Oyster Tissue Research Articles

Realistic environmental exposure to microplastics does not induce biological effects in the Pacific oyster Crassostrea gigas.

The aim of the present study was to evaluate the presence and potential toxic effects of plastic fragments (<400 μm) of polyethylene and polypropylene on the Pacific oyster Crassostrea gigas. Oysters were exposed to environmentally relevant concentrations (0, 0.008, 10, 100 μg of particles/L) during 10 days, followed by a depuration period of 10 days in clean seawater. Effects of microplastics were evaluated on the clearance rate of organisms, tissue alteration, antioxidant defense, immune alteration and DNA damage. Detection and quantification of microplastics in oyster's tissues (digestive gland, gills and other tissues) and biodeposits using infrared microscopy were also conducted. Microplastics were detected in oyster's biodeposits following exposure to all tested concentrations: 0.003, 0.006 and 0.05 particles/mg of biodeposits in oysters exposed to 0.008, 10 and 100 μg of particles/L, respectively. No significant modulation of biological markers was measured in organisms exposed to microplastics in environmentally relevant conditions.

Differential responses in the biotransformation systems of the oyster Crassostrea gasar (Adanson, 1757) elicited by pyrene and fluorene: molecular, biochemical and histological approach - Part I

Polycyclic aromatic hydrocarbons (PAHs) are among the main contaminants in aquatic environments. PAHs can affect organisms due to their carcinogenic, mutagenic and/or teratogenic characteristics. Depending on the PAHs, concentration, and period of exposure, biological damage can occur leading to histopathologic alterations. This study aimed to evaluate the molecular, biochemical and histological responses of the oyster Crassostrea gasar exposed to pyrene (0.25 and 0.5 μM) and fluorene (0.6 and 1.2 μM), after exposure for 24 and 96 h. Concentrations of both PAHs were quantified in the water and in oyster tissues. Transcript levels of phase I (CYP3475C1, CYP2-like, CYP2AU1 and CYP356A) and phase II (GSTO-like, MGST-like and SULT-like) biotransformation-related genes and the activities of ethoxyresorufin-O-deethylase (EROD), total and microsomal glutathione S-transferase (GST and MGST) were evaluated in the gills. Also, histological changes and localization of mRNA transcripts CYP2AU1 in gills, mantle, and digestive diverticula were evaluated. Both PAHs accumulated in oyster tissues. Pyrene half-life in water was significantly lower than fluorene. Transcript levels of all genes were higher in oysters exposed to of pyrene 0.5 μM (24 h). Only CYP2AU1 gene was up-regulated by fluorene exposure. EROD and MGST activities were higher in oysters exposed to pyrene. Tubular atrophy in the digestive diverticula and an increased number of mucous cells in the mantle were observed in oysters exposed to pyrene. CYP2AU1 transcripts were observed in different tissues of pyrene-exposed oysters. A significant correlation was observed between tubular atrophy and the CYP2AU1 hybridization signal in oysters exposed to pyrene, suggesting the sensibility of the species to this PAH. These results suggest an important role of biotransformation-related genes and enzymes and tissue alterations associated to pyrene metabolism but not fluorene. In addition, it reinforces the role of CYP2AU1 gene in the biotransformation process of PAHs in the gills of C. gasar.

Effects of graphene oxide nanomaterial exposures on the marine bivalve, Crassostrea virginica

Since its discovery in 2004, graphene has been used in a wide variety of fields including biomedicine, electronics, filtration materials, and surface coatings. The rapidly expanding consumer market for graphene family nanomaterials (GFNs), such as graphene oxide (GO), raises concern regarding their environmental toxicity. The aim of this study was to evaluate the effects of GO exposures in a marine filter-feeding bivalve (Crassostrea virginica) using sublethal biomarker approaches that can contribute to the development of an adverse outcome pathway (AOP). A 14-day study was conducted to identify tissue-specific molecular markers of GO toxicity using a static renewal design. Elevated lipid peroxidation and changes in glutathione-s-transferase (GST) activities were observed in gills and digestive gland tissues of the GO-exposed oysters. These cellular changes were noted for 2.5 and 5 mg/L GO exposures in seawater. Based on our results, reactive oxygen species (ROS)-induced oxidative damage is identified as a key event in the proposed AOP. Additionally, detoxification enzymes, such as GST, are thought to be involved in stress signaling leading to adverse effects on cellular health. This study is a part of our two-tier approach towards the identification of short- and long-term effects of GO exposures. This work, together with our previous 72 h exposure, represents the application of biomarker-based investigations in the process of AOP development for graphene family nanomaterials.

Viral uptake and stability in Crassostrea gigas oysters during depuration, storage and steaming.

More stable than bacteria in environmental samples, enteric viruses are generally related to outbreaks of gastroenteritis caused by the consumption of contaminated oysters. This study evaluated: i) the dynamic processes of enteric viral models bioaccumulation by Crassostrea gigas oysters artificially contaminated; ii) the stability of these viruses in oysters in controlled temperature conditions and iii) the effect of UV light in inactivating these viruses in depurated oysters. Plaque assay (PA) was used to assess the infectivity of both viral models. Cell culture coupled with RT-qPCR (ICC-RT-qPCR) was used to measure infectious adenovirus type 2 (HAdV-2) genomes and qPCR to measure genome copies of murine norovirus (MNV-1). The virus uptake through bioaccumulation behave differently: HAdV-2 reached its peak of uptake faster than MNV-1. Both viruses showed high stability in oysters when maintained under 4 °C, but were completely inactivated in steamed oysters. The HAdV-2 was completely inactivated after 12 h of depuration with UV light and after 24 h without UV light. After 72 h of depuration, MNV-1 was still detected in both tanks, probably due to the stronger interaction of this virus with the oyster's tissues. This study demonstrated the importance of a secure depuration time in ensuring a clean and safe product, and that the steaming process is the safest way to prepare oysters for consumption.

Differential Gene expression related to morphological variation in the adductor muscle tissues of diploid and triploid fujian oysters, Crassostrea angulata

The Fujian oyster, Crassostrea angulata, accounts for approximately 50% of the total oyster production in China and is one of the most economically important marine products. In this study, we investigated differential gene expression profiles between diploid (n = 2) and triploid (n = 3) oysters that may indicate the specific adaptation associated with ploidy. A total of 14,626,126 and 15,492,551 reads were generated from diploid and triploid oyster muscle transcriptomes using Illumina RNA-Seq respectively. Gene expression analysis revealed a total of 2,045 differentially expressed genes between 2n and 3n individuals, from which 814 genes were significantly up-regulated in the 2n transcriptome while 1,231 were down-regulated. In addition, 24 differentially expressed genes (DEGs) were uniquely expressed in 2n individuals while 28 DEGs were uniquely expressed in 3n individuals. Furthermore, our pathway analysis suggested that the DEGs up-regulated in 2n oysters were enriched in the pathways related to normal growth, development and reproduction, whereas DEGs up-regulated in 3n individuals may be involved in faster growth and metabolism. Finally, we validated the expression levels of 15 genes using quantitative real-time PCR (qRT-PCR), and 14 out of 15 genes showed consistent expression patterns with RNA-Seq data. This study provides an important foundation for further investigations on distinct gene expression patterns and signalling pathways that differ between diploid and triploid oysters.

Determination of essential and potentially toxic elements and their estimation of bioaccessibility in honeys

In this work, the evaluation of the mineral profile and estimation of the bioaccessibility of essential and potentially toxic elements in honey samples was proposed. The experimental design and multivariate data analysis were applied as tool chemometrics. The acidic honey decomposition procedure using a cold finger digester block was proposed for analysis by inductively coupled plasma optical emission spectrometry (ICP OES). The analytical method presented good precision (<10%). The accuracy was confirmed through the analysis of a certified reference material (CRM) of tomato leaves (AGRO C1003a) and oyster tissue (NIST 1566b). The concentration ranges (in mg kg−1) of the analytes quantified in honey samples were As (<LoQ, 3.25), B (<LoQ, 6.72), Ba (<LoQ, 0.25), Ca (28.4–216), Cd (<LoQ, 0.29), Cu (<LoQ, 0.29), Fe (<LoQ, 47.0), K (27.2–1210), Mg (2.82–96.0), Mn (0.17–4.62), Na (16.4–260), P (<LoQ, 128), Se (<LoQ, 0.45), Sr (<LoQ, 1.08), and Zn (<LoQ, 2.94). Higher concentrations of Cu and Zn were observed in dark and amber honeys. From the multivariate analysis of the data, it was verified that the samples of urban predominance presented higher mineral content than the samples of honey of predominance rural. The bioaccessibility estimation was performed using the in vitro extraction method using the simple bioaccessibility extraction test (SBET). The bands of the bioaccessible contents (in %) for the analytes were Ca (<LoQ, 21.4), Fe (<LoQ, 21.8), K (9.4–20.0), Mg (15.0–25.0), and Na (<LoQ, 20.2). These results provide relevant information about the mineral composition of honey based on the visual coloration, geographical origin and bioaccessible content of the analytes in the gastric phase, contributing to the control of honey quality.

Dual-cloud point extraction for speciation of mercury in water and fish samples by electrothermal atomic absorption spectrometry

A dual-cloud point extraction (d-CPE) technique was established for preconcentration and speciation of mercury ions (Hg(II) and CH3Hg) in water and fresh water fish samples by electrothermal atomic absorption spectrometry (ETAAS). In the first step of d-CPE, Hg(II) was complexed with Dithizone (DTZ) and transferred into a nonionic surfactant (Triton X-114) solution. In the second step, the complexes of Hg(II)-DTZ in the surfactant phase were back-extracted with an aqueous solution of thiourea in nitric acid combined with heating and ultrasonication. Then, final amount of Hg(II) was detected by ETAAS. The CH3Hg concentration was calculated by subtracting the concentration of Hg(II) from the total mercury concentration after the conversion of CH3Hg to Hg(II) by acid microwave-assisted digestion. The conditions affecting the extraction of Hg(II) were investigated in detail and fully optimized. The enrichment factor for this method was 17.8, with a linear range of 0.40–15.0 μg L-1 and a correlation coefficient of 0.9997 along with a detection limit of 0.23 μg L-1. Relative standard deviations (RSDs, when concentration = 1.0 μg L-1 and n = 6) was 4.85%. Standard reference materials (SRM-1566b, oyster tissue) was used to find out the accuracy of the proposed method, and the developed method was successfully applied for speciation and determination of mercury within several water and freshwater fish samples.

Dynamics of the Pacific oyster pathobiota during mortality episodes in Europe assessed by 16S rRNA gene profiling and a new target enrichment next-generation sequencing strategy.

SummaryInfectious agents such as the bacteria Vibrio aestuarianus or Ostreid herpesvirus 1 have been repeatedly associated with dramatic disease outbreaks of Crassostrea gigas beds in Europe. Beside roles played by these pathogens, microbial infections in C. gigas may derive from the contribution of a larger number of microorganisms than previously thought, according to an emerging view supporting the polymicrobial nature of bivalve diseases. In this study, the microbial communities associated with a large number of C. gigas samples collected during recurrent mortality episodes at different European sites were investigated by real‐time PCR and 16SrRNA gene‐based microbial profiling. A new target enrichment next‐generation sequencing protocol for selective capturing of 884 phylogenetic and virulence markers of the potential microbial pathogenic community in oyster tissue was developed allowing high taxonomic resolution analysis of the bivalve pathobiota. Comparative analysis of contrasting C. gigas samples conducted using these methods revealed that oyster experiencing mortality outbreaks displayed signs of microbiota disruption associated with the presence of previously undetected potential pathogenic microbial species mostly belonging to genus Vibrio and Arcobacter. The role of these species and their consortia should be targeted by future studies aiming to shed light on mechanisms underlying polymicrobial infections in C. gigas.

First Record of Stephanostomum Sp. Looss, 1899 (Digenea: Acanthocolpidae) Metacercariae Parasitising the Pleasure Oyster Crassostrea Corteziensis (Hertlein) from the Mexican Pacific Coast.

SummaryThe aim of this investigation was to identify the parasites present in the largely understudied pleasure oyster Crassostrea corteziensis in Sinaloa state in the northwestern Mexican Pacific coast. Inspection of twenty-eight oysters collected on “Ceuta” lagoon revealed the presence of the digenean Stephanostomum sp. (Digenea: Acanthocolpidae) cysts. Metacercariae were found encapsulated and embedded in the digestive gland and mantle tissue of oysters. The prevalence of infection revealed that 84.6 % were infected, the abundance was 13.62, with a mean intensity of 16.09 per host. The members of this genus are characterized by a double crown of spines in the cephalic region surrounding the buccal opening of the worm. Significantly, we report the first incidence of the digenean Stephanostomum sp of the family Acanthocolpidae parasitizing Crassostrea corteziensis. Further we report that this bivalve is now considered a new intermediate host, and the northwestern Mexican Pacific coast is a new geographical distribution area for this digenean. The findings contribute to our understanding of the biology, biodiversity and host preference of these parasites, with implications for health risks posed by human consumption of the pleasure oyster.

Imidacloprid and formulated product impacts the fatty acids and enzymatic activities in tissues of Sydney rock oysters, Saccostrea glomerata

The use of imidacloprid (IMI) and its formulated products in agriculture is a risk to aquatic organisms due to deposition into waterways from runoff and aerial spraying. However, there is limited information on the potential effects of this pesticide on commercially important shellfish, such as oysters. We investigated the impacts of IMI and Spectrum 200SC (IMI formulation) on the activity of the enzymes Glutathione-S-transferase (GST), Catalase (CAT) and Acetylcholinesterase (AChE), in different oyster tissues including the gill, adductor muscle and digestive gland. We also investigated the condition index and fatty acid composition of the flesh of oysters after 2 weeks exposure. The concentrations of IMI in the different tissues was assessed using Liquid Chromatography-Mass Spectrometry (LC-MS) after QuEChERS extraction. Higher concentrations of IMI residues were detected in the adductor muscle of the oysters, followed by the gills and with the lowest amounts recovered from the digestive gland across all the concentrations tested. IMI and Spectrum 200SC significantly affected the gill AChE activity at 2 mg/L, but digestive gland CAT, and gill and digestive gland GST were impacted at environmentally relevant concentrations (0.01 and 0.05 mg/L). In the whole oyster, 2 weeks exposure to IMI (≥0.01 mg/L) resulted in a proportional increase in saturated fatty acids (SFA), altered the polyunsaturated fatty acid (PUFA) to SFA ratio and altered the omega 3 fatty acids (n-3) to omega 6 fatty acids (n-6) ratio, but there were no effects on the condition index of the oyster. Although the oysters responded differently to the formulated product, there was no consistent difference in the sublethal effects of analytical IMI and Spectrum 200SC. This study showed that exposure to IMI and Spectrum 200SC can significantly affect the biochemical processes and metabolites in oysters, with implications for food quality and safety.

Development of a liquid chromatography-quadrupole-time-of-flight-mass spectrometry based method for the targeted and suspect screening of contaminants in the pearl oyster Pinctada imbricata radiata

A rapid method based on solvent extraction followed by direct injection in liquid chromatography-quadrupole-time-of-flight-mass spectrometry (LC-Q-TOF-MS) was developed for the targeted and suspect screening of contaminants in the soft tissues of the pearl oyster Pinctada imbricata radiata. The quantification method was first validated for the targeted analysis of 21 contaminants including some pharmaceutically active compounds, with the relative recoveries ranging from 88 to 123%, and method detection limits generally below 1 ng g−1 on the wet weight (ww) basis. This targeted analysis method was then applied to oyster samples collected around the Qatari coast between 2017/2018, and none of the 21 compounds were detected in these samples. The post-acquisition data treatment based on the accurate mass measurement in both full MS scan and All Ions MS/MS was further used for mining other contaminants in oyster extracts, as well as 21 targeted compounds spiked in oyster extracts (suspect screening). The 21 spiked compounds were identified successfully and the estimated limit of identification for the individual 21 compounds ranged from 0.5 to 117 ng g−1 ww of oyster tissues. A phthalate, di(2-ethylhexyl) phthalate (DEHP) was identified to be present in oyster extracts from 2018 batches, at a concentration level significantly higher than that in procedure blanks. These results confirmed that high resolution MS data obtained using the targeted method can be exploited through suspect screening workflows to identify contaminants in the tissues of bioindicator mollusks. However, a number of false identifications could be obtained and future work will be on improving the success rate of the correct identifications using this workflow.

Preparation and evaluation of a new reference material for macro- and micronutrients in fish feed

A new reference material for inorganic constituents analysis in fish feed was produced and characterized according to the ISO Guides 30-35. The evaluation of the estimated minimum mass, homogeneity, and short- and long-term stability of the material was performed using microwave-assisted digestion and inductively coupled plasma optical emission spectroscopy as an in-house validated technique for Ca, Cu, Fe, K, Mg, Mn, Na, P, and Zn analysis. The accuracy of the measurements results was confirmed by analyzing the certified reference materials Oyster Tissue and Tomato Leaves, NIST SRM 1566b and SRM 1573a. The chemical characterization concerning elemental composition was performed through a collaborative trial with the participation of 35 laboratories by using spectroscopic and instrumental neutron activation analysis techniques. One-way analysis of variance (ANOVA) and regression analysis were the statistical methods applied to achieve the consensus values and their combined uncertainty, and the homogeneity and stability of the material presented averages within the 95% confidence interval. The expanded uncertainty considered all the instability observed during the stability, homogeneity, and characterization tests. The certified values and expanded uncertainties (k = 2) for the candidate reference were (28.65 ± 1.84) g kg−1 Ca, (10.51 ± 0.87) mg kg−1 Cu, (231.97 ± 20.17) mg kg−1 Fe, (5.86 ± 0.31) mg kg−1 K, (1.55 ± 0.16) g kg−1 Mg, (19.46 ± 2.12) mg kg−1 Mn, (2.16 ± 0.19) g kg−1 Na, (16.06 ± 0.97) g kg−1 P, and (129.56 ± 6.79) mg kg−1 Zn. The produced material proved its suitability for internal instrumental quality control or for the quality assurance of Ca, Cu, Fe, Mg, Mn, Na, P, and Zn analysis in fish feed for evaluation the laboratories quality control.

Trace metal biomarker based Cancer diagnostics in body tissue by energy dispersive X-ray fluorescence and scattering spectrometry

Direct diagnosis and characterization of cancer in tissue via trace biometals analyzed by energy dispersive X-ray fluorescence (EDXRF) spectrometry is challenging, as it requires sensitive detection and accurate quantitative analysis of the appropriate cancer biomarkers. The EDXRF spectrometry technique is not directly applicable due to the complexity of the tissue biopsy samples that are of limited size and irregular geometry, enhanced scatter from the sample dark matrix and extreme matrix effects as well as spectral overlaps and prominent Bremsstrahlung that masks the subtle biomarker analyte peaks. We report on the direct determination of biometals namely Cu, Mn, Fe, Zn; Mg, Co and Na and associated speciation (for Cu, Mn, Fe) in soft body tissue in the context of disease diagnostics utilizing a robust chemometrics enabled energy dispersive X-ray fluorescence and scattering (EDXRFS) spectrometric method. The EDXRFS method exploits, in addition to multiple fluorescence spectral signatures, scatter profiles associated with the trace metals and dark matrix to determine through hybridized multivariate chemometrics calibration models, the biometals in thin (10 μm) tissue sections. Wavelet transform (WT), principal component analysis (PCA) and independent component analysis (ICA) were used for spectral preprocessing towards model optimization using con-jointly artificial neural network (ANN) and partial least squares (PLS) based on paraffin wax ‘standards’ spiked with the cancer biomarker trace metals. Results obtained from applying oyster tissue standard validated models (to ≤6% accuracy) to dog tissues (used here as human body tissue analogues) show that both prostate and mammary malignant tissues have significantly high concentration of Zn i.e. 301 ± 4 μg/g and 301 ± 4 μg/g respectively when compared to benign tissues i.e. 160 ± 3 μg/g and 171 ± 10 μg/g. The same is the case for Fe and Cu. The concentrations of Fe, Zn, Cu and Mg in malignant (mammary) as compared to benign tissues occur in the ratios 3:1, 2:1, 3:1 and 2:1. On the other hand, for prostate malignant compared to benign tumor the corresponding ratios are 5:2, 2:1, 2:1 and 2:1 respectively. Prostate cancer was found to be characterized by strong positive correlation between Cu and Mg (0.999) and Mn and Fe (0.999) while mammary cancer is characterized by strong negative correlations between Cu and Mg (−0.994), Mn and Fe (−0.974). ICA and PCA were further used to successfully discriminate the dog tissue to 97% accuracy as either cancerous or non-cancerous based on validated pattern recognition PCA-ICA models for the determination of speciation of Cu, Fe and Mn in soft body tissue. For both mammary and prostate cancer malignancy was characterized by higher speciation of Cu, Fe and Mn (i.e. Cu2+, Fe3+, and Mn7+) compared to the benign. The results of this study demonstrate that robust chemometrics enabled EDXRFS spectrometry not only determine directly and rapidly but also accurately in a diagnostics manner cancer biomarker trace metals in soft body tissue. The technique has an additional advantage in that it has inbuilt multivariate capability to model the determined levels, their ratios and correlations as well as alterations in the speciation of the biometals to detect and characterize cancer (according to severity) as well discriminate among different types of cancer in the same tissue in a simple methodology that has potential for clinical applications.

Ecological risk of potentially toxic elements (PTEs) in sediments, seawater, wastewater, and benthic macroinvertebrates, Persian Gulf

In the current study, ecological risk of potentially toxic elements (PTEs) in sediments, seawater, wastewater, soft tissues and shell of a major fouling species Callista florida (C. florida) and soft tissue of rocky oyster, Saccostrea cucullata (S. cucullata) are investigated. For this purpose, 25 sediment samples, 24 seawater samples, 28 wastewater samples, and 100 bivalve samples were collected for PTEs analysis. Risk index (RI) and sediment quality guidelines along with calculated enrichment factors (EF) and PTEs profiles revealed that Musa Estuary is threatened by contamination, especially with respect to Hg, Cu, and Zn. The decreasing trend of average element enrichment factor is: Hg > Cu > Ni > Cd > Zn > Co > Cr > Mn > Fe > As > Pb > Mo > Sb. Among the investigated elements, Hg indicated the highest potential ecological risk factor in sediment (RI and EF are 1341.6 and 214.66 close to the industrial area). The Ficklin chart results demonstrated that seawater samples almost plot in regions with high metal load and pH values were the same. Mean concentrations of PTEs in water samples were 1.2 (for Cu) to 6565 (for Hg) times higher than world seawater. Regarding wastewater, pH values changed from very acidic to alkaline while PTEs load ranged from low to high load. In general, PTEs concentration in water samples was higher compared to those of the world seawater. Based on the results obtained in this biomonitoring study, elevated concentrations of Al, Fe, Cu, and Zn were found in soft tissue of C. florida and S. cucullata. Statistical analysis revealed significant differences in PTEs concentration between the two studied species. Generally, most PTEs concentration including Al, Co, Cr, Fe, Mn, Ni, Pb, and Sb in soft tissue fall between water and sediment samples i.e., sediment > biota > water.

Metal concentrations in Sydney Cockle (Anadara trapezia) tissue and ambient sediment in a highly-modified estuary (Sydney estuary, Australia)

The influence of sedimentary metals on the cockle A. trapezia tissue was examined using a strong difference in sedimentary metal concentrations in an embayment (Hen and Chicken Bay) highly contaminated in Cu and an adjacent cove (Iron Cove), strongly enriched in Cd, Cu, Pb and Zn within the heavily-urbanised Sydney estuary catchment (Australia). Statistically significant differences were recorded for cockle tissue metal concentrations between the study locations reflecting differences in surficial sediment metal concentrations. Low metal uptake was apparent in A. trapezia tissue, which were considerably less than background sedimentary concentrations and was of low-risk for human consumption. Dissimilar bioconcentration of Cd, Cu, Pb and Zn was apparent in tissue of cockles (A. trapezia), prawns (Metapenaeus bennettae), mussels (Mytilus galloprovincialis) and oysters (Saccostrea glomerata), due possibly to different feeding patterns and biogeochemical conditions in bottom sediments.

Defining Dermo resistance phenotypes in an eastern oyster breeding population

Perkinsus marinus, the causative agent of Dermo disease, is responsible for mass mortalities and negatively impacts aquaculture production of the eastern oyster, Crassostrea virginica. Selective breeding is a viable option for Dermo disease management; however, fluctuations in natural selection pressure and environmental noise hinder accumulation of genetic gains acquired through field performance trials. The purpose of this study was to adapt and apply laboratory disease challenge methods to eastern oysters, better characterize resistance-specific traits and assess the potential for genetic variation in Dermo resistance among distinct families within a breeding population. Two challenge experiments were conducted, one in 2014 and the other in 2015. Significant treatment (control vs. challenged) and family effects on survival (measured as per cent survival and days to death) were detected in the 2014 challenge, while overall high survival precluded the detection of a significant family effect in the 2015 challenge. An alternate measure of resistance, parasite elimination rate, was also measured in the 2015 challenge, and this varied significantly among families. Thus, both survival and the change in parasite concentration in oyster tissues over time represent Dermo resistance phenotypes that can be measured accurately with the adapted laboratory disease challenge protocol described here. The obvious next step is to incorporate the challenge protocol in eastern oyster breeding programmes to assess whether well-defined, accurately measured, Dermo-resistant phenotypes result in enhanced genetic improvement for this commercially important trait.

Uptake, depuration and sublethal effects of the neonicotinoid, imidacloprid, exposure in Sydney rock oysters

The broad utilisation of imidacloprid (IMI) in agriculture poses an increasing risk to aquatic organisms. However, the potential impacts on commercially important shellfish and chemical residues after exposure, are yet to be assessed. We investigated the levels of IMI in Sydney rock oyster (SRO) tissue during a three-day uptake and four-day depuration cycle using liquid chromatography-mass spectrometry. IMI was absorbed from the water, with significantly higher concentrations in the adductor muscles than the gills and digestive glands. Depuration was also fast with a significant drop in tissue concentrations after one day in clean water and complete elimination from all tissues except the digestive gland after four days. The distribution of IMI in SRO after direct exposure using mass spectrometry imaging demonstrated uptake and spatially resolved metabolism to hydroxyl-IMI in the digestive gland and IMI-olefin in the gills. We assessed the effects of IMI on filtration rate (FR), acetylcholinesterase (AChE) activity in the gills, and gene expression profiles in the digestive gland using transcriptomics. Exposure to 2 mg/L IMI reduced the FR of oysters on the first day, while exposure to 0.5 and 1 mg/L reduced FR on day four. IMI reduced the gill AChE activity and altered the digestive gland gene expression profile. This study indicates that commercially farmed SRO can uptake IMI from the water, but negative impacts were only detected at concentrations higher than currently detected in estuarine environments and the chemical residues can be effectively eliminated using simple depuration in clean water.

Biomarker responses in oysters Crassostrea hongkongensis in relation to metal contamination patterns in the Pearl River Estuary, southern China

The Pearl River Estuary (PRE) is the third largest estuary in China, where estuarine organisms are under metal stress at various biological levels. Based on the metal concentrations measured in oyster Crassostrea hongkongensis, we documented a change in dominance of metal contamination from Cd, Cr, Cu, Ni and Zn to Ag, Cd, Cu and Zn. In general, metal concentrations were higher in upstream stations and displayed a clear up-downstream gradient. Compared to the historical values, we noted the reductions in Cd, Cr and Ni concentrations, and the changing inputs due to evolving industrial activities were responsible for shaping the metal contamination profile in the PRE region. Along with metal concentrations, a suite of biomarkers was analyzed. Among the metals measured in the oyster tissues, Ag, Cd, Cu, Ni and Zn showed the strongest associations with pro-oxidant and oxidative stress responses (superoxide dismutase, lipid peroxidation and lysosomal membrane destabilization) and detoxification responses (glutathione and metallothionein), suggesting that the present metal contamination still exerts significant amount of stress in biota in the PRE. Metal contamination in estuaries in China is still severe compared to other countries, therefore continuous efforts should be taken to monitor the changing metal profiles with necessary control and remediation measures.

Multiplex qPCR Detection of Nonpathogenic and Pathogenic Vibrio parahaemolyticus and Vibrio vulnificus from Oysters Cultured in Massachusetts and Rhode Island: The Reliability of MPN as an Indicator

The leading cause of seafood-borne illness in the United States is Vibrio parahaemolyticus, whereas Vibrio vulnificus has the highest case fatality rate from wound infections of any foodborne pathogen. The Food and Drug Administration (FDA) uses a method of bacterial determination in oyster tissues termed “most probable number” (MPN) to enumerate Vibrio spp. abundance. This study used the development of two multiplex qPCR methods for the enumeration of total and pathogenic V. parahaemolyticus and V. vulnificus in oyster tissue using plasmid controls. Oysters, Crassostrea virginica, were collected from aquaculture farms in Wareham, MA, and Portsmouth, RI, from May to October 2014. Food and Drug Administration MPN and MPN-qPCR methods were compared and showed that FDA methods had higher values for both locations than MPN-qPCR methods. Throughout the summer at both locations, V. parahaemolyticus was most abundant with a majority being trh⁺ strains. Although serotypes were not directly tested for, tdh⁺ and trh⁺ strains were identified, suggesting a low possible occurrence of O4: K12 and no possible occurrence of O3:K6. In some samples, Vibrio alginolyticus was detected, indicating that trh⁺ percentages may be attributed to either V. parahaemolyticus or V. alginolyticus because of the similarity of the trh gene. The results from this study indicate the poor specificity and quantifiability of the FDA MPN method. This work also demonstrated that the use of plasmid controls for routine molecular methods to be used in a laboratory provided a reliable and consistent source of positive controls to compare against unknown samples for the abundance of V. parahaemolyticus and V. vulnificus.

Chlorothalonil induces oxidative stress and reduces enzymatic activities of Na+/K+-ATPase and acetylcholinesterase in gill tissues of marine bivalves.

Chlorothalonil is a thiol-reactive antifoulant that disperses widely and has been found in the marine environment. However, there is limited information on the deleterious effects of chlorothalonil in marine mollusks. In this study, we evaluated the effects of chlorothalonil on the gill tissues of the Pacific oyster, Crassostrea gigas and the blue mussel, Mytilus edulis after exposure to different concentrations of chlorothalonil (0.1, 1, and 10 μg L−1) for 96 h. Following exposure to 1 and/or 10 μg L−1 of chlorothalonil, malondialdehyde (MDA) levels significantly increased in the gill tissues of C. gigas and M. edulis compared to that in the control group at 96 h. Similarly, glutathione (GSH) levels were significantly affected in both bivalves after chlorothalonil exposure. The chlorothalonil treatment caused a significant time- and concentration-dependent increase in the activity of enzymes, such as catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione reductase (GR), in the antioxidant defense system. Furthermore, 10 μg L−1 of chlorothalonil resulted in significant inhibitions in the enzymatic activity of Na+/K+-ATPase and acetylcholinesterase (AChE). These results suggest that chlorothalonil induces potential oxidative stress and changes in osmoregulation and the cholinergic system in bivalve gill tissues. This information will be a useful reference for the potential toxicity of chlorothalonil in marine bivalves.