SUMMARY In order to understand the role of metabolic regulation in environmental stress tolerance, a comprehensive analysis of demand-side effects (i.e. changes in energy demands for basal maintenance) and supply-side effects (i.e. metabolic capacity to provide ATP to cover the energy demand) of environmental stressors is required. We have studied the effects of temperature (12, 20 and 28°C) and exposure to a trace metal, cadmium (50 μg l–1), on the cellular energy budget of a model marine poikilotherm, Crassostrea virginica (eastern oysters), using oxygen demand for ATP turnover, protein synthesis, mitochondrial proton leak and non-mitochondrial respiration in isolated gill and hepatopancreas cells as demand-side endpoints and mitochondrial oxidation capacity, abundance and fractional volume as supply-side endpoints. Cadmium exposure and high acclimation temperatures resulted in a strong increase of oxygen demand in gill and hepatopancreas cells of oysters. Cd-induced increases in cellular energy demand were significant at 12 and 20°C but not at 28°C,possibly indicating a metabolic capacity limitation at the highest temperature. Elevated cellular demand in cells from Cd-exposed oysters was associated with a 2–6-fold increase in protein synthesis and, at cold acclimation temperatures, with a 1.5-fold elevated mitochondrial proton leak. Cellular aerobic capacity, as indicated by mitochondrial oxidation capacity,abundance and volume, did not increase in parallel to compensate for the elevated energy demand. Mitochondrial oxidation capacity was reduced in 28°C-acclimated oysters, and mitochondrial abundance decreased in Cd-exposed oysters, with a stronger decrease (by 20–24%) in warm-acclimated oysters compared with cold-acclimated ones (by 8–13%). These data provide a mechanistic basis for synergism between temperature and cadmium stress on metabolism of marine poikilotherms. Exposure to combined temperature and cadmium stress may result in a strong energy deficiency due to the elevated energy demand on one hand and a reduced mitochondrial capacity to cover this demand on the other hand, which may have important implications for surviving seasonally and/or globally elevated temperatures in polluted estuaries.

No abstract

No abstract

ABSTRACT-Lysosomal destabilization indices (based on a neutral red retention assay) were evaluated for digestive gland cells of oysters Crassostrea virginica. Laboratory studies were conducted to evaluate the effects of variable salinity regimes (a natural stressor) and copper exposures (a pollutant stres-SOT) on lysosomal destabilization. Field studies were also conducted with native oysters and hatcheryreared juvenile oysters deployed in situ at reference and polluted sites. Lysosomal integrity was not affected by either short-term or longer-term variations in salinity during laboratory experiments, and the destabilization indices were similar to those of deployed and native oysters from reference sites characterized by a range of salinlties. However, laboratory Cu exposures (ranging from 2.5 to 20 pg Cu 1-') caused significant adverse effects on lysosomal destabilization after only 18 h, and the eiiecis were sustained or worsened with increasing exposure time and concentration. Hatchery-reared oysters deployed at or native oysters collected from polluted sites had significantly higher lysosomal destabilization indices. Estuanne habitats are characterized by variable sal~nity regimes, so cellular responses that are sensitive to salinity stress as well as contaminant stress would be difficult to interpret if salinity effects cannot be distinguished from pollutant effects. A robust biomarker of anthropogenic effects should be insensitive to natural stressors such as salinity, but should be sensitive to pollutants. The salinity and contaminant studies described in this paper indicate that lysosomal destabilization responses in oysters are insensitive to salinity variations and sensitive to pollutants, and are therefore potentially valuable biomarkers of anthropogenic stress.

Metallothioneins are typically low relative molecular mass (6000-7000), sulfhydryl-rich metal-binding proteins with characteristic repeating cysteine motifs (Cys-X-Cys or Cys-X n -Cys) and a prolate ellipsoid shape containing single a-and b-domains. While functionally diverse, they play important roles in the homeostasis, detoxification and stress response of metals. The originally reported metallothionein of the American oyster, Crassostrea virginica showed the canonical molluscan ab-domain structure. Oyster metallothioneins have been characterized as cDNA and as expressed proteins, and here it is shown that the previously reported metallothionein is a prototypical member of a subfamily (designated as CvMT-I) of ab-domain metallothioneins. A second extensive subfamily of oyster metallothioneins (designated as CvMT-II) has apparently arisen from (a) a stop mutation that truncates the protein after the a-domain, and (b) a subsequent series of duplication and recombination events that have led to the development of metallothionein isoforms containing one to four a-domains and that lack a b-domain. Analysis of metallothioneins revealed that certain CvMT-I isoforms showed preferential association either with cadmium or with copper and zinc, even after exposure to cadmium. These data extend our knowledge of the evolutionary diversification of metallothioneins, and indicate differences in metal-binding preferences between isoforms within the same family.Keywords: cadmium; gene expression; MALDI-TOF; metallothionein; oyster.Metallothioneins (MTs) are a superfamily of ubiquitously expressed metal-binding proteins that can be upregulated by metal exposure, oxidative stress and immune challenge. Typical MTs are low relative molecular mass (M r ) (6000-7000) proteins of high thiol content that lack histidine and aromatic amino acids [1,2]. While they are functionally diverse, they play major roles in metal homeostasis and detoxification. The defining characteristic of MTs is the high cysteine content ( 30%) and conserved Cys-X n -Cys motifs, where X can be any amino acid other than cysteine. The proteins typically have a one-or two-domain structure and bind multiple mono-and divalent metal ions. The structure of MTs, and the nature of their metal-binding, reveal extensive evolutionary diversification. While fungi and early diverged metazoans have small, single-domain MT proteins capable of binding up to eight monovalent metal ions [3][4][5][6], most MTs are comprised of two domains, designated a and b, which are capable of binding metals independently and are separated by a short linker region [7,8]. The a-domain typically contains 11 or 12 cysteines, binds four divalent metal cations, and is believed to convey structure and stability to the protein [9]. In contrast, the b-domain contains nine cysteines, binds three divalent metal cations and participates in metal exchange reactions involving glutathione-shuttling with zinc-and copper-requiring apoproteins [10][11][12]. Some crustacean MTs deviate from this canonical struc...

Oysters are an ecologically important group of filter-feeders, and a valuable toxicology model for characterizing the potential impacts of nanoparticles to marine organisms. Fullerene (C60) exposure studies with oysters, Crassostrea virginica, were conducted with a variety of biological levels, e.g., developmental studies with embryos, whole organism exposures with adults, and isolated hepatopancreas cells. Significant effects on embryonic development and lysosomal destabilization were observed at concentrations as low as 10 ppb. Moreover, based on our extensive experience with the lysosomal assay, the lysosomal destabilization rates at fullerene concentrations > or = 100 ppb were regarded as biologically significant as they are associated with reproductive failure. Interestingly, there was no significant increase in lipid peroxidation levels in hepatopancreas tissues. Oyster hepatopancreas tissues are composed of lysosomal rich cells, and confocal microscopy studies indicated thatthe fullerene particles readily accumulated inside hepatopancreas cells within 4 h. Fullerene aggregates tended to be localized and concentrated into lysosomes. The microscopic work in conjunction with the lysosomal function assays supports the premise that endocytotic and lysosomal pathways may be major targets of fullerenes and other nanoparticles. Nanoparticles that affect normal lysosomal and autophagic processes may contribute to long-term, chronic problems for individual health as well as ecosystem health.

A pilot program was initiated to identify genes from the American oyster, Crassostrea virginica, that are potentially involved in the stress response for use as bioindicators of exposure to environmental pollutants and to toxic and infectious agents. A PCR-based method was used to construct cDNA libraries from pooled embryos and the hemocytes of a single individual. A total of 998 randomly selected clones (expressed sequence tags, ESTs) were sequenced. Approximately 40% of the ESTs are novel sequences. Several potential biomarkers identified include an antimicrobial peptide, recognition molecules (lectin receptors), proteinases and proteinase inhibitors, and a novel metallothionein. Diversity analysis shows that 363 and 286 unique genes were identified from the hemocyte and embryo libraries, respectively, indicating that full-scale EST collection is a valuable approach for the discovery of new genes of potential significance in the molluscan stress response.

No abstract