Cockle Shell-Derived Aragonite CaCO3 Nanoparticles for Co-Delivery of Doxorubicin and Thymoquinone Eliminates Cancer Stem Cells.

Cancer stem cells CSCs (tumour-initiating cells) are responsible for cancer metastasis and recurrence associated with resistance to conventional chemotherapy. This study generated MBA MD231 3D cancer stem cells enriched spheroids in serum-free conditions and evaluated the influence of combined doxorubicin/thymoquinone-loaded cockle-shell-derived aragonite calcium carbonate nanoparticles. Single loaded drugs and free drugs were also evaluated. WST assay, sphere forming assay, ALDH activity analysis, Surface marker of CD44 and CD24 expression, apoptosis with Annexin V-PI kit, cell cycle analysis, morphological changes using a phase contrast light microscope, scanning electron microscopy, invasion assay and migration assay were carried out; The combination therapy showed enhanced apoptosis, reduction in ALDH activity and expression of CD44 and CD24 surface maker, reduction in cellular migration and invasion, inhibition of 3D sphere formation when compared to the free drugs and the single drug-loaded nanoparticle. Scanning electron microscopy showed poor spheroid formation, cell membrane blebbing, presence of cell shrinkage, distortion in the spheroid architecture; and the results from this study showed that combined drug-loaded cockle-shell-derived aragonite calcium carbonate nanoparticles can efficiently destroy the breast CSCs compared to single drug-loaded nanoparticle and a simple mixture of doxorubicin and thymoquinone.

Formulation of a Sustained Release Docetaxel Loaded Cockle Shell-Derived Calcium Carbonate Nanoparticles against Breast Cancer.

PURPOSE: Here, we explored the formulation of a calcium carbonate nanoparticle delivery system aimed at enhancing docetaxel (DTX) release in breast cancer. METHODS: The designed nano- anticancer formulation was characterized thorough X-ray diffraction (XRD), Fourier transformed infrared (FTIR), transmission electron microscopy (TEM) and field emission scanning electron microscopy (FESEM) and Brunauer-Emmett-Teller (BET) methods. The nano- anticancer formulation (DTX- CaCO3NP) was evaluated for drug delivery properties thorough in vitro release study in human body simulated solution at pH 7.4 and intracellular lysosomal pH 4.8. RESULTS: Characterization revealed the successful synthesis of DTX- CaCO3NP, which had a sustained release at pH 7.4. TEM showed uniformly distributed pleomorphic shaped pure aragonite particles. The highest entrapment efficiency (96%) and loading content (11.5%) were obtained at docetaxel to nanoparticles ratio of 1:4. The XRD patterns revealed strong crystallizations in all the nanoparticles formulation, while FTIR showed chemical interactions between the drug and nanoparticles with negligible positional shift in the peaks before and after DTX loading. BET analysis showed similar isotherms before and after DTX loading. The designed DTX- CaCO3NP had lower (p < 0.05) cytotoxity against MCF-7 cells than DTX at 24 h but comparable (p > 0.05) effects at 48 h and 72 h. However, the DTX- CaCO3NP released less than 80% of bond DTX at 48 and 72 h but showed comparable effects with free DTX. CONCLUSIONS: The results showed that the developed DTX- CaCO3NP released DTX slower at pH 7.4 and had comparable cytotoxicity with free DTX at 48 and 72 h in MCF-7 cells.

Concentration of Mercury in Cockles (Anadara granosa and A. antiquata) Harvested from Estuaries of Western Lombok, Indonesia, and Potential Risks to Human Health.

This study measured the levels of total mercury (tHg) in the whole tissues of cockles (Anadara granosa and A. antiquata) harvested from three estuaries of Western Lombok Island (WLI), Indonesia. This paper also evaluated the hazard level posed by the mercury in relation to the maximum residual limit for human consumption and to estimate the weekly intake and compare it with the provisional tolerable weekly intake (PTWI). The tHg concentrations in A. granosa ranged from 0.020 to 0.070 mg kg(-1), and those in A. antiquata were between 0.032 and 0.077 mg kg(-1) at all locations. All samples of cockles harvested from WLI contain tHg below the permissible limit for human consumption. The maximum weekly intakes for total mercury by coastal people range from 0.28 to 1.08 µg kg(-1) b.w., and they are below the recommended values of PTWI (5.6 µg kg(-1) b.w.). If it is assumed that 100% of the Hg in cockles is methyl mercury (MeHg), consumption of the indicated amounts at the measured values wouldn't exceed the MeHg PTWI (1.6 µg kg(-1) b.w.).

Partitioning of paralytic shellfish toxins in sub-cellular fractions of the digestive gland of the cockle Cerastoderma edule: Changes under post-bloom natural conditions.

Concentrations of paralytic shellfish toxins (C1+2, B1, dcGTX2+3, dcSTX, GTX2+3 and STX) were determined by LC-FLD in composite samples of digestive glands of the cockle Cerastoderma edule and in each sub-cellular particulate fractions obtained after differential centrifugation (nuclei+debris, mitochondria, lysosomes and microsomes). The specimens were sampled during the exposure to a bloom of Gymnodinium catenatum (day 0) and in the subsequent 8, 12, 14, 19, 21 and 25 days under natural depuration conditions. Toxin profiles of digestive glands were dominated by C1+2 followed by B1 and dcGTX2+3, although the proportion between C1+2 and B1 contents decreased with the time, indicating a slower elimination of B1. All toxins, except GTX2+3 and STX, were quantified in the four sub-cellular fractions. The content of the quantified toxins decreased most markedly in nuclei+debris and microsomal fractions, during the first eight and 12 days, respectively. Conversely, different patterns were observed among toxins in mitochondrial and lysosomal fractions. The less accentuated decreases of dcGTX2+3 and dcSTX contents in the mitochondrial fraction may have resulted from the conversion of other toxins, like C1+2 and B1, associated with enzymatic activities in that fraction. The largest discrepancy was registered in lysosomal fraction for B1, since its content increased after eight days of post-bloom conditions. Input of B1 may come from the conversion of other toxins, like the abundant B2 and C1+2. These transformations are associated to the major role of lysosomes in the intra-cellular digestive process of materials acquired through vesicular transport.

Characteristics of minerals and oxide compounds in sediment collected from blood cockle culture areas at Bandon Bay, Thailand.

Bandon Bay is a very fertile bay for coastal aquaculture, especially for blood cockles (Anadara granosa). Its structural pattern supports the flow of nutrients which directly sent from many rivers resulted the high production capacity of blood cockle at the top level in the country. Besides organic compounds present in sediment, inorganic substances are essential for growth, survival and shell development of blood cockles. A comparative study of minerals and oxide compounds which accumulated in the sediments at eight stations around the cockle culture area was conducted. These stations are located along the estuaries at Tha Thong, Tha Chang, Phum Riang, and Tapi. The proportion of oxide compounds were determinedusing X-Ray Fluorescence (XRF) technique and minerals were analyzed by Atomic Absorption Spectroscopy (AAS). Results showed that sediment characteristics, oxide composition and the amount of minerals among the stations are different from each other. The sediments of the eastern and the western coasts were characterized as crumble clay and muddy sand, respectively. Twelve types of oxide compounds, namely SiO2, Al2O3, Fe2O3, K2O, Cl, MgO, Na2O, SO3, CaO, TiO2, MnO, P2O5 were found in various quantities, with SiO2, Al2O3, and Fe2O3 were the fundamental minerals ranging from 85.64-90.82%. Tha Thong estuary in the east coast showed highly significant quantities (P<0.05) of potassium, calcium and manganese compared to the other estuaries.

Partitioning of metals in the tissues and cytosolic fraction of Cerastoderma edule.

The concentrations of Cd, Cu and Zn have been determined in the tissues and the cytosolic fraction of the common cockle, Cerastoderma edule, collected from sediments in the Tamar, Plym and Avon estuaries (South West, England). Metal concentrations in the tissues of C. edule from the Avon were lower than those from the Tamar and Plym, except for Cu in the digestive gland. Significant statistical relationships were only obtained between the total sedimentary metal concentrations and Cd in the body of C. edule and Cu in the digestive gland. The cytosolic fraction was extracted from each of the tissues and separated for protein analysis thereby allowing determination of the metal contents in high molecular weight (HMW) compounds, metallothionein-like proteins (MTLP) and very low molecular weight (VLMW) compounds. The digestive glands of C. edule from the Avon had relatively low concentrations of MTLP, whereas MTLP concentrations in the digestive gland of cockles from the Tamar and Plym were higher. The cytosolic fraction of C. edule had relatively low total Cd and Cu concentrations associated with MTLP, whereas Zn was preferentially associated with the HMW and the VLMW components. The results are relevant to metal distributions in C. edule and the role of cytosols in the management of metals by C. edule and other invertebrates.

Calcium oxide derived from waste shells of mussel, cockle, and scallop as the heterogeneous catalyst for biodiesel production.

The waste shell was utilized as a bioresource of calcium oxide (CaO) in catalyzing a transesterification to produce biodiesel (methyl ester). The economic and environmen-friendly catalysts were prepared by a calcination method at 700-1,000°C for 4 h. The heterogeneous catalysts were characterized by X-ray diffraction (XRD), X-ray fluorescence (XRF), scanning electron microscopy (SEM), and the Brunauer-Emmett-Teller (BET) method. The effects of reaction variables such as reaction time, reaction temperature, methanol/oil molar ratio, and catalyst loading on the yield of biodiesel were investigated. Reusability of waste shell catalyst was also examined. The results indicated that the CaO catalysts derived from waste shell showed good reusability and had high potential to be used as biodiesel production catalysts in transesterification of palm oil with methanol.

Are metallothioneins equally good biomarkers of metal and oxidative stress?

Several researchers investigated the induction of metallothioneins (MTs) in the presence of metals, namely Cadmium (Cd). Fewer studies observed the induction of MTs due to oxidizing agents, and literature comparing the sensitivity of MTs to different stressors is even more scarce or even nonexistent. The role of MTs in metal and oxidative stress and thus their use as a stress biomarker, remains to be clearly elucidated. To better understand the role of MTs as a biomarker in Cerastoderma edule, a bivalve widely used as bioindicator, a laboratory assay was conducted aiming to assess the sensitivity of MTs to metal and oxidative stressors. For this purpose, Cd was used to induce metal stress, whereas hydrogen peroxide (H2O2), being an oxidizing compound, was used to impose oxidative stress. Results showed that induction of MTs occurred at very different levels in metal and oxidative stress. In the presence of the oxidizing agent (H2O2), MTs only increased significantly when the degree of oxidative stress was very high, and mortality rates were higher than 50 percent. On the contrary, C. edule survived to all Cd concentrations used and significant MTs increases, compared to the control, were observed in all Cd exposures. The present work also revealed that the number of ions and the metal bound to MTs varied with the exposure conditions. In the absence of disturbance, MTs bound most (60-70 percent) of the essential metals (Zn and Cu) in solution. In stressful situations, such as the exposure to Cd and H2O2, MTs did not bind to Cu and bound less to Zn. When organisms were exposed to Cd, the total number of ions bound per MT molecule did not change, compared to control. However the sort of ions bound per MT molecule differed; part of the Zn and all Cu ions where displaced by Cd ions. For organisms exposed to H2O2, each MT molecule bound less than half of the ions compared to control and Cd conditions, which indicates a partial oxidation of thiol groups in the cysteine residues through ROS scavenging. The present results suggest that MTs are excellent markers of metal stress, but not of oxidative stress.

Transcriptomic Profile of the Cockle Cerastoderma edule Exposed to Seasonal Diarrhetic Shellfish Toxin Contamination.

Bivalves constitute an important source of proteins for human consumption, but some accumulate biotoxins such as diarrhetic shellfish toxins (DSTs), constituting a risk to human health. The cockle Cerastoderma edule is one of the most important species harvested in the Portuguese coast but also one of the most affected species due to recurrent DSTs exposure. However, little is known regarding the effects of the toxins produced by blooming dinoflagellates on C. edule. Herein, we explore the Differentially Expressed Genes (DEGs) of two tissues (gills and digestive gland) from wild cockles sampled in Portugal, through their whole transcriptomic response in two different seasons (exposed and not exposed to DSTs). The de novo transcriptome assembly returned 684,723 contigs, N50 of 1049, and 98.53% completeness. Altogether, 1098 DEGs were identified, of which 353 DEGs were exclusive for the digestive gland, 536 unique for the gills and 209 DEGs were common. Among DEGs were identified known DSTs-biomarkers including glutathione peroxidase, glutathione S-transferase, superoxide dismutase, cytochrome P450, ABC transporters, actin and tubulin-related proteins, Heat shock proteins and complement C1Q-like proteins. This study provides the first transcriptomic profile of C. edule, giving new insights about its molecular responses under different environmental conditions of DSTs exposure.

Single laboratory validation of a surface plasmon resonance biosensor screening method for paralytic shellfish poisoning toxins.

A research element of the European Union (EU) sixth Framework project BioCop focused on the development of a surface plasmon resonance (SPR) biosensor assay for the detection of paralytic shellfish poisoning (PSP) toxins in shellfish as an alternative to the increasingly ethically unacceptable mouse bioassay. A biosensor assay was developed using both a saxitoxin binding protein and chip surface in tandem with a highly efficient simple extraction procedure. The present report describes the single laboratory validation of this immunological screening method, for this complex group of toxins with differing toxicities, according to the European Decision 2002/657/EC in conjunction with IUPAC and AOAC single laboratory validation guidelines. The different performance characteristics (detection capability CCbeta, specificity/selectivity, repeatability, reproducibility, stability, and applicability) were determined in relation to the EU regulatory limit of 800 microg of saxitoxin equivalents (STX eq) per kg of shellfish meat. The detection capability CCbeta was calculated to be 120 microg/kg. Intra-assay repeatability was found to be between 2.5 and 12.3% and interassay reproducibility was between 6.1 and 15.2% for different shellfish matrices. Natural samples were also evaluated and the resultant data displayed overall agreements of 96 and 92% with that of the existing AOAC approved methods of mouse bioassay (MBA) and high performance liquid chromatography (HPLC), respectively.

In-house validation of a liquid chromatography tandem mass spectrometry method for the analysis of lipophilic marine toxins in shellfish using matrix-matched calibration.

A liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the quantitative analysis of lipophilic marine toxins in shellfish extracts (mussel, oyster, cockle and clam) was validated in-house using European Union (EU) Commission Decision 2002/657/EC as a guideline. The validation included the toxins okadaic acid (OA), yessotoxin (YTX), azaspiracid-1 (AZA1), pectenotoxin-2 (PTX2) and 13-desmethyl spirolide-C (SPX1). Validation was performed at 0.5, 1 and 1.5 times the current EU permitted levels, which are 160 microg kg(-1) for OA, AZA1 and PTX2 and 1,000 microg kg(-1) for YTX. For SPX1, 400 microg kg(-1) was chosen as the target level as no legislation has been established yet for this compound. The method was validated for determination in crude methanolic shellfish extracts and for extracts purified by solid-phase extraction (SPE). Extracts were also subjected to hydrolysis conditions to determine the performance of the method for OA and dinophysistoxin esters. The toxins were quantified against a set of matrix-matched standards instead of standard solutions in methanol. To save valuable standard, methanolic extract instead of the homogenate was spiked with the toxin standard. This was justified by the fact that the extraction efficiency is high for all relevant toxins (above 90%). The method performed very well with respect to accuracy, intraday precision (repeatability), interday precision (within-laboratory reproducibility), linearity, decision limit, specificity and ruggedness. At the permitted level the accuracy ranged from 102 to 111%, the repeatability from 2.6 to 6.7% and the reproducibility from 4.7 to 14.2% in crude methanolic extracts. The crude extracts performed less satisfactorily with respect to the linearity (less than 0.990) and the change in LC-MS/MS sensitivity during the series (more than 25%). SPE purification resulted in greatly improved linearity and signal stability during the series. Recently the European Food Safety Authority (EFSA) has suggested that to not exceed the acute reference dose the levels should be below 45 microg kg(-1) OA equivalents and 30 microg kg(-1) AZA1 equivalents. A single-day validation was successfully conducted at these levels. If the regulatory levels are lowered towards the EFSA suggested values, the official methods prescribed in legislation (mouse and rat bioassay) will no longer be sensitive enough. The validated LC-MS/MS method presented has the potential to replace these animal tests.

Single-laboratory validation of a refined AOAC HPLC method 2005.06 for oysters, cockles, and clams in U.K. shellfish.

In 2009, a refined HPLC method based on AOAC Official Method 2005.06 was developed and validated for the determination of paralytic shellfish poisoning (PSP) in mussels. A single-laboratory validation study of this method was undertaken here for the analysis of PSP toxins in oysters, cockles, clams, and razor clams. The method was characterized for selectivity, sensitivity, linearity, precision, repeatability, recovery, ruggedness, and uncertainty of measurement. Validation data were utilized to determine method performance characteristics for non-mussel bivalves for all commercially available certified reference toxins, extending the method to dcNEO and dcGTX2,3, where available. A period of parallel testing of oysters, cockles, and clams enabled a comparison of sample toxicities obtained using mouse bioassay (MBA) and HPLC methodologies, although only a very low number of PSP-positive samples were obtained through the United Kingdom official control monitoring program. Results from the MBA and HPLC methods were well-correlated for PSP-negative samples, but the low number of naturally contaminated PSP-positive samples has prevented any comparative statistical assessment of method performance for non-mussels between the two official methods. However, some evidence for potentially significant differences in total saxitoxin equivalents obtained by the two methods in some species has highlighted the need for further comparative testing in non-mussel samples to be conducted prior to implementation of the HPLC method in routine official control monitoring programs.

Oxidative stress responses in bivalves (Scrobicularia plana, Cerastoderma edule) from the Oued Souss estuary (Morocco).

The aim of this work was to study the responses of oxidative-stress biomarkers (catalase: CAT, thiobarbituric acid reactive substances: TBARS) in bivalves (Scrobicularia plana and Cerastoderma edule) from the Oued Souss estuary (Morocco). This estuary, subjected to large amounts of sewage discharges and industrial effluents, do not receive since November 2002 any waste outlets due to their connection to a wastewater purification plant. High CAT and TBARS values were recorded in both species collected before the implantation of wastewater treatment. These results confirm that these bivalves were submitted to various contaminants generating oxidative stress and membrane lipid peroxidation. The diminution of these two biomarkers following the setting up of the purification plant indicates probably a reduction in contaminants in the Oued Souss estuary. We recommend the use of CAT and TBARS as oxidative-stress biomarkers in S. plana and C. edule.

Comparison of amnesic, paralytic and lipophilic toxins profiles in cockle (Acanthocardia tuberculata) and smooth clam (Callista chione) from the central Adriatic Sea (Croatia).

Searching for Amnesic (ASP), Paralytic (PSP) and Lipophilic (LT) toxins in seafood is of great importance for consumer protection. Studies are usually focused on the most aquacultured species, the mussel. But, there are a number of potentially commercially important shellfish species as rough cockle Acanthocardia tuberculata (Linnaeus, 1758) and smooth clam Callista chione (Linnaeus, 1758) which are common in the Croatian Adriatic Sea. Investigation of marine biotoxins accumulation in these two species of shellfish from the Adriatic Sea has not been conducted up to now. In order to detect the potential marine biotoxin profile of A. tuberculata and C. chione wild populations, samples were taken monthly during one-year survey from the estuarine area in the central Adriatic Sea. HPLC-FLD with pre-column oxidation and HPLC-UV-DAD methods were employed for PSP and ASP toxins determination, respectively, while LTs were determined by LC-MS/MS. This research had revealed the differences in the accumulation of ASP, PSP and LT toxins between the two studied species, as Acanthocardia tuberculata showed more diverse profile with higher concentrations of analysed toxins. Both investigated shellfish species had shown levels of these biotoxins under the legal limits set by the European Commission.

Microplastic contamination and pollutant levels in mussels and cockles collected along the channel coasts.

Nowadays, environmental pollution by microplastics (<5 mm; MP) is a major issue. MP are contaminating marine organisms consumed by humans. This work studied MP contamination in two bivalve species of commercial interest: blue mussel (Mytilus edulis) and common cockle (Cerastoderma edule) sampled on the Channel coastlines (France). In parallel, 13 plastic additives and 27 hydrophobic organic compounds (HOC) were quantified in bivalves flesh using SBSE-TD-GS-MS/MS to explore a possible relationship between their concentrations and MP contamination levels. MP were extracted using a 10% potassium hydroxide digestion method then identified by µ-Raman spectroscopy. The proportion of contaminated bivalves by MP ranged from 34 to 58%. Blue mussels and common cockles exhibited 0.76 ±â€¯0.40 and 2.46 ±â€¯1.16 MP/individual and between 0.15 ±â€¯0.06 and 0.74 ±â€¯0.35 MP/g of tissue wet weight. Some HOC and plastic additives were detected in bivalves. However, no significant Pearson or Spearman correlation was found between MP loads and plastic additives or HOC concentrations in bivalve tissues for the two species.

Mineral and physichocemical characterization of cockle (Anadara granosa) shells as an alternative biomaterial for bone tissue engineering.

This study was conducted based on the hypothesis that mineral and physicochemical properties of cockle shells similarly resemble the properties of corals (Porites sp.). Hence, the mineral and physicochemical evaluations of cockle shells were conducted to support the aforementioned hypothesis. The results indicated that cockle shells and coral exoskeleton shared similar mineral and physicochemical properties.

Organic contamination in clams, Venerupis aurea laeta and Cerastoderma edule glaucum, from Sicily (Italy).

Two species of edible clams Venerupis aurea laeta and Cerastoderma edule glaucum from Ganzirri Lake (Sicily, Italy) were investigated to determine OCP and PCB residues by GC-MS. Thirty-five samples were examined for two years in order to perform: a) their actual contamination; b) the daily dietary exposure of seafood consumers to the pollutants in question. In all the clams analysed in this study, PCB and OCP (4,4'-DDE) residues are always below the limits fixed by law. It seems that there is some seasonality of concentrations observed. In fact the more high concentrations of residues are detected in the hot months, for both species and years. Given the consumption of clams and the values obtained in this study for the two species analysed, it is possible estimate that the average daily dietary intake of NDL-PCB and of 4,4'-DDE for adults is really low.

First evidence of altered vitellogenin-like protein levels in clam Tapes philippinarum and in cockle Cerastoderma glaucum from the Lagoon of Venice.

Possible xenoestrogenic effects were investigated, for the first time, in two bivalve species from the Lagoon of Venice (Italy): the clam Tapes philippinarum and the cockle Cerastoderma glaucum. Bivalves were collected far from their reproductive phase at the very early stage of gametogenesis (January), and in the pre-spawning period (June) in six sites. Vitellogenin (Vg)-like proteins (a biomarker of exposure to estrogenic compounds) were measured by the alkali-labile phosphate method (ALP), in both haemolymph and digestive gland from males and females (when it was possible to distinguish sex by microscopic observation of gonadal tissue). Haemolymph calcium concentrations (a parameter considered closely related to the presence of Vg-like proteins) and the bivalve condition index (CI) were also measured. In both seasons, bivalves collected at Campalto (near a sewage treatment plant) and Marghera (a highly contaminated area) had higher Vg-like protein levels, particularly in haemolymph, than animals from the other sampling sites. Interestingly, CI had high values in these polluted sites. In June only, Vg-like proteins and Ca(2+) levels in haemolymph exhibited similar trends in both bivalve species at most sampling sites. The responsiveness of bivalves to environmental xenoestrogens was higher in June, allowing better discrimination among sites. The present study demonstrates that animals from highly polluted areas have increased Vg-like protein levels. As endocrine disruption due to exposure to estrogenic compounds may cause fertility reduction, alterations in the sex ratio, and a decrease in reproductive rate, a condition of potential risk for bivalve populations in estuarine areas is highlighted.

Spatio-temporal variability of trace elements fingerprints in cockle (Cerastoderma edule) shells and its relevance for tracing geographic origin.

Understanding spatio-temporal variability of trace elements fingerprints (TEF) in bivalve shells is paramount to determine the discrimination power of this analytical approach and secure traceability along supply chains. Spatio-temporal variability of TEF was assessed in cockle (Cerastoderma edule) shells using inductively coupled plasma-mass spectrometry (ICP-MS). Four elemental ratios (Mg/Ca, Mn/Ca, Sr/Ca and Ba/Ca) were measured from the shells of specimens originating from eight different ecosystems along the Portuguese coast, as well as from four different areas, within one of them, over two consecutive years (2013 and 2014). TEF varied significantly in the shells of bivalves originating from the eight ecosystems surveyed in the present study. Linear discriminant function analyses assigned sampled cockles to each of the eight ecosystems with an average accuracy of 90%. Elemental ratios also displayed significant differences between the two consecutive years in the four areas monitored in the same ecosystem. Overall, while TEF displayed by cockle shells can be successfully used to trace their geographic origin, a periodical verification of TEF (>6 months and <1 year) is required to control for temporal variability whenever comparing specimens originating from the same area collected more than six months apart.

Impact of digenean parasite infection on metallothionein synthesis by the cockle (Cerastoderma edule): a multivariate field monitoring.

Metallothioneins (MT) are proteins that play an important role in metabolism of essential metals and detoxification of trace metals from living organisms. Their synthesis is induced by metal pollution but can also be exacerbated by other factors such as reproduction processes. In this context, we monitored MT concentrations in a cockle Cerastoderma edule (marine bivalve) population and highlighted the effect of a castrating digenean parasite, Labratrema minimus. In spent cockles, MT levels were low (ca. 5 nmol sites g(-1), fresh weight) but slightly higher in parasitized individuals. During gametogenesis, MT synthesis increased in all cockles, but concentrations were lower in parasitized individuals (18 against 27 nmol sites g(-1), fw in unparasitized cockles) in relation with gonad damage by parasites. Therefore, it is suggested that parasite infection in cockles can modulate MT synthesis that could consequently interfere with the response of these protective proteins in case of metal contamination.