Unraveling the interplay between environmental microplastics and salinity stress on Mytilus galloprovincialis larval development: A holistic exploration.

The rise of plastic production has triggered a surge in plastic waste, overwhelming marine ecosystems with microplastics. The effects of climate change, notably changing salinity, have shaped the dynamics of coastal lagoons. Thus, understanding the combined impact of these phenomena on marine organisms becomes increasingly crucial. To address these knowledge gaps, we investigated for the first time the interactive effects of environmental microplastics (EMPs) and increased salinity on the early development of Mytilus galloprovincialis larvae. Morphological assessments using the larval embryotoxicity test revealed larval anomalies and developmental arrests induced by EMPs and increased salinity. Transcriptomic analyses targeting 12 genes involved in oxidative stress, apoptosis, DNA repair, shell formation, and stress proteins were conducted on D-larvae uncovered the potential effects of EMPs on shell biomineralization, highlighting the role of Histidine Rich Glycoproteine (HRG) and tubulin as crucial adaptive mechanisms in Mytilus sp. in response to environmental shifts. Furthermore, we explored oxidative stress and neurotoxicity using biochemical assays. Our findings revealed a potential interaction between EMPs and increased salinity, impacting multiple physiological processes in mussel larvae. Our data contribute to understanding the cumulative effects of emerging anthropogenic pollutants and environmental stressors, emphasizing the need for a holistic approach to assessing their impact on marine ecosystems.

Molecular and behavioural responses of the mussel Mytilus edulis exposed to a marine heatwave.

Marine heatwaves (MHW) threaten marine organisms and tend to increase in frequency and intensity. We exposed the blue mussel Mytilus edulis to a MHW lasting 23 days, including two 10-d periods of thermal intensity increase of +5 °C (20 °C-25 °C) interspersed by 1 day back to 20 °C, followed by a 4-d recovery period. We investigated behaviour responses of mussels and gene expression changes relative to the circadian rhythm (Per), oxidative stress (SOD), cellular apoptosis (CASP3), energy production (ATPs), and general stress response (hsp70). Results showed that the MHW disturbed the valve activity of mussels. Particularly, mussels increased the number of valve micro-closures, showing a stressful state of organisms. Mussels also decreased Per, CASP3, ATPs, and Hsp70 gene expression. Some behavioural and molecular effects persisted after the MHW, suggesting a limited recovery capacity of individuals. This work highlighted the vulnerability of M. edulis to a realistic MHW.

Testing the physiological capacity of the mussel Mytilus chilensis to establish into the Southern Ocean.

The Southern Ocean and the Antarctic Circumpolar Current create environmental conditions that serve as an efficient barrier to prevent the colonization of non-native species (NNS) in the marine ecosystems of Antarctica. However, warming of the Southern Ocean and the increasing number of transport opportunities are reducing the physiological and physical barriers, increasing the chances of NNS arriving. The aim of this study was to determine the limits of survival of the juvenile mussels, M. chilensis, under current Antarctic conditions and those projected under climate change. These assessments were used to define the mussels potential for establishment in the Antarctic region. Experimental mussels were exposed to four treatments: -1.5 °C (Antarctic winter), 2 °C (Antarctic summer), 4 °C (Antarctic projected) and 8 °C (control) for 80 days and a combination of physiological and transcriptomics approaches were used to investigate mussel response. The molecular responses of mussels were congruent with the physiological results, revealing tolerance to Antarctic winter temperatures. However, a higher number of regulated differentially expressed gene (DEGs) were reported in mussels exposed to Antarctic winter temperatures (-1.5 °C). This tolerance was associated with the activation of the biological processes associated with apoptosis (up regulated) and both cell division and cilium assembly (down regulated). The reduced feeding rate and the negative scope for growth, for a large part of the exposure period at -1.5 °C, suggests that Antarctic winter temperatures represents an environmental barrier to M. chilensis from the Magellanic region settling in the Antarctic. Although M. chilensis are not robust to current Antarctica thermal conditions, future warming scenarios are likely to weaken these physiological barriers. These results strongly suggest that the West Antarctic Peninsula could become part of Mytilus distributional range, especially with dispersal aided by increasing maritime transport activity across the Southern Ocean.

Evaluating quaternium-15 effects on Mytilus galloprovincialis: New insights on physiological and cellular responses.

Among personal care products, quaternium-15 is prominently featured as a preservative in items such as shampoos, soaps, shaving products, and cosmetics. The widespread use of these products in people's daily routines contributes to quaternium-15 release into aquatic ecosystems. In this context, the primary aim of the study was to assess the physiological and cellular responses of the digestive gland and gills in Mytilus galloprovincialis to quaternium-15 exposure. Cell viability and the ability of digestive gland cells to regulate their volume were evaluated. Additionally, the expression of the genes involved in oxidative stress response was assessed to further substantiate the compound's harmful effects. Results indicated a significant decrease in both the viability of digestive gland cells and their RVD (regulatory volume decrease) capacity when exposed to a hypotonic solution. Furthermore, impairment of digestive gland cell function was corroborated by the modulation of oxidative stress-related gene expression, including SOD, Cat, as well as Hsp70 and CYP4Y1. Similar gene expression alterations were observed in the gills, reflecting impaired functionality in this vital organ as well. In summary, the outcomes of the study provide conclusive evidence of the toxicity of quaternium-15. This underscores the urgent need to further investigate the toxicological effects of this contaminant on aquatic ecosystems and emphasises the necessity of limiting the use of products containing quaternium-15.

Yttrium effects on the Mediterranean mussel under a scenario of salinity shifts and increased temperature.

Climate change (CC) induces significant worldwide alterations in salinity and temperature, impacting ecosystems and their services. Marine organisms, susceptible to these changes, may experience modified vulnerability to anthropogenic contaminants, including rare-earth elements (REEs) such as yttrium (Y) derived from electronic waste. This study investigated the influence of temperature and salinity changes on the impacts of Y in Mytilus galloprovincialis mussels. Organisms were subjected to Y (0 and 10 µg/L) for 28 days under three salinity scenarios (20, 30 (control), and 40, at a control temperature of 17 °C) or to two temperatures (17 and 22 °C, at the control salinity of 30). Under these conditions, Y bioaccumulation and different biomarkers were evaluated. Results showed that salinity and temperature did not affect Y accumulation, indicating effective detoxification mechanisms and physiological adaptations in the exposed organisms. However, in Y-exposed mussels effects were intensified under decreased salinity, evidenced by increased metabolism, defense enzyme activities, and acetylcholinesterase (AChE) levels. Similar responses occurred under heat stress with enhanced metabolic capacity, AChE activity, and activation of defense mechanisms such as glutathione S-transferases. These defense mechanisms mitigated cellular damage caused by Y, but under the highest temperature and especially lower salinity, Y-exposed mussels exhibited increased oxidative stress and decreased efficiency of activated defense enzymes, resulting in cellular damage compared to their uncontaminated counterpart. The present study sheds light on the effects that interactions between temperature, salinity, and the presence of emerging contaminants like REEs may have on marine organisms. Such assessments are crucial for developing effective strategies to mitigate the impacts of CC and protect the long-term health and resilience of marine ecosystems.

Size-dependent effects of plastic particles on antioxidant and immune responses of the thick-shelled mussel Mytilus coruscus.

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 (H2O2) 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.

The combined effects of phenanthrene and micro-/nanoplastics mixtures on the cellular stress responses of the thick-shell mussel Mytilus coruscus.

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.

The molecular response of Mytilus coruscus mantle to shell damage under acute acidified sea water revealed by iTRAQ based quantitative proteomic analysis.

Mytilus coruscus is an economically important marine bivalve that lives in estuarine sea areas with seasonal coastal acidification and frequently suffers shell injury in the natural environment. However, the molecular responses and biochemical properties of Mytilus under these conditions are not fully understood. In the present study, we employed tandem mass spectrometry combined with isobaric tagging to identify differentially expressed proteins in the mantle tissue of M. coruscus under different short-term treatments, including shell-complete mussels raised in normal seawater (pH 8.1), shell-damaged mussels raised in normal seawater (pH 8.1), and acidified seawater (pH 7.4). A total of 2694 proteins were identified in the mantle, and analysis of their relative abundance from the three different treatments revealed alterations in the proteins involved in immune regulation, oxidation-reduction processes, protein folding and processing, energy provision, and cytoskeleton. The results obtained by quantitative proteomic analysis of the mantle allowed us to delineate the molecular strategies adopted by M. coruscus in the shell repair process in acidified environments, including an increase in proteins involved in oxidation-reduction processes, protein processing, and cell growth at the expense of proteins involved in immune capacity and energy metabolism. SIGNIFICANCE: The impact of global ocean acidification on calcifying organisms has become a major ecological and environmental problem in the world. Mytilus coruscus is an economically important marine bivalve living in estuary sea area with seasonal coastal acidification, and frequently suffering shell injury in natural environment. Molecular responses of M coruscus under the shell damage and acute acidification is still largely unknown. For this reason, iTRAQ based quantitative proteomic and histological analysis of the mantle from M. coruscus under shell damage and acute acidification were performed, for revealing the proteomic response and possible adaptation mechanism of Mytilus under combined shell damage and acidified sea water, and understanding how the mussel mantle implement a shell-repair process under acidified sea water. Our study provides important data for understanding the shell repair process and proteomic response of Mytilus under ocean acidification, and providing insights into potential adaptation of mussels to future global change.

Metabolic profiling of Mytilus coruscus mantle in response of shell repairing under acute acidification.

Mytilus coruscus is an economically important marine bivalve mollusk found in the Yangtze River estuary, which experiences dramatic pH fluctuations due to seasonal freshwater input and suffer from shell fracture or injury in the natural environment. In this study, we used intact-shell and damaged-shell M. coruscus and performed metabolomic analysis, free amino acids analysis, calcium-positive staining, and intracellular calcium level tests in the mantle to investigate whether the mantle-specific metabolites can be induced by acute sea-water acidification and understand how the mantle responds to acute acidification during the shell repair process. We observed that both shell damage and acute acidification induced alterations in phospholipids, amino acids, nucleotides, organic acids, benzenoids, and their analogs and derivatives. Glycylproline, spicamycin, and 2-aminoheptanoic acid (2-AHA) are explicitly induced by shell damage. Betaine, aspartate, and oxidized glutathione are specifically induced by acute acidification. Our results show different metabolic patterns in the mussel mantle in response to different stressors, which can help elucidate the shell repair process under ocean acidification. furthermore, metabolic processes related to energy supply, cell function, signal transduction, and amino acid synthesis are disturbed by shell damage and/or acute acidification, indicating that both shell damage and acute acidification increased energy consumption, and disturb phospholipid synthesis, osmotic regulation, and redox balance. Free amino acid analysis and enzymatic activity assays partially confirmed our findings, highlighting the adaptation of M. coruscus to dramatic pH fluctuations in the Yangtze River estuary.

The effect of interspecific and intraspecific diversity on microplastic ingestion in two co-occurring mussel species in South Africa.

Interspecific and intraspecific diversity are essential components of biodiversity with far-reaching implications for ecosystem function and service provision. Importantly, genotypic and phenotypic variation within a species can affect responses to anthropogenic pressures more than interspecific diversity. We investigated the effects of interspecific and intraspecific diversity on microplastic ingestion by two coexisting mussel species in South Africa, Mytilus galloprovincialis and Perna perna, the latter occurring as two genetic lineages. We found significantly higher microplastic abundance in M. galloprovincialis (0.54 ± 0.56 MP items g-1WW) than P. perna (0.16 ± 0.21 MP items g-1WW), but no difference between P. perna lineages. Microbeads were the predominant microplastic (76 % in P. perna, 99 % in M. galloprovincialis) and polyethylene the prevalent polymer. Interspecific differences in microplastic abundance varied across locations, suggesting diverse sources of contamination. We suggest that microplastic ingestion can be species-specific even in organisms that coexist and play similar functional roles within ecosystems.

Toxic effects of four emerging pollutants on cardiac performance and associated physiological parameters of the thick-shell mussel (Mytilus coruscus).

Robust cardiac performance is critical for the health and even survival of an animal; however, it is sensitive to environmental stressors. At present, little is known about the cardiotoxicity of emerging pollutants to bivalve mollusks. Thus, in this study, the cardiotoxic effects of four emergent pollutants, carbamazepine (CBZ), bisphenol A (BPA), tetrabromobisphenol A (TBBPA), and tris(2-chloroethyl) phosphate (TCEP), on the thick-shell mussel, Mytilus coruscus, were evaluated by heartbeat monitoring and histological examinations. In addition, the impacts of these pollutants on parameters that closely related to cardiac function including neurotransmitters, calcium homeostasis, energy supply, and oxidative status were assessed. Our results demonstrated that 28-day exposure of the thick-shell mussel to these pollutants resulted in evident heart tissue lesions (indicated by hemocyte infiltration and myocardial fibrosis) and disruptions of cardiac performance (characterized by bradyrhythmia and arrhythmia). In addition to obstructing neurotransmitters and calcium homeostasis, exposure to pollutants also led to constrained energy supply and induced oxidative stress in mussel hearts. These findings indicate that although do differ somehow in their effects, these four pollutants may exert cardiotoxic impacts on mussels, which could pose severe threats to this important species and therefore deserves more attention.

The influence of temperature on the effects of lead and lithium in Mytilus galloprovincialis through biochemical, cell and tissue levels: Comparison between mono and multi-element exposures.

Lead (Pb) and lithium (Li) are metals which have been detected in the environment and, at high concentrations, can induce toxic effects that disturb the growth, metabolism or reproduction of organisms along the entire trophic chain. The impacts of these metals have scarcely been investigated using marine bivalves, especially when acting as a mixture. The present study aimed to investigate the influence of temperature on the ecotoxicological effects caused by Pb and Li, acting alone and as a mixture, on the mussel species Mytilus galloprovincialis after 28 days of exposure. The impacts were evaluated under actual (17 °C) and projected (+4 °C) warming conditions, to understand the influence of temperature rise on the effects of the metals (both acting alone or as a mixture). The results obtained showed that the increased temperature did not influence the accumulation of metals. However, the biomarkers evaluated showed greater responses in mussels that are exposed to metals under increased temperature (21 °C). The IBR index showed that there is a comparable toxic effect of Li and Pb separately, while exposure to a mixture of both pollutants causes a significantly higher stress response. Overall, the results obtained revealed that temperature may cause extra stress on the mussels and exposure to the metal mixture caused the greatest impacts compared to each metal acting alone.

Nano-TiO2 aggravates the adverse effect of pentachlorophenol on antioxidant and immune response in anti-predatory mussels.

Nano-TiO2 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-TiO2 and PCP on the mussel Mytilus coruscus in the presence of its natural predator, the swimming crab Portunus trituberculatus. Exposure to n-TiO2, 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-TiO2 exposure. Integrated biomarker (IBR) response values showed the effect of PCP was concentration dependent. Of the two used n-TiO2 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-TiO2 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-TiO2, 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.

Outer membrane vesicles induce the mussel plantigrade settlement via regulation of c-di-GMP.

Despite the importance of outer membrane vesicles (OMVs) in benthic animal settlement, the underlying molecular mechanism remains elusive. Here, the impact of OMVs and OMVs synthesis-related tolB gene in Mytilus coruscus plantigrade settlement was tested. The OMVs were extracted from Pseudoalteromonas marina through density gradient centrifugation, and a tolB knockout strain, achieved by homologous recombination, was utilized for the investigation. Our results demonstrated that OMVs could significantly enhance M. coruscus plantigrades settlement. Deleting the tolB resulted in downregulation of c-di-GMP, accompanied by a reduction of OMV production, a decline in bacterial motility and increasing biofilm-forming ability. Enzyme treatment resulted in a 61.11% reduction in OMV-inducing activity and a 94.87% reduction in LPS content. Thus, OMVs regulate mussel settlement via LPS, and c-di-GMP is responsible for the OMV-inducing capacity. These findings provide new insights into the interactions between bacteria and mussels.

Effects of ifosfamide and cisplatin exposure combined with a climate change scenario on the transcriptome responses of the mussel Mytilus galloprovincialis.

Coastal ecosystems are currently exposed to pollutants and climate change. Namely, the increasing consumption of antineoplastic drugs and their potential release to aquatic ecosystems are raising concerns. Nevertheless, information regarding the toxicity of these drugs towards non-target species is scarce, especially considering climate change scenarios. Ifosfamide (IF) and cisplatin (CDDP) are among the antineoplastics already detected in aquatic compartments and due to their mode of action (MoA) can negatively affect aquatic organisms. This study evaluates the transcription of 17 selected target genes related to the MoA of IF and CDDP in Mytilus galloprovincialis gills exposed to environmentally relevant and toxicological meaningful concentrations (IF - 10, 100, 500 ng/L; CDDP - 10, 100, 1000 ng/L), under an actual (17 °C) and predicted warming scenario (21 °C). Results showed an upregulation of the cyp4y1 gene when exposed to the highest concentrations of IF, regardless of the temperature. Both drugs upregulated genes related to DNA damage and apoptosis (p53, caspase 8 and gadd45), especially under warmer conditions. Increased temperature also downregulated genes related to stress and immune responses (krs and mydd88). Therefore, the present results showed a gene transcriptional response of mussels to increasing concentrations of antineoplastics and that warmer temperatures modulated those effects.

Mytilus galloprovincialis releases immunologically functional haemocytes to the intervalvar space in response to tissue injury and infection.

Haemocytes of Mytilus galloprovincialis represent the main component of the internal self-defence system. Although haemocytes from haemolymph are usually studied to analyse these animals' immune response, the presence of haemocytes in the intervalvar liquid, which is essentially sea water, led us to characterize them. Several functional (ROS production, phagocytosis, gene expression, travel velocity and distance) and morphological (area, size and granularity) assays were performed by applying different stimuli to the mussels (waterborne infection, shell injury and their combination). Our results revealed that intervalvar liquid haemocytes share common characteristics with haemolymph haemocytes (for instance, the cell morphology and the cell population structure divided in three main groups) but also show significant differences in size (usually smaller in the intervalvar liquid), mobility (commonly faster in the intervalvar liquid), ROS production (higher in non-stimulated intervalvar liquid cells) and gene expression (IL17, Myd88 and CathL are over expressed in liquid intervalvar cells compared to haemolymph cells). Moreover, differences were observed when mussels were subjected to the mentioned treatments. These free intervalvar haemocytes could constitute the first line of defence as external sentinels extending the immunological alert system outside of the mussel body.

Polybrominated diphenyl ether-47 and food shortage impair the byssal attachment and health of marine mussels.

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.

Physiological and biochemical responses to caffeine and microplastics in Mytilus galloprovincialis.

Caffeine (Caff) is one of the most widely used substances in the human diet and a well-recognized drug. Its input into surface waters is remarkable, but biological effects on aquatic organisms are unclear, particularly in combination with pollutants of suspected modulatory activity, like microplastics (MP). The aim of this study was to reveal the impact on the marine mussel Mytilus galloprovincialis (Lamark, 1819) of Caff (20.0 µg L-1) in the environmentally relevant combination (Mix) with MP 1 mg L-1 (size 35-50 µm) after the exposure for 14 days. Untreated and exposed to Caff and MP separately groups were also examined. Cell viability and cell volume regulation in hemocytes and digestive cells, as well as the indexes of oxidative stress, glutathione (GSH/GSSG) and metallothioneins levels, and caspase-3 activity in digestive gland were assessed. MP and Mix reduced Mn-superoxide dismutase, catalase, and glutathione S-transferase activities and level of lipid peroxidation, but increased the digestive gland cell viability, GSH/GSSG ratio (by 1.4-1.5-fold), metallothioneins level and their Zn content, while Caff did not affect oxidative stress indexes and metallothionein-related Zn chelation. Protein carbonyls were not targeted in all exposures. The distinguishing feature of the Caff group was the decline (2-fold) in caspase-3 activity and low cell viability. The multi-stress effect of Mix was shown by the worsening of the volume regulation of digestive cells and confirmed by discriminant analysis of biochemical indexes. The special capabilities of M. galloprovincialis as a sentinel organism make it an excellent bio-indicator reflecting the multi-stress effects in sub-chronic exposures to potentially harmful substances. The identification of the modulation of individual effects in combined exposure increases the need to base monitoring programs on studies of multi-stress effects in sub-chronic exposures.

Combined toxic effects of nanoplastics and norfloxacin on mussel: Leveraging biochemical parameters and gut microbiota.

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.

Modeling integrated biomarker response (IBR) index for the mussel Mytilus galloprovincialis (Lamark 1819) exposed to heavy metal mixture using the CCF design.

Marine pollutants such as heavy metals (HMs) are considered among the most copious oxidative stress (OS) inducers in marine organisms which leads to reactive oxygen species (ROS) formation. Complementary to our previous bioassays studies, the present research focuses on Catalase (CAT), Glutathione S-transferase (GST) and Malondialdehyde (MDA) as oxidative stress biomarkers and the integrated biomarker response (IBR) indexes (IBR1 and IBR2) as an ecotoxicological assessment tool in Mytilus galloprovincialis using central composite face centered (CCF) design. The oxidative stress biomarkers were measured in adult mussels (45-55 mm) on 3 days-exposed under different sub-lethal concentrations of cadmium (Cd), zinc (Zn), and copper (Cu). Using multiple regressions, ANOVA analysis revealed that experimental data fitted to second-order (quadratic) polynomial equations. The results showed that types, concentrations and metals combinations has a direct effect in CAT and GST activities, MDA level and IBR indexes. Additionally, metal-metal interactions were found synergistic (supra-additive), antagonistic (infra-additive) or zero interaction in the toxicological effect. As necessary, the optimization of the experimental results was done in order to determine the optimal conditions for the oxidative stress responses and IBR indexes. It was demonstrated that the CCF design combined with the multi-biomarker approach and IBR index can be used as an appropriate tool in ecotoxicological modulation and prediction of oxidative stress and antioxidant status by heavy metals in the mussels Mytilus galloprovincialis.