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.

Two branchial pathological conditions, nuclear hypertrophy and abnormal epithelial proliferation, in Mya arenaria from northwest Russian coasts.

Histopathological analysis of soft-shell clams Mya arenaria collected from 2 northwest Russian locations disclosed high prevalence of 2 pathological gill conditions. One involved the occurrence of more or less extended gill areas in which the branchial filaments showed hyperchromatic (basophilic) epithelium with some hypertrophied nuclei, which were considered presumptive signs of viral infection. Another pathological condition involved abnormal proliferation of the branchial epithelium, which lost the main differential features of the normal branchial epithelium (ciliated and simple cell layer structure), becoming non-ciliated, pseudostratified or stratified hyperchromatic epithelium with abundant mitotic figures and frequent apoptotic cells. The most complex cases involved loss of the normal branchial filament architecture, which was replaced with tumour-like growths consisting of branching, convoluted epithelial projections with a connective stroma. Images suggesting migration (invasion) of cells from the abnormally proliferating epithelium to the subjacent connective tissue, which would involve malignancy, were observed in one individual. The occurrence of both pathological conditions in clams from both locations and their co-occurrence in one clam suggest the possibility of a common, possibly viral, aetiology. Furthermore, the high prevalence of the abnormal proliferative disorder in non-polluted areas suggests an infectious aetiology. Additional studies are needed to assess a viral aetiology for the nuclear hypertrophy and/or the abnormal epithelial proliferation as well as the malignancy of the latter condition.

The first high-density genetic map of common cockle (Cerastoderma edule) reveals a major QTL controlling shell color variation.

Shell color shows broad variation within mollusc species and despite information on the genetic pathways involved in shell construction and color has recently increased, more studies are needed to understand its genetic architecture. The common cockle (Cerastoderma edule) is a valuable species from ecological and commercial perspectives which shows important variation in shell color across Northeast Atlantic. In this study, we constructed a high-density genetic map, as a tool for screening common cockle genome, which was applied to ascertain the genetic basis of color variation in the species. The consensus genetic map comprised 19 linkage groups (LGs) in accordance with the cockle karyotype (2n = 38) and spanned 1073 cM, including 730 markers per LG and an inter-marker distance of 0.13 cM. Five full-sib families showing segregation for several color-associated traits were used for a genome-wide association study and a major QTL on chromosome 13 associated to different color-traits was detected. Mining on this genomic region revealed several candidate genes related to shell construction and color. A genomic region previously reported associated with divergent selection in cockle distribution overlapped with this QTL suggesting its putative role on adaptation.

Mitochondrial genome sequencing of marine leukaemias reveals cancer contagion between clam species in the Seas of Southern Europe.

Clonally transmissible cancers are tumour lineages that are transmitted between individuals via the transfer of living cancer cells. In marine bivalves, leukaemia-like transmissible cancers, called hemic neoplasia (HN), have demonstrated the ability to infect individuals from different species. We performed whole-genome sequencing in eight warty venus clams that were diagnosed with HN, from two sampling points located more than 1000 nautical miles away in the Atlantic Ocean and the Mediterranean Sea Coasts of Spain. Mitochondrial genome sequencing analysis from neoplastic animals revealed the coexistence of haplotypes from two different clam species. Phylogenies estimated from mitochondrial and nuclear markers confirmed this leukaemia originated in striped venus clams and later transmitted to clams of the species warty venus, in which it survives as a contagious cancer. The analysis of mitochondrial and nuclear gene sequences supports all studied tumours belong to a single neoplastic lineage that spreads in the Seas of Southern Europe.

In humans and other animals, cancer cells divide excessively, forming tumours or flooding the blood, but they rarely spread to other individuals. However, some animals, including dogs, Tasmanian devils and bivalve molluscs like clams, cockles and mussels, can develop cancers that are transmitted from one individual to another. Despite these cancers being contagious, each one originates in a single animal, meaning that even when the cancer has spread to many individuals, its origins can be traced through its DNA. Cancer contagion is rare, but transmissible cancers seem to be particularly common in the oceans. In fact, 7 types of contagious cancer have been described in bivalve species so far. These cancers are known as 'hemic neoplasias', and are characterized by the uncontrolled division of blood-like cells, which can be released by the host they developed in, and survive in ocean water. When these cells encounter individuals from the same species, they can infect them, causing them to develop hemic neoplasia too There are still many unanswered questions about contagious cancers in bivalves. For example, how many species do the cancers affect, and which species do the cancers originate in? To address these questions, Garcia-Souto, Bruzos, Díaz et al. gathered over 400 specimens of a species of clam called the warty venus clam from the coastlines of Europe and examined them for signs of cancer. Clams collected in two regions of Spain showed signs of hemic neoplasia: one of the populations was from the Balearic Islands in the Mediterranean Sea, while the other came from the Atlantic coast of northwestern Spain. Analyzing the genomes of the tumours from each population showed that the cancer cells from both regions had likely originated in the same animal, indicating that the cancer is contagious and had spread through different populations. The analysis also revealed that the cancer did not originally develop in warty venus clams: the cancer cells contained DNA from both warty venus clams and another species called striped venus clams. These two species live close together in the Mediterranean Sea, suggesting that the cancer started in a striped venus clam and then spread to a warty venus clam. To determine whether the cancer still affected both species, Garcia-Souto, Bruzos, Díaz et al. screened 200 striped venus clams from the same areas, but no signs of cancer were found in these clams. This suggests that currently the cancer only affects the warty venus clam. These findings confirm that contagious cancers can jump between clam species, which could be threat to the marine environment. The fact that the cancer was so similar in clams from the Atlantic coast and from the Mediterranean Sea, however, suggests that it may have emerged very recently, or that human activity helped it to spread from one place to another. If the latter is the case, it may be possible to prevent further spread of these sea-borne cancers through human intervention.

Assessing the elemental fingerprints of cockle shells (Cerastoderma edule) to confirm their geographic origin from regional to international spatial scales.

Geographic origin is directly linked to the quality and commercial value of bivalves. The globalization of the seafood trade and the increasing number of fraudulent practices in the bivalves industry has prompted consumers to become increasingly aware on the geographic origin of the seafood they consume. To enhance consumers' confidence and allow authorities to effectively enforce regulations and contain risks that threaten public health, fast and accurate tools must be made available to confirm claims along the trade chain on the geographic origin of bivalves. In the present study the efficiency of using the elemental fingerprints of a small-homogenized subsample of the shell of common cockles (Cerastoderma edule) to confirm their harvesting location is evaluated at different spatial scales: i) regional (along the Galician coast (Spain) - Espasante, Barallobre, Rio Anllóns, Camariñas, Muros, Noia, Carril, Grove, Combarro, Placeres, Moaña, and Baiona), ii) national (along the Portuguese coast - Ria de Aveiro, Óbidos lagoon, Tagus estuary, Sado estuary and Ria Formosa), and iii) international (along the Northeast Atlantic coast - Hejeltefjorden (Norway), Nykobing Mors (Denmark), Sylt (Germany), Slikken van Viane (Netherlands), Roscoff (France), Plymouth (England), Swansea (Wales), Ria de Aveiro (Portugal) and Oualidia (Morocco). Results confirm that elemental fingerprints of bivalve shells are significantly different among locations and that they can be successfully used with high accuracy to discriminate the geographic origin of cockles at all spatial scales surveyed (97.2% at regional scale, 99.3% at national scale and 100% at international scale). Overall, elemental fingerprints of a small-homogenized subsample of the shell showed to be a replicable, low cost and fast tool to reliably trace the place of origin of cockles sampled at different spatial scales, with success rate of discrimination directly increasing with distance between collection sites.

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.

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.

Disseminated neoplasia in clams Venerupis aurea from Galicia (NW Spain): histopathology, ultrastructure and ploidy of the neoplastic cells, and comparison of diagnostic procedures.

This study reports evidence of a neoplastic disorder in the clam Venerupis aurea. In the first stage of the disease, masses of neoplastic cells were mainly observed in connective tissue of gills. These masses of neoplastic cells appeared more compact than in cases of disseminated neoplasia of other bivalve molluscs. As disease progresses, masses of abnormal cells were also observed in the connective tissue of all organs, thus alteration of vital functions in late stage is likely. The neoplastic cells had aneuploid DNA content, ranging from 1.6n to 7.8 n. The ploidy level increased with the severity of the disease. A comparison of three light microscopy diagnostic procedures was performed. Histology was the most sensitive diagnostic test, whereas two different haematological techniques assayed presented unsatisfactory low levels of sensitivity. Specificity was high for the three assayed tests.

Disseminated neoplasia in cockles Cerastoderma edule: ultrastructural characterisation and effects on haemolymph cell parameters.

Disseminated neoplasia (DN) has been detected in cockles from various beds in Galicia (NW Spain). A study was performed to characterise cockle neoplastic cell ultrastructure and to evaluate the effect of this disease at different severity stages on various haemolymph cell parameters. Examination of cockle neoplastic cells with transmission electron microscopy (TEM) showed round shapes and a lack of pseudopods, a high nucleus:cytoplasm diameter ratio, Golgi complexes, abundant mitochondria, ribosomes, and numerous endoplasmic reticulum tubes and electron-lucent vesicles. Various haemolymph cell parameters (cell mortality, non-specific esterase and lysosome biovolume, reactive oxygen intermediates [ROI] production, phagocytosis ability, intracellular Ca2+ and actin levels) were compared between DN severity categories by flow cytometry; haemocyte mortality, non-specific esterase activities and lysosome biovolume were found to be higher with increasing DN severity. The phagocytic ability of neoplastic cells was sharply reduced with regard to haemocytes. The cytoplasmic-free Ca2+ level was higher and actin content lower in haemolymph cells of diseased cockles compared to unaffected ones. A significant increase in ROI production was detected in later stages of disease progression.

Evidence of retroviral etiology for disseminated neoplasia in cockles (Cerastoderma edule).

Epizootiologic outbreaks of disseminated neoplasia have been reported in association with massive mortalities of various bivalve species. In cockles, Cerastoderma edule, this pathological condition was described in Ireland and France. Since 1997, different populations affected by this pathology have been detected in Galicia (NW Spain). Transmission electron microscopy allowed the visualization of virus-like particles in neoplastic cells, resembling a retrovirus-like agent. To confirm this hypothesis, we used a commercial kit for detection and quantification of reverse transcriptase (RT) activity, based on the use of bromo-deoxyuridine triphosphate (BrdUTP) and a BrdU binding antibody conjugated to alkaline phosphatase. In addition, we developed a product-enhanced RT assay using RNA of hepatitis A virus as a template. These two assays showed positive RT activity in 90.9 and 81.8% of samples, respectively, from cockles displaying disseminated neoplasia as determined by light microscopy. These results strongly support the hypothesis of retroviral etiology for this pathological condition.

Urosporidium sp. hyperparasite of the turbellarian Paravortex cardii in the cockle Cerastoderma edule.

Cysts with spores showing different degree of maturity and a single plasmodium were observed in the connective tissue of the turbellarian Paravortex cardii located in the digestive lumen of the cockle Cerastoderma edule. The study of spore morphology by transmission electron microscopy revealed that they correspond to an haplosporidian belonging to the genus Urosporidium. Spore ornaments were similar to those described from Urosporidium spisuli, infecting a nematode parasite of the Atlantic surf clam, Spisula solidissima.