anthropogenic CO 2 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 CO 2 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. [ABSTRACT FROM AUTHOR]

• The anti-predation behavior of mussels is induced by predator. • Low pH and nano-ZnO synergistically reduced the anti-predation behavior of mussels. • Nano-ZnO and seawater acidification may change the predator-prey dynamics in the environment. Artificial nanoparticles and ocean acidification (OA) caused by the rapid increase of CO 2 absorbed by the ocean are both ecologically hazardous to marine organisms. The combined effects of the two environmental stressors on the anti-predation ability of marine mussels were studied. Mytilus coruscus was exposed to three different gradient concentrations of nano-ZnO (0, 2.5, 10 mg/L) in combination of two pH levels (7.7 and 8.1). The crab Charybdis japonica was used as its predator. During the experiment, anti-predator indexes, including number of byssus threads (NBT), shell-closing strength (SCS), diameter of byssus thread (BTD), length of byssus thread (BTL), cumulative length of byssus thread (CBTL) and cumulative volume of byssus thread (CBTV) were studied. The results showed that predator induced the anti-predation responses in M. coruscus , and NBT, SCS, BTL, CBTL and CBTV were significantly increased. Under the conditions of pH 7.7 and 10 mg/L nano-ZnO, NBT, SCS, BTD, BTL, CBTL, and CBTV were significantly reduced. What's more, significant interactions among pH, nano-ZnO and predator were observed in CBTL and CBTV. Therefore, the joint treatment of nano-ZnO and low pH reduces the adhesion strength of byssus thread and may increase the probability of mussels being preyed. [ABSTRACT FROM AUTHOR]

Abstract Transgenerational effects of multiple stressors on marine organisms are emerging environmental themes. We thus experimentally tested for transgenerational effects of seawater acidification and hypoxia on the early development traits of the mussel Mytilus edulis. Fertilization rate, embryo deformity rate, and larval shell length were negatively impacted by acidification, while hypoxia had little effect except for increasing deformity rates under control pH conditions. Offspring from low pH/O 2 parents were less negatively affected by low pH/O 2 conditions than offspring from control parents; however, low pH/O 2 conditions still negatively affected developmental traits in offspring from acclimated parents compared to control seawater conditions. Our results demonstrate that experimental seawater acidification and hypoxia can adversely affect early developmental traits of M. edulis and that parental exposure can only partially alleviate these impacts. If experimental observations hold true in nature, it is unlikely that parental exposure will confer larval tolerance to ocean acidification for M. edulis. Graphical abstract Image 1 Highlights • Effects of ocean acidification and hypoxia on the early development of the mussel M. edulis were investigated. • Positive carry-over effects of adult mussels exposed to low pH and hypoxia were observed on larvae performance. • Low pH showed key negative effects on the early development of the mussel M. edulis. [ABSTRACT FROM AUTHOR]

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 10 mg l − 1 ) at two pH levels (7.3 and 8.1) for 14 days following a recovery period of 7 days. 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 10 mg L −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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 O 2 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 O 2 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. [ABSTRACT FROM AUTHOR]

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. Image 1 • Low pH can enhance the toxicity of TiO 2 NPs to mussels. • The feeding and metabolism of mussels are impaired by TiO 2 NPs and low pH. • TiO 2 NPs and low pH have significant interactions and carry-over effects on mussels. [ABSTRACT FROM AUTHOR]

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. Image 1 • Effects of OA and nano-TiO 2 on the digestive enzyme activity of mussels were studied. • The Seven-day recovery was not sufficient for digestive enzymes to fully recover. • Nano-TiO 2 showed greater negative effects compared with low pH. • Significant interactions between low pH and nano-TiO 2 occurred. [ABSTRACT FROM AUTHOR]