Unravelling epigenetic mechanisms in Cerastoderma edule genome: a comparison of healthy and neoplastic cockles.

Cancer is a multifaceted genetic disease characterized by the acquisition of several essential hallmarks. Notably, certain cancers exhibit horizontal transmissibility, observed across mammalian species and diverse bivalves, the latter referred to as hemic neoplasia. Within this complex landscape, epigenetic mechanisms such as histone modifications and cytosine methylation emerge as fundamental contributors to the pathogenesis of these transmissible cancers. Our study delves into the epigenetic landscape of Cerastoderma edule, focusing on whole-genome methylation and hydroxymethylation profiles in heathy specimens and transmissible neoplasias by means of Nanopore long-read sequencing. Our results unveiled a global hypomethylation in the neoplastic specimens compared to their healthy counterparts, emphasizing the role of DNA methylation in these tumorigenic processes. Furthermore, we verified that intragenic CpG methylation positively correlated with gene expression, emphasizing its role in modulating transcription in healthy and neoplastic cockles, as also highlighted by some up-methylated oncogenic genes. Hydroxymethylation levels were significantly more elevated in the neoplastic samples, particularly within satellites and complex repeats, likely related to structural functions. Additionally, our analysis also revealed distinct methylation and activity patterns in retrotransposons, providing additional insights into bivalve neoplastic processes. Altogether, these findings contribute to understanding the epigenetic dynamics of bivalve neoplasias and shed light on the roles of DNA methylation and hydroxymethylation in tumorigenesis. Understanding these epigenetic alterations holds promise for advancing our broader understanding of cancer epigenetics.

Somatic evolution of marine transmissible leukemias in the common cockle, Cerastoderma edule.

Transmissible cancers are malignant cell lineages that spread clonally between individuals. Several such cancers, termed bivalve transmissible neoplasia (BTN), induce leukemia-like disease in marine bivalves. This is the case of BTN lineages affecting the common cockle, Cerastoderma edule, which inhabits the Atlantic coasts of Europe and northwest Africa. To investigate the evolution of cockle BTN, we collected 6,854 cockles, diagnosed 390 BTN tumors, generated a reference genome and assessed genomic variation across 61 tumors. Our analyses confirmed the existence of two BTN lineages with hemocytic origins. Mitochondrial variation revealed mitochondrial capture and host co-infection events. Mutational analyses identified lineage-specific signatures, one of which likely reflects DNA alkylation. Cytogenetic and copy number analyses uncovered pervasive genomic instability, with whole-genome duplication, oncogene amplification and alkylation-repair suppression as likely drivers. Satellite DNA distributions suggested ancient clonal origins. Our study illuminates long-term cancer evolution under the sea and reveals tolerance of extreme instability in neoplastic genomes.

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.

Genomic Survey and Resources for the Boring Giant Clam Tridacna crocea.

The boring giant clam Tridacna crocea is an evolutionary, ecologically, economically, and culturally important reef-dwelling bivalve targeted by a profitable ornamental fishery in the Indo-Pacific Ocean. In this study, we developed genomic resources for T. crocea. Using low-pass (=low-coverage, ~6×) short read sequencing, this study, for the first time, estimated the genome size, unique genome content, and nuclear repetitive elements, including the 45S rRNA DNA operon, in T. crocea. Furthermore, we tested if the mitochondrial genome can be assembled from RNA sequencing data. The haploid genome size estimated using a k-mer strategy was 1.31-1.39 Gbp, which is well within the range reported before for other members of the family Cardiidae. Unique genome content estimates using different k-mers indicated that nearly a third and probably at least 50% of the genome of T. crocea was composed of repetitive elements. A large portion of repetitive sequences could not be assigned to known repeat element families. Taking into consideration only annotated repetitive elements, the most common were classified as Satellite DNA which were more common than Class I-LINE and Class I-LTR Ty3-gypsy retrotransposon elements. The nuclear ribosomal operon in T. crocea was partially assembled into two contigs, one encoding the complete ssrDNA and 5.8S rDNA unit and a second comprising a partial lsrDNA. A nearly complete mitochondrial genome (92%) was assembled from RNA-seq. These newly developed genomic resources are highly relevant for improving our understanding of the biology of T. crocea and for the development of conservation plans and the fisheries management of this iconic reef-dwelling invertebrate.

A Useful SNP Panel to Distinguish Two Cockle Species, Cerastoderma edule and C. glaucum, Co-Occurring in Some European Beds, and Their Putative Hybrids.

Cockles are highly appreciated mollusks and provide important services in coastal areas. The two European species, edible (Cerastodermaedule) and lagoon (Cerastodermaglaucum) cockles, are not easily distinguishable, especially when young. Interestingly, the species show different resistance to Marteilia cochillia, the parasite responsible for marteiliosis outbreaks, which is devastating cockle production in some areas. C.edule is severely affected by the parasite, while C. glaucum seems to be resistant, although underlying reasons are still unknown. Hybrids between both species might be interesting to introgress allelic variants responsible for tolerance, either naturally or through artificial selection, from lagoon into edible cockle. Here, we used 2b restriction site-associated DNA sequencing (2b-RAD) to identify single nucleotide polymorphisms (SNP) diagnostic for cockle discrimination (fixed for alternative allelic variants). Among the nine diagnostic SNPs selected, seven were validated using a SNaPshot assay in samples covering most of the distribution range of both species. The validated SNPs were used to check cockles that were suggested to be hybrids by a claimed diagnostic tool based on the internal transcribed spacers of the ribosomal RNA. Although these were shown to be false positives, we cannot rule out the fact that hybrids can occur and be viable. The SNP tool here developed will be valuable for their identification and management.

Resolving the relationships of clams and cockles: dense transcriptome sampling drastically improves the bivalve tree of life.

Bivalvia has been the subject of extensive recent phylogenetic work to attempt resolving either the backbone of the bivalve tree using transcriptomic data, or the tips using morpho-anatomical data and up to five genetic markers. Yet the first approach lacked decisive taxon sampling and the second failed to resolve many interfamilial relationships, especially within the diverse clade Imparidentia. Here we combine dense taxon sampling with 108 deep-sequenced Illumina-based transcriptomes to provide resolution in nodes that required additional study. We designed specific data matrices to address the poorly resolved relationships within Imparidentia. Our results support the overall backbone of the bivalve tree, the monophyly of Bivalvia and all its main nodes, although the monophyly of Protobranchia remains less clear. Likewise, the inter-relationships of the six main bivalve clades were fully supported. Within Imparidentia, resolution increases when analysing Imparidentia-specific matrices. Lucinidae, Thyasiridae and Gastrochaenida represent three early branches. Gastrochaenida is sister group to all remaining imparidentians, which divide into six orders. Neoheterodontei is always fully supported, and consists of Sphaeriida, Myida and Venerida, with the latter now also containing Mactroidea, Ungulinoidea and Chamidae, a family particularly difficult to place in earlier work. Overall, our study, by using densely sampled transcriptomes, provides the best-resolved bivalve phylogeny to date.

Effects of metal contamination on the gene expression profile of two benthic species: Cerastoderma edule and Ruditapes philippinarum.

This study aimed to identify new biomarkers for metal exposure in two bivalve species. Suppressive Subtractive Hybridization (SSH) was employed to evaluate the transcriptomic response of Cerastoderma edule and Ruditapes philippinarum to metal pollution. Protein synthesis and catalytic activity were the most affected metabolic processes in C. edule and R. philippinarum, respectively. Also, different genes responded to the effect of contamination in each species. The different response observed in both species reinforces the importance of including more than one bioindicator species in risk assessment studies. These results provide the basis for new studies, which are necessary for further validation of the use of the identified genes as molecular biomarkers for metal exposure.

Molecular phylogenetics and historical biogeography amid shifting continents in the cockles and giant clams (Bivalvia: Cardiidae).

Reconstructing historical biogeography of the marine realm is complicated by indistinct barriers and, over deeper time scales, a dynamic landscape shaped by plate tectonics. Here we present the most extensive examination of model-based historical biogeography among marine invertebrates to date. We conducted the largest phylogenetic and molecular clock analyses to date for the bivalve family Cardiidae (cockles and giant clams) with three unlinked loci for 110 species representing 37 of the 50 genera. Ancestral ranges were reconstructed using the dispersal-extinction-cladogenesis (DEC) method with a time-stratified paleogeographic model wherein dispersal rates varied with shifting tectonics. Results were compared to previous classifications and the extensive paleontological record. Six of the eight prior subfamily groupings were found to be para- or polyphyletic. Cardiidae originated and subsequently diversified in the tropical Indo-Pacific starting in the Late Triassic. Eastern Atlantic species were mainly derived from the tropical Indo-Mediterranean region via the Tethys Sea. In contrast, the western Atlantic fauna was derived from Indo-Pacific clades. Our phylogenetic results demonstrated greater concordance with geography than did previous phylogenies based on morphology. Time-stratifying the DEC reconstruction improved the fit and was highly consistent with paleo-ocean currents and paleogeography. Lastly, combining molecular phylogenetics with a rich and well-documented fossil record allowed us to test the accuracy and precision of biogeographic range reconstructions.

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.

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.

A novel widespread cryptic species and phylogeographic patterns within several giant clam species (Cardiidae: Tridacna) from the Indo-Pacific Ocean.

Giant clams (genus Tridacna) are iconic coral reef animals of the Indian and Pacific Oceans, easily recognizable by their massive shells and vibrantly colored mantle tissue. Most Tridacna species are listed by CITES and the IUCN Redlist, as their populations have been extensively harvested and depleted in many regions. Here, we survey Tridacna crocea and Tridacna maxima from the eastern Indian and western Pacific Oceans for mitochondrial (COI and 16S) and nuclear (ITS) sequence variation and consolidate these data with previous published results using phylogenetic analyses. We find deep intraspecific differentiation within both T. crocea and T. maxima. In T. crocea we describe a previously undocumented phylogeographic division to the east of Cenderawasih Bay (northwest New Guinea), whereas for T. maxima the previously described, distinctive lineage of Cenderawasih Bay can be seen to also typify western Pacific populations. Furthermore, we find an undescribed, monophyletic group that is evolutionarily distinct from named Tridacna species at both mitochondrial and nuclear loci. This cryptic taxon is geographically widespread with a range extent that minimally includes much of the central Indo-Pacific region. Our results reinforce the emerging paradigm that cryptic species are common among marine invertebrates, even for conspicuous and culturally significant taxa. Additionally, our results add to identified locations of genetic differentiation across the central Indo-Pacific and highlight how phylogeographic patterns may differ even between closely related and co-distributed species.

Biomarkers and transcription levels of cancer-related genes in cockles Cerastoderma edule from Galicia (NW Spain) with disseminated neoplasia.

Disseminated neoplasia (DN) is a pathological condition reported for several species of marine bivalves throughout the world, but its aetiology has not yet been satisfactorily explained. It has been suggested that chemical contamination could be a factor contributing to neoplasia. The aim of the present study was to compare cell and tissue biomarkers and the transcription level of cancer-related genes in cockles (Cerastoderma edule) affected by DN with those of healthy cockles in relation to chemical contaminant burdens. For this, cockles were collected from a natural bed in Cambados (Ria de Arousa, Galicia) in May 2009. The prevalence of DN was 12.36% and 3 degrees of DN severity were distinguished. No significant differences in metal accumulation, non-specific inflammatory responses and parasites were observed between healthy and DN-affected cockles. Lysosomal membrane stability was significantly reduced in cockles affected by DN, which indicates a poorer health condition. Very low frequencies of micronuclei were recorded and no significant differences were detected between DN severity groups. Haemolymph analyses showed a higher frequency of mitotic figures and binucleated cells in cockles affected by moderate and heavy DN than in healthy ones. Neoplastic animals showed significantly higher transcription levels of p53 and ras than healthy cockles and mutational alterations in ras gene sequence were detected. Low concentrations of metals, polycyclic aromatic hydrocarbons, polychlorinated biphenyls and phthalate esters were measured in cockles from Cambados. In conclusion, cockles affected by DN suffer a general stress situation and have altered patterns of cancer-related gene transcription. Further studies are in progress to elucidate mechanisms of carcinogenesis in this species.

Cross-habitat interactions among bivalve species control community structure on intertidal flats.

Increasing evidence shows that spatial interactions between sedentary organisms can structure communities and promote landscape complexity in many ecosystems. Here we tested the hypothesis that reef-forming mussels (Mytilus edulis L.), a dominant intertidal ecosystem engineer in the Wadden Sea, promote abundances of the burrowing bivalve Cerastoderma edule L. (cockle) in neighboring habitats at relatively long distances coastward from mussel beds. Field surveys within and around three mussel beds showed a peak in cockle densities at 50-100 m toward the coast from the mussel bed, while cockle abundances elsewhere in the study area were very low. Field transplantation of cockles showed higher survival of young cockles (2-3 years old) and increased spat fall coastward of the mussel bed compared to within the bed and to areas without mussels, whereas growth decreased within and coastward of the mussel bed. Our measurements suggest that the observed spatial patterns in cockle numbers resulted from (1) inhibition effects by the mussels close to the beds due to preemptive algal depletion and deteriorated sediment conditions and (2) facilitation effects by the mussels farther away from the beds due to reduction of wave energy. Our results imply that these spatial, scale-dependent interactions between reef-forming ecosystem engineers and surrounding communities of sedentary benthic organisms can be an important determinant of the large-scale community structure in intertidal ecosystems. Understanding this interplay between neighboring communities of sedentary species is therefore essential for effective conservation and restoration of soft-bottom intertidal communities.

Disseminated neoplasia causes changes in ploidy and apoptosis frequency in cockles Cerastoderma edule.

A proliferative disease, usually referred as disseminated neoplasia (DN), shows high prevalence in some cockle Cerastoderma edule beds of Galicia (NW Spain). Chromosome counts, examination of chromosome morphology, DNA quantification by flow cytometry and estimation of apoptosis frequency by TUNEL assay and flow cytometry were performed in cockles with different DN severity. Metaphases obtained from gills of DN-affected cockles displayed a chromosome number ranging from 41 to 145, while normal number is 38; changes in chromosome morphology were also evident, with numerous microchromosomes occurring. Haemolymph flow cytometry analysis revealed difference in DNA content between healthy and DN-affected cockles. Aneuploid peaks ranged from 1.3n to 8.9n. Apoptosis frequency was determined on histological sections (TUNEL assay) and haemolymph samples (flow cytometry). Both techniques revealed neoplastic cells in apoptosis. The higher DN severity, the lower the percentage of apoptotic cells. According to flow cytometry results, the negative association between DN severity and apoptosis frequency only affected the neoplastic cells, whereas DN did not significantly affect the percentage of apoptotic hyalinocytes or apoptotic granulocytes.

Cloning, characterization and gene expression of a metallothionein isoform in the edible cockle Cerastoderma edule after cadmium or mercury exposure.

Metallothionein (MT) genes encode crucial metal-binding proteins ubiquitously expressed in living organisms and which play important roles in homeostasis of essential metals and detoxification processes. Here, the molecular organization of the first metallothionein gene of the edible cockle Cerastoderma edule and its expression after cadmium (Cd) or mercury (Hg) exposures were determined. The resulting sequence (Cemt1) exhibits unusual features. The full length cDNA encodes a protein of 73 amino acids with nine classical Cys-X((1-3))-Cys motifs, but also one Cys-Cys not generally found in molluscan MT. Moreover, characterization of the molecular organization of the Cemt1 gene revealed two different alleles (A1 and A2) with length differences due to large deletion events in their intronic sequences involving direct Short Interspersed repeated Elements (SINE), while their exonic sequences were identical. To our knowledge, such large excision mechanisms have never before been reported in a bivalve gene sequence. After 10 days of Cd exposure at environmentally relevant doses, quantitative real-time PCR revealed a strong induction of Cemt1 in gills of C. edule. Surprisingly, neither induction of the Cemt1 gene nor of MT protein was shown after Hg exposure, despite the fact that this organism is able to bioaccumulate a high amount of this trace metal which is theoretically one of the most powerful inducers of MT biosynthesis.

Phylogenetic study and barcoding of the blood cockle, Tegillarca granosa, found on the west coast of peninsular Malaysia using the COI gene.

Blood cockles are among the most economically important brackish water invertebrates found in Malaysia. However, our knowledge of blood cockle phylogeny and systematics is rudimentary, especially for the species Tegillarca granosa. It is unclear, for instance, whether the cockles occurring on the west coast of peninsular Malaysia constitute a single species, or multiple, phylogenetically distinct species. We performed the first DNA molecular phylogenetic analysis of T. granosa to distinguish it from other related species found in other parts of the world and to create a DNA database for the species. An approximately 585-nucleotide fragment of the mitochondrial DNA (cytochrome oxidase I, COI) was sequenced for 150 individual cockles, representing 10 populations: three from the north, four from the central part and three from the southern part of peninsular Malaysia. Phylogenetic analyses of the resulting dataset yielded tree topologies that not only showed the relationship between T. granosa and its closest relatives but its position in the evolutionary tree. Three mitochondrial clades were evident, each containing an individual genus. Using the mutation rate of the COI gene, the divergence time between T. granosa and its closest related species was estimated to be 460 thousand years ago. This study provides a phylogenetic framework for this ecologically prominent and commercially important cockle species.

Utilization of molecular markers for the conservation of blood cockles, Anadara granosa (Arcidae).

We examined genetic variation in blood cockles in an effort to obtain information useful for the sustainability, management, and the stability of this species as a major commodity in the fisheries sector. Ten populations of cockles were sampled from the north to the south of the west coast of peninsular Malaysia. The cockles were collected in collaboration with the Fisheries Research Institute, Penang. The population genetic analysis of the cockles were studied via RAPD-PCR and mtDNA sequencing. Three hundred individuals were analyzed with RAPD-PCR experiments. High gene diversity over all loci was observed (Shannon index = 0.549 ± 0.056 and Nei's gene diversity = 0.4852 ± 0.0430 among 35 loci). The second method, mtDNA sequencing, was employed to complement the information obtained from RAPD-PCR. The gene selected for mtDNA sequencing was cytochrome c oxidase I (COI). One hundred and fifty individuals were sequenced, yielding a partial gene of 585 bp. Statistical analysis showed homogeneity in general but did reveal some degree of variability between the populations in Johor and the rest of the populations. The Mantel test showed a positive but nonsignificant correlation between geographic and genetic distances (r = 0.2710, P = 0.622), as in the RAPD analysis. We propose that the homogeneity between distant populations is caused by two factors: 1) the translocation of the spats; 2) larvae are carried by current movement from the north of the peninsula to the south. The different genetic composition found in Johor could be due to pollution, mutagenic substances or physical factors such as the depth of the water column. This population genetic study is the first for this species in peninsular Malaysia. The data from this study have important implications for fishery management, conservation of blood cockles and translocation policies for aquaculture and stock enhancement programs.

Isolation and characterization of the first microsatellite markers for the endangered relict mussel Hypanis colorata (Mollusca: Bivalvia: Cardiidae).

Hypanis colorata (Eichwald, 1829) (Cardiidae: Lymnocardiinae) is a bivalve relict species with a Ponto-Caspian distribution and is under strict protection in Romania, according to national regulations. While the species is depressed in the western Black Sea lagoons from Romania and Ukraine, it is also a successful invader in the middle Dniepr and Volga regions. Establishing a conservation strategy for this species or studying its invasion process requires knowledge about the genetic structure of the species populations. We have isolated and characterized nine polymorphic microsatellite markers in H. colorata. The number of alleles per locus ranged from 4 to 28 and the observed heterozygosity ranged from 0.613 to 1.000. The microsatellites developed in the present study are highly polymorphic and they should be useful for the assessment of genetic variation within this species.

Morphological and molecular studies on life cycle stages of Diphtherostomum brusinae (Digenea: Zoogonidae) from northern Portugal.

Diphtherostomum brusinae was first recorded by the present study in the north of Portugal. Sporocysts, containing cercariae and encysted metacercariae, were observed in the gonads and digestive gland of the gastropod Nassarius reticulatus. Metacercariae were also found infecting the foot, mantle border and gills of the cockle Cerastoderma edule. The adult form was lodged in the rectum of the definitive host Diplodus sargus. The morphology of the three parasitic stages was studied by light (LM) and scanning electron microscopy (SEM). Despite the close similarity between cercaria and metacercaria, SEM data provided information that allowed their differentiation, namely the presence of a dense crown of microvilli around the oral cavity of the cercariae, which was absent in the metacercariae. In addition, the metacercariae presented a specific pre-acetabular rectangular band with conspicuous triangular spines. The adult showed characteristics of D. brusinae species, in particular the presence of acetabular lips, compact vitellaria and large elliptical eggs. Sequenced ITS1 data clearly demonstrated that the cercariae and metacercarial cysts from N. reticulatus, the cysts from C. edule and the adult isolated from D. sargus were life cycle stages that belonged to the same species, i.e. D. brusinae. Two transmission strategies in the life cycle of this species were observed: (1) cercariae encyst within the sporocysts of N. reticulatus and await ingestion by the definitive host; and (2) N. reticulatus naturally emits cercariae; they encyst in C. edule or the environment and are ingested by the definitive host.

An evaluation of the relative sensitivity of two marine bivalve mollusc species using the Comet assay.

The aim of this study was to (a) evaluate the potential for the 'Comet assay' to be used as a method for detecting genetic damage in the common cockle Cerastoderma edule; and (b) to compare the relative sensitivity with Mytilus edulis as the bivalve widely used as a sentinel species in biomonitoring studies. In vitro validation studies were carried out on haemocytes from each species using hydrogen peroxide (H2O2), a known oxidant and the induced DNA damage was measured using the Comet assay. On exposure to 0, 100, 500 and 1000 microM H2O2, a significant concentration-dependent increase was observed in both species. Use of an additional concentration of 5000 microM H2O2 showed that while DNA damage could be assessed in M. edulis at this concentration, only a few cells from C. edule were amenable to measurements owing to extensive DNA damage ("hedgehog cells"). The evidence also suggested that the cells from C. edule are more sensitive to oxidative damage induced by H2O2 when compared with M. edulis. Bearing in mind that sediments are the ultimate sink for many contaminants, this study demonstrates the potential application of sediment-dwelling C. edule as a useful biomonitoring species.