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Chromosomal, copy number and structural variation in cockle BTN. A, FISH of telomeric peptide nucleic acid probes (TEL, shown in green) onto healthy (left) and representative type A (centre) and type B neoplastic metaphase spreads. All chromosomes, including the smallest neoplastic chromosomes, hold telomeric signals on all chromatid ends. Scale bars, 10 µm. B, CN profiles of representative healthy, CedBTN1 and CedBTN2 samples. Grey dots represent estimates of unrounded CN for 10-kb windows along the reference genome (x-axis); blue segments indicate inferred segments of integer CN. Distributions of unrounded CN are shown on the right margin. C, Phylogenetic tree inferred from BTN-specific SVs. The number of SVs per branch is indicated, and branches corresponding to sets of ancestral or pre-divergence variants (A0, A1, A2) are labelled. Bootstrap support values (n=1000 replicates) are ≥99.9 for all nodes except that marked with symbol Ä (91.6). D, Copy number profiles in a 500-kb region around the MGMT gene locus in healthy (n=3) and CedBTN1 (n=7) samples. Each sample is represented by a line. The highest CedBTN1 CN estimate at the gene locus (CN=0.4) corresponds to sample EICE18/889H. E, Numbers of sequence reads aligning to five satellite DNA elements identified in a diverse set of healthy cockles (n=30) and CedBTN1 (n=7) and CedBTN2 (n=3) tumours. Each dot represents a sample. Boxes represent first and third quartiles; middle line within each box denotes the median; whiskers indicate values within 1.5× interquartile range from the first and third quartiles. Monomer size is provided for each satellite. F, FISH of DNA probe for satellite CeS4 (red) onto representative metaphases of healthy (left) and neoplastic specimens. Scale bars, 10 µm; DAPI, 4′,6-diamidino-2-phenylindole.
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Transmissible cancers are malignant cell clones that spread among individuals through transfer of living cancer cells. Several such cancers, collectively known as bivalve transmissible neoplasia (BTN), are known to infect and cause leukaemia in marine bivalve molluscs. This is the case of BTN clones affecting the common cockle, Cerastoderma edule ,...
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... (Table S3). Gene annotation resulted in a 42-Mb exome with 14,055 protein-coding genes. While this protein-coding exome constitutes 5.3% of the total nuclear genome size, repetitive sequences comprise 46.2% of the genome, with long interspersed nuclear elements (LINEs) being the most frequent type of transposable element among annotated repeats ( Fig. S4; Table ...
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... of changes in mtDNA VAF across different tissues of the same animal revealed three cases in which two CedBTN mtDNA lineages coexisted within the same host (Fig. S14). In one remarkable animal (EICE18/910), VAF analysis revealed the presence of mtDNA haplotypes from both CedBTN1 and CedBTN2 lineages (Fig. 2E), with co-infection by both clones being confirmed through histopathological identification of cell morphologies matching DN types A and B (Fig. ...
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... within individual tumours. For instance, neoplastic metaphase spreads from sample PACE17/478H contained 11-354 chromosomes of variable size and structure. Fluorescent in situ hybridization (FISH) probes targeting telomeric sequences showed that, despite such karyotypic plasticity, all the chromosomes in CedBTN cells present a canonical structure (Fig. 4A). These results suggest that the shifting karyotypes of CedBTN are probably the outcome of extensive chromosomal reorganization and frequent chromosome mis-segregation during anaphase. It therefore seems likely that a fraction of cell divisions may produce inviable cells in these ...
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... we inferred copy number (CN) profiles from whole-genome sequencing data for each tumour in our 'golden set'. The profiles were marked by a ubiquitous pattern of highly complex CN alterations along every reference chromosome, with lower CN levels visibly underrepresented (Fig. 4B). CN distributions were consistent with a modal CN of 4n, suggestive of ancestral tetraploidy, except for one tumour (UGCE17/2401H) presenting a modal CN of 5n. Profiles were loosely conserved across tumours from each lineage, with a combination of shared and sample-specific CN features (Fig. ...
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... (6,916 in CedBTN1, 10,925 in CedBTN2), with deletions being the most frequent type of event (80%, 14,589/18,272; Fig. S17). A maximum-parsimony phylogenetic tree reconstructed from these variants confirmed the CedBTN nuclear phylogeny inferred from SNVs, supporting two divergent lineages with a minimal fraction of shared structural variants (Fig. ...
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... encoding a cyclin that promotes the G1/S cell cycle transition ( Table S13). Recurrent amplification of these genes has been observed in multiple cancer types, and is thought to prevent cell cycle arrest and apoptosis under conditions of genomic instability (42-45). Notably, we also identified an ancestral homozygous deletion of MGMT in CedBTN1 ( Fig. 4D; Table S13). The enzyme encoded by this gene, O 6 -methylguanine-DNA methyltransferase, is essential for repair of alkylated DNA bases, and its inactivation results in hypersensitivity to the toxic and mutagenic effects of alkylating agents (46)(47)(48). Given the cumulative and virtually lineage-specific activity of signature SBS-D (Fig. 3D), ...
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... relevant for genome stability, exhibiting long-term conservation and propensity for rapid copy number changes (49). Our method identified 34 satellite DNA candidates in the common cockle reference genome (Table S14), four of which varied in frequency between non-neoplastic and BTN genomes, providing further insights into the origins of cockle BTN (Fig. 4E). Two satellites, named CeS4 and CeS14, were found at high frequency in all samples from a genetically diverse cohort of non-neoplastic cockles, yet were entirely absent from both BTN clones. We designed FISH probes to target satellite CeS4, which confirmed the results obtained from sequencing data (Fig. 4F). This finding suggests that ...
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... insights into the origins of cockle BTN (Fig. 4E). Two satellites, named CeS4 and CeS14, were found at high frequency in all samples from a genetically diverse cohort of non-neoplastic cockles, yet were entirely absent from both BTN clones. We designed FISH probes to target satellite CeS4, which confirmed the results obtained from sequencing data (Fig. 4F). This finding suggests that both CedBTN1 and CedBTN2 may be ancient cancer lineages that diverged from the cockle population before the emergence and expansion of CeS4 and CeS14 in the C. edule germ line. Another satellite, CeS6, was found in cockle populations and CedBTN2 samples, while being absent from CedBTN1 (Fig. ...
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... from sequencing data (Fig. 4F). This finding suggests that both CedBTN1 and CedBTN2 may be ancient cancer lineages that diverged from the cockle population before the emergence and expansion of CeS4 and CeS14 in the C. edule germ line. Another satellite, CeS6, was found in cockle populations and CedBTN2 samples, while being absent from CedBTN1 (Fig. ...
Citations
... Bowtie 1.1.2 (Langmead et al. 2009) was used to align reads to the cockle's genome (Bruzos et al. 2022) allowing a maximum of three mismatches and a unique valid alignment (-v 3 -m 1). The reference-based mode with default parameters in the gstacks module of STACKS 2.0 (Catchen et al. 2013) was used for SNP calling. ...
... RAD-tags including divergent outlier SNPs were mapped in the C. edule genome (Bruzos et al. 2022) and their position compared with the consistent genomic windows under divergent selection previously reported by Vera et al. (2022) in the Northeast Atlantic Ocean. The very low genetic differentiation with neutral markers in the studied areas precluded the detection of consistent genomic regions under stabilising selection. ...
Knowledge of genetic structure at the finest level is essential for the conservation of genetic resources. Despite no visible barriers limiting gene flow, significant genetic structure has been shown in marine species. The common cockle (Cerastoderma edule) is a bivalve of great commercial and ecological value inhabiting the Northeast Atlantic Ocean. Previous population genomics studies demonstrated significant structure both across the Northeast Atlantic, but also within small geographic areas, highlighting the need to investigate fine-scale structuring. Here, we analysed two geographic areas that could represent opposite models of structure for the species: (1) the SW British Isles region, highly fragmented due to biogeographic barriers, and (2) Galicia (NW Spain), a putative homogeneous region. A total of 9250 SNPs genotyped by 2b-RAD on 599 individuals from 22 natural beds were used for the analysis. The entire SNP dataset mostly confirmed previous observations related to genetic diversity and differentiation; however, neutral and divergent SNP outlier datasets enabled disentangling physical barriers from abiotic environmental factors structuring both regions. While Galicia showed a homogeneous structure, the SW British Isles region was split into four reliable genetic regions related to oceanographic features and abiotic factors, such as sea surface salinity and temperature. The information gathered supports specific management policies of cockle resources in SW British and Galician regions also considering their particular socioeconomic characteristics; further, these new data will be added to those recently reported in the Northeast Atlantic to define sustainable management actions across the whole distribution range of the species. Heredity; https://doi.
... Genomic resources of common cockle have recently increased in the framework of the COCKLES Interreg (EAPA_458/2016) and the Scuba Cancers (ERC-2016-STG) projects, which ensured a robust genetic baseline for that purpose. A population genomics approach using 2b-RADseq along with the chromosome-level genome assembly of the species (Bruzos et al., 2022) was applied to disentangle the demographic and environmental factors underlying the common cockle structure in the Northeast Atlantic (Vera et al., 2022). Furthermore, RNAseq was applied to identify differentially expressed genes (DEG) in the digestive gland across the different infection stages. ...
... 2b-RAD libraries were constructed at the Genomics Platform of Universidad de Santiago de Compostela (USC) and delivered to the FISABIO Platform (Valencia, Spain) for sequencing in a NextSeq 500 sequencer (Illumina). Then, reads from each individual were aligned to the common cockle genome (Bruzos et al., 2022) using Bowtie 1.1.2 (Langmead et al., 2009), and SNP calling was performed with Stacks 2.0 (Catchen et al., 2013;Rochette et al., 2019), following the parameters described by Vera et al. (2022). ...
... (Langmead et al., 2009), and SNP calling was performed with Stacks 2.0 (Catchen et al., 2013;Rochette et al., 2019), following the parameters described by Vera et al. (2022). The RAD-tag SNP panel reported by Vera et al. (2022) mapped in the common cockle genome (Bruzos et al., 2022) was used as reference for genotyping to make feasible comparison with previous studies. Finally, some SNPs/RAD-tags or individuals were removed from the data using Plink 1.9 (Purcell et al., 2007) according to the following criteria: i) SNPs deviated from Hardy-Weinberg proportions (p < 0.05) in at least two sampling sites; ii) RAD-tags with more than 3 SNPs; iii) SNPs with missing data in > 50% NS and AS: naive (samples collected before 1 st marteiliosis detection) and affected (samples collected after six years of marteiliosis outbreaks), respectively; NES and ES: non-exposed (samples collected before detection of the 2018/19 marteiliosis outbreak) and exposed (samples collected during the 2018/19 marteiliosis outbreak), respectively. ...
The common cockle (Cerastoderma edule) plays an important role in marine ecosystems and represents a valuable socioeconomic resource for coastal communities. In 2012, the cockle beds from Rı́a de Arousa (Galicia, NW Spain) were seriously decimated by the protozoan Marteilia cochillia responsible for marteiliosis. We aimed to identify single nucleotide polymorphisms (SNP) markers potentially associated with resilience to marteiliosis to be used in marker-assisted selection programs for restoring affected cockle beds and recovering their production. For this, we carried out a population genomics approach using 2b-RADseq, where 38 naive samples (before the first detection of M. cochillia in 2012) from two beds of Rı́a de Arousa were compared with 39 affected samples collected in 2018/2019 (after several years of marteiliosis occurring in the area), collected either before (15 non-exposed samples) or during (24 exposed samples) the marteiliosis outbreak. Additionally, 767 differentially expressed genes (DEG) from a previous transcriptomic study addressed during the aforementioned 2018/19 marteiliosis outbreak, were evaluated to identify SNPs showing signals of selection. Using 2b-RADseq, 9,154 SNPs were genotyped and among them, 110 consistent outliers for divergent selection were identified. This set of SNPs was able to discriminate the samples according to their marteiliosis status (naive vs affected; exposed vs non-exposed), while another 123 SNPs were identified linked to DEGs associated with the level of infection across a temporal series. Finally, combining the population genomics and transcriptomics information, we selected the 60 most reliable SNPs associated with marteiliosis resilience. These SNPs were close to or within DEGs, and many of them were related to immune response (phagocytosis and cell adhesion), defence, such as apoptosis, stress, and cellular cycle, among other functions. This set of SNPs will eventually be validated to develop a cost-effective genotyping tool for their application for obtaining cockle-resilient strains for marteiliosis.
... Illumina-specific adaptors were clipped from the reads; ii) the read removed, if a sliding window average Phred score over five bases was < 20; and iii) only reads where both pair-ends were longer than 50 bp post-filtering were retained. Filtered reads were aligned against the cockle genome (Bruzos et al., 2022) using a custom script with SAMtools v1.9 (Li et al., 2009) and STAR v2.7.0e (Dobin et al., 2013). Bam files from aligned reads were merged with SAMtools and a cockle transcriptome draft was finally obtained. ...
... After filtering, 1991 million total reads were retained, 1609 million from the digestive gland and 381 million from whole meat. Among the 33 million pair-end filtered reads per sample, 28.8 million were consistently aligned to the chromosome-level assembled cockle genome (Bruzos et al., 2022) by retaining only the single best hit when more than one position matched. The common cockle transcriptome, particularly enriched on the digestive gland, was constructed taking as reference the cockle genome assembly and using a conservative bioinformatic pipeline (Fig. 2): i) overlapping genes with the same annotation were collapsed to a single gene and the length of the overlapping region was considered as their full length; ii) those genes with no annotation were discarded; and iii) only those genes with ≥ 5 TPM in at least one sample were retained. ...
The edible cockle ( Cerastoderma edule ) is a widely cultivated bivalve with relevant ecological value roles and high value for shellfisheries in different European regions. The emergence of new threats, such as the parasite Marteilia cochillia , has impaired the production and ecosystem of shellfish beds where the parasite was detected. Knowledge of the molecular mechanisms involved in cockle immune response to this parasite is essential to devise strategies for its control. With this aim, a transcriptomic study of the digestive gland (target organ of the parasite) and the whole cockle meat in response to M. cochillia infection was carried out in heavily impacted area in the Northwest of Spain (Lombos do Ulla, Ria de Arousa). A total of 2079 million raw RNA-seq reads were obtained after filtering and used for annotation of 9049 genes following a conservative bioinformatic pipeline using the chromosome-level cockle genome as reference. Gene expression analysis identified a total of 973 consistent differentially expressed genes (DEGs) between comparisons across a temporal series involving cockles with different degrees of infection. DEGs increased with the level of infection within each temporal sample, but the higher DEGs number were detected when comparing temporal samples. Enrichment analysis of DEGs showed an increased expression of molecular functions related to hydrolase, peptidase activity, carbohydrate binding and active transmembrane transporter activity; cellular components such as extracellular matrix and extracellular regions; and a few biological functions associated with immunity and defence response. This information will be valuable for further studies focused on DEGs and associated SNP markers to develop reliant cockle strains to marteiliosis.
Bivalve transmissible neoplasia (BTN) is a “leukemia-like” cancer
affecting marine bivalve species such as soft-shell clam (Mya
arenaria) (MarBTN) and the common cockle (Cerastoderma
edule). Although cancer is generally considered non-transmissible,
MarBTN is thought to spread through the water column. To test
for MarBTN in clams, multiple techniques, such as qPCR and
qualitative assessment via light microscopy were used.
Transfection and primary neoplastic cell culture or healthy
hemocytes is not yet available for MarBTN, which has limited the
ability of researchers to further characterize the disease. For this
study, we instead transfected by electroporation a plasmid DNA
vector to express green fluorescent protein (GFP) in the protist,
Perkinsus marinus, which is responsible for ‘Dermo’ disease in
Eastern oysters (Crassostrea virginica). Fluorescence microscopy
was performed less than 24 hours after electroporation and was
observed in each P. marinus culture. Research on the pathology of
marine bivalves is of particular interest to coastal communities
and fishing industries and may have relevance as a model for
researching human leukemic diseases. Ten SMCC students
selected for this project were part of a residential biology research
experience short course hosted at MDI Biological Laboratory with
funding from Maine INBRE.
Transmissible cancer cell lines are rare biological entities giving rise to diseases at the crossroads of cancer and parasitic diseases. These malignant cells have acquired the amazing capacity to spread from host to host. They have been described only in dogs, Tasmanian devils and marine bivalves. The Mytilus trossulus bivalve transmissible neoplasia 2 (MtrBTN2) lineage has even acquired the capacity to spread inter-specifically between marine mussels of the Mytilus edulis complex worldwide. To identify the oncogenic processes underpinning the biology of these atypical cancers we performed transcriptomics of MtrBTN2 cells. Differential expression, enrichment, protein–protein interaction network, and targeted analyses were used. Overall, our results suggest the accumulation of multiple cancerous traits that may be linked to the long-term evolution of MtrBTN2. We also highlight that vertebrate and lophotrochozoan cancers could share a large panel of common drivers, which supports the hypothesis of an ancient origin of oncogenic processes in bilaterians.
Ecology and biogeography of bivalve transmissible neoplasia (BTN) are underexplored due to its recent discovery and a challenging diagnostics. Blue mussels harbour two evolutionary lineages of BTN, MtrBTN1 and MtrBTN2, both derived from Mytilus trossulus. MtrBTN1 has been found only in M. trossulus from North Pacific. MtrBTN2 parasitizes different Mytilus spp. worldwide. BTN in M. trossulus in the Atlantic sector has never been studied. We looked for BTN in mussels from the Barents Sea using flow cytometry of cells, qPCR with primers specific to cancer-associated alleles and sequencing of mtDNA and nuclear loci. Both MtrBTN1 and MtrBTN2 were present in our material, though their prevalence was low (~0.4%). All cancers parasitized M. trossulus except one, MtrBTN1, which was found in a hybrid between M. trossulusand M. edulis. The mtDNA haplotypes found in both lineages were nearly identical to those known from the Northwest Pacific but not from elsewhere. Our results suggest that these two lineages may have arrived in the Barents Sea in recent decades with the maritime transport along the Northern Sea Route. A young evolutionary age of MtrBTN1 seems to indicate that it is an emerging disease in the process of niche ex-pansion. Comparing the new and the published sequence data on tumour suppressor p53, we proved that the prevalence of BTN in mussels can reach epizootic levels. The finding of diverse recombinants between paternally and maternally inherited mtDNAs in somatic tissues of M. trossulus was an unexpected result of our study.
