Seasonal changes in mRNA encoding for cell stress markers in the oyster Crassostrea gigas exposed to radioactive discharges in their natural environment

Abstract

The North Cotentin area (Normandy, France) hosts several nuclear facilities among which the AREVA reprocessing plant of La Hague is responsible for controlled discharges of liquid radioactive wastes into the marine environment. The resulting increase in radioactivity is very small compared to natural radioactivity. However, concerns about environment protection prompted the scientific community to focus on the effects of the chronic exposure to low concentrations of radionuclides in non-human biota. This study contributes to the evaluation of the possible impact of radioactive discharges on the oyster Crassostrea gigas in the field. Real-time polymerase chain reaction was used to quantify the expression levels of genes involved in cell stress in the oyster. They included members of the heat shock protein family (Hsp70, Hsc72, Hsp90), superoxide dismutase (SOD) and metallothionein (MT). Times series measurements were built from periodic samplings in the natural environment in order to characterize the natural variability as well as possible seasonal fluctuations. The genes studied exhibited a general seasonal expression pattern with a peak value in winter. The data inversely correlated with seawater temperature and the nature of the relationship between gene expression and temperature is discussed. In parallel, oysters were collected in four locations on the French shores, exposed or not to radioactive liquid wastes from the nuclear facilities hosted in the North Cotentin. The comparison of data obtained in the reference location on the Atlantic coast (not exposed) and data from oysters of the English Channel (exposed) gave no evidence for any statistical difference. However, because of the complexity of the natural environment, we cannot rule out the possibility that other parameters may have masked the impact of radioactive discharges. This dense set of data is a basis for the use of the expression levels of those genes as biomarkers to address the question of the possible effects of chronic exposure of the oyster to low concentrations of radionuclides in controlled laboratory experimental conditions.

Transcriptional expression levels of cell stress marker genes in the Pacific oyster Crassostrea gigas exposed to acute thermal stress

“…In September, mRNA levels returned to control values 48 h after stress, while in March, mRNA levels were still elevated 48 h after the heat shock. In the environment, because of natural seasonal variations, higher levels of mRNA encoding the studied genes were observed in oysters collected in March-April, compared to September (Farcy et al 2007). Higher HSP70 protein concentrations were also observed in horse mussels during winter compared to summer (Lesser and Kruse 2004).…”

“…Firstly, the levels observed in laboratory control conditions were similar to those observed at the oyster farm, indicating that acclimation of the animals did not induce any additional stress due to handling in the laboratory. Secondly, the ranges of variation in field data, which were documented in greater detail in our previous study regarding seasonal expression patterns (Farcy et al 2007), provided a valuable basis for assessing the significance of observed changes in response to experimental exposure to stress. In the present work, heat stress values were clearly outside the ranges of control environmental and laboratory values.…”

“…In the English Channel, minimal and maximal seawater temperatures are observed in March and September, respectively (see Farcy et al 2007 for a detailed seawater temperature monitoring profile). Oysters were obtained from a local oyster farm (Normandy, France); all oysters used were diploid, 3-4 years old and had spent their entire growing life at the farm.…”

“…Controls without template cDNA were included on PCR plates. Gene-specific mRNA quantification was performed by normalizing data to the amount of mRNA encoding for actin, a cytoskeleton protein taken as a housekeeping gene (Farcy et al 2007). For each analysed specific DNA, realtime PCR provides a cycle threshold (Ct) value where the fluorescence signal is detectable above the background.…”

“…The transcription of several cell stress marker genes was previously monitored in oyster during the annual cycle (Farcy et al 2007). This study showed that the mRNA levels exhibited a seasonal pattern with maximal observed values generally coinciding with lowest seawater temperature during winter and, for some genes, a secondary peak during summer.…”