Differentiation of sympatric zebra and quagga mussels in ecotoxicological studies: A comparison of morphometric data, gene expression, and body metal concentrations.

The zebra mussel is among the best studied freshwater molluscs in ecotoxicology, but information on the quagga mussel is lacking. Considering its potential spread, we selected a river in France in which zebra and quagga mussels coexisted, and then we used genetic markers to differentiate the two species and compared morphological parameters. cDNA sequencing assays of ten genes already used in zebra mussels were performed on quagga mussels to obtain functional specific primers. Then we analyzed the expression of genes involved in cellular metabolic activities (Cytochrome-c-oxidase - cox, and ATP synthase - atp), detoxification processes (Glutathione-S-Transferase - gst), oxidative stress (Catalase - cat), and digestive functions (Amylase - amy) on the two species. Whereas morphometric analysis underlined similarities in shape between the two species, relative gene expression profiles and metal concentrations evidenced strong differences. Quagga mussels notably presented half as high concentrations in Cd and Pb, two particularly toxic elements, as zebra mussels. These results imply that i) particular attention should be paid to properly distinguish the two species considering their similar external appearance, and ii) zebra mussels cannot be replaced by quagga mussels in ecotoxicological studies without preliminary investigations on biomarker response patterns. To our knowledge, this study is the first to have undertaken such an approach in gene expression analysis in quagga mussels, and more generally to have compared such biomarker responses of zebra and quagga mussels in the field.

Variations in gene expression levels in four European zebra mussel, Dreissena polymorpha, populations in relation to metal bioaccumulation: A field study.

The present study was performed to validate the suitability of using gene expression in zebra mussels, Dreissena polymorpha, for biomonitoring of freshwater environment. Mussels were collected in four French rivers (Meuse, Moselle, Oise and Vilaine) in spring and autumn. Relative gene expression of 9 candidate genes involved in cellular metabolic activities (Cytochrome-c-oxidase - cox, and ATP synthase - atp), detoxification process (Metallothionein - mt and Glutathion-S-Transferase - gst), oxidative stress (Catalase - cat, Superoxyde Dismutase - sod and Glutathion peroxidase - gpx) and digestive functions (Amylase - amy and Cellulase - ghf) were measured in digestive gland. Metal bioaccumulation in tissues and morphometric parameters were also analyzed to interpret molecular responses. All our results are consistent with different physiological reactions to environmental condition between zebra mussel populations. In spring, the levels of mt, sod, gpx, cat, atp, amy and ghf relative expression were significantly higher in mussels with the lowest metal bioaccumulation (the Meuse) compared to at least one of the other sites. In autumn, this higher expression levels in Meuse River were still observed for gpx, cat, atp and amy. This study has also pointed out different sources of variability in gene expression (individual size, season, trophic resources and origin of mussels) which are inevitable in natural fluctuant environment. This underlines the importance to take them into account in field study to propose a correct interpretation of biomarker responses.

Zebra mussel as a new tool to show evidence of freshwater contamination by waterborne Toxoplasma gondii.

AIMS: The objective of this study was to evaluate if freshwater bivalves can be used to detect the presence of Toxoplasma gondii in water bodies. METHODS AND RESULTS: Zebra mussels (Dreissena polymorpha) were caged for 1 month upstream and downstream of the discharge points of wastewater treatment plants (WWTPs). Physiological status was assessed to assure good health of bivalves during transplantation. The presence of T. gondii was investigated in mussel tissues by qPCR. In autumn, T. gondii was detected in mussels caged downstream of the discharge points of two WWTPs. In spring, it was detected upstream of one WWTP. CONCLUSIONS: For the first time, T. gondii DNA has been shown in a continental mollusc in environmental conditions. This highlights the interest of an active approach that could be applied independently of the presence or accessibility of autochthonous populations, and underlines the presence of T. gondii in natural waters under pressure of WWTP discharge at a certain time of the year. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that transplanted zebra mussels could be used as biosamplers to reveal contamination of freshwater systems by T. gondii.

Effects of chemical stress and food limitation on the energy reserves and growth of turbot, Scophthalmus maximus.

The objective of the present study is to examine the growth and energetic performance of juvenile turbot after exposure to contaminated sediment and during the subsequent recovery period with or without food limitation. We designed a two-step experiment by first exposing juvenile turbot to harbour sediment for 26 days and then transferring them to clean sea water with different frequencies of feeding for 35 days. Without food limitation, fish previously exposed to contaminated sediment compensated for weight, length and lipid reserve losses; we did not record any differences in size, Fulton's K condition index and triacylglycerol/sterol (TAG/ST) ratio after the 35-day depuration period compared to the reference fish. This result could be related to the compensatory growth mechanism observed in a wide range of fish species following a period of growth depression. With food limitation during the 35-day depuration period, recovery growth was not sufficient to restore length and weight values similar to the reference fish. Moreover, turbot previously exposed to contaminated sediment and subsequently fed twice or once a week exhibited extremely low TAG/ST ratios, but the reference fish submitted to the same restrictive feeding conditions did not. This study indicates that juvenile fish affected by chemical pollution can improve their biological performance if pollution events are followed by a period of abundant food. However, if pollution events occur during periods of food scarcity, e.g. in winter, storage of energy reserves will be compromised.

A combined measurement of metal bioaccumulation and condition indices in juvenile European flounder, Platichthys flesus, from European estuaries.

Condition indices and metal bioaccumulation of early life stages of juvenile flounder (5-10 cm) were determined in three anthropogenic estuaries (the Scheldt, Seine and Loire) and compared to a reference site (the Canche). Significant correlations were found between metal concentrations in sediment and (i) fish liver for Cd, Cu, Ni, Pb, V and Zn and (ii) fish gills for Cd and Mn. Metal accumulation in juvenile flounder from the three anthropogenic estuaries coincided with significantly lower Fulton's K indices (from 0.99 ± 0.03 to 1.06 ± 0.01 mg mm(-3)) compared to those from the Canche estuary (from 1.02 ± 0.01 to 1.13 ± 0.01 mg mm(-3)). This discrepancy in fish condition index increased with fish size and therefore, strongly depends on the time juvenile spend in estuary. Muscle lipid contents and Triacylglycerol to Sterol ratios were significantly lower in fish collected in the Scheldt (lipid content: 21.3 ± 3.6%), Seine (17.9 ± 19.8%) and Loire (19.5 ± 2.4%) estuaries compared to those originating from the Canche (38.3 ± 4.6%). This study highlights that combined measures of both fish metal contents and condition indices gives a relevant assessment of juvenile fish health growing in anthropogenic estuaries.

Metal concentrations, growth and condition indices in European juvenile flounder (Platichthys flesus) relative to sediment contamination levels in four Eastern English Channel estuaries.

The aim of the present study was to evaluate the effects of metal contamination on the biological responses of 0-group juvenile European flounder and to assess and compare the quality of four estuarine habitats located in the Eastern English Channel. Fish otolith growth and condition indices (RNA : DNA ratio, Fulton's K condition index) were measured and found to be significantly lower in individuals from the Seine estuary compared to those of the Canche, Authie and Somme estuaries. No obvious effects of hydrological condition or food availability on the flounder biological responses were observed. Sediments from the Seine showed the highest metal concentrations, bioavailable proportion and enrichment factors. Higher metal concentrations were observed in fish from the Seine compared to the other ones caught in less polluted estuaries. These results suggest that contaminants may have a negative impact on the early life history stage of flounder.

A multibiomarker approach in juvenile turbot, Scophthalmus maximus, exposed to contaminated sediments.

Juvenile turbot were exposed in laboratory conditions to a mixture of chemical contaminants associated with harbour and estuarine sediments for seven and 21 days. Several molecular biomarkers of exposure were then measured in fish liver: two biotransformation parameters [ethoxyresorufin-O-deethylase (EROD) and phase II glutathione S-transferase (GST) activities] and an antioxidant enzyme activity [catalase (CAT)]. Modifications at the histological level were analysed by the measurement of the number and size of melanomacrophage centres (MMCs) and disturbances to the immune function by the measurement of cytokine transforming growth factor-beta (TGF-ß) and development of the thymus. The responses of these molecular and immunological biomarkers were correlated with metal and PAH concentrations measured in sediments and with the physiological performance of turbot analysed in a previous study on the same fish (growth rates, condition factor K, RNA:DNA ratio and lipid index). While no difference was found in thymus analysis, some molecular and immunological responses were observed in fish exposed to contaminated sediments. Weak relationships between molecular biomarkers' responses and PAH concentrations were recorded, while their responses were significantly correlated with some metals. MMC and aggregates were weakly related to chemical contaminants whereas some significant correlations were found between TGF-b1 responses and some metal concentrations. However, molecular and immunological biomarkers were weakly related to fish physiological damages since low responses were observed in the condition which led to the lowest growth and condition indices. These data suggest the complexity of cause-effect relationships between exposure to pollutants, metabolisms and health damages. Precautions should be considered in the use of molecular and immunological biomarkers alone in biomonitoring programs. Their complementary use with physiological biomarkers, such as fish growth and condition indices, could improve their utilisation.

Growth and condition indices of juvenile turbot, Scophthalmus maximus, exposed to contaminated sediments: effects of metallic and organic compounds.

Since sediments have the potential to form associations with several classes of pollutants, they have been recognized as a possible and significant source of contamination for the benthic environment. Flatfish maintain a close association with sediments for food and cover, and are therefore more likely to be exposed to contaminated sediments, especially in coastal areas (e.g. nursery grounds). The assessment of these potential biological effects involves the use of adapted biomonitoring tools. The main objective of this study was to assess and compare the response of several physiological biomarkers measured on juvenile turbot (Scophthalmus maximus) exposed to contaminated sediments. Sediments were collected from three stations in a harbour in northern France (Boulogne-sur-Mer), in an anthropogenic French estuary (the Seine), and in a reference site (exposed sandy beach of Wimereux). Unexposed lab-reared juvenile turbots were exposed to sediments for 7 and 21 days in laboratory conditions. Sediments were analysed for metals, PAH and PCB contamination. Several fish growth and condition indices were individually analysed in fish according to the chemical contaminant availability in sediment, the metal concentrations in gills and the estimation of PAH metabolites in their bile. Significant decreases in growth rates, morphometric index, RNA:DNA ratio and the lipid storage index, based on the ratio of the quantity of triacylglycerols on sterols (TAG:ST), were observed with increasing level of chemical contamination. This decrease in the fish's physiological status could be related to the significant increase of several metal concentrations in contaminated fish gills and the significant increase of PAH metabolites in bile. In a field situation, such a reduction in growth and energetic status of juvenile fish could dramatically decrease their over-winter survival in contaminated nursery grounds.

Responses of juvenile sea bass, Dicentrarchus labrax, exposed to acute concentrations of crude oil, as assessed by molecular and physiological biomarkers.

In the present study, juvenile sea bass were exposed for 48 and 96 h to an Arabian light crude oil and their responses were assessed at the molecular and physiological levels. The aim of the study was therefore to assess (i) the short term effects of crude oil exposure by the measurement of several molecular biomarkers, (ii) the consequences of this short term exposure on fish health by using growth and condition indices measured after a decontamination period of 28 and 26 d in seawater. Hydrocarbon petroleum concentrations was monitored during the 96 h experiments and an increase of PAH concentrations were found in fish following both exposure times. An 7-ethoxyresorufin-O-deethylase (EROD) induction was observed after 48 h of exposure, while a significant decrease in the sea bass specific growth rate in length and for the RNA:DNA ratio was observed 28 d after that exposure ceased. The EROD induction doubled after the 96 h exposure, and a significant increase in GST activities was observed. A significant decrease in the specific growth rates, the otolith recent growth, the RNA:DNA ratio and the Fulton's K condition index were then observed in sea bass 26 d after the 96 h exposure to mechanically dispersed crude oil compared to the control. The present study shows that growth and condition indices can prove useful in assessing fish health status following an oil spill. Their complementary analysis with sensitive molecular biomarkers as EROD could improve the determination of oil spill impact on fish populations.

Are biochemical biomarker responses related to physiological performance of juvenile sea bass (Dicentrarchus labrax) and turbot (Scophthalmus maximus) caged in a polluted harbour?

Biomarker responses to toxic exposure have been used for decades to indicate stress in aquatic organisms, or the magnitude of environmental pollution. However, little has been done to compare the simultaneous responses of both biochemical and physiological biomarkers. The purpose of this study was twofold. Firstly to analyse the responses of several biochemical biomarkers measured on juvenile sea bass and turbot caged in a northern France harbour at a reference and contaminated stations. Several biotransformation parameters (Ethoxyresorufin-O-deethylase - EROD - and Glutathione S-transferase -GST) and an antioxidant enzyme (Catalase -CAT) were analysed. Secondly, to compare their responses to several growth and condition indices, measured on the same fish. In the contaminated station, EROD and GST activities were found to be significantly higher, and a decrease of CAT activity was observed for both species. For individual sea bass, biochemical biomarkers showed numerous significant correlations with growth and condition indices, such as the Fulton's K condition index, the RNA:DNA ratio and the lipid storage index. On the contrary, there were only a few significant correlations for turbot, suggesting a species-specific response. Our study indicates that the analysis of the simultaneous responses of both biochemical and physiological biomarkers can be useful for monitoring complex exposure and to assess habitat quality.