Gonad differential proteins revealed with proteomics in oyster (Saccostrea cucullata) using alga as food contaminated with cadmium

As mercury and lead, cadmium (Cd) is one of the highly toxic metals in both the ocean and land environments, but its toxicological mechanism in organisms including human is still unclear because of the complex toxicological pathways in vivo. Here, the alga Chlorella vulgaris were cultivated at room temperature under the stress of cadmium (1 mg L⁻¹) to obtain a toxic food, and then the contaminated food were directly supplied to oyster (Saccostrea cucullata) in seawater. After feeding with C. vulgaris contaminated with Cd (C. vulgaris-Cd), the differential proteins in the oyster gonad (OG) were effectively separated and identified with proteomic approaches. Eleven protein spots were observed to be significantly changed in the OG feeding with C. vulgaris-Cd, which seven spots of these differential proteins were down-regulated while four spots were up-regulated. These altered spots were further excised in gels and identified by a combined technique of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS) and database searching. A portion of these differential proteins were further proofed by real-time PCR and Western blotting. The results indicate that the major functions of these differential proteins were described as follows: binding, protein translocation, catalysis, regulation of energy metabolism, reproductive function and skeleton structure. These differential proteins in part may effectively provide a few novel biomarkers for the evaluation of Cd pollution level via a food pathway for harming halobios, mammal and human health, and for understanding the complex mechanisms of Cd toxicity in vivo.     

The recovery of oyster (Saccostrea glomerata) populations in Sydney estuary (Australia)

The current work documented a significant and widespread increase in the abundance of the Sydney rock oyster Saccostrea glomerata, in Sydney estuary (Australia) by undertaking surveys of oyster density in the estuary in 1989 and annually from 1994 to 2006. Oyster density at six control sites located in nearby National Parks unaffected by boating and stormwater discharges were compared to 17 study sites widely distributed within Sydney estuary. No oyster populations were evident in Sydney estuary in 1989; however, by 1994 oysters had colonised areas of the lower and central estuary and by 2002 densities were statistically similar to control sites. The timing of estuary-wide increases in oyster abundance suggests that the partial banning of tributyltin in 1989 for vessels under 25 m long may have played a major role in the increase of S. glomerata in this estuary.     

Di- and tribromoindoles in the common oyster (Crassostrea virginica)

The sources/origins, fate and impacts of naturally occurring organobromine compounds in the marine environment are largely unknown. Soft tissue composites of the common oyster (Crassostrea virginica) collected from coastal Georgia (USA) were analyzed for organobromines by gas chromatography. Three simple bromoindoles (BIs)--two dibromo- and one tribromo congener--were detected and their molecular formulas elucidated by electron and negative chemical ionization GC-MS. Semi-quantitative estimates of BI concentrations in these samples using GC-ECD indicated that oysters sampled in November 1999 contained 5-10 times more BIs than those sampled in August 2000 and March 2001. Although their bromine substitution patterns are presently unknown, this first ever report of bromoindoles in C. virginica, a prolific and important commercial and ecological species, underscores its potential utility as a bioindicator of organobromines in the coastal marine environment.     

Proteomic analysis in the lichen Physcia adscendens exposed to cadmium stress

This work was undertaken to explore the potential of proteomics to dissect parallel and consecutive events of cadmium stress response in the lichen Physcia adscendens (Fr.) H. Olivier. Thalli were exposed to 0 (control) and 36 μM Cd for 6, 18, 24 and 48 h. Two-dimensional electrophoresis and mass spectrometry analyses showed an 80-85% spot identity between 6 and 18 h vs. 24 and 48 h of Cd exposure. Putative heat-shock proteins and glutathione S-transferase generally increased their expression all over the Cd treatments. By contrast, ABC transporters were underexpressed after 6-18 h, but in some cases induced after 24-48 h of Cd exposure. The cytochrome P450 appeared to have a variable expression pattern over time. Overall these data suggest that a considerable importance in the response of P. adscendens thalli to Cd stress can be assumed by differential expression of various protein families.     

Proteomic analysis of chromate-induced modifications in Pseudokirchneriella subcapitata

In this work, we have analyzed the changes in the protein expression profile elicited by chromium (Cr) exposure in the freshwater green alga Pseudokirchneriella subcapitata, a well known bio-indicator of water pollution. We tested two experimental conditions, namely 0.2 and 1 ppm of potassium dichromate; this concentration range includes the environmentally-relevant concentrations. Results show that neither concentration of potassium dichromate tested inhibited algal growth. However, the proteomic approach allowed the identification of relevant modifications in protein expression. In fact, among 800 protein spots detected by two-dimensional electrophoresis, 16 Cr-regulated proteins, including predicted and novel ones, were identified using tandem mass spectromic protein analysis.The results demonstrate a Cr-specific action in altering several photosynthetic proteins, such as ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), RuBisCO activase, Light Harvesting Chla/b protein complex, and stress related Chla/b binding protein1. Although Cr toxicity with respect to photosynthesis has been already documented, here we have identified, for the first time, the target proteins of this toxicity. Cr also induced a modulation of some proteins involved in the metabolism of the amino acids glutamine, arginine and methionine. These data are supported by changes in cellular polyamine (PA) accumulation. Present findings provide new insight into the molecular mechanisms underlying Cr toxicity in P. subcapitata.     

Bioaccumulation and metabolisation of (14)C-pyrene by the Pacific oyster Crassostrea gigas exposed via seawater

The first objective of this study was to determine the bioaccumulation kinetics of pyrene in the soft tissues of Crassostrea gigas (mantle, muscle, gills, digestive gland, and the remaining soft tissues). As bivalves can biotransform hydrocarbons in more polar compounds (metabolites) that are more easily excreted, the second objective was to investigate the oyster capacity to metabolize pyrene into its metabolite, the 1-hydroxypyrene. To these ends, oysters were exposed 24 h to waterborne ¹⁴C-pyrene then placed in depuration conditions for 15 d. Oysters efficiently bioaccumulated pyrene in their soft tissues and equilibrium was reached within the exposure time. The metabolite1-hydroxypyrene was also detected in oyster tissues but represented only 4-14% of the parent pyrene. At the end of the exposure period, the gills and the mantle showed the highest pyrene proportion of total soft tissue content, i.e. 47% and 26%, respectively. After 15 d of depuration, the mantle contained 32% and 30% of the remaining pyrene and 1-hydroxypyrene, respectively. As C. gigas did not display a high capacity for metabolizing pyrene, it can be considered as a good bioindicator species to survey and monitor pyrene contamination in the coastal marine environment.     

Weight and salinity effects on zinc uptake and accumulation for the American oyster (Crassostrea virginica Gmelin)

The power relationships of short-term net uptake and of in-situ body burden with body weight were examined. The accumulation of soft tissue zinc in the American oyster (Crassostrea virginica) was related to time integration of uptake. Short term uptake of (65)Zn was measured in the laboratory. It was (i) a function of the (dry soft-tissue) weight of the oyster, (ii) inversely related to the salinity of the ambient water, and (iii) increased linearly with ambient concentrations. When in-situ soft-tissue zinc body burdens of oysters from the James River and the Rappahannock River were fit to power functions of body weights (y = aWb), the values of b for all sites were larger by 1 than the powers for (65)Zn uptake when b was adjusted for the oyster-bed salinities. The soft-tissue zinc concentration (y/W) of an oyster increased continuously, but the rate of the increase was reduced as the oyster grows larger. Both short-term uptake and in-situ body burden varied with salinity. The soft-tissue zinc concentrations of hooked mussels (Ischadium recurvum) from the Rappahannock River oyster beds, contrary to the oysters, appeared to be in equilibrium with ambient concentrations.     

Characterization of a fatty acid-binding protein from the Pacific oyster (Crassostrea gigas): pharmaceutical and toxicological implications

Environmental Science and Pollution Research - Pharmaceuticals and their metabolites constitute a class of xenobiotics commonly found in aquatic environments which may cause toxic effects in...     

Distribution of butyltin and derivatives in oyster shells and trapped sediments of two estuaries in Cantabria (Northern Spain)

Distribution of butyltin compounds (BTs) and derivatives (monobutyltin, MBT; dibutyltin, DBT and tributyltin, TBT) was analysed in Crassostrea gigas oyster shells and the sediments trapped in the shell chambers, from two different estuaries in Cantabria (Northern Spain), with very different environmental conditions, where previous data have not been reported. Inorganic tin analysis in oyster shells was performed in order to study the degradation of BTs. Shell thickening and losses in biological growth are related with the presence of TBT, and were determined using three morphological indexes. Total BTs concentrations, expressed as Sn, ranged from 18.0 ng g(-1) to 176.7 ng g(-1) in sediments, and from 2.4 ng g(-1) to 11.1 ng g(-1) in oyster shells. Total inorganic tin concentrations ranged from 1775.0 ng g(-1) to 4781.3 ng g(-1) in oyster shells. The amount of BTs in oyster shells has been associated with the concentrations in the sediments trapped in the shell chambers. Partition coefficients between oyster sediments and oyster shells show the affinity of BTs by the sediments and the higher inorganic tin in oyster shells does not seem to be related to the BTs pollution.     

Pacific oyster (Crassostrea gigas) hemocyte are not affected by a mixture of pesticides in short-term in vitro assays

Pesticides are frequently detected in estuaries among the pollutants found in estuarine and coastal areas and may have major ecological consequences. They could endanger organism growth, reproduction, or survival. In the context of high-mortality outbreaks affecting Pacific oysters, Crassostrea gigas, in France since 2008, it appears of importance to determine the putative effects of pesticides on oyster susceptibility to infectious agents. Massive mortality outbreaks reported in this species, mainly in spring and summer, may suggest an important role played by the seasonal use of pesticides and freshwater input in estuarine areas where oyster farms are frequently located. To understand the impact of some pesticides detected in French waters, their effects on Pacific oyster hemocytes were studied through short-term in vitro experiments. Bivalve immunity is mainly supported by hemocytes eliminating pathogens by phagocytosis and producing compounds including lysosomal enzymes and antimicrobial molecules. In this study, oyster hemocytes were incubated with a mixture of 14 pesticides and metaldehyde alone, a molecule used to eliminate land mollusks. Hemocyte parameters including dead/alive cells, nonspecific esterase activities, intracytoplasmic calcium, lysosome number and activity, and phagocytosis were monitored by flow cytometry. No significant effect of pesticides tested at different concentrations was reported on oyster hemocytes maintained in vitro for short-term periods in the present study. It could be assumed that these oyster cells were resistant to pesticide exposure in tested conditions and developing in vivo assays appears as necessary to better understand the effects of pollutants on immune system in mollusks.     

Proteomic analysis in Daphnia magna exposed to As(III), As(V) and Cd heavy metals and their binary mixtures for screening potential biomarkers

In this study, the effects of three widespread heavy metals, As(III), As(V) and Cd, and their binary mixtures on the proteomic profile in D. magna were examined to screen novel protein biomarkers using the two-dimensional gel electrophoresis method (2DE). Ten 20d daphnia were exposed to the LC20 concentrations for each of a total of 8 treatments, including the control, As(III), As(V), Cd, [As(III)+As(V)], [As(III)+Cd], [As(V)+Cd], and [As(III), As(V), Cd], for 24 h before protein isolation. Three replicates were performed for each treatment. These protein samples were employed for 2DE experiments with a pH gradient gel strip from pH 3 to pH 10. The protein spots were detected by a silver staining process and their intensities were analyzed by Progenesis software to discover the differentially expressed proteins (DEPs) in response to each heavy metal. A total of 117 differentially expressed proteins (DEPs) were found in daphnia responding to the 8 treatments and mapped onto a 2D proteome map, which provides some information of the molecular weight (MW) and pI value for each protein. All of these DEPs are considered as potential candidates for protein biomarkers in D. magna for detecting heavy metals in the aquatic ecosystem. Comparing the proteomic results among these treatments suggested that exposing D. magna to binary mixtures of heavy metals may result in some complex interactive molecular responses within them, rather than just the simple sum of the proteomic profiles of the individual chemicals, (As(III), As(V), and Cd).     

Uptake and depuration of toxic halogenated aromatic hydrocarbons by the American oyster (Crassostrea virginica): a field study

Uptake and depuration of toxic chlorinated compounds such as planar polychlorinated biphenyls (PCBs 77, 126, 169), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 2,3,7,8-tetrachlorodibenzofuran (TCDF) were monitored during a 50-day field study where American oysters (Crassostrea virginica) were transplanted back and forth between a heavily polluted area (Houston Ship Channel, SC) and a relatively unimpacted area (Hanna's Reef, HR) within Galveston Bay, TX. In general, low molecular weight, less lipophilic compounds accumulated in the oysters to a larger extent than high molecular weight ones. Estimated half-lives for planar PCB congeners 77 and 126 were 28 and 51 days, respectively for depuration of newly contaminated oysters (HR-SC-HR) while longer half-lives (42 and 60 days, respectively) were observed for the same compounds as they were eliminated from chronically contaminated individuals (SC-HR). Estimated half-lives for 2,3,7,8-TCDD and 2,3,7,8-TCDF were 35 and 36 days, respectively and were similar to the tetrachlorinated biphenyls (PCBs 77 and 81). Compared with ortho-substituted PCB congeners of the same chlorination level, the more toxic PCBs take longer to depurate from the oysters. With few exceptions, elimination of all toxic compounds investigated proceeded at a slower rate from the chronically exposed population that from the newly contaminated one.     

Correction to: Characterization of a fatty acid-binding protein from the Pacific oyster (Crassostrea gigas): pharmaceutical and toxicological implications

Environmental Science and Pollution Research - A Correction to this paper has been published: https://doi.org/10.1007/s11356-021-13135-x     

The integrated use of chemical analysis, cytochrome P450 and stress proteins in mussels to assess pollution along the Galician coast (NW Spain)

Aliphatic and polycyclic aromatic hydrocarbons (PAHs) as well as biochemical markers, such as cytochrome P450, benzo(a)pyrene hydroxylase (BPH) and stress-70 proteins, were determined in mussels (Mytilus galloprovincialis) collected in October 1995 from several locations along the Galician coast (NW Spain). The survey proved the existence of a pollution gradient among sampling sites and a chronic exposure to petrogenic and pyrolitic hydrocarbons. This was reflected in mussel tissue levels of PAHs which ranged from 22 to 562 ng/g wet weight. High levels of total cytochrome P450 were detected in mussels from the most polluted sites, but differences were not statistically significant. BPH activity did not show either differences among sampling sites. Conversely, stress-70 proteins were significantly induced in the most polluted locations, and levels of the 72-kDa band correlated well (r2 = 0.854) with the sum of the PAHs from four to six rings accumulated by mussels.     

Level of contamination and impact of pesticides in cupped oyster, Crassostrea gigas, reared in a shellfish production area in Normandy (France)

Over the last decade, oysters in the Bay of Veys (Northwest France) have sporadically experienced significant summer mortality events which appear to be due to a combination of extrinsic and intrinsic factors. In the present work, the involvement of pesticides (as additional stressors) was investigated using both artificial exposure and field studies (at two sites). Six herbicides were detected in seawater following tests for a total of 15 herbicides. The most estuarine site was the most contaminated, showing relatively high values compared with those recorded in a neighboring river. No pesticides were detected in the flesh of oysters in the field but exposure experiments led to an accumulation of two substituted ureas (diuron and isoproturon, from 0.5 micro g/L and 1 micro gL(- 1) respectively). Some physiological effects were observed in terms of reproduction (partial spawning) and histopathology (atrophy of the digestive tubule epithelium) but results related to other tissue alterations and to neutral red retention (NRR) assays were not conclusive and require further investigation.     

Detection of early effects of a single herbicide (diuron) and a mix of herbicides and pharmaceuticals (diuron, isoproturon, ibuprofen) on immunological parameters of Pacific oyster (Crassostrea gigas) spat

In the context of massive summer mortality events of the Pacific oyster Crassostrea gigas, the aim of this study was to investigate the early effects on genes, enzymes and haemocyte parameters implicated in immune defence mechanisms in C. gigas oysters exposed to a potentially hostile environment, i.e. to an herbicide alone or within a mixture. Following 2 h of exposure to the herbicide diuron at 1 μg L⁻¹, the repression of different genes implicated in immune defence mechanisms in the haemocytes and the inhibition of enzyme activities, such as laccase-type phenoloxidase (PO) in the plasma, were observed. The inhibition of superoxide dismutase (SOD) activity in the plasma was also observed after 6 and 24 h of exposure. In the mixture with the herbicides diuron and isoproturon, and the pharmaceutical ibuprofen, catecholase-type PO activity in the plasma and the percentage of phagocytosis in the haemocytes were reduced after 6 h of exposure. Our results showed that early effects on molecular, biochemical and cellular parameters can be detected in the presence of diuron alone or within a mixture, giving an insight of its potential effect in situations that can be found in natural environments, i.e. relatively high concentrations for short periods of time.     

Methodology for the analysis of Peroxyacetyl nitrate (PAN) in the unpolluted atmosphere

A light weight electron capture, gas chromatograph has been laboratory- and field-tested to conduct surface and airborne PAN measurements in the unpolluted troposphere. A dynamic calibration system based on CH₃CHO/NO₂/Cl₂ photolysis studies by Gay et al. (1976) was constructed and successfully tested. PAN was cryogenically preconcentrated prior to analysis. A sensitivity of 5 parts per trillion (ppt) and an overall accuracy of ± 20 % is estimated. It is shown that gas phase coulometry (GPC) is unsuited for absolute PAN analysis—principally, because a significant fraction of PAN is destroyed prior to coulometric detection. The kinetics of this destruction process are nonlinear. PAN measurements at a marine Pacific site, and aboard an aircraft, show that PAN is always present at a concentration range of 10–100 ppt, although concentrations as high as 400 ppt were measured at an altitude of 4.6 km over the Pacific Ocean. Surface PAN measurements at a Pacific marine site indicate a distinct diurnal behavior, tentatively attributed to photochemistry involving alkenes, alkanes and NO_x. There was no evidence of PAN diurnal variation in the free troposphere, but the data are currently too sparse. Measurements in the global atmosphere are needed to accurately describe the distribution and the role of PAN in the chemistry of the natural atmosphere.     

Vitellogenin and zona radiata proteins as biomarkers of endocrine disruption in peregrine falcon (Falco peregrinus)

The aim of this study was to test a specific method for the detection of Vitellogenin (Vtg) and Zona Radiata Proteins (Zrp) in plasma from peregrine falcon (Falco peregrinus) as specific biomarkers for the evaluation of the effects of endocrine disruptors. The method was assayed with different peregrine falcon individuals (including mature and immature birds of both sexes) from a Spanish population being studied in terms of their contamination with organochlorine compounds with endocrine disrupting properties. This study shows that mouse anti bird Vtg monoclonal antibody ND3C3 (Biosense) seems to be the most specific antibody in binding plasmatic lipoproteins in peregrine falcon when compared to other anti Vtg antibodies. Rabbit anti salmon Zrp polyclonal antibodies O146 (Biosense) show cross-reactivity with Zrp in the samples studied. These preliminary results confirm the applicability of both of these diagnostic tools assayed (induction of Vtg and Zrp) in detecting exposure to Endocrine Disrupting Chemicals (EDCs) in this species. The increase of Vtg and Zrp detected in male specimens suggest a potential hazard to EDCs in the peregrine falcon which represents a species still affected by organochlorine compounds, and in particular those with estrogenic activity.