Mussel Hemocytes Research Articles

Transcriptomic and functional analysis of the antiviral response of mussels after a poly I:C stimulation

The study of mussels (Mytilus galloprovincialis) has grown in importance in recent years due to their high economic value and resistance to pathogens. Because of the biological characteristics revealed by mussel genome sequencing, this species is a valuable research model. The high genomic variability and diversity, particularly in immune genes, may be responsible for their resistance to pathogens found in seawater and continuously filtered and internalized by them. These facts, combined with the lack of proven mussel susceptibility to viruses in comparison to other bivalves such as oysters, result in a lack of studies on mussel antiviral response. We used RNA-seq to examine the genomic response of mussel hemocytes after they were exposed to poly I:C, simulating immune cell contact with viral dsRNA. Apoptosis and the molecular axis IRFs/STING-IFI44/IRGC1 were identified as the two main pathways in charge of the response but we also found a modulation of lncRNAs. Finally, in order to obtain new information about the response of mussels to putative natural challenges, we used VHSV virus (Viral Hemorrhagic Septicemia Virus) to run some functional analysis and confirm poly I:C's activity as an immunomodulator in a VHSV waterborne stimulation. Both, poly I:C as well as an injury stimulus (filtered sea water injection) accelerated the viral clearance by hemocytes and altered the expression of several immune genes, including IL-17, IRF1 and viperin.

Photoaging Elevated the Genotoxicity of Polystyrene Microplastics to Marine Mussel Mytilus trossulus (Gould, 1850).

Micro-sized particles of synthetic polymers (microplastics) are found in all parts of marine ecosystems. This fact requires intensive study of the degree of danger of such particles to the life activity of hydrobionts and needs additional research. It is evident that hydrobionts in the marine environment are exposed to microplastics modified by biotic and abiotic degradation. To assess the toxic potential of aging microplastic, comparative studies were conducted on the response of cytochemical and genotoxic markers in hemocytes of the mussel Mytilus trossulus (Gould, 1850) after exposure to pristine and photodegraded (UV irradiation) polystyrene microparticles (µPS). The results of cytochemical tests showed that UV-irradiated µPS strongly reduced metabolism and destabilized lysosome membranes compared to pristine µPS. Using a Comet assay, it was shown that the nuclear DNA of mussel hemocytes showed high sensitivity to exposure to both types of plastics. However, the level of DNA damage was significantly higher in mussels exposed to aging µPS. It is suggested that the mechanism of increased toxicity of photo-oxidized µPS is based on free-radical reactions induced by the UV irradiation of polymers. The risks of toxic effects will be determined by the level of physicochemical degradation of the polymer, which can significantly affect the mechanisms of toxicity.

Exposure to nanopollutants (nZnO) enhances the negative effects of hypoxia and delays recovery of the mussels’ immune system

Aquatic environments face escalating challenges from multiple stressors like hypoxia and nanoparticle exposure, with impact of these combined stressors on mussel immunity being poorly understood. We investigated the individual and combined effects of short-term and long-term hypoxia and exposure to zinc oxide nanoparticles (nZnO) on immune system of the mussels (Mytilus edulis). Hemocyte functional traits (mortality, adhesion capacity, phagocytosis, lysosomal abundance, and oxidative burst), and transcript levels of immune-related genes involved in pathogen recognition (the Toll-like receptors, the complement system components, and the adaptor proteins MyD88) were assessed. Short-term hypoxia minimally affected hemocyte parameters, while prolonged exposure led to immunosuppression, impacting hemocyte abundance, viability, phagocytosis, and defensin gene expression. Under normoxia, nZnO stimulated immune responses of mussel hemocytes. However, combined nZnO and hypoxia induced more pronounced and rapid immunosuppression than hypoxia alone, indicating a synergistic interaction. nZnO exposure hindered immune parameter recovery during post-hypoxic reoxygenation, suggesting persistent impact. Opposing trends were observed in pathogen-sensing and pathogen-elimination mechanisms, with a positive correlation between pathogen-recognition system activation and hemocyte mortality. These findings underscore a complex relationship and potential conflict between pathogen-recognition ability, immune function, and cell survival in mussel hemocytes under hypoxia and nanopollutant stress, and emphasize the importance of considering multiple stressors in assessing the vulnerability and adaptability of mussel immune system under complex environmental conditions of anthropogenically modified coastal ecosystems.

Differential cellular uptake and trafficking of nanoplastics in two hemocyte subpopulations of mussels Perna viridis

Nanoplastics (NPs) could be ingested by bivalves and translocated to their hemocytes, which played critical roles in immune-defense. In this study, highly fluorescent aggregation-induced emission nanoplastics (AIE-NPs) of two sizes (50 nm and 200 nm) were utilized to explore the dynamic systems of cellular uptake, intracellular trafficking, and ultimate localization in the granulocyte and agranulocyte subpopulations of hemocytes in the green mussels Perna viridis. We firstly revealed their differential cellular uptake and trafficking of nanoplastics in two hemocyte subpopulation systems. Specifically, agranulocytes performed relatively faster uptake rate for nanoplastics of two sizes, whereas granulocytes had about tenfold higher accumulation capacity. Unexpectedly, there was little removal of nanoplastics in the two subpopulation systems, and such persistence probably could trigger more severe cytotoxicity. Considerable amounts of 200 nm NPs without plasma membrane wrapping were present in the agranulocytes, which may be related to their unique caveolae-mediated uptake pathway. We further demonstrated that the internalized NPs accelerated the differentiation of agranulocytes into granulocytes with higher immunoreactivity, which may be a crucial detoxification mechanism. Our results highlighted the size-driven fate of NPs in different subpopulations of mussel hemocytes, contributing to a more comprehensive understanding of the toxicity of NPs to aquatic organisms.

Genotoxicand biomarker response of mussel gill cells, haemocyte and A549 human lung cells to radiation from X-rays

A comparative study on the genotoxic effect of radiation from X-rays of different levels on Mussel gill cells, haemocyte and human lung cell A549 using the comet assay was carried out. The results revealed an increasing DNA damage with increasing doses of X-ray, single strand breaks and alkali labile sites in the haemocyte of the control had a mean value of 5.92 ± 1.27 while the highest treatment group 15Gray (Gy) had a mean value of 78.65 ± 4.36,withsignificant differences (P <0.05), A mean value of 21.10 ± 13.10 was recorded for Mussel gill cells in the control group while the highest treatment group 15 Gray (Gy) had 85.55 ± 1.43. Statistical analysis revealed significant differences (P <0.05) for univariate tests of the control group and the treatment groups. The A549 human lung cells had a minimal damage in the control group 2.30 ± 0.30 while the highest exposed group had a mean value from the median % tail intensity of 43.10 ± 2.81. Pair wise comparison of gills cells, haemocytes from mussels and A549 human lung cells exposed to 15 Gy of X-rays shows that the highest damaging effect was observed on the gill cells followed by the haemocytes with the least damaging effect on the A549 human lung cells exposed to the same dose rate of X-rays with significant differences (P <0.05) between them.This study revealed that A549 human lung cells were more resistant to DNA damage induced by the X-rays when compared to mussel gill and haemocyte cells and also indicates that mussel gill cells and haemocytes can serve as biomarkers of genotoxicity of radiation exposure using the comet assay.

Short communication: ROS production and mitochondrial membrane potential in hemocytes of marine bivalves, Mytilus galloprovincialis and Magallana gigas, under hypoosmotic stress.

Bivalve mollusks that inhabit low-depth coastal and estuarine areas frequently experience osmotic stress that may be also associated with alterations of antioxidant enzyme activities and markers of oxidative stress. Mitochondria are a major source of reactive oxygen species (ROS) in eucaryotic cells. Overpoduction of ROS induces oxidative stress leading to a damage of intracellular compartments and cell death. In euryhaline bivalves, information concerning cellular ROS production upon osmotic stress and changes in mitochondrial membrane potential is scarce. The present study investigates osmotic stability and hemocytes` regulatory volume decrease (RVD) of Mediterranean mussel (Mytilus galloprovincialis) and the Pacific oyster (Magallana gigas). We also studied dynamic changes in intracellular ROS levels and mitochondrial membrane potential in hemocytes undergoing the RVD response following hypoosmotic swelling. Our data revealed that osmotic stability of mussel and oyster hemocytes did not significantly differ. Loss of environmental osmolarity from 460.0±2.0mOsml-1 to 216.0±4.0mOsml-1 resulted in an increase of hemocyte volume by 60% of the initial cellular volume in mussels and by 28% in oysters. After rapid hypoosmotic swelling hemocytes of both species demonstrated the RVD response. At the end of 60min exposure to hypoosmotic environment, hemocyte volume significantly decreased in both species by 10-12% compared to the maximal hemocyte volume. Hypoosmotic shock induced an increase of mitochondrial membrane potential in hemocytes of mussels and oysters. In mussels, increased mitochondrial membrane potential was accompanied with decreased ROS levels in hemocytes, whereas oyster hemocytes showed enhanced ROS production.

Impact of face masks weathering on the mussels Mytilus galloprovincialis

The COVID-19 pandemic has triggered an unprecedented need for single-use face masks, leading to an alarming increase in plastic waste globally. Consequently, the improper disposal of face masks has added to the existing burden of plastic pollution in the oceans. However, the complete environmental and marine ecotoxicological impact remains unclear. This study aims to investigate the ecotoxicological impact caused by the weathering of disposable face masks (DFMs) in the marine environment on mussels Mytilus galloprovincialis (M. galloprovincialis ) by assessing biochemical, cytotoxic, and genotoxic effects. The mask leachate was analysed for the presence of nano and microplastics. Furthermore, the leachate was used in in vivo and in vitro toxicity bioassays to assess its impacts on M. galloprovincialis . The in vivo exposure of M. galloprovincialis to face mask leachate for 14 days induced a significant increase in catalase (CAT) activity in mussel gills, although not enough to prevent oxidative damage to cell membranes. DNA damage was also registered in mussel haemocytes after in vivo exposure to mask leachate. The in vitro Neutral Red (NR) cytotoxicity assay indicated that leachate concentrations ≤ 0.5 g/L-1 pose a significant risk to the health of mussel haemocytes, which seems a reliable tool for the cytotoxicity impact assessment of face masks in the marine environment. Therefore, the leachate obtained from face masks in seawater causes oxidative stress, oxidative damage, cytotoxicity, and genotoxicity in M. galloprovincialis , indicating that the plastic burden generated by DFMs in the ocean and its subsequent weathering represents a ubiquitous and invisible threat to the marine biota.

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.

Metabolomics and lipidomics reveal the effects of the toxic dinoflagellate Alexandrium catenella on immune cells of the blue mussel, Mytilus edulis

The increasing occurrence of harmful algal blooms, mostly of the dinoflagellate Alexandrium catenella in Canada, profoundly disrupts mussel aquaculture. These filter-feeding shellfish feed on A. catenella and accumulate paralytic shellfish toxins, such as saxitoxin, in tissues, making them unsafe for human consumption. Algal toxins also have detrimental effects upon several physiological functions in mussels, but particularly on the activity of hemocytes – the mussel immune cells. The objective of this work was to determine the effects of experimental exposure to A. catenella upon hemocyte metabolism and activity in the blue mussel, Mytilus edulis. To do so, mussels were exposed to cultures of the toxic dinoflagellate A. catenella for 120 h. The resulting mussel saxitoxin load had measurable effects upon survival of hemocytes and induced a stress response measured as increased ROS production. The neutral lipid fraction of mussel hemocytes decreased two-fold, suggesting a differential use of lipids. Metabolomic 1H nuclear magnetic resonance (NMR) analysis showed that A. catenella modified the energy metabolism of hemocytes as well as hemocyte osmolyte composition. The modified energy metabolism was reenforced by contrasting plasma metabolomes between control and exposed mussels, suggesting that the blue mussel may reduce feed assimilation when exposed to A. catenella.

The Comet Assay, a Sensitive Biomarker of Water Quality Improvement Following Adoption of Beneficial Agricultural Practices?

Numerous actions have been undertaken by farmers to attenuate the impact of agricultural activities on aquatic ecosystems. The identification of biomarkers that respond quickly to water quality improvement could facilitate the assessment of adopted alternative practices and help maintain mobilization among stakeholders. We evaluated the potential of the comet assay, a biomarker of genotoxic effects, using a freshwater mussel, Elliptio complanata, as a model animal. The frequency of DNA damage was assessed in hemocytes of mussels collected from a pristine habitat and caged for 8 weeks in the Pot au Beurre River, a tributary of the fluvial Lake St.-Pierre (Quebec, Canada) impacted by agricultural activities. We found that the level of DNA damage naturally induced in mussel hemocytes was low and showed very limited variations over time. Compared with these baseline levels and to laboratory controls, we observed a doubling in DNA alterations in mussels exposed to agricultural runoff in the third branch of the Pot au Beurre River. The genotoxic response was significantly lower in mussels caged in the first branch of the Pot au Beurre River, where longer stretches of shoreline have been restored as buffer strips. Glyphosate, mesotrione, imazethapyr, and metolachlor were the main discriminant pesticides between these two branches. Metolachlor was found in sufficient concentrations to induce DNA damage, but it is more likely that the observed genotoxicity was the result of a "cocktail effect," that is, the cumulative contribution of coexisting genotoxicants including the above-mentioned herbicides and ingredients in their formulation. Our findings suggest that the comet assay is a sensitive tool for the early detection of changes in water toxicity following the adoption of agricultural beneficial practices. Environ Toxicol Chem 2023;42:2201-2214. © 2023 Crown copyright and The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC. This article is published with the permission of the Controller of HMSO and the King's Printer for Scotland.

Different isoforms of parabens into marine environment: Biological effects on the bacterium Aliivibrio fischeri and the marine mussel Mytilus galloprovincialis

Different isoforms of alkyl esters of p-Hydroxybenzoic acid, also known as parabens, are of great concern due to their widespread presence into the aquatic environment, their high concentrations in wastewater discharges, as well as their ability to induce adverse effects on aquatic organisms. Considering the imperative need for assessing their fate and risk to aquatic environment, the present study investigated the biological effects of two isoforms of parabens, methyl- (MeP) and propyl- (PrP), on the bacterium Aliivibrio fischeri (using the Bioluminescence Inhibition/Microtox® bioassay) and the mussel Mytilus galloprovincialis (in terms of mussel mortality, cellular, oxidative and genotoxic stress indices). The assessment of MeP and PrP behavior into aquatic media (artificial sea water/ASW and 2 % NaCl), primarily performed by UHPLC-UV-MS analysis, showed only a slight hydrolysis of PrP to 4-Hydrobenzoic acid (4-HBA). Furthermore, exposure of both species to different concentrations of each paraben revealed differences among their toxic potential, as well as their ability to cause cellular, oxidative and genotoxic effects on hemocytes of challenged mussels. Interestingly, the Microtox® bioassay showed that PrP mediated toxicity in A. fischeri were more pronounced than MeP, as revealed by the estimated toxic endpoints (in terms of concentration that promote 50 % of bioluminescence inhibition, EC50). Similarly, in challenged mussels, a significant disturbance of mussel hemocytes' lysosomal membrane integrity, as well as enhanced levels of superoxides, nitric oxides, lipid peroxidation byproducts, and micronuclei formation were observed. These findings are of great interest, since MeP and PrP differential toxic potential, as well their ability to induce pre-pathological alterations in marine species, like mussels, give new evidence for their risk to aquatic biota.

Lysosomal Membrane Stability of Mussel (Mytilus galloprovincialis Lamarck, 1819) as a Biomarker of Cellular Stress for Environmental Contamination.

The lysosomal membrane stability (LMS) of hemocytes in wild mussels (Mytilus galloprovincialis) as a biomarker of cellular stress for chemical pollution was tested by neutral red retention time (NRRT) assays. To assess the environmental contamination in the study area, seawater quality and pollutant bioaccumulation throughout the soft tissue of mussels were investigated. The samples were collected in July 2022 at four sites on the Romanian Black Sea coast considered to be differently affected by contamination. To support the suitability of LMS as a biomarker of contaminant-induced stress, the contaminant body burdens of the mussels were compared with the NRRT values. The results showed a significantly reduced NRRT in all investigated locations, particularly in port areas (mean retention time between 11 and 14 min). The elevated bioaccumulation of organochlorinated pesticides (OCPs) and polychlorinated biphenyls (PCBs) and low NRRTs were observed at the most contaminated sites (i.e., ports). The low lysosomal stability reflected stress and damage in the hemocytes of mussels and could be related to the body burdens of contaminants. LMS is an effective indicator of health status in mussels and could be considered a sensitive biomarker of cellular stress induced by contaminant exposure.

Polystyrene nanoplastics in the marine mussel Mytilus galloprovincialis.

Concerns about plastic pollution and its toxicity towards animals and people are growing. Polystyrene (PS) is a plastic polymer highly produced in Europe for packaging purposes and building insulation amongst others. Whatever their source—illegal dumping, improper waste management, or a lack of treatment for the removal of plastic debris from wastewater treatment plants—PS products ultimately end up in the marine environment. Nanoplastics (<1000 nm) are the new focus for plastic pollution, gaining broad interest. Whether primary or secondary, their small size permits nanoparticles to cross cellular boundaries, consequently leading to adverse toxic effects. An in vitro assay of Mytilus galloprovincialis haemocytes exposed to 10 μg/L of polystyrene nanoplastics (PS-NPs; 50 nm) for 24 h was used to test cellular viability along with the luminescence inhibition (LC50) of Aliivibrio fischeri bacteria to evaluate acute toxicity. Cellular viability of mussel haemocytes decreased significantly after a 24 h exposure and PS-NPs LC50 range from 180 to 217, μg/L. In addition, a 28-day exposure of the marine bivalve M. galloprovincialis to PS-NPs (10 μg/L; 50 nm) was performed to evaluate the neurotoxic effects and the uptake of these plastic particles in three bivalve tissues (gills, digestive gland, and gonads). The ingestion of PS-NPs was time- and tissue-specific, suggesting that PS-NPs are ingested through the gills and then translocated through the mussel bloodstream, to the digestive gland and gonads where the highest amount of ingested PS-NPs was reported. Ingested PS-NPs may compromise the digestive glands' key metabolic function and impair mussels' gametogenic and reproductive success. Data on acetylcholinesterase inhibition and those previously obtained on a wide range of cellular biomarkers were elaborated through weighted criteria providing a synthetic assessment of cellular hazard from PS-NPs.

Multiple responses of Mytilus galloprovincialis to plastic microfibers

Plastic microfibers (MF) represent the major source of MF found in the environment, the majority made of polyester (PES). Marine bivalves, suspension feeders widespread in coastal areas subjected to higher anthropogenic input, can accumulate MF from the water column in their tissues. This raised some concern about their possible impact on bivalve health and potential transfer along the food chain. In this work, the effects of PES-MF on the mussel Mytilus galloprovincialis were investigated, utilizing MF obtained by cryo-milling of a fleece cover. Fiber characterization indicated the polymer composition as polyethylene terephthalate (PET); the size distribution was in a length range resembling that of MF released from textile washing, and including those that can be ingested by mussels. MF were first screened for short-term in vitro immune responses in mussel hemocytes. The effects of in vivo exposure (96 h, 10 and 100 μg/L, corresponding to about 150 and 1500 MF/mussel/L, respectively), were then evaluated. Data are presented on hemolymph immune biomarkers (Reactive Oxygen Species and nitric oxide production, lysozyme activity), and on antioxidant biomarkers (catalase and glutathione S-transferase) and histopathology in gills and digestive gland. Tissue MF accumulation was also evaluated.MF exposure stimulated extracellular immune responses both in vitro and in vivo, indicating induction of immune/inflammatory processes. In both tissues, stimulation of antioxidant enzyme activities, suggesting oxidative stress conditions, and histopathological changes were observed, with stronger effects often observed at lower concentration. Although mussel retained a very small fraction of MF, their accumulation was higher in the digestive gland than in gills, and in both tissues of mussels exposed to the lowest concentration. Selective accumulation of shorter MF was also observed, particularly in gills. Overall, the results demonstrate that at environmental exposure levels, PET-MF have a significant impact on mussel physiology, affecting multiple processes in different tissues.

In vitro assessment of the genotoxicity and immunotoxicity of treated and untreated municipal effluents and receiving waters in freshwater organisms

Municipal wastewater effluent is one of the largest sources of pollution entering surface waters in the Laurentian Great Lakes. Exposure to wastewater effluent has been associated with impaired immune systems and induction of genotoxicity to aquatic animals. Due to habitat degradation and environmental pollution linked to industrial development and population growth, several regions of the Great Lakes have been designated Areas of Concern (AOCs). In this study, we assessed the effect of extracts of sewage influent, (treated) effluent and receiving surface waters from the Hamilton Harbour AOC and the Toronto and Region AOC (Ontario, Canada) on the phagocytic immune response of rainbow trout (Oncorhynchus mykiss) kidney leukocytes and the genotoxicity (DNA strand breaks) of these extracts on freshwater mussel (Eurynia dilatata) hemocytes. We identified and quantified numerous chemicals present in the various samples extracted for exposure. In freshwater mussels, extracts from Hamilton Harbour AOC induced DNA damage with the most frequency (12 out of 28 samples) regardless of sample type, reflecting past and present industrial activities. In contrast, extracts from Toronto and Region AOC induced DNA damage infrequently (2 out of 32 (summer) and 5 out of 32 (fall) samples, respectively) and from different WWTPs at different times. None of the extracts induced any significant effect on phagocytosis of rainbow trout kidney leukocytes. The present study indicates that despite overall improvements to effluent quality, treatment of influent by WWTPs may not result in a corresponding improvement of the genotoxicity of effluents. In vitro bioassays are useful and cost-effective rapid-screening tools for preliminary assessments of contamination of aquatic ecosystems.

Comparative immune responses of blue mussel and zebra mussel haemocytes to simultaneous chemical and bacterial exposure

Biomonitoring at the scale of the aquatic continuum and based on biomarkers, requires various representative species and a knowledge of their sensitivity to contaminants. Mussel immunomarkers are established tools for evaluating immunotoxic stress, but little is known about the consequences of an immune activation by local microorganisms on their response to pollution. This study aims to compare the sensitivity of cellular immunomarkers in two mussel species from different environments, the marine mussel Mytilus edulis (blue mussel) and the freshwater mussel Dreissena polymorpha (zebra mussel), to chemical stressors combined with bacterial challenge. Haemocytes were exposed ex vivo to the contaminants (bisphenol A, caffeine, copper chloride, oestradiol, ionomycin) for 4 h. The chemical exposures were coupled with simultaneous bacterial challenges (Vibrio splendidus and Pseudomonas fluorescens) to trigger activation of the immune response. Cellular mortality, phagocytosis efficiency and phagocytosis avidity were then measured by flow cytometry. The two mussel species had different basal levels since D. polymorpha showed higher cell mortality than M. edulis (23.9 ± 11% and 5.5 ± 3% dead cells respectively), and lower phagocytosis efficiency (52.6 ± 12% and 62.2 ± 9%), but similar phagocytosis avidity (17.4 ± 5 and 13.4 ± 4 internalised beads). Both bacterial strains led to an increase in cellular mortality (+8.4% dead cells in D. polymorpha, +4.9% in M. edulis), as well an activation of phagocytosis (+9.2% of efficient cells in D. polymorpha, +6.2% efficient cells and +3 internalised beads per cell in M. edulis). All chemicals triggered an increase in haemocyte mortality and/or phagocytotic modulations, except for bisphenol A. The two species differed in the amplitude of their response. The addition of a bacterial challenge significantly altered cell responses to chemicals with synergetic and antagonistic variations compared to a single exposure, depending on the compound used and the mussel species. This work highlights the species-specific sensitivity of mussel immunomarkers to contaminants, with or without bacterial challenge, and the necessity of considering the presence of in natura non-pathogenic microorganisms for future in situ applications of immunomarkers.

Trophic Transfer of Single-Walled Carbon Nanotubes at the Base of the Food Chain and Toxicological Response.

The potential for trophic transfer of single-walled carbon nanotubes (SWCNTs) was assessed using the green algae Tetraselmis suecica and the blue mussel Mytilus edulis in a series of laboratory experiments. Swanee River Natural Organic Matter (SRNOM)-dispersed SWCNTs were introduced into growing algal cultures. Light microscopical observations, confirmed by scanning electronic microscopy (SEM) and Raman spectroscopy, showed that SWCNT agglomerates adhered to the external algal cell walls and transmission electronic microscopy (TEM) results suggested internalization. A direct effect of SWCNT exposure on the algae was a significant decrease in growth, expressed as chlorophyll a concentration and cell viability. Mussels, fed with algae in the presence of SWCNTs, led to significantly increased pseudofaeces production, indicating selective feeding. Nevertheless, histological sections of the mussel digestive gland following exposure showed evidence of SWCNT-containing algae. Furthermore, DNA damage and oxidative stress biomarker responses in the mussel haemocytes and gill tissue were significantly altered from baseline values and were consistent with previously observed responses to SWCNT exposure. In conclusion, the observed SWCNT-algal interaction demonstrated the potential for SWCNT entrance at the base of the food chain, which may facilitate their trophic transfer with potential consequences for human exposure and health.

Responses of Mytilus galloprovincialis to challenge with environmental isolates of the potential emerging pathogen Malaciobacter marinus

Bacteria of the Arcobacter-like spp. represent emerging foodborne zoonotic pathogens in humans and animals. Their increasing presence in seafood, suggesting higher occurrence in seawater due to marine pollution, is raising some environmental concern. Although Arcobacter is frequently detected in diseased oysters and stressed bivalve species, no data are available so far on its potential pathogenicity or interactions with the immune system of the bivalve host.In this work, responses to challenge with two strains of Malaciobacter marinus IRTA-19-131 and IRTA-19-132, R1 and R2), isolated from adult Crassostrea gigas during a mortality event in 2019 in Spain, were investigated in the mussel Mytilus galloprovincialis.In vivo experiments were performed in larvae (48 h post-fertilization), and in adult mussels at 24 h post-injection, in order to evaluate the pathogenicity for early developmental stages, and the hemolymph immune responses, respectively. Both R1 and R2 were moderately pathogenic to early larvae, with significant decreases in the development of normal D-veligers from 104 and 103 CFU/mL, respectively. In adults, both strains decreased hemocyte lysosomal membrane stability (LMS), and stimulated extracellular defense responses (ROS production and lysozyme activity).The interactions between mussel hemocytes and M. marinus were investigated in in vitro short-term experiments (30–90 min) using the R1 strain (106–108 CFU/mL). R1 decreased LMS and induced lysosomal enlargement, but not cell detachment or death, and stimulated extracellular ROS production and lysozyme release, confirming in vivo data. Moreover, lysosomal internalization and degradation of bacteria were observed, together with changes in levels of activated mTor and LC3, indicating phagocytic activity. Overall, the results indicate the activation of both extracellular and intracellular immune defenses against M. marinus R1. Accordingly, these responses resulted in a significant hemolymph bactericidal activity, with a large contribution of hemolymph serum.The results represent the first data on the potential pathogenicity of Arcobacter isolated from a shellfish mortality to bivalve larvae and adults, and on their interactions with the immune system of the host.

Bioaccumulation, release and genotoxicity of stainless steel particles in marine bivalve molluscs.

During the decommissioning and removal of radioactive material in nuclear facilities, fine, tritiated dusts of stainless steel, cement or tungsten are generated that could be accidently released to the environment. However, the potential radio- and ecotoxicological effects these tritiated particles may have are unknown. In this study, stainless steel particles (SSPs) representative of those likely to be tritiated are manufactured by hydrogenation and their tissue-specific bioaccumulation, release (depuration) and subsequent genotoxic response have been studied in the marine mussel, Mytilus galloprovincialis, as a baseline for future assessments of the potential effects of tritiated SSPs. Exposure to 1000μgL-1 of SSPs and adopting Cr as a proxy for stainless steel revealed relatively rapid accumulation (∼5h) in the various mussel tissues but mostly in the digestive gland. Over longer periods up to 18 days, SSPs were readily rejected and egested as faecal material. DNA strand breaks, as a measure of genotoxicity, were determined at each time point in mussel haemocytes using single cell gel electrophoresis, or the comet assay. Lack of chemical genotoxicity was attributed to the rapid processing of SSP particles and limited dissolution of elemental components of steel. Further work employing tritiated SSPs will enable radio-toxicology to be studied without the confounding effects of chemical toxicity.

Effects of microplastics alone or with sorbed oil compounds from the water accommodated fraction of a North Sea crude oil on marine mussels (Mytilus galloprovincialis)

Microplastics (MPs) can adsorb persistent organic pollutants such as oil hydrocarbons and may facilitate their transfer to organisms (Trojan horse effect). The aim of this study was to examine the effects of a 21 day dietary exposure to polystyrene MPs of 4.5 μm at 1000 particles/mL, alone and with sorbed oil compounds from the water accommodated fraction (WAF) of a naphthenic North Sea crude oil at two dilutions (25 % and 100 %), on marine mussels. An additional group of mussels was exposed to 25 % WAF for comparison. PAHs were accumulated in mussels exposed to WAF but not in those exposed to MPs with sorbed oil compounds from WAF (MPs-WAF), partly due to the low concentration of PAHs in the studied crude oil. Exposure to MPs or to WAF alone altered the activity of enzymes involved in aerobic (isocitrate dehydrogenase) and biotransformation metabolism (glutathione S-transferase). Prevalence of oocyte atresia and volume density of basophilic cells were higher and absorption efficiency lower in mussels exposed to MPs and to WAF than in controls. After 21 days MPs caused DNA damage (Comet assay) in mussel hemocytes. In conclusion, a Trojan horse effect was not observed but both MPs and oil WAF caused an array of deleterious effects on marine mussels at different levels of biological organization.