Metal Contamination and Biomarkers in Cerastoderma glaucum: A Multi-level Approach.

In this study, we focused on evaluating the responses of the cockle, Cerastoderma glaucum to in situ exposures to metals at three sites in the Gulf of Gabes in the coastal zone of Tunisia differing in levels of metal contamination. Firstly, we examined the general physiological state of the organisms. Secondly, we evaluated the bioaccumulation of several metals (Cd, Cu, Zn, Ni) in the cockles. Thirdly, we focused on evaluating histologically changes in gametogenesis and sexual maturity of the organisms. Finally, we determined the expression of seven key genes encoding enzymes or proteins involved in responses to different types of environmental stressors. Results showed a decrease in the general physiological status of the cockles, including a reduced condition index, sex ratios skewed to females (70% and 80% females in the intermediate and the contaminated site, respectively) and greater mortalities in tests under anoxic conditions (i.e., stress on stress test) in cockles collected from the most contaminated site (LT50 = 2.88 days) compared to the cockles from the intermediate site (LT50 = 5 days) and the less contaminated site (LT50 = 6 days). Results for metal bioaccumulation showed that the levels of Cd, Cu, Zn and Ni in cockles were consistent with the contaminant gradient, with the highest levels in cockles from the most contaminated site (1.04; 4.92; 52.76 and 13.81 µg/g dw, respectively), followed by those from the intermediate site (0.34; 2.94; 36.94; 17.40 µg/g dw, respectively) and then the less contaminated site (0.065; 1.27; 21.62 and 5.40 µg/g dw, respectively). Results from the gametogenesis and maturity index showed few differences in the reproductive cycle of cockles collected from the three study sites. There were different patterns of gene expression that were divided into three groups in terms of responses: (1) expression of genes involved in metal detoxification, ATP Binding Cassette Subfamily B Member 1 (ABCB1) and metallothionein MT) and genes for superoxide dismutases (i.e., Mn SOD and CuZn SOD), which did not show any difference in their levels of expression; (2) heat shock protein 70 (HSP70) gene expression, which decreased in cockles according to the pollution gradient, and (3) expression of catalase (CAT) and cytochrome oxidase subunit 1 (COI) genes was threefold and 1000-fold higher in cockles from intermediate and most contaminated sites compared to the less contaminated site. Therefore, changes in overall physiological condition, sex ratios and expression of HSP70, CAT and COI genes may be appropriate biomarkers for in situ studies of the impacts of metals in cockles. However, these biomarkers should be coupled to proteomics studies.

Responses of primary cultured haemocytes derived from the marine gastropod Haliotis tuberculata to an industrial effluent exposure.

This study assessed the responses of primary cultured haemocytes from the marine gastropod Haliotis tuberculata exposed to the increasing concentrations of industrial effluent (0, 0.5, 1, 10, 15 and 20%) discharged into the Tunisian coastal area. Analyses showed the presence of metals such as cadmium (Cd), chromium (Cr), copper (Cu), manganese (Mn), zinc (Zn), nickel (Ni) and lead (Pb) in the effluent. The effects of this mixture of pollutants on abalone haemocyte parameters were reflected by a significant decrease of cell viability, phagocytotic activity and reactive oxygen species (ROS) production as well as morphological and lysosomal membrane alterations. Thus, these results indicated that our primary culture system represents a suitable in vitro model for monitoring of anthropogenic contaminants in aquatic environments.

The reproductive cycle, condition index, and glycogen reserves of the cockles Cerastoderma glaucum from the Gulf of Gabès (Tunisia).

The gametogenic cycle of the Cerastoderma glaucum was analyzed using both qualitative and semi-quantitative methods. The condition index and glycogen concentrations were determined in order to provide information on energy storage. The cockles were collected monthly from a Bayyadha site located 15 km south of Sfax City (Gulf of Gabès) between January 2007 and January 2008. From histological point of view, we applied two approaches: (i) the qualitative method describing the various stages of gamete development for males and females during a cycle of 13 months, and (ii) the semi-quantitative method concerning the estimation of different tissue surfaces. The results showed that there is evidence of three periods of reproduction in this population. A comparison between the surfaces occupied by the three organs showed that the foot and the gonad surfaces are higher than the surface of the adductor muscle. This could suggest that these two organs are more involved in the process of glycogen reserve storage. The results of the glycogen concentrations in the different tissues (gonad, adductor muscle, and "remainders") show that during the second and third periods of reproduction, glycogen was stored in the adductor muscle and in the remainder during sexual rest, and in the gonad during the gametogenesis phases in order to supply the reproductive effort. On the contrary, in the first period of reproduction, the low concentrations of glycogen recorded in the gonad coincided with its high degree of development. This fact could be related to environmental conditions (low temperature and food) recorded during this period.

Transcriptional response of stress-regulated genes to cadmium exposure in the cockle Cerastoderma glaucum from the gulf of Gabès area (Tunisia).

This study investigates cadmium effects on key messenger RNA (mRNA) expression (MT, MnSOD, CuZnSOD, CAT, ABCB1, HSP70, and CO1) by qPCR in the cockle Cerastoderma glaucum after chronic exposure to two high but environmentally relevant concentrations of CdCl2 (50 µg/L and 5 mg/L) for 12 h to 18 days. Cd accumulation measured in cockles' tissues is significantly higher in both treatment conditions compared to controls and in a dose-dependent manner. Stress on stress tests performed at different times of the experiment clearly demonstrated that exposure to both concentrations of Cd significantly affects cockle survival time in air. Important changes in gene transcription were also highlighted. In particular, MT, HSP70, CAT, and CuZnSOD seem to be relevant biomarkers of Cd exposure because (1) their mRNA levels increase upon exposure and (2) they are highly correlated to Cd accumulation in tissues. Results may be useful for control strategies and for the use of cockles as sentinel organisms.

Transcriptional response of stress-regulated genes to industrial effluent exposure in the cockle Cerastoderma glaucum.

This study assessed the responses of molecular biomarkers and heavy metal levels in Cerastoderma glaucum exposed for 1 week to two industrial effluents (1%) discharged into the Tunisian coastal area, F1 and F2, produced by different units of production of a phosphate treatment plant. A significant uptake of metals (Cd, Cu, Zn, and Ni) was observed in exposed cockles compared to controls, with an uptake higher for F1 than for F2. A decrease in LT50 (stress on stress test) was also observed after an exposure to the effluent F1. Treatments resulted in different patterns of messenger RNA (mRNA) expression of the different genes tested in this report. Gene transcription monitoring performed on seven genes potentially involved in the tolerance to metal exposure showed that for both exposures, mechanisms are rapidly and synchronically settled down to prevent damage to cellular components, by (1) handling and exporting out metal ions through the up-regulation of ATP-binding cassette xenobiotic transporter (ABCB1) and metallothionein (MT), (2) increasing the mRNA expression of antioxidant enzymes (catalase (CAT), superoxide dismutases, CuZnSOD and MnSOD), (3) protecting and/or repairing proteins through the expression of heat shock protein 70 (HSP70) mRNAs, and (4) increasing ATP production (through the up-regulation of cytochrome c oxidase 1 (CO1)) to provide energy for cells to tolerate stress exposure. The tools developed may be useful both for future control strategies and for the use of the cockle C. glaucum as a sentinel species.