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

Article Figures and data Abstract Editor's evaluation eLife digest Introduction Results and discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract 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. Editor's evaluation This paper describes a previously unknown lineage of transmissible cancer in a clam, in which the cancer arose from a different, but related, species. The data are clear and overall, the conclusions well-supported, and this finding increases our understanding of transmissible cancers in nature and will be of broad interest. https://doi.org/10.7554/eLife.66946.sa0 Decision letter Reviews on Sciety eLife's review process eLife digest 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. Introduction Cancers are clonal cell lineages that arise due to somatic changes that promote cell proliferation and survival (Stratton et al., 2009). Although natural selection operating on cancers favours the outgrowth of malignant clones with replicative immortality, the continued survival of a cancer is generally restricted by the lifespan of its host. However, clonally transmissible cancers – from now on, transmissible cancers – are somatic cell lineages that are transmitted between individuals via the transfer of living cancer cells, meaning that they can survive beyond the death of their hosts (Murchison, 2008). Naturally occurring transmissible cancers have been identified in dogs (Murgia et al., 2006; Murchison et al., 2014; Baez-Ortega et al., 2019), Tasmanian devils (Murchison et al., 2012; Pye et al., 2016) and, more recently, in marine bivalves (Metzger et al., 2015; Metzger et al., 2016; Yonemitsu et al., 2019). Hemic neoplasia (HN), also called disseminated neoplasia, is a type of leukaemia cancer found in multiple species of bivalves, including oysters, mussels, cockles, and clams (Carballal et al., 2015). Although these leukaemias represent different diseases across bivalve species, they have been classically grouped under the same term because neoplastic cells share morphological features (Carballal et al., 2015). Some HNs have been proven to have a clonal transmissible behaviour (Metzger et al., 2015), in which neoplastic cells, most likely haemocytes (i.e. the cells that populate the haemolymph and play a role in the immune response), are likely to be transmitted through marine water. In late stages of the disease, leukaemic cells invade the surrounding tissues and, generally, animals die because of the infection (Carballal et al., 2015), although remissions have also been described (Burioli et al., 2019). Despite the observation that leukaemic cells are typically transmitted between individuals from the same species, on occasion they can infect and propagate across populations from a second, different bivalve species (Metzger et al., 2016; Yonemitsu et al., 2019). Hence, these cancers represent a potential threat for the ecology of the marine environment, which argues for the necessity of their identification and characterization for their monitoring and prevention. Here, we use multiplatform next-generation genome sequencing technologies, including Illumina short reads and Oxford Nanopore long reads, together with cytogenetics, electron microscopy, and cytohistological approaches to identify, characterize, and decipher the evolutionary origin of a new marine leukaemia that is transmitted between two different clam species that inhabit the Seas of Southern Europe, namely warty venus (Venus verrucosa) and striped venus (Chamelea gallina) (Video 1). Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Mitochondrial genome sequencing of marine leukaemias reveals cancer contagion between clam species in the Seas of Southern Europe. Infographic video outlining the main findings of the research carried out. Results and discussion We investigated the prevalence of HN in the warty venus clam (V. verrucosa), a saltwater bivalve found in the Atlantic Coast of Europe and the Mediterranean Sea. We collected 345 clam specimens from six sampling regions in the Atlantic and the Mediterranean coasts of Europe across five different countries, including Spain, Portugal, France, Ireland, and Croatia (Figure 1a; Supplementary file 1). Cytohistological examination identified HN-like tumours in eight specimens from two sampling points in Spain (Figure 1b–e; Figure 1—figure supplement 1). Three HN-positive specimens (ERVV17-2995, ERVV17-2997, and ERVV17-3193) were collected in Galicia, northwest of the Iberian Peninsula in the Atlantic Ocean, and another five specimens (EMVV18-373, EMVV18-376, EMVV18-391, EMVV18-395, and EMVV18-400) were collected in the Balearic Islands, bathed by the Mediterranean Sea (Figure 1a; Supplementary file 1). Four of these specimens (ERVV17-2995, ERVV17-3193, EMVV18-391, and EMVV18-395) showed a severe form of the disease – classified as N3 stage – which is characterized by high levels of neoplastic cells infiltrating the gills, different levels of infiltration of the digestive gland and gonad, and low/very low infiltration of the mantle and foot (Figure 1d,e; Figure 1—figure supplement 1); one specimen (EMVV18-400) was found that was affected with an intermediate form of the disease – N2 stage – characterized by low levels of neoplastic cells infiltrating the gill vessels, digestive gland, and gonad, but not the foot (Figure 1—figure supplement 1); and three specimens (ERVV17-2997, EMVV18-373, and EMVV18-376) were diagnosed with a light form of the disease – N1 stage – characterized by low levels of neoplastic cells infiltrating the gills vessels only, and no infiltration in the remaining tissues (Figure 1—figure supplement 1). Electron microscopy analysis through gill's ultrathin sections from two neoplastic warty venus specimens (ERVV17-2995 and ERVV17-3193) revealed tumour cells with a round shape and a pleomorphic nucleus, which are morphological features that generally characterize bivalves' HN (Figure 1f; Figure 1—figure supplement 2). Finally, one additional neoplastic warty venus specimen (EVVV11-02) was included in the study. The animal, which was sampled in 2011 in Galicia and came from a private collection, showed abnormal metaphases in the gills that were suggestive of HN. Although the species typically shows a 2n = 38 karyotype with metacentric chromosomes that are homogeneous in size (García-Souto et al., 2015), the tumoural metaphases from this individual showed around 100 chromosomes that were variable in size and shape (Figure 1g). Figure 1 with 2 supplements see all Download asset Open asset Geographical location of warty venus (V. verrucosa) specimens and diagnosis of hemic neoplasia. (a) Locations of V. verrucosa clams collected for this study and specimens diagnosed with hemic neoplasia. Size of the pie charts correlates with the number of samples collected (number of samples 'n' is shown together with each pie chart). Pie charts show the proportion of samples with hemic neoplasia (black, no neoplastic specimens; red, neoplastic specimens). Codes of neoplastic samples are shown. Top-right corner shows a representative specimen of the species V. verrucosa. (b) Cytological examination of haemolymph smear (Hemacolor stain) from a healthy (N0) specimen, ERVV17-2963, shows normal haemocytes. (c) Haemolymph smear of a V. verrucosa specimen with high-grade (N3 stage) hemic neoplasia, ERVV17-3193, shows neoplastic cells that replaced normal haemocytes. (d) Detail of haematoxylin and eosin-stained of histological section from the gills of the healthy (N0) specimen ERVV17-2990. (e) Same for ERVV17-2995, a specimen infected with a high-grade (N3 stage) hemic neoplasia, showing neoplastic cells infiltrating the gills. (f) Transmission electron microscopy analysis of a V. verrucosa hemic neoplasia tumour cell shows a round shape, pseudopodia 'p', pleomorphic nucleus 'n' with scattered heterochromatin, and mitochondria 'm'. (g) Metaphase chromosomes from a neoplastic cell found in the gills of the V. verrucosa specimen EVVV11-02, showing abnormal chromosome number (>19 pairs) and abnormal chromosome morphology. Chromosomes stained with 4′,6-DiAmidino-2-PhenylIndole (DAPI) and Propidium Iodide (PI). To obtain some biological insights into the clonal dynamics of this cancer, we carried out whole-genome sequencing with Illumina paired-ends in DNA samples isolated from the tumoural haemolymph from eight out of nine neoplastic specimens mentioned above (Table 1). Their feet were also sequenced, as foot typically represents the tissue with lower infiltration of neoplastic cells, making it a good candidate tissue to act as 'matched-normal' (i.e. host tissue). As for the animal with an abnormal karyotype (EVVV11-02) that was compatible with HN, we sequenced the only tissue available, which were gills (Table 1). Only one neoplastic specimen (EMVV18-373) that had a very low proportion of tumour cells in its haemolymph was excluded from the sequencing. Then, we mapped the paired-end reads onto a dataset containing non-redundant mitochondrial Cytochrome C Oxidase subunit 1 (Cox1) gene references from 118 Venerid clam species. In six out of eight sequenced neoplastic specimens, the results revealed an overrepresentation (>99%) of reads in the sequenced tissues mapping to Cox1 DNA sequences that exclusively identified two different clam species (Figure 2a): the expected one, warty venus clam (V. verrucosa), and a second, unexpected one, the striped venus (C. gallina), a clam that inhabits the Mediterranean Sea (Figure 2b). Preliminary analysis by PCR and capillary sequencing of Cox1 in the haemolymph of two neoplastic specimens, EMVV18-373 and EVVV11-02, revealed an electropherogram with overlapping peaks apparently containing two different haplotypes that match the reference Cox1 sequences for warty and striped venus (Figure 2c). Figure 2 with 2 supplements see all Download asset Open asset Mitochondrial DNA sequencing and phylogenetic analyses reveal cancer contagion between warty venus (V. verrucosa) and striped venus (C. gallina) clam species. (a) In eight warty venus specimens sequenced with Illumina paired-ends, the pie charts show the proportion of reads mapping Cox1 reference sequences from 137 different Verenidae species, including V. verrucosa (red), C. gallina (blue), and the remaining species (grey). Two different tissues were sequenced: the tumour tissue (left pie chart), typically haemolymph, and the host/matched-normal tissue (right pie chart), typically foot. Note that for specimen EVVV11-02 only the host/matched-normal tissue (gills) was available. 'n' denotes the total number of reads mapping the Cox1 reference for the tumour tissue (left), and the host tissue (right). (b) Representative specimen of the species C. gallina. (c) Capillary sequencing electropherograms of mitochondrial Cox1 gene fragments from two neoplastic V. verrucosa specimens (EMVV18-373 and EVVV11-02) and two healthy reference specimens from V. verrucosa and C. gallina. The results show overlapping peaks (arrows) in the sequenced tissues from the neoplastic animals, which suggest coexistence of mitochondrial DNA (mtDNA) haplotypes from two clam species. (d) In V. verrucosa neoplastic (N2-stage) specimen EMVV18-400, mtDNA read depth shows different proportion of warty venus and striped venus mtDNA haplotypes in the tumour tissue (haemolymph) and the matched-normal tissue (foot). (e) Molecular phylogeny using Bayesian inference inferred on the alignment of all mitochondria coding genes and rRNA gene sequences (15 loci) that includes six neoplastic V. verrucosa specimens with evidence of cancer contagion from C. gallina. Bootstrap values are shown above the branches. Table 1 Clam specimens and tissues sequenced with Illumina paired-ends. Sixteen specimens (eight neoplastic and eight non-neoplastic) from three different clam species (V. verrucosa, C. gallina, and C. striatula) were sequenced with Illumina paired-ends. Columns 5 and 6 show the number of reads generated for the host tissue (when neoplastic, matched-normal tissue was foot) and the tumoural haemolymph, respectively. (*) denotes the only available tissue from this neoplastic animal, collected in 2011, were gills. (#) denotes hemic neoplasia stage was not determined because cytohistological examination was not possible in this individual, which was diagnosed by cytogenetics. Clam speciesSpecimen originSpecimen codeDiagnosisFoot readsHaemolymph readsV. verrucosaGalicia, SpainERVV17-2995N3833 M919 MV. verrucosaGalicia, SpainERVV17-2997N1766 M598 MV. verrucosaGalicia, SpainERVV17-3193N3739 M850 MV. verrucosaBalearic Islands, SpainEMVV18-376N1784 M849 MV. verrucosaBalearic Islands, SpainEMVV18-391N3617 M623 MV. verrucosaBalearic Islands, SpainEMVV18-395N3697 M679 MV. verrucosaBalearic Islands, SpainEMVV18-400N1782 M1133 MV. verrucosaGalicia, SpainEVVV11-02N#743 M*–*V. verrucosaSplit, CroatiaCSVV18-1052Healthy161 M–V. verrucosaBalearic Islands, SpainEMVV18-385Healthy143 M–V. verrucosaGranville, FranceFGVV18-183Healthy752 M–V. verrucosaCarna, IrelandIGVV19-666Healthy155 M–V. verrucosaOeiras, PortugalPLVV18-2249Healthy163 M–C. gallinaS.Benedetto, ItalyIMCG15-69Healthy147 M–C. gallinaCadiz, SpainECCG15-201Healthy752 M–C. striatulaGalicia, SpainEVCS14-09Healthy706 M– These results suggested cancer contagion between the two clam species of the family Veneridae. Hence, to decipher the origins of this clam neoplasia, we further analysed the mitochondrial DNA (mtDNA) from the two species involved and the tumours. Firstly, we performed multiplatform genome sequencing, including Illumina short reads and Oxford Nanopore long reads, on canonical individuals from the two species to obtain a preliminary assembly of the mitogenomes of V. verrucosa and C. gallina. These reconstructions resulted in 18,092- and 17,618-bp long mtDNA genomes for the warty venus and the striped venus clam, respectively (Figure 2—figure supplement 1). The comparative analysis of the nucleotide sequences from both mitogenomes confirms that, although both species are relatively close within the subfamily Venerinae (Canapa et al., 1996), they represent distinct sister species, showing a Kimura's two-parameter nucleotide distance (K2P) equal to 21.13%. Then, we mapped the paired-end sequencing data from the six neoplastic specimens with evidence of interspecies cancer transmission onto the two reconstructed species-specific mtDNA genomes. This approach confirmed the coexistence of two different mtDNA haplotypes in the six examined neoplastic samples, matching the canonical mtDNA genomes from the two clam species. For example, in a N2-stage specimen (EMVV18-400), this analysis revealed different proportion of tumour and host mtDNA molecules in the two tissue types sequenced (Figure 2d). Here, the striped venus mtDNA results the most abundant in the haemolymph, in which tumour cells are dominant over the remaining cell types, and the lower in the matched-normal tissue (i.e. infiltrated foot), where tumour cells represent a minor fraction of the total. Similar results were obtained for the remaining five neoplastic individuals (Figure 2—figure supplement 2). To further investigate the evolutionary origins and geographic spread of this cancer, we sequenced with Illumina paired-ends an additional set of eight healthy (i.e. non-neoplastic) clams from three different Veneridae species, including five more warty venus specimens (EMVV18-385, IGVV19-666, FGVV18-183, CSVV18-1052, and PLVV18-2249) from five different countries, two striped venus specimens (IMCG15-69 and ECCG15-201) from two countries, and one specimen (EVCS14-09) from its sibling species Chamelea striatula, a type of striped venus clam that inhabits the Atlantic Ocean from Norway to the Gulf of Cadiz in Spain. This made a total of 16 Veneridae specimens sequenced, all listed in Table 1 (see also Supplementary file 1). The complete mitochondrial genomes from all tumoural and healthy V. verrucosa specimens (13 individuals), 2 C. gallina, and 1 from its sibling species C. striatula, were individually de novo assembled from the sequencing reads. As expected, this approach reconstructed two different haplotypes in six out eight sequenced neoplastic animals, supporting the presence of mtDNA from two different species. Despite the high sequencing coverage obtained for these individuals (Table 1), we did not find foreign reads in the N1 tumours (ERVV17-2997 and EMVV18-373), most likely due to a low proportion of neoplastic cells in the haemolymph and the matched-normal tissue. Then, we performed a phylogenetic analysis based on the alignment of these mitochondrial genomes (13 coding and 2 RNA gene sequences, altogether encompassing ~14 kb). The results show that tumour and non-tumour sequences from neoplastic warty venus specimens define two well-differentiated clades, and that tumoural warty venus sequences are all identical and closer to striped venus mtDNA than to its own (warty venus) (Figure 2e). Overall, these data support the existence of a single cancer clone originated in the striped venus clam C. gallina that was transmitted to V. verrucosa. Transmissible cancers are known to occasionally acquire mitochondria from transient hosts (Strakova et al., 2016; et al., which can to of their evolutionary we for nuclear markers to confirm the striped venus origin of this cancer We performed a preliminary assembly of the warty venus and the striped venus nuclear using the paired-end sequencing data from two Then, we approaches to find single nuclear genes that were between the two species, two candidate regions (see a long from a gene that for an RNA and a long from the the of finding between both species, we performed PCR and capillary sequencing on a from the and a from in 2 of warty venus specimens for and for 2 of striped venus for and for and 1 specimen of its sister species C. This analysis and for the and the with that to between the species and the tumour (Figure These were to the Illumina reads from each sequenced warty venus neoplastic specimens that were for warty venus or striped venus, which to obtain the sequences that to the tumour tissue and the tissue from each neoplastic the of this we performed phylogenetic reconstructions from these individual nuclear the one the phylogeny for the confirmed both the of the tumoural sequences and their closer to C. gallina than to the host species (Figure which were also in the mtDNA However, the phylogeny from the showed that, although the tumours they were in a to C. gallina and V. verrucosa (Figure Hence, to these we also obtained a species based on the alignment of both the mtDNA and the two nuclear This new phylogeny confirmed that warty venus tumours are closer to striped venus specimens than to warty venus sequences from the same specimens, while the sequences a more warty venus lineage (Figure Figure with 1 supplement see all Download asset Open asset DNA sequencing and phylogenetic analyses confirm a single cancer lineage in populations of the warty venus (V. verrucosa) that originated in the striped venus (C. (a) between V. verrucosa tumours and the three canonical species (V. verrucosa, C. gallina, and C. striatula) a and a long fragments of nuclear genes and respectively. (b) based on the two fragments of the nuclear DNA markers and Bootstrap support values from analyses above are shown on the branches. (c) of V. verrucosa, their tumours and Chamelea based on the mitochondrial DNA (mtDNA) and the two nuclear and is with the and of as The shown includes 1000 and represents the of in which is the most common set of the most common one, and the (d) in to the DNA in one V. verrucosa tumour and healthy specimens from the species C. gallina and V. verrucosa shows in in and from the chromosomes of the tumour and the healthy C. gallina but not in healthy V. verrucosa. To obtain further evidence on the striped venus origin of this neoplasia, we performed a comparative of in the genomes of C. gallina and V. verrucosa using in (Figure Figure supplement 1). We on two DNA namely and The represent of and long and were identified in a preliminary of the striped venus reference genome (see This approach revealed that the mentioned are very abundant in regions from the genomes of the canonical striped venus and the neoplastic warty venus specimens (Figure Figure supplement 1). However, the were in the metaphases from all the healthy warty venus individuals (Figure Figure supplement 1). These results suggest that the chromosomes with and found in neoplastic warty venus specimens from C. gallina, supporting that a tumour originated in C. gallina was transmitted to V. verrucosa. To find out whether this cancer is in the clam species where it we performed a for its presence in natural populations of striped venus clams from the species C. gallina = and C. = five additional sampling points across two file 1), including Spain = and = analyses did not show of HN in these The of this tumour in natural populations of striped venus clams may suggest that this leukaemia is being not transmitted between specimens of the species, warty However, further sampling in other regions across the striped venus of may be to confirm these Overall, the results reveal the existence of a transmissible leukaemia originated in a striped venus clam, most likely C. gallina, which was transmitted to a species, the warty venus clam (V. verrucosa), and specimens it currently We identified this cancer in warty venus clams from two sampling points that are more than 1000 nautical miles away in the coasts of Spain, bathed by two different the Atlantic Ocean and the Mediterranean Sea. The analysis of mitochondrial and nuclear gene sequences revealed no nucleotide within the tumours sequenced, which supports that all belong to the same neoplastic lineage that spreads between Veneridae clams in the Seas of Southern Europe. Although we the of this cancer we can confirm it arose 2011, when the neoplastic warty venus specimen EVVV11-02 was The of between all even from sampling suggests that this cancer is very or that it may have been scattered by the of a of transmission that has been for other bivalve transmissible cancers et al., 2019). Materials and methods of clam specimens a We collected clam specimens from three different species, from the and file 1). V. verrucosa clams were collected in Spain = Balearic Islands, = = Croatia = = and = C. gallina clams were collected in Spain = = and = = C. clams were collected in Spain = we samples from the specimens from private one V. verrucosa clam collected in 2011 in Spain C. gallina collected in in