1. Cancer Biology
  2. Microbiology and Infectious Disease
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A single clonal lineage of transmissible cancer identified in two marine mussel species in South America and Europe

  1. Marisa A Yonemitsu
  2. Rachael M Giersch
  3. Maria Polo-Prieto
  4. Maurine Hammel
  5. Alexis Simon
  6. Florencia Cremonte
  7. Fernando T Avilés
  8. Nicolás Merino-Véliz
  9. Erika AV Burioli
  10. Annette F Muttray
  11. James Sherry
  12. Carol Reinisch
  13. Susan A Baldwin
  14. Stephen P Goff
  15. Maryline Houssin
  16. Gloria Arriagada
  17. Nuria Vázquez
  18. Nicolas Bierne
  19. Michael J Metzger  Is a corresponding author
  1. Pacific Northwest Research Institute, United States
  2. Howard Hughes Medical Institute, United States
  3. ISEM, Université de Montpellier, CNRS- EPHE-IRD, France
  4. IHPE, Université de Montpellier, CNRS-Ifremer-UPVD, France
  5. Instituto de Biología de Organismos Marinos (IBIOMAR) (CCT CONICET - CENPAT), Argentina
  6. Universidad Andres Bello, Chile
  7. LABÉO Frank Duncombe, France
  8. Environmental Resources Management, Canada
  9. Environment and Climate Change Canada, Canada
  10. University of British Columbia, Canada
  11. Columbia University Medical Center, United States
  12. FRE BOREA, MNHN, UPMC, UCN, CNRS-7208, IRD-207, Université de Caen Normandie, France
Research Article
Cite this article as: eLife 2019;8:e47788 doi: 10.7554/eLife.47788
7 figures and 4 additional files

Figures

Phylogenetic analysis of EF1α alleles from normal and diseased mussels.

The EF1α locus was amplified, and multiple alleles were cloned from different individual normal and diseased mussels of different species and locations: M. trossulus from BC (black), M. chilensis from Argentina and Chile (red), and M. edulis from France and the Netherlands (blue). (A) A list of cloned alleles is shown, with filled boxes marking cancer-associated alleles. (B) Phylogenetic analysis of aligned alleles shows groups of related alleles (see Figure 1—source data 1). Names of alleles on the tree specify individual ID and allele ID. Open circles mark alleles from normal individuals and host alleles from diseased individuals. Filled circles mark cancer-associated alleles on the tree (colored by host species). The tree was rooted at the midpoint, with bootstrap values below 50 removed. Model used was HKY85+G. The scale bar marks genetic distance.

https://doi.org/10.7554/eLife.47788.003
Figure 1—source data 1

FASTA formatted text file of EF1α sequence alignment.

https://doi.org/10.7554/eLife.47788.004
Phylogenetic analysis of H4 alleles from normal and diseased mussels.

The H4 locus was amplified, and multiple alleles were cloned from different individual normal and diseased mussels of different species and locations: M. trossulus from BC (black), M. chilensis from Argentina and Chile (red), and M. edulis from France and the Netherlands (blue). (A) A list of cloned alleles is shown, with filled boxes marking cancer-associated alleles. (B) Phylogenetic analysis of aligned alleles shows groups of related alleles (see Figure 2—source data 1). Names of alleles on the tree specify individual ID and allele ID. Open circles mark alleles from normal individuals and host alleles from diseased individuals. Closed circles mark cancer-associated alleles (colored by host species). The tree was rooted at the midpoint, with bootstrap values below 50 removed. Model used was HKY85+I. The scale bar marks genetic distance. Three sequences (AR7-2I, BA30-2J, and Castro26-2J) are consistent with recombination between the two cancer-associated alleles and were removed from the tree for clarity. The two alleles identified in the Mytilus BTN1 lineage (2A and 2A’) are distinguished by a unique single base deletion in 2A’, which does not show up as a difference in the tree, as gaps are treated as missing data.

https://doi.org/10.7554/eLife.47788.005
Figure 2—source data 1

FASTA formatted text file of H4 sequence alignment.

https://doi.org/10.7554/eLife.47788.006
Phylogenetic analysis of mitochondrial CR alleles from normal and diseased mussels.

The CR region of mitochondrial DNA was amplified, and multiple alleles were cloned from different individual normal and diseased mussels of different species and locations: M. trossulus from BC (black), M. chilensis from Argentina and Chile (red), and M. edulis from France and the Netherlands (blue). (A) A list of cloned alleles is shown, with filled boxes marking cancer-associated alleles. (B) A schematic of the rearrangement found in the cancer-associated alleles is shown with black boxes representing female-derived sequence and white boxes representing male-derived sequence (gray bars represent the likely recombination region). Parentheses mark the repeat region in allele D. (C) Phylogenetic analysis of aligned alleles shows groups of related alleles (see Figure 3—source data 1). Names specify individual ID and allele ID. Alleles from normal individuals and host alleles from diseased individuals are marked with open circles (female allele) and open squares (male allele). Closed circles mark cancer-associated alleles (colored by host species). The tree was rooted at the midpoint, with bootstrap values below 50 removed. Model used was GTR+I. The scale bar marks genetic distance. Multiple male alleles were identified in M. chilensis samples, and a representative with the greatest number of clones was chosen for each individual. Reference sequences include female M. trossulus (AY823625.1), male M. trossulus (HM462081.1), female M. edulis (DQ198231.2), male M. edulis (AY823623.1), and the M. trossulus recombinant 62cm10 (KM192133.1).

https://doi.org/10.7554/eLife.47788.007
Figure 3—source data 1

FASTA formatted text file of mtCR sequence alignment.

https://doi.org/10.7554/eLife.47788.008
Phylogenetic analysis of mitochondrial COI alleles from normal and diseased mussels.

The mtCOI locus was amplified, and multiple alleles were cloned from different individual normal and diseased mussels of different species and locations: M. trossulus from BC (black), M. chilensis from Argentina and Chile (red), and M. edulis from France and the Netherlands (blue). (A) A list of cloned alleles is shown, with filled boxes marking cancer-associated alleles. (B) Phylogenetic analysis of aligned alleles shows groups of related alleles (see Figure 4—source data 1). Names of alleles on the tree specify individual ID and allele ID. Open circles mark alleles from normal individuals and host alleles from diseased individuals. Closed circles mark cancer-associated alleles (colored by host species). The tree was rooted at the midpoint, with bootstrap values below 50 removed. Model used was GTR+G. The scale bar marks genetic distance. An additional Chilean sample was used for cloning (Castro52) as Castro26 showed difference from the other samples at this locus and a second sample was needed to infer the cancer-associated allele and confirm the mitochondrial replacement. One clone obtained from Castro26 was a recombinant between a M. trossulus-like sequence and an M. chilensis-like sequence, and it was excluded from the phylogenetic tree for clarity.

https://doi.org/10.7554/eLife.47788.009
Figure 4—source data 1

FASTA formatted text file of mtCOI sequence alignment.

https://doi.org/10.7554/eLife.47788.010
qPCR of multiple loci confirms the presence of the cancer lineage in M. trossulus and M. edulis from multiple populations.

Allele-specific qPCR was conducted to determine the fraction of cancer-associated alleles at the four loci: allele H at EF1α, allele RM and KNS at H4, allele C and D at mtCR, and allele B at mtCOI (see Figure 5—source data 1). Boxes show whether the cancer-specific allele is undetectable or detectible, with a heatmap showing the fraction of the total alleles at each locus that correspond to the specific allele (the quantity of amplification with an allele-specific qPCR reaction divided by the quantity amplified with ‘universal’ Mytilus primers). Each individual mussel is marked with its ID, species, and location, and each animal that was diagnosed as containing disseminated neoplasia is marked ‘Cancer’.

https://doi.org/10.7554/eLife.47788.011
Figure 5—source data 1

Tab separated text file with qPCR data (U means undetectable).

https://doi.org/10.7554/eLife.47788.012
Identification of M. trossulus-specific SNPs in M. chilensis and M. edulis samples.

Cancer and normal samples of (A) M. chilensis from Argentina and (B) M. edulis from Europe were analyzed for the presence of 13 SNPs that are diagnostic for M. trossulus alleles. The fraction of the fluorescence attributable to the M. trossulus allele is shown for each SNP, and the mean multi-locus estimate is shown on the right. SNP IDs indicate shortened names of the specific nuclear SNP loci used (see Supplementary file 3). Red points indicate M. chilensis and blue indicate M. edulis. Dark filled circles represent cancer samples, dark open circles represent normal samples which were a part of the sequence and qPCR analysis (Figures 14), and light open circles represent samples from an extended set of normal individuals from the same populations. Cancer samples included: Mch23, Mch41, and Mch42 for M. chilensis, and BA30, HO8, Ch5, Ch13, LA84, and STBRI43 from M. edulis. Normal samples included Mch3, Mch5, and Mch7 for M. chilensis, and HO9, HO19, Ch10, and Ch11 for M. edulis, as well as 109 additional normal M. chilensis and 62 additional normal M. edulis. See Figure 6—source data 1 for M. chilensis data and Figure 6—source data 2 for M. edulis data.

https://doi.org/10.7554/eLife.47788.013
Figure 6—source data 1

Comma separated text file with SNP data from M. chilensis samples.

https://doi.org/10.7554/eLife.47788.014
Figure 6—source data 2

Comma separated text file with SNP data from M. edulis samples.

https://doi.org/10.7554/eLife.47788.015
Map of the spread of Mytilus BTN1 and 2.

The locations of all collections in the current study are shown with circles. Diseased animals were found at all collection locations. The species collected at each location is marked. The Mytilus BTN1 lineage (gray) was only identified in M. trossulus populations in BC, Canada, while the Mytilus BTN2 lineage (purple) was identified in both M. edulis and M. chilensis populations in both the Pacific and Atlantic Oceans. Both cancer lineages originated in an M. trossulus individual, although the exact locations of the origins are unknown. The direction of spread of these cancers is unknown. Winkel-Tripel map was created by Flappiefh (Wikimedia Commons) using gringer's perlshaper script, and Natural Earth (naturalearthdata.com).

https://doi.org/10.7554/eLife.47788.016

Additional files

Supplementary file 1

Table of PCR primers used for amplification and sequencing.

https://doi.org/10.7554/eLife.47788.017
Supplementary file 2

Table of qPCR primers used.

https://doi.org/10.7554/eLife.47788.018
Supplementary file 3

Table of diagnostic SNP loci used to discriminate M. trossulus from M. edulis and M. chilensis.

https://doi.org/10.7554/eLife.47788.019
Transparent reporting form
https://doi.org/10.7554/eLife.47788.020

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