Effect of temperature on the toxicokinetics and gene expression of the pacific cupped oyster Crassostrea gigas exposed to cadmium.

In this study, we investigated the influence of temperature on the bioaccumulation and depuration of Crassostrea gigas exposed to Cd associated with its molecular responses. Oysters were acclimatized to different temperatures (10 °C, 15 °C, 20 °C, 25 °C, and 30 °C) for 14 d and then exposed to 10 µg/L Cd for 28 d, followed by a depuration period of 35 d. Oysters were sampled for chemical analysis by inductively coupled plasma mass spectrometry (ICP-MS) and for mRNA quantification by qPCR. In the digestive gland, gill, and mantle, the cadmium concentration at 10 °C was significantly lower than that at 25 °C and 30 °C in both the whole experiments. The use of a two-compartment model showed that the uptake rate k1 in the above three tissues increased with increasing temperatures ranging from 15 to 25 °C. The fastest elimination rates and shortest half-lives were observed at 15-25 °C. The induction of metallothionein (MT) only occurred in the digestive gland at 15 °C and 20 °C at the end of the accumulation phase. In the mantle and gills, the expression of P-glycoprotein (P-gp) was significantly induced at the end of the accumulation phase and significantly inhibited at the end of the depuration phase. In the digestive gland, the expression of P-gp was induced at the end of both the accumulation and depuration phases. Heat shock protein (hsp70) expression exhibited an overall increasing trend throughout the experiment.

Immune responses of oyster hemocyte subpopulations to in vitro and in vivo zinc exposure.

Oysters are an excellent biomonitor of coastal pollution and the hyper-accumulator of toxic metals such as copper and zinc (Zn). One unique feature of molluscs is their hemocytes which are mainly involved in immune defenses. Different subpopulations of hemocytes have been identified, but their functions in metal transport and detoxification are not clear. In this study, we examined the immune responses of different subpopulations of oyster Crassostrea hongkongensis hemocytes under different periods of Zn exposure by using flow cytometer and confocal microscopy. In vitro exposure to Zn resulted in acute immune responses by increasing the reactive oxygen species (ROS) production and phagocytosis and decreased number of granulocytes and mitochondrial membrane potential (MMP) within 3 h. Granulocyte mortality and lysosomal pH increased whereas glutathione (GSH) decreased within 1 h of in vitro exposure, indicating the immune stimulation of granulocytes. Within the first 7 days of in vivo exposure, immunocompetence of granulocytes was inhibited with increasing granulocyte mortality but decreasing ROS production and phagocytosis. However, with a further extension of Zn exposure to 14 days, both phagocytosis and lysosomal content increased with an increasing number of granulocytes, indicating the increase of hemocyte-mediated immunity. Our study demonstrated that granulocytes played important roles in oyster immune defenses while other subpopulations may also participate in immune functions. The degranulation and granulation due to transition between semigranulocytes and granulocytes after Zn exposure were important in metal detoxification. The study contributed to our understanding of the immune phenomena and the adaptive capability of oysters in metal contaminated environments.

Intergenerational effects of environmentally-aged microplastics on the Crassostrea gigas.

This study focused on the impacts of aged aquaculture microplastics (MPs) on oysters (Crassostrea gigas). Adult oysters were exposed for two months to a cocktail of MPs representative of the contamination of the Pertuis Charentais area (Bay of Biscay, France) and issuing from oyster framing material. The MPs mixture included 28% of polyethylene, 40% of polypropylene and 32% of PVC (polyvinyl chloride). During the exposure, tissues were sampled for various analyzes (MP quantification, toxicity biomarkers). Although no effect on the growth of adult oysters was noted, the mortality rate of bivalves exposed to MPs (0.1 and 10 mg. L-1 MP) increased significantly (respectively 13.3 and 23.3% of mortalities cumulative). On the one hand, the responses of biomarkers revealed impacts on oxidative stress, lipid peroxidation and environmental stress. At 56 days of exposure, significant increases were noted for Glutathione S-Transferase (GST, 10 mg. L-1 MP), Malondialdehyde (MDA, 10 mg. L-1 MP) and Laccase (LAC, 0.1 and 10 mg. L-1 MP). No variations were observed for Superoxyde Dismutase (SOD). Besides, ingestion of MPs in oyster tissues and the presence in biodeposits was highlighted. In addition, in vitro fertilisations were performed to characterize MPs effects on the offspring. Swimming behavior, development and growth of D-larvae were analysed at 24-, 48- and 72-h after fertilisation. D-larvae, from exposed parents, demonstrated reduced locomotor activity. Developmental abnormalities and arrest as well as growth retardation were also noted. This study highlighted direct and intergenerational effects of MPs from aged plastic materials on Pacific oysters.

Virulence of Vibrio alginolyticus Accentuates Apoptosis and Immune Rigor in the Oyster Crassostrea hongkongensis.

Vibrio species are ubiquitously distributed in marine environments, with important implications for emerging infectious diseases. However, relatively little is known about defensive strategies deployed by hosts against Vibrio pathogens of distinct virulence traits. Being an ecologically relevant host, the oyster Crassostrea hongkongensis can serve as an excellent model for elucidating mechanisms underlying host-Vibrio interactions. We generated a Vibrio alginolyticus mutant strain (V. alginolyticus△vscC ) with attenuated virulence by knocking out the vscC encoding gene, a core component of type III secretion system (T3SS), which led to starkly reduced apoptotic rates in hemocyte hosts compared to the V. alginolyticusWT control. In comparative proteomics, it was revealed that distinct immune responses arose upon encounter with V. alginolyticus strains of different virulence. Quite strikingly, the peroxisomal and apoptotic pathways are activated by V. alginolyticusWT infection, whereas phagocytosis and cell adhesion were enhanced in V. alginolyticus△vscC infection. Results for functional studies further show that V. alginolyticusWT strain stimulated respiratory bursts to produce excess superoxide (O2•-) and hydrogen peroxide (H2O2) in oysters, which induced apoptosis regulated by p53 target protein (p53tp). Simultaneously, a drop in sGC content balanced off cGMP accumulation in hemocytes and repressed the occurrence of apoptosis to a certain extent during V. alginolyticus△vscC infection. We have thus provided the first direct evidence for a mechanistic link between virulence of Vibrio spp. and its immunomodulation effects on apoptosis in the oyster. Collectively, we conclude that adaptive responses in host defenses are partially determined by pathogen virulence, in order to safeguard efficiency and timeliness in bacterial clearance.

Metaplasia of respiratory and digestive tissues in the eastern oyster Crassostrea virginica associated with the Deepwater Horizon oil spill.

Metaplasia is a well documented and deleterious effect of crude oil components on oysters. This reversible transformation of one cell type to another is a common response to petroleum-product exposure in molluscs. It has been shown experimentally in previous work that eastern oysters (Crassostrea virginica) exposed to petroleum products will exhibit metaplasia of digestive tissues. Here we document for the first time that wild adult oysters inhabiting coastal waters in the northern Gulf of Mexico during and in the aftermath of the Deepwater Horizon oil spill (2010) exhibited metaplasia in both ctenidial (respiratory and suspension feeding) and digestive tract tissues at significantly higher frequencies than geographic controls of C. virginica from Chesapeake Bay. Metaplasia included the loss of epithelial cilia, transformations of columnar epithelia, hyperplasia and reduction of ctenidial branches, and vacuolization of digestive tissues. Evidence for a reduction of metaplasia following the oil spill (2010-2013) is suggestive but equivocal.

The cGAS/STING-TBK1-IRF Regulatory Axis Orchestrates a Primitive Interferon-Like Antiviral Mechanism in Oyster.

Interferon (IFN) system is considered as the first defense line against viral infection, and it has been extensively studied in vertebrates from fish to mammals. In invertebrates, Vagos from arthropod and IFN-like protein (CgIFNLP) from Crassostrea gigas appeared to function as IFN-like antiviral cytokines. In the present study, the CgIFNLP protein in hemocytes was observed to increase after Poly (I:C) stimulation. After CgIFNLP was knocked down by RNAi, the mRNA expression of IFN-stimulated genes (CgISGs) was significantly inhibited. Both cyclic GMP-AMP synthase (CgcGAS) and stimulator of interferon gene (CgSTING) identified from oyster were able to recognize the double-stranded nucleic acid [Poly (I:C) and dsDNA] and expressed at high level after Poly (I:C) stimulation. The expression of CgIFNLP and interferon regulatory factors (CgIRF1/8) and the nuclear translocation of CgIRF8 were all suppressed in CgcGAS-RNAi or CgSTING-RNAi oysters after Poly (I:C) stimulation. The expression level of CgSTING and TANK binding kinase1 (CgTBK1) did not decrease in CgcGAS-RNAi oysters. After CgSTING was knocked down, the high expression of CgTBK1 induced by Poly (I:C) was prevented significantly. These results indicated that there was a primitive IFN-like antiviral mechanism dependent on the cGAS/STING-TBK1-IRFs regulatory axis in mollusks, which was different from the classic cGAS-STING-TBK1 signal pathway in mammals.

Effects of the toxic dinoflagellates Prorocentrum lima and Ostreopsis cf. ovata on immune responses of cultured oysters Crassostrea gasar.

Oyster production in Brazil has been highlighted as an important economic activity and is directly impacted by the quality of the environment, which is largely the result of human interference and climate change. Harmful algal blooms occur in aquatic ecosystems worldwide, including coastal marine environments which have been increasing over the last decades as a result of global change and anthropogenic activities. In this study, the native oysters Crassostrea gasar from Northeast of Brazil were exposed to two toxic benthic dinoflagellate species, Prorocentrum lima and Ostreopsis cf. ovata. Their respective effects on C. gasar physiology and defense mechanisms were investigated. Oyster hemocytes were first exposed in vitro to different concentrations of both dinoflagellate species to assess their effects on hemocyte functions, such as phagocytosis, production of reactive oxygen species, as well as mortality. Results highlighted an alteration of hemocyte phagocytosis and viability in presence of O. cf. ovata, whereas P. lima did not affect the measured hemocyte functions. In a second experiment, oysters were exposed for 4 days in vivo to toxic culture of O. cf. ovata to assess its effects on hemocyte parameters, tissues damages and pathogenic Perkinsus spp. infection. An increase in hemocyte mortality was also observed in vivo, associated with a decrease of ROS production. Histopathological analyses demonstrated a thinning of the epithelium of the digestive tubules of the digestive gland, inflammatory reaction and a significant increase in the level of infection by Perkinsus spp. in oysters exposed to O. cf. ovata. These results indicate that oysters C. gasar seem to be pretty resilient to an exposure to P. lima and may be more susceptible to O. cf. ovata. Furthermore, the latter clearly impaired oyster physiology and defense mechanisms, thus highlighting that harmful algal blooms of O. cf. ovata could potentially lead to increased susceptibility of C. gasar oysters to parasite infections.

Seasonal modulation of oxidative stress biomarkers in mangrove oyster (Crassostrea gasar) from an Amazon estuary.

Estuaries are the final destination of many pollutants derived from anthropogenic activity. Therefore, it is difficult to find this kind of ecosystem in a pristine condition. In this context, biomonitoring studies that characterize the organism's conditions against the environment' s natural variation are essential for future impact analysis due to anthropic activity. The present study aims to characterize the natural modulation of biochemical biomarkers in oysters Crassostrea gasar. The research was conducted in Japerica Bay, an estuary region located in the Eastern Amazon (Pará, Brazil), which has remained in pristine condition for many years. The samplings were carried out throughout one year during the rainy-dry transition period (June/2013), dry period (September/2013), dry-rainy transition period (November / 2013), and rainy period (February / 2014) in the lower and upper estuary. The activity of glutathione-S-transferase (GST) and total antioxidant capacity (ACAP) were evaluated as biomarkers of exposure and lipid peroxidation (LPO) as an effect biomarker. In gills, GST decreased during the rainy season in both sites and increased during the salinity peak (dry-rainy transition period) for the upper estuary's organisms. In this organ, the lowest levels of LPO occurred during the dry season for both points. There was an induction of ACAP in muscle during the rainy-dry transition period compared to the dry and dry-rainy transition periods for the lower estuary's organisms, and there were no differences for GST suggesting low tissue sensitivity. There was an increase in LPO during the rainy season compared to the rainy-dry transition period for the lower estuaries animals. Biomarkers in gills suggest a metabolic challenge to the rainy season and stability during the dry season. The species shows high viability of use in biomonitoring programs. However, these seasonality-induced alterations in biomarkers responses must be taken into account to interpret the results.

In vitro Evaluation of Programmed Cell Death in the Immune System of Pacific Oyster Crassostrea gigas by the Effect of Marine Toxins.

Programmed cell death (PCD) is an essential process for the immune system's development and homeostasis, enabling the remotion of infected or unnecessary cells. There are several PCD's types, depending on the molecular mechanisms, such as non-inflammatory or pro-inflammatory. Hemocytes are the main component of cellular immunity in bivalve mollusks. Numerous infectious microorganisms produce toxins that impair hemocytes functions, but there is little knowledge on the role of PCD in these cells. This study aims to evaluate in vitro whether marine toxins induce a particular type of PCD in hemocytes of the bivalve mollusk Crassostrea gigas during 4 h at 25°C. Hemocytes were incubated with two types of marine toxins: non-proteinaceous toxins from microalgae (saxitoxin, STX; gonyautoxins 2 and 3, GTX2/3; okadaic acid/dynophysistoxin-1, OA/DTX-1; brevetoxins 2 and 3, PbTx-2,-3; brevetoxin 2, PbTx-2), and proteinaceous extracts from bacteria (Vibrio parahaemolyticus, Vp; V. campbellii, Vc). Also, we used the apoptosis inducers, staurosporine (STP), and camptothecin (CPT). STP, CPT, STX, and GTX 2/3, provoked high hemocyte mortality characterized by apoptosis hallmarks such as phosphatidylserine translocation into the outer leaflet of the cell membrane, exacerbated chromatin condensation, DNA oligonucleosomal fragments, and variation in gene expression levels of apoptotic caspases 2, 3, 7, and 8. The mixture of PbTx-2,-3 also showed many apoptosis features; however, they did not show apoptotic DNA oligonucleosomal fragments. Likewise, PbTx-2, OA/DTX-1, and proteinaceous extracts from bacteria Vp, and Vc, induced a minor degree of cell death with high gene expression of the pro-inflammatory initiator caspase-1, which could indicate a process of pyroptosis-like PCD. Hemocytes could carry out both PCD types simultaneously. Therefore, marine toxins trigger PCD's signaling pathways in C. gigas hemocytes, depending on the toxin's nature, which appears to be highly conserved both structurally and functionally.

Regulatory Role of Sugars on the Settlement Inducing Activity of a Conspecific Cue in Pacific Oyster Crassostrea gigas.

This study evaluated the larval settlement inducing effect of sugars and a conspecific cue from adult shell extract of Crassostrea gigas. To understand how the presence of different chemical cues regulate settlement behavior, oyster larvae were exposed to 12 types of sugars, shell extract-coated and non-coated surfaces, and under varied sugar exposure times. Lectin-glycan interaction effects on settlement and its localization on oyster larval tissues were investigated. The results showed that the conspecific cue elicited a positive concentration dependent settlement inducing trend. Sugars in the absence of a conspecific cue, C. gigas adult shell extract, did not promote settlement. Whereas, in the presence of the cue, showed varied effects, most of which were found inhibitory at different concentrations. Sugar treated larvae exposed for 2 h showed significant settlement inhibition in the presence of a conspecific cue. Neu5Ac, as well as GlcNAc sugars, showed a similar interaction trend with wheat germ agglutinin (WGA) lectin. WGA-FITC conjugate showed positive binding on the foot, velum, and mantle when exposed to GlcNAc sugars. This study suggests that a WGA lectin-like receptor and its endogenous ligand are both found in the larval chemoreceptors and the shell Ethylenediaminetetraacetic acid (EDTA) extract that may complementarily work together to allow the oyster larva greater selectivity during site selection.

Effects of a bacteria-produced algicide on non-target marine invertebrate species.

Harmful algal blooms (HABs) affect both freshwater and marine systems. Laboratory experiments suggest an exudate produced by the bacterium Shewanella sp. IRI-160 could be used to prevent or mitigate dinoflagellate blooms; however, effects on non-target organisms are unknown. The algicide (IRI-160AA) was tested on various ontogenetic stages of the copepod Acartia tonsa (nauplii and adult copepodites), the blue crab Callinectes sapidus (zoea larvae and megalopa postlarvae), and the eastern oyster Crassostrea virginica (pediveliger larvae and adults). Mortality experiments with A. tonsa revealed that the 24-h LC50 was 13.4% v/v algicide for adult females and 5.96% for early-stage nauplii. For C. sapidus, the 24-h LC50 for first-stage zoeae was 16.8%; results were not significant for megalopae or oysters. Respiration rates for copepod nauplii increased in the 11% concentration, and in the 11% and 17% concentrations for crab zoeae; rates of later stages and oysters were unaffected. Activity level was affected for crab zoeae in the 1%, 11%, and 17% treatments, and for oyster pediveliger larvae at the 17% level. Activity of later stages and of adult copepods was unaffected. Smaller, non-target biota with higher surface to volume could be negatively impacted from IRI-160AA dosing, but overall the taxa and stages assayed were tolerant to the algicide at concentrations required for dinoflagellate mortality (EC50 = ~ 1%).

Bivalves are NO different: nitric oxide as negative regulator of metamorphosis in the Pacific oyster, Crassostrea gigas.

BACKGROUND: Nitric oxide (NO) is presumed to be a regulator of metamorphosis in many invertebrate species, and although NO pathways have been comparatively well-investigated in gastropods, annelids and crustaceans, there has been very limited research on the effects of NO on metamorphosis in bivalve shellfish. RESULTS: In this paper, we investigate the effects of NO pathway inhibitors and NO donors on metamorphosis induction in larvae of the Pacific oyster, Crassostrea gigas. The nitric oxides synthase (NOS) inhibitors s-methylisothiourea hemisulfate salt (SMIS), aminoguanidine hemisulfate salt (AGH) and 7-nitroindazole (7-NI) induced metamorphosis at 75, 76 and 83% respectively, and operating in a concentration-dependent manner. Additional induction of up to 54% resulted from exposures to 1H-[1,2,4]Oxadiazole[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylyl cyclase, with which NO interacts to catalyse the synthesis of cyclic guanosine monophosphate (cGMP). Conversely, high concentrations of the NO donor sodium nitroprusside dihydrate in combination with metamorphosis inducers epinephrine, MK-801 or SMIS, significantly decreased metamorphosis, although a potential harmful effect of excessive NO unrelated to metamorphosis pathway cannot be excluded. Expression of CgNOS also decreased in larvae after metamorphosis regardless of the inducers used, but intensified again post-metamorphosis in spat. Fluorescent detection of NO in competent larvae with DAF-FM diacetate and localisation of the oyster nitric oxide synthase CgNOS expression by in-situ hybridisation showed that NO occurs primarily in two key larval structures, the velum and foot. cGMP was also detected in the foot using immunofluorescent assays, and is potentially involved in the foot's smooth muscle relaxation. CONCLUSION: Together, these results suggest that the NO pathway acts as a negative regulator of metamorphosis in Pacific oyster larvae, and that NO reduction induces metamorphosis by inhibiting swimming or crawling behaviour, in conjunction with a cascade of additional neuroendocrine downstream responses.

Uptake and Biological Effects of Perfluorooctane Sulfonate Exposure in the Adult Eastern Oyster Crassostrea virginica.

Perfluorooctane sulfonate (PFOS) is a legacy contaminant that has been detected globally within the environment and throughout numerous species, including humans. Despite an international ban on its use, this unique contaminant continues to persist in organisms and their surroundings due to PFOS's inability to breakdown into nontoxic forms resulting in bioaccumulation. In this study, we analyzed the effects of a technical mixture of PFOS (linear and branched isomers) in the adult Eastern oyster, Crassostrea virginica, at 2 days and 7 days exposure. Biomarker analysis (lysosomal destabilization, lipid peroxidation, and glutathione assays) in oyster tissue along with chemical analysis (liquid chromatography tandem mass spectrometry) of PFOS in oyster tissue and water samples revealed the oysters' ability to overcome exposures without significant damage to lipid membranes or the glutathione phase II enzyme system; however, significant cellular lysosomal damage was observed. The oysters were able to eliminate up to 96% of PFOS at 0.3 mg/L and 3 mg/L exposures when allowed to depurate for 2 days in clean seawater. Chemical analysis showed the linear isomer to be the prevailing fraction of the residual PFOS contained in oyster tissue. Results provide insight into possible detrimental cellular effects of PFOS exposure in addition to offering insight into contaminant persistence in oyster tissue.

Nanopolystyrene beads affect motility and reproductive success of oyster spermatozoa (Crassostrea gigas).

Oysters are keystone species that use external fertilization as a sexual mode. The gametes are planktonic and face a wide range of stressors, including plastic litter. Nanoplastics are of increasing concern because their size allows pronounced interactions with biological membranes, making them a potential hazard to marine life. In the present study, oyster spermatozoa were exposed for 1 h to various doses (from 0.1 to 25 µg mL-1) of 50-nm polystyrene beads with amine (50-NH2 beads) or carboxyl (50-COOH beads) functions. Microscopy revealed adhesion of particles to the spermatozoa membranes, but no translocation of either particle type into cells. Nevertheless, the 50-NH2 beads at 10 µg mL-1 induced a high spermiotoxicity, characterized by a decrease in the percentage of motile spermatozoa (-79%) and in the velocity (-62%) compared to control spermatozoa, with an overall drop in embryogenesis success (-59%). This major reproduction failure could be linked to a homeostasis disruption in exposed spermatozoa. The 50-COOH beads hampered spermatozoa motility only when administered at 25 µg mL-1 and caused a decrease in the percentage of motile spermatozoa (-66%) and in the velocity (-38%), but did not affect embryogenesis success. Microscopy analyses indicated these effects were probably due to physical blockages by microscale aggregates formed by the 50-COOH beads in seawater. This toxicological study emphasizes that oyster spermatozoa are a useful and sensitive model for (i) deciphering the fine interactions underpinning nanoplastic toxicity and (ii) evaluating adverse effects of plastic nanoparticles on marine biota while waiting for their concentration to be known in the environment.

Differential responses in the biotransformation systems of the oyster Crassostrea gigas (Thunberg, 1789) elicited by pyrene and fluorene: Molecular, biochemical and histological approach - Part II.

Pyrene (PYR) and fluorene (FLU) are among the sixteen priority Polycyclic Aromatic Hydrocarbons (PAH) of the United States Environmental Protection Agency and are both frequently detected in contaminated sites. Due to the importance of bivalve mollusks in biomonitoring programs and the scarce information on the biotransformation system in these organisms, the aim of this study was to investigate the effect of PYR and FLU at the transcriptional level and the enzymatic activities of some biotransformation systems in the Pacific oyster Crassostrea gigas, and to evaluate the histological effects in their soft tissues. Oysters C. gigas were exposed for 24 h and 96 h to PYR (0.25 and 0.5 µM) and FLU (0.6 and 1.2 µM). After exposure, transcript levels of cytochrome P450 coding genes (CYP1-like, CYP2-like, CYP2AU2, CYP356A1, CYP17α-like), glutathione S tranferase genes (omega GSTO-like and microsomal, MGST-like) and sulfotransferase gene (SULT-like), and the activity of ethoxyresorufin O-deethylase (EROD), Glutathione S-transferase (GST) and microssomal GST (MGST) were evaluated in gills. Histologic changes were also evaluated after the exposure period. PYR and FLU bioconcentrated in oyster soft tissues. The half-life time of PYR in water was lower than fluorene, which is in accordance to the higher lipophilicity and bioconcentration of the former. EROD activity was below the limit of detection in all oysters exposed for 96 h to PYR and FLU. The reproductive stage of the oysters exposed to PYR was post-spawn. Exposure to PYR caused tubular atrophy in digestive diverticula, but had no effect on transcript levels of biotransformation genes. However, the organisms exposed for 96 h to PYR 0.5 µM showed higher MGST activity, suggesting a protective role against oxidative stress in gills of oysters under higher levels of PYR in the tissues. Increased number of mucous cells in mantle were observed in oysters exposed to the higher FLU concentration, suggesting a defense mechanisms. Oysters exposed for 24 h to FLU 1.2 µM were in the ripe stage of gonadal development and showed higher transcript levels of CYP2AU2, GSTO-like and SULT-like genes, suggesting a role in the FLU biotransformation. In addition, after 96 h of exposure to FLU there was a significant increase of mucous cells in the mantle of oysters but no effect was observed on the EROD, total GST and MGST activities. These results suggest that PAH have different effects on transcript levels of biotransformation genes and enzyme activities, however these differences could also be related to the reproductive stage.

The effects of atrazine on the microbiome of the eastern oyster: Crassostrea virginica.

Long-standing evidence supports the importance of maintaining healthy populations of microbiota for the survival, homeostasis, and complete development of marine mollusks. However, the long-term ecological effects of agricultural runoff on these populations remains largely unknown. Atrazine (6-Chloro-n-ethyl-n'-(1-methylethyl)-triazine-2,4-diamine), a prevalent herbicide in the United States, is often used along tributaries of the Chesapeake Bay where oyster breeding programs are concentrated. To investigate any potential effects atrazine maybe having on mollusk-prokaryote interactions, we used 16S rRNA gene amplicons to evaluate how microbial compositions shift in response to exposure of environmentally relevant concentrations of atrazine previously found within the Chesapeake Bay. The dominant bacterial genera found within all groups included those belonging to Pseudoalteromonas, Burkholderia, Bacteroides, Lactobacillis, Acetobacter, Allobaculum, Ruminococcus, and Nocardia. Our results support previously published findings of a possible core microbial community in Crassostrea virginica. We also report a novel finding: oysters exposed to atrazine concentrations as low as 3 µg/L saw a significant loss of a key mutualistic microbial species and a subsequent colonization of a pathogenic bacteria Nocardia. We conclude that exposure to atrazine in the Chesapeake Bay may be contributing to a significant shift in the microbiomes of juvenile oysters that reduces fitness and impedes natural and artificial repopulation of the oyster species within the Bay.

Effects of Microplastics Associated with Triclosan on the Oyster Crassostrea brasiliana: An Integrated Biomarker Approach.

Urban waste is a complex mixture of different substances, including microplastics and pharmaceuticals and personal care products. Microplastics have a high affinity for hydrophobic substances. One of these substances is triclosan, a bactericide used in a variety of hygiene products. Therefore, microplastics (MPs) may serve as a vector between triclosan and aquatic organisms. The current study sought to evaluate the effects of the interaction between microplastics and triclosan based on a mechanistic approach in which the oyster Crassostrea brasiliana was used as a model. The organisms were exposed to three conditions: the control, microplastic (MP), and microplastic contaminated with triclosan (MPT). The organisms were exposed for 3 or 7 days. After the exposure time, hemolymph was sampled for performing the neutral red retention time assay and, subsequently, the gills, digestive glands, and adductor muscles were dissected for measuring biomarkers responses (EROD, DBF, GST, GPx, GSH, lipid peroxidation, DNA strand breaks, and AChE). Our results demonstrate combined effects of MPs associated with triclosan on oyster physiology and biochemistry, as well as on lysosomal membrane stability. These results contribute to understanding the effects of contaminants of emerging concern and microplastics on aquatic organisms.

Transcriptomic profile of oyster Crassostrea gigas hemocyte after short-term cadmium exposure and bacteria stimulation.

Oyster Crassostrea gigas, is considered as a useful environmental indicator since it is widely distributed along the intertidal zone whereby it tends to accumulate cadmium and is always exposed to various pathogen agents. However, its molecular responses to both cadmium and pathogen stimulation remain unclear. In the present study, transcriptome data of hemocytes from oysters were analyzed to reveal specific molecular responses of oyster to cadmium or cadmium/bacteria stimulation. A total of 21591, 22872 and 20107 genes were detected in the BLANK, Cd24h and Cd/Bac24h group, respectively. Among them, there were 685 differentially expressed genes collected in the comparison of Cd24h versus BLANK. GO analysis of these genes found that sixteen terms into the Molecular Function category displayed transporter activities, and were all over-enrichment by cadmium exposure, whereas twelve terms into Biological Process category involved mainly in metabolic process of the various cellular components and two terms into Cellular Component category were all under-enrichment. The 330 immune responsive genes were shared by two gene lists of CdBac24h versus BLANK and CdBac24h versus Cd24h, and seven out of thirty terms in GO analysis were related to the immune process. Further annotation of these genes from the KEGG database revealed fourteen pathways, including two nervous system related pathways, arachidonic acid pathway, four immune pathways, MAPK cascade and other four cell signaling pathways, and two energy related pathways. Twenty-two differentially expressed genes were identified to responsive to both cadmium exposure and bacteria stimulation, but in different expression patterns, suggesting that bilateral responsive genes, such as alkaline phosphatase and sodium and chloride-dependent glycine transporter gene, could be candidate biomarkers for early warning of cadmium pollution. The present results collectively indicated that a profound neuro-endocrine-immune regulatory network was activated in response to cadmium and bacteria stimulation in oyster C. gigas, and the expression pattern of some cadmium responsive genes may be either reversed or strengthened by bacteria stimulation. The results provide knowledge on the transcriptomic response profile of oyster after short-term cadmium exposure and bacteria stimulation, which would be useful for future studies on stress response mechanism of mollusc, and some cadmium-bacteria responsive genes may be explored as potential biomarkers for monitoring marine pollution.

Identification of a Novel Pattern Recognition Receptor DM9 Domain Containing Protein 4 as a Marker for Pro-Hemocyte of Pacific Oyster Crassostrea gigas.

DM9 refers to an uncharacterized protein domain that is originally discovered in Drosophila melanogaster. Two proteins with DM9 repeats have been recently identified from Pacific oyster Crassostrea gigas as mannose-specific binding pattern-recognition receptors (PRRs). In the present study, a novel member of DM9 domain containing protein (designated as CgDM9CP-4) was identified from C. gigas. CgDM9CP-4, about 16 kDa with only two tandem DM9 domains, was highly enriched in hemocytes and gill. The transcripts level of CgDM9CP-4 in circulating hemocytes were decreased after LPS, PGN and Vibrio splendidus stimulations. The recombinant protein of CgDM9CP-4 (rCgDM9CP-4) displayed a broad binding spectrum towards various pathogen-associated molecular patterns (PAMPs) (LPS, PGN, ß-glucan and Mannose) and microorganisms (Staphylococcus aureus, Micrococcus luteus, V. splendidus, V. anguillarum, Escherichia coli, Pichia pastoris and Yarrowia lipolytica). CgDM9CP-4 was mostly expressed in gill and some of the hemocytes. Flow cytometry analysis demonstrated that the CgDM9CP-4-positive hemocytes accounted for 7.3% of the total hemocytes, and they were small in size and less in granularity. CgDM9CP-4 was highly expressed in non-phagocytes (~82% of total hemocytes). The reactive oxygen species (ROS) and the expression levels of cytokines in CgDM9CP-4-positive hemocytes were much lower than that in CgDM9CP-4-negative hemocytes. The mRNA expression level of CgDM9CP-4 in hemocytes was decreased after RNAi of hematopoietic-related factors (CgGATA, CgRunt, CgSCL, and CgNotch). In addition, CgDM9CP-4-positive cells were found to be much more abundant in hemocytes from gill than that from hemolymph, with most of them located in the gill filament. All these results suggested that CgDM9CP-4 was a novel member of PRR that expressed in undifferentiated pro-hemocytes to mediate immune recognition of pathogens.

Assessment of swimming behavior of the Pacific oyster D-larvae (Crassostrea gigas) following exposure to model pollutants.

This study describes an image analysis method that has been used to analyze the swimming behavior of native oyster D-larvae (Crassostrea gigas) from the Arcachon Bay (SW, France). In a second time, this study evaluated the impact of copper and S-metolachlor pollutants on D-larvae swimming activity and the possible relationship between developmental malformations and abnormal swimming behavior. Analyses in wild and cultivated oyster D-larvae were investigated during two breeding-seasons (2014 and 2015) at different sampling sites and dates. In controlled conditions, the average speed of larvae was 144 µm s-1 and the maximum speed was 297 µm s-1 while the trajectory is mainly rectilinear. In the presence of environmental concentration of copper or S-metolachlor, no significant difference in maximum or average larval speed was observed compared to the control condition but the percentage of circular trajectory increased significantly while the rectilinear swimming larvae significantly declined. The current study demonstrates that rectilinear trajectories are positively correlated to normal larvae while larvae with shell anomalies are positively correlated to circular trajectories. This abnormal behavior could affect the survival and spread of larvae, and consequently, the recruitment and colonization of new habitats.