The queen scallop Aequipecten opercularis: A slow domoic acid depurator?

Domoic acid (DA) is a dangerous phycotoxin produced by several strains of diatoms of the genus Pseudo-nitzschia, and responsible for Amnesic Shellfish Poisoning (ASP) in humans. The increasingly intense ASP-outbreaks along the English Channel over the last three decades have forced persistent harvest closures of economically important and highly contaminated bivalve stocks exhibiting slow DA-depuration rates, like the king scallop Pecten maximus. Under this scenario, other pectinid species, such as the queen scallop Aequipecten opercularis have been empirically proposed as alternative resources to redress the high economic losses due to the banning of the exploitation of P. maximus. Nevertheless, the kinetics of DA depuration in A. opercularis have not been assessed so far, and its direct extraction after ASP-episodes could represent a serious threat to public health. Hence, the main objective of this work was to estimate the DA-depuration rate in the digestive gland (DG) of naturally contaminated scallops A. opercularis after a toxic Pseudo-nitzschia australis bloom subjected to experimental depuration in the laboratory for 30 days. This study also intended to go further in the knowledge about the anatomical distribution of DA in scallop tissues, and corroborate the implications of autophagy in DA-sequestration in the DG of this species as recently hypothesized. In the DG, the DA-depuration rate (0.018 day-1) suggested that even with toxin burdens as low as 40 mg⋅kg-1 in the DG, queen scallops may remain contaminated for about 70 days, thus longer under intensely contamination scenarios. The subcellular analyses corroborated DA-sequestration mainly through late-autophagy within residual bodies in the DG, without differences in the frequencies of anti-DA labeled residual bodies across the entire depuration process. These results revealed that A. opercularis cannot be considered a fast DA-depurator, and represent a baseline knowledge for decision-making about harvesting natural beds of queen scallops after toxic Pseudo-nitzschia blooms. The findings of this work also represent a cornerstone for further research to accelerate DA-depuration in this species.

The amnesic shellfish poisoning toxin, domoic acid: The tattoo of the king scallop Pecten maximus.

Domoic acid (DA) is a potent neurotoxin produced by diatoms of the genus Pseudo-nitzschia and is responsible for Amnesic Shellfish Poisoning (ASP) in humans. Some fishery resources of high commercial value, such as the king scallop Pecten maximus, are frequently exposed to toxic Pseudo-nitzschia blooms and are capable of accumulating high amounts of DA, retaining it for months or even a few years. This poses a serious threat to public health and a continuous economical risk due to fishing closures of this resource in the affected areas. Recently, it was hypothesized that trapping of DA within autophagosomic-vesicles could be one reason explaining the long retention of the remaining toxin in P. maximus digestive gland. To test this idea, we follow the kinetics of the subcellular localization of DA in the digestive glands of P. maximus during (a) the contamination process - with sequential samplings of scallops reared in the field during 234 days and naturally exposed to blooms of DA-producing Pseudo-nitzschia australis, and (b) the decontamination process - where highly contaminated scallops were collected after a natural bloom of toxic P. australis and subjected to DA-depuration in the laboratory for 60 days. In the digestive gland, DA-depuration rate (0.001 day-1) was much slower than contamination kinetics. The subcellular analyses revealed a direct implication of early autophagy in DA sequestration throughout contamination (r = 0.8, P < 0.05), while the presence of DA-labeled residual bodies (late autophagy) appeared to be strongly and significantly related to slow DA-depuration (r = -0.5) resembling an analogous DA-tattooing in the digestive glands of P. maximus. This work provides new evidence about the potential physiological mechanisms involved in the long retention of DA in P. maximus and represents the baseline to explore procedures to accelerate decontamination in this species.

Comparative study of domoic acid accumulation, isomer content and associated digestive subcellular processes in five marine invertebrate species.

Despite the deleterious effects of the phycotoxin domoic acid (DA) on human health, and the permanent threat of blooms of the toxic Pseudo-nitzschia sp. over commercially important fishery-resources, knowledge regarding the physiological mechanisms behind the profound differences in accumulation and depuration of this toxin in contaminated invertebrates remain very scarce. In this work, a comparative analysis of accumulation, isomer content, and subcellular localization of DA in different invertebrate species was performed. Samples of scallops Pecten maximus and Aequipecten opercularis, clams Donax trunculus, slippersnails Crepidula fornicata, and seasquirts Asterocarpa sp. were collected after blooms of the same concentration of toxic Pseudo-nitzschia australis. Differences (P < 0.05) in DA accumulation were found, wherein P. maximus showed up to 20-fold more DA in the digestive gland than the other species. Similar profiles of DA isomers were found between P. maximus and A. opercularis, whereas C. fornicata was the species with the highest biotransformation rate (∼10 %) and D. trunculus the lowest (∼4 %). DA localization by immunohistochemical analysis revealed differences (P < 0.05) between species: in P. maximus, DA was detected mainly within autophagosome-like vesicles in the cytoplasm of digestive cells, while in A. opercularis and C. fornicata significant DA immunoreactivity was found in post-autophagy residual bodies. A slight DA staining was found free within the cytoplasm of the digestive cells of D. trunculus and Asterocarpa sp. The Principal Component Analysis revealed similarities between pectinids, and a clear distinction of the rest of the species based on their capabilities to accumulate, biotransform, and distribute the toxin within their tissues. These findings contribute to improve the understanding of the inter-specific differences concerning the contamination-decontamination kinetics and the fate of DA in invertebrate species.

Microbiota of the Digestive Glands and Extrapallial Fluids of Clams Evolve Differently Over Time Depending on the Intertidal Position.

The Manila clam (Ruditapes philippinarum) is the second most exploited bivalve in the world but remains threatened by diseases and global changes. Their associated microbiota play a key role in their fitness and acclimation capacities. This study aimed at better understanding the behavior of clam digestive glands and extrapallial fluids microbiota at small, but contrasting spatial and temporal scales. Results showed that environmental variations impacted clam microbiota differently according to the considered tissue. Each clam tissue presented its own microbiota and showed different dynamics according to the intertidal position and sampling period. Extrapallial fluids microbiota was modified more rapidly than digestive glands microbiota, for clams placed on the upper and lower intertidal position, respectively. Clam tissues could be considered as different microhabitats for bacteria as they presented different responses to small-scale temporal and spatial variabilities in natural conditions. These differences underlined a more stringent environmental filter capacity of the digestive glands.

First subcellular localization of the amnesic shellfish toxin, domoic acid, in bivalve tissues: Deciphering the physiological mechanisms involved in its long-retention in the king scallop Pecten maximus.

Domoic acid (DA), the phycotoxin responsible for amnesic shellfish poisoning (ASP), is an excitatory amino acid naturally produced by at least twenty-eight species of the bloom-forming marine diatoms Pseudo-nitzschia spp. Suspension feeders, such as bivalve mollusks, can accumulate and lengthy retain high amounts of DA in their tissues, threatening human health and leading to extensive-prolonged fishery closures, and severe economic losses. This is particularly problematic for the king scallop Pecten maximus, which retains high burdens of DA from months to years compared to other fast-depurator bivalves. Nonetheless, the physiological and cellular processes responsible for this retention are still unknown. In this work, for the first time, a novel immunohistochemical techniques based on the use of an anti-DA antibody was successfully developed and applied for DA-detection in bivalve tissues at a subcellular level. Our results show that in naturally contaminated P. maximus following a Pseudo-nitzschia australis outbreak, DA is visualized mainly within small membrane-bounded vesicles (1 - 2.5 µm) within the digestive gland cells, identified as autophagosomic structures by means of immune-electron microscopy, as well as in the mucus-producing cells, particularly those from gonad ducts and digestive tract. Trapping of DA in autophagososomes may be a key mechanism in the long retention of DA in scallops. These results and the development of DA-immunodetection are essential to provide a better understanding of the fate of DA, and further characterize DA contamination-decontamination kinetics in marine bivalves, as well as the main mechanisms involved in the long retention of this toxin in P. maximus.

Electrophysiological Evaluation of Pacific Oyster (Crassostrea gigas) Sensitivity to Saxitoxin and Tetrodotoxin.

Pacific oysters (Crassostrea gigas) may bio-accumulate high levels of paralytic shellfish toxins (PST) during harmful algal blooms of the genus Alexandrium. These blooms regularly occur in coastal waters, affecting oyster health and marketability. The aim of our study was to analyse the PST-sensitivity of nerves of Pacific oysters in relation with toxin bio-accumulation. The results show that C. gigas nerves have micromolar range of saxitoxin (STX) sensitivity, thus providing intermediate STX sensitivity compared to other bivalve species. However, theses nerves were much less sensitive to tetrodotoxin. The STX-sensitivity of compound nerve action potential (CNAP) recorded from oysters experimentally fed with Alexandrium minutum (toxic-alga-exposed oysters), or Tisochrysis lutea, a non-toxic microalga (control oysters), revealed that oysters could be separated into STX-resistant and STX-sensitive categories, regardless of the diet. Moreover, the percentage of toxin-sensitive nerves was lower, and the STX concentration necessary to inhibit 50% of CNAP higher, in recently toxic-alga-exposed oysters than in control bivalves. However, no obvious correlation was observed between nerve sensitivity to STX and the STX content in oyster digestive glands. None of the nerves isolated from wild and farmed oysters was detected to be sensitive to tetrodotoxin. In conclusion, this study highlights the good potential of cerebrovisceral nerves of Pacific oysters for electrophysiological and pharmacological studies. In addition, this study shows, for the first time, that C. gigas nerves have micromolar range of STX sensitivity. The STX sensitivity decreases, at least temporary, upon recent oyster exposure to dinoflagellates producing PST under natural, but not experimental environment.

The toxic dinoflagellate Alexandrium minutum affects oyster gamete health and fertilization potential.

Dinoflagellates from the globally distributed genus Alexandrium are known to produce both paralytic shellfish toxins (PST) and uncharacterized bioactive extracellular compounds (BEC) with allelopathic, ichthyotoxic, hemolytic and cytotoxic activities. In France, blooms of Alexandrium minutum appear generally during the spawning period of most bivalves. These blooms could therefore alter gametes and/or larval development of bivalves, causing severe issues for ecologically and economically important species, such as the Pacific oyster Crassostrea (=Magallana) gigas. The aim of this work was to test the effects of three strains of A. minutum producing either only PST, only BEC, or both PST and BEC upon oyster gametes, and potential consequences on fertilization success. Oocytes and spermatozoa were exposed in vitro for 2 h to a range of environmentally realistic A. minutum concentrations (10-2.5 × 104 cells mL-1). Following exposure, gamete viability and reactive oxygen species (ROS) production were assessed by flow cytometry, spermatozoa motility and fertilization capacities of both spermatozoa and oocytes were analysed by microscopy. Viability and fertilization capacity of spermatozoa and oocytes were drastically reduced following exposure to 2.5 × 104 cells mL-1 of A. minutum. The BEC-producing strain was the most potent strain decreasing spermatozoa motility, increasing ROS production of oocytes, and decreasing fertilization, from the concentration of 2.5 × 103 cells mL-1. This study highlights the significant cellular toxicity of the BEC produced by A. minutum on oyster gametes. Physical contact between gametes and motile thecate A. minutum cells may also contribute to alter oyster gamete integrity. These results suggest that oyster gametes exposure to A. minutum blooms could affect oyster fertility and reproduction success.

Biological rhythms in the deep-sea hydrothermal mussel Bathymodiolus azoricus.

Biological rhythms are a fundamental property of life. The deep ocean covers 66% of our planet surface and is one of the largest biomes. The deep sea has long been considered as an arrhythmic environment because sunlight is totally absent below 1,000 m depth. In the present study, we have sequenced the temporal transcriptomes of a deep-sea species, the ecosystem-structuring vent mussel Bathymodiolus azoricus. We reveal that tidal cycles predominate in the transcriptome and physiology of mussels fixed directly at hydrothermal vents at 1,688 m depth at the Mid-Atlantic Ridge, whereas daily cycles prevail in mussels sampled after laboratory acclimation. We identify B. azoricus canonical circadian clock genes, and show that oscillations observed in deep-sea mussels could be either a direct response to environmental stimulus, or be driven endogenously by one or more biological clocks. This work generates in situ insights into temporal organisation in a deep-sea organism.

Cultures of Dinophysis sacculus, D. acuminata and pectenotoxin 2 affect gametes and fertilization success of the Pacific oyster, Crassostrea gigas.

Harmful algal blooms (HABs) of toxic species of the dinoflagellate genus Dinophysis are a threat to human health as they are mainly responsible for diarrheic shellfish poisoning (DSP) in the consumers of contaminated shellfish. Such contamination leads to shellfish farm closures causing major economic and social issues. The direct effects of numerous HAB species have been demonstrated on adult bivalves, whereas the effects on critical early life stages remain relatively unexplored. The present study aimed to determine the in vitro effects of either cultivated strains of D. sacculus and D. acuminata isolated from France or their associated toxins (i.e. okadaic acid (OA) and pectenotoxin 2 (PTX2)) on the quality of the gametes of the Pacific oyster Crassostrea gigas. This was performed by assessing the ROS production and viability of the gametes using flow cytometry, and fertilization success using microscopic counts. Oocytes were more affected than spermatozoa and their mortality and ROS production increased in the presence of D. sacculus and PTX2, respectively. A decrease in fertilization success was observed at concentrations as low as 0.5 cell mL-1 of Dinophysis spp. and 5 nM of PTX2, whereas no effect of OA could be observed. The effect on fertilization success was higher when both gamete types were concomitantly exposed compared to separate exposures, suggesting a synergistic effect. Our results also suggest that the effects could be due to cell-to-cell contact. These results highlight a potential effect of Dinophysis spp. and PTX2 on reproduction and recruitment of the Pacific oyster.

The marine intertidal zone shapes oyster and clam digestive bacterial microbiota.

Digestive microbiota provide a wide range of beneficial effects on host physiology and are therefore likely to play a key role in marine intertidal bivalve ability to acclimatize to the intertidal zone. This study investigated the effect of intertidal levels on the digestive bacterial microbiota of oysters (Crassostrea gigas) and clams (Ruditapes philippinarum), two bivalves with different ecological niches. Based on 16S rRNA region sequencing, digestive glands, seawater and sediments harbored specific bacterial communities, dominated by operational taxonomic units assigned to the Mycoplasmatales,Desulfobacterales and Rhodobacterales orders, respectively. Field implantation modified digestive bacterial microbiota of both bivalve species according to their intertidal position. Rhodospirillales and Legionellales abundances increased in oysters and clams from the low intertidal level, respectively. After a 14-day depuration process, these effects were still observed, especially for clams, while digestive bacterial microbiota of oysters were subjected to more short-term environmental changes. Nevertheless, 3.5 months stay on an intertidal zone was enough to leave an environmental footprint on the digestive bacterial microbiota, suggesting the existence of autochthonous bivalve bacteria. When comparing clams from the three intertidal levels, 20% of the bacterial assemblage was shared among the levels and it was dominated by an operational taxonomic unit affiliated to the Mycoplasmataceae and Spirochaetaceae families.

Author Correction: Proteinaceous secretion of bioadhesive produced during crawling and settlement of Crassostrea gigas larvae.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The toxic dinoflagellate Alexandrium minutum impairs the performance of oyster embryos and larvae.

The dinoflagellate genus Alexandrium comprises species that produce highly potent neurotoxins known as paralytic shellfish toxins (PST), and bioactive extracellular compounds (BEC) of unknown structure and ecological significance. The toxic bloom-forming species, Alexandrium minutum, is distributed worldwide and adversely affects many bivalves including the commercially and ecologically important Pacific oyster, Crassostrea gigas. In France, recurrent A. minutum blooms can co-occur with C. gigas spawning and larval development, and may endanger recruitment and population renewal. The present study explores how A. minutum affects oyster early development by exposing embryos and larvae, under controlled laboratory conditions, to two strains of A. minutum, producing only BEC or both PST and BEC. Results highlight the major role of BEC in A. minutum toxicity upon oyster development. The BEC strain caused lysis of embryos, the most sensitive stage to A. minutum toxicity among planktonic life stages. In addition, the non-PST-producing A. minutum strain inhibited hatching, disrupted larval swimming behavior, feeding, growth, and induced drastic decreases in survival and settlement of umbonate and eyed larvae (9 and 68 %, respectively). The findings indicated PST accumulation in oyster larvae (e.g. umbonate stages), possibly impairing development and settlement of larvae in response to the PST-producing strain. This work provides evidences that A. minutum blooms could hamper settlement of shellfish.

The dinoflagellate Alexandrium minutum affects development of the oyster Crassostrea gigas, through parental or direct exposure.

Harmful algal blooms are a threat to aquatic organisms and coastal ecosystems. Among harmful species, the widespread distributed genus Alexandrium is of global importance. This genus is well-known for the synthesis of paralytic shellfish toxins which are toxic for humans through the consumption of contaminated shellfish. While the effects of Alexandrium species upon the physiology of bivalves are now well documented, consequences on reproduction remain poorly studied. In France, Alexandrium minutum blooms have been recurrent for the last decades, generally appearing during the reproduction season of most bivalves including the oyster Crassostrea gigas. These blooms could not only affect gametogenesis but also spawning, larval development or juvenile recruitment. This study assesses the effect of toxic A. minutum blooms on C. gigas reproduction. Adult oysters were experimentally exposed to A. minutum, at environmentally realistic concentrations (102 to 103 cells mL-1) for two months during their gametogenesis and a control group, not exposed to A. minutum was fed with a non-toxic dinoflagellate. To determine both consequences to next generation and direct effects of A. minutum exposure on larvae, the embryo-larval development of subsequent offspring was conducted with and without A. minutum exposure at 102 cells mL-1. Effects at each stage of the reproduction were investigated on ecophysiological parameters, cellular responses, and offspring development. Broodstock exposed to A. minutum produced spermatozoa with decreased motility and larvae of smaller size which showed higher mortalities during settlement. Embryo-larval exposure to A. minutum significantly reduced growth and settlement of larvae compared to non-exposed offspring. This detrimental consequence on larval growth was stronger in larvae derived from control parents compared to offspring from exposed parents. This study provides evidence that A. minutum blooms, whether they occur during gametogenesis, spawning or larval development, can either affect gamete quality and/or larval development of C. gigas, thus potentially impacting oyster recruitment.

Proteinaceous secretion of bioadhesive produced during crawling and settlement of Crassostrea gigas larvae.

Bioadhesion of marine organisms has been intensively studied over the last decade because of their ability to attach in various wet environmental conditions and the potential this offers for biotechnology applications. Many marine mollusc species are characterized by a two-phase life history: pelagic larvae settle prior to metamorphosis to a benthic stage. The oyster Crassostrea gigas has been extensively studied for its economic and ecological importance. However, the bioadhesive produced by ready to settle larvae of this species has been little studied. The pediveliger stage of oysters is characterized by the genesis of a specific organ essential for adhesion, the foot. Our scanning electron microscopy and histology analysis revealed that in C. gigas the adhesive is produced by several foot glands. This adhesive is composed of numerous fibres of differing structure, suggesting differences in chemical composition and function. Fourier transformed infrared spectroscopy indicated a mainly proteinaceous composition. Proteomic analysis of footprints was able to identify 42 proteins, among which, one uncharacterized protein was selected on the basis of its pediveliger transcriptome specificity and then located by mRNA in situ hybridization, revealing its potential role during substrate exploration before oyster larva settlement.

Oyster transcriptome response to Alexandrium exposure is related to saxitoxin load and characterized by disrupted digestion, energy balance, and calcium and sodium signaling.

Harmful Algal Blooms are worldwide occurrences that can cause poisoning in human seafood consumers as well as mortality and sublethal effets in wildlife, propagating economic losses. One of the most widespread toxigenic microalgal taxa is the dinoflagellate Genus Alexandrium, that includes species producing neurotoxins referred to as PST (Paralytic Shellfish Toxins). Blooms cause shellfish harvest restrictions to protect human consumers from accumulated toxins. Large inter-individual variability in toxin load within an exposed bivalve population complicates monitoring of shellfish toxicity for ecology and human health regulation. To decipher the physiological pathways involved in the bivalve response to PST, we explored the whole transcriptome of the digestive gland of the Pacific oyster Crassostrea gigas fed experimentally with a toxic Alexandrium minutum culture. The largest differences in transcript abundance were between oysters with contrasting toxin loads (1098 transcripts), rather than between exposed and non-exposed oysters (16 transcripts), emphasizing the importance of toxin load in oyster response to toxic dinoflagellates. Additionally, penalized regressions, innovative in this field, modeled accurately toxin load based upon only 70 transcripts. Transcriptomic differences between oysters with contrasting PST burdens revealed a limited suite of metabolic pathways affected, including ion channels, neuromuscular communication, and digestion, all of which are interconnected and linked to sodium and calcium exchanges. Carbohydrate metabolism, unconsidered previously in studies of harmful algal effects on shellfish, was also highlighted, suggesting energy challenge in oysters with high toxin loads. Associations between toxin load, genotype, and mRNA levels were revealed that open new doors for genetic studies identifying genetically-based low toxin accumulation.

Long dsRNAs promote an anti-viral response in Pacific oyster hampering ostreid herpesvirus 1 replication.

Double-stranded RNA (dsRNA)-mediated genetic interference (RNAi) is a widely used reverse genetic tool for determining the loss-of-function phenotype of a gene. Here, the possible induction of an immune response by long dsRNA was tested in a marine bivalve (Crassostrea gigas), as well as the specific role of the subunit 2 of the nuclear factor κB inhibitor (IκB2). This gene is a candidate of particular interest for functional investigations in the context of oyster mass mortality events, as Cg-IκB2 mRNA levels exhibited significant variation depending on the amount of ostreid herpesvirus 1 (OsHV-1) DNA detected. In the present study, dsRNAs targeting Cg-IκB2 and green fluorescent protein genes were injected in vivo into oysters before being challenged by OsHV-1. Survival appeared close to 100% in both dsRNA-injected conditions associated with a low detection of viral DNA and a low expression of a panel of 39 OsHV-1 genes as compared with infected control. Long dsRNA molecules, both Cg-IκB2- and GFP-dsRNA, may have induced an anti-viral state controlling the OsHV-1 replication and precluding the understanding of the specific role of Cg-IκB2 Immune-related genes including Cg-IκB1, Cg-Rel1, Cg-IFI44, Cg-PKR and Cg-IAP appeared activated in the dsRNA-injected condition, potentially hampering viral replication and thus conferring a better resistance to OsHV-1 infection. We revealed that long dsRNA-mediated genetic interference triggered an anti-viral state in the oyster, emphasizing the need for new reverse genetics tools for assessing immune gene function and avoiding off-target effects in bivalves.

Remodeling of the cycling transcriptome of the oyster Crassostrea gigas by the harmful algae Alexandrium minutum.

As a marine organism, the oyster Crassostrea gigas inhabits a complex biotope governed by interactions between the moon and the sun cycles. We used next-generation sequencing to investigate temporal regulation of oysters under light/dark entrainment and the impact of harmful algal exposure. We found that ≈6% of the gills' transcriptome exhibits circadian expression, characterized by a nocturnal and bimodal pattern. Surprisingly, a higher number of ultradian transcripts were also detected under solely circadian entrainment. The results showed that a bloom of Alexandrium minutum generated a remodeling of the bivalve's temporal structure, characterized by a loss of oscillations, a genesis of de novo oscillating transcripts, and a switch in the period of oscillations. These findings provide unprecedented insights into the diurnal landscape of the oyster's transcriptome and pleiotropic remodeling due to toxic algae exposure, revealing the intrinsic plasticity of the cycling transcriptome in oysters.

Molecular Characterization of Voltage-Gated Sodium Channels and Their Relations with Paralytic Shellfish Toxin Bioaccumulation in the Pacific Oyster Crassostrea gigas.

Paralytic shellfish toxins (PST) bind to voltage-gated sodium channels (Nav) and block conduction of action potential in excitable cells. This study aimed to (i) characterize Nav sequences in Crassostrea gigas and (ii) investigate a putative relation between Nav and PST-bioaccumulation in oysters. The phylogenetic analysis highlighted two types of Nav in C. gigas: a Nav1 (CgNav1) and a Nav2 (CgNav2) with sequence properties of sodium-selective and sodium/calcium-selective channels, respectively. Three alternative splice transcripts of CgNav1 named A, B and C, were characterized. The expression of CgNav1, analyzed by in situ hybridization, is specific to nervous cells and to structures corresponding to neuromuscular junctions. Real-time PCR analyses showed a strong expression of CgNav1A in the striated muscle while CgNav1B is mainly expressed in visceral ganglia. CgNav1C expression is ubiquitous. The PST binding site (domain II) of CgNav1 variants possess an amino acid Q that could potentially confer a partial saxitoxin (STX)-resistance to the channel. The CgNav1 genotype or alternative splicing would not be the key point determining PST bioaccumulation level in oysters.

Exposure of marine mussels Mytilus spp. to polystyrene microplastics: Toxicity and influence on fluoranthene bioaccumulation.

The effects of polystyrene microbeads (micro-PS; mix of 2 and 6 µm; final concentration: 32 µg L(-1)) alone or in combination with fluoranthene (30 µg L(-1)) on marine mussels Mytilus spp. were investigated after 7 days of exposure and 7 days of depuration under controlled laboratory conditions. Overall, fluoranthene was mostly associated to algae Chaetoceros muelleri (partition coefficient Log Kp = 4.8) used as a food source for mussels during the experiment. When micro-PS were added in the system, a fraction of FLU transferred from the algae to the microbeads as suggested by the higher partition coefficient of micro-PS (Log Kp = 6.6), which confirmed a high affinity of fluoranthene for polystyrene microparticles. However, this did not lead to a modification of fluoranthene bioaccumulation in exposed individuals, suggesting that micro-PS had a minor role in transferring fluoranthene to mussels tissues in comparison with waterborne and foodborne exposures. After depuration, a higher fluoranthene concentration was detected in mussels exposed to micro-PS and fluoranthene, as compared to mussels exposed to fluoranthene alone. This may be related to direct effect of micro-PS on detoxification mechanisms, as suggested by a down regulation of a P-glycoprotein involved in pollutant excretion, but other factors such as an impairment of the filtration activity or presence of remaining beads in the gut cannot be excluded. Micro-PS alone led to an increase in hemocyte mortality and triggered substantial modulation of cellular oxidative balance: increase in reactive oxygen species production in hemocytes and enhancement of anti-oxidant and glutathione-related enzymes in mussel tissues. Highest histopathological damages and levels of anti-oxidant markers were observed in mussels exposed to micro-PS together with fluoranthene. Overall these results suggest that under the experimental conditions of our study micro-PS led to direct toxic effects at tissue, cellular and molecular levels, and modulated fluoranthene kinetics and toxicity in marine mussels.