Identification and expression analysis of two steamer-like retrotransposons in the Chilean blue mussel (Mytilus chilensis).

BACKGROUND: Disseminated neoplasia (DN) is a proliferative cell disorder of the circulatory system of bivalve mollusks. The disease is transmitted between individuals and can also be induced by external chemical agents such as bromodeoxyuridine. In Mya arenaria, we have cloned and characterized an LTR-retrotransposon named Steamer. Steamer mRNA levels and gene copy number correlates with DN and can be used as a marker of the disease. So far, the only mollusk where a retrotransposon expression relates to DN is Mya arenaria. On the other hand, it has been reported that the Chilean blue mussel Mytilus chilensis can also suffers DN. Our aim was to identify retrotransposons in Mytilus chilensis and to study their expression levels in the context of disseminated neoplasia. RESULTS: Here we show that 7.1% of individuals collected in August 2018, from two farming areas, presents morphological characteristics described in DN. Using Steamer sequence to interrogate the transcriptome of M. chilensis we found two putative retrotransposons, named Steamer-like elements (MchSLEs). MchSLEs are present in the genome of M. chilensis and MchSLE1 is indeed an LTR-retrotransposon. Neither expression, nor copy number of the reported MchSLEs correlate with DN status but both are expressed at different levels among individual animals. We also report that in cultured M. chilensis haemocytes MchSLEs1 expression can be induced by bromodeoxyuridine. CONCLUSIONS: We conclude that SLEs present in Mytilus chilensis are differentially expressed among individuals and do not correlate with disseminated neoplasia. Treatment of haemocytes with a stressor like bromodeoxyuridine induces expression of MchSLE1 suggesting that in Mytilus chilensis environmental stressors can induce activation of LTR-retrotransposon.

Centuries of genome instability and evolution in soft-shell clam, Mya arenaria, bivalve transmissible neoplasia.

Transmissible cancers are infectious parasitic clones that metastasize to new hosts, living past the death of the founder animal in which the cancer initiated. We investigated the evolutionary history of a cancer lineage that has spread though the soft-shell clam (Mya arenaria) population by assembling a chromosome-scale soft-shell clam reference genome and characterizing somatic mutations in transmissible cancer. We observe high mutation density, widespread copy-number gain, structural rearrangement, loss of heterozygosity, variable telomere lengths, mitochondrial genome expansion and transposable element activity, all indicative of an unstable cancer genome. We also discover a previously unreported mutational signature associated with overexpression of an error-prone polymerase and use this to estimate the lineage to be >200 years old. Our study reveals the ability for an invertebrate cancer lineage to survive for centuries while its genome continues to structurally mutate, likely contributing to the evolution of this lineage as a parasitic cancer.

Horizontal transmission of disseminated neoplasia in the widespread clam Macoma balthica from the Southern Baltic Sea.

Disseminated neoplasia (DN) is one of the most challenging and unrecognised diseases occurring in aquatic fauna. It has been diagnosed in four bivalve species from the Gulf of Gdansk (Southern Baltic Sea) with the highest frequency in Macoma balthica (formerly Limecola balthica), reaching up to 94% in some populations. The aetiology of DN in the Baltic Sea has not yet been identified, with earlier studies trying to link its occurrence with environmental pollution. Taking into account recent research providing evidence that DN is horizontally transmitted as clonal cells between individuals in some bivalve species, we aimed to test whether DN is a bivalve transmissible neoplasia (BTN) in the population of M. balthica from the Gulf of Gdansk highly affected with cancer. We examined mitochondrial cytochrome c oxidase I (mtCOI) and elongation factor 1α (EF1α) sequences of genomes obtained from haemolymph and tissues of neoplastic and healthy individuals. Sequence analysis resulted in detection of an independent transmissible cancer lineage occurring in four neoplastic clams that is not present in healthy animals. This study describes the first case of BTN in the clam M. balthica (MbaBTN), providing further insights for studies on this disease.

Survival and Detection of Bivalve Transmissible Neoplasia from the Soft-Shell Clam Mya arenaria (MarBTN) in Seawater.

Many pathogens can cause cancer, but cancer itself does not normally act as an infectious agent. However, transmissible cancers have been found in a few cases in nature: in Tasmanian devils, dogs, and several bivalve species. The transmissible cancers in dogs and devils are known to spread through direct physical contact, but the exact route of transmission of bivalve transmissible neoplasia (BTN) has not yet been confirmed. It has been hypothesized that cancer cells from bivalves could be released by diseased animals and spread through the water column to infect/engraft into other animals. To test the feasibility of this proposed mechanism of transmission, we tested the ability of BTN cells from the soft-shell clam (Mya arenaria BTN, or MarBTN) to survive in artificial seawater. We found that MarBTN cells are highly sensitive to salinity, with acute toxicity at salinity levels lower than those found in the native marine environment. BTN cells also survive longer at lower temperatures, with 50% of cells surviving greater than 12 days in seawater at 10 °C, and more than 19 days at 4 °C. With one clam donor, living cells were observed for more than eight weeks at 4 °C. We also used qPCR of environmental DNA (eDNA) to detect the presence of MarBTN-specific DNA in the environment. We observed release of MarBTN-specific DNA into the water of laboratory aquaria containing highly MarBTN-diseased clams, and we detected MarBTN-specific DNA in seawater samples collected from MarBTN-endemic areas in Maine, although the copy numbers detected in environmental samples were much lower than those found in aquaria. Overall, these data show that MarBTN cells can survive well in seawater, and they are released into the water by diseased animals. These findings support the hypothesis that BTN is spread from animal-to-animal by free cells through seawater.

Prevalence and polymorphism of a mussel transmissible cancer in Europe.

Transmissible cancers are parasitic malignant cell lineages that have acquired the ability to infect new hosts from the same species, or sometimes related species. First described in dogs and Tasmanian devils, transmissible cancers were later discovered in some marine bivalves affected by a leukaemia-like disease. In Mytilus mussels, two lineages of bivalve transmissible neoplasia (BTN) have been described to date (MtrBTN1 and MtrBTN2), both of which emerged in a Mytilus trossulus founder individual. Here, we performed extensive screening of genetic chimerism, a hallmark of transmissible cancer, by genotyping 106 single nucleotide polymorphisms of 5,907 European Mytilus mussels. Genetic analysis allowed us to simultaneously obtain the genotype of hosts - Mytilus edulis, M. galloprovincialis or hybrids - and the genotype of tumours of heavily infected individuals. In addition, a subset of 222 individuals were systematically genotyped and analysed by histology to screen for possible nontransmissible cancers. We detected MtrBTN2 at low prevalence in M. edulis, and also in M. galloprovincialis and hybrids although at a much lower prevalence. No MtrBTN1 or new BTN were found, but eight individuals with nontransmissible neoplasia were observed at a single polluted site on the same sampling date. We observed a diversity of MtrBTN2 genotypes that appeared more introgressed or more ancestral than MtrBTN1 and reference healthy M. trossulus individuals. The observed polymorphism is probably due to somatic null alleles caused by structural variations or point mutations in primer-binding sites leading to enhanced detection of the host alleles. Despite low prevalence, two sublineages divergent by 10% fixed somatic null alleles and one nonsynonymous mtCOI (mitochondrial cytochrome oxidase I) substitution are cospreading in the same geographical area, suggesting a complex diversification of MtrBTN2 since its emergence and host species shift.

Identifying key questions in the ecology and evolution of cancer.

The application of evolutionary and ecological principles to cancer prevention and treatment, as well as recognizing cancer as a selection force in nature, has gained impetus over the last 50 years. Following the initial theoretical approaches that combined knowledge from interdisciplinary fields, it became clear that using the eco-evolutionary framework is of key importance to understand cancer. We are now at a pivotal point where accumulating evidence starts to steer the future directions of the discipline and allows us to underpin the key challenges that remain to be addressed. Here, we aim to assess current advancements in the field and to suggest future directions for research. First, we summarize cancer research areas that, so far, have assimilated ecological and evolutionary principles into their approaches and illustrate their key importance. Then, we assembled 33 experts and identified 84 key questions, organized around nine major themes, to pave the foundations for research to come. We highlight the urgent need for broadening the portfolio of research directions to stimulate novel approaches at the interface of oncology and ecological and evolutionary sciences. We conclude that progressive and efficient cross-disciplinary collaborations that draw on the expertise of the fields of ecology, evolution and cancer are essential in order to efficiently address current and future questions about cancer.

Intercellular viral spread and intracellular transposition of Drosophila gypsy.

It has become increasingly clear that retrotransposons (RTEs) are more widely expressed in somatic tissues than previously appreciated. RTE expression has been implicated in a myriad of biological processes ranging from normal development and aging, to age related diseases such as cancer and neurodegeneration. Long Terminal Repeat (LTR)-RTEs are evolutionary ancestors to, and share many features with, exogenous retroviruses. In fact, many organisms contain endogenous retroviruses (ERVs) derived from exogenous retroviruses that integrated into the germ line. These ERVs are inherited in Mendelian fashion like RTEs, and some retain the ability to transmit between cells like viruses, while others develop the ability to act as RTEs. The process of evolutionary transition between LTR-RTE and retroviruses is thought to involve multiple steps by which the element loses or gains the ability to transmit copies between cells versus the ability to replicate intracellularly. But, typically, these two modes of transmission are incompatible because they require assembly in different sub-cellular compartments. Like murine IAP/IAP-E elements, the gypsy family of retroelements in arthropods appear to sit along this evolutionary transition. Indeed, there is some evidence that gypsy may exhibit retroviral properties. Given that gypsy elements have been found to actively mobilize in neurons and glial cells during normal aging and in models of neurodegeneration, this raises the question of whether gypsy replication in somatic cells occurs via intracellular retrotransposition, intercellular viral spread, or some combination of the two. These modes of replication in somatic tissues would have quite different biological implications. Here, we demonstrate that Drosophila gypsy is capable of both cell-associated and cell-free viral transmission between cultured S2 cells of somatic origin. Further, we demonstrate that the ability of gypsy to move between cells is dependent upon a functional copy of its viral envelope protein. This argues that the gypsy element has transitioned from an RTE into a functional endogenous retrovirus with the acquisition of its envelope gene. On the other hand, we also find that intracellular retrotransposition of the same genomic copy of gypsy can occur in the absence of the Env protein. Thus, gypsy exhibits both intracellular retrotransposition and intercellular viral transmission as modes of replicating its genome.

A single clonal lineage of transmissible cancer identified in two marine mussel species in South America and Europe.

Transmissible cancers, in which cancer cells themselves act as an infectious agent, have been identified in Tasmanian devils, dogs, and four bivalves. We investigated a disseminated neoplasia affecting geographically distant populations of two species of mussels (Mytilus chilensis in South America and M. edulis in Europe). Sequencing alleles from four loci (two nuclear and two mitochondrial) provided evidence of transmissible cancer in both species. Phylogenetic analysis of cancer-associated alleles and analysis of diagnostic SNPs showed that cancers in both species likely arose in a third species of mussel (M. trossulus), but these cancer cells are independent from the previously identified transmissible cancer in M. trossulus from Canada. Unexpectedly, cancers from M. chilensis and M. edulis are nearly identical, showing that the same cancer lineage affects both. Thus, a single transmissible cancer lineage has crossed into two new host species and has been transferred across the Atlantic and Pacific Oceans and between the Northern and Southern hemispheres.

From the raw bar to the bench: Bivalves as models for human health.

Bivalves, from raw oysters to steamed clams, are popular choices among seafood lovers and once limited to the coastal areas. The rapid growth of the aquaculture industry and improvement in the preservation and transport of seafood have enabled them to be readily available anywhere in the world. Over the years, oysters, mussels, scallops, and clams have been the focus of research for improving the production, managing resources, and investigating basic biological and ecological questions. During this decade, an impressive amount of information using high-throughput genomic, transcriptomic and proteomic technologies has been produced in various classes of the Mollusca group, and it is anticipated that basic and applied research will significantly benefit from this resource. One aspect that is also taking momentum is the use of bivalves as a model system for human health. In this review, we highlight some of the aspects of the biology of bivalves that have direct implications in human health including the shell formation, stem cells and cell differentiation, the ability to fight opportunistic and specific pathogens in the absence of adaptive immunity, as source of alternative drugs, mucosal immunity and, microbiome turnover, toxicology, and cancer research. There is still a long way to go; however, the next time you order a dozen oysters at your favorite raw bar, think about a tasty model organism that will not only please your palate but also help unlock multiple aspects of molluscan biology and improve human health.

Horizontal transfer of retrotransposons between bivalves and other aquatic species of multiple phyla.

The LTR retrotransposon Steamer is a selfish endogenous element in the soft-shell clam genome that was first detected because of its dramatic amplification in bivalve transmissible neoplasia afflicting the species. We amplified and sequenced related retrotransposons from the genomic DNA of many other bivalve species, finding evidence of horizontal transfer of retrotransposons from the genome of one species to another. First, the phylogenetic tree of the Steamer-like elements from 19 bivalve species is markedly discordant with host phylogeny, suggesting frequent cross-species transfer throughout bivalve evolution. Second, sequences nearly identical to Steamer were identified in the genomes of Atlantic razor clams and Baltic clams, indicating recent transfer. Finally, a search of the National Center for Biotechnology Information sequence database revealed that Steamer-like elements are present in the genomes of completely unrelated organisms, including zebrafish, sea urchin, acorn worms, and coral. Phylogenetic incongruity, a patchy distribution, and a higher similarity than would be expected by vertical inheritance all provide evidence for multiple long-distance cross-phyla horizontal transfer events. These data suggest that over both short- and long-term evolutionary timescales, Steamer-like retrotransposons, much like retroviruses, can move between organisms and integrate new copies into new host genomes.

Evidence of horizontal transmission of the cancer-associated Steamer retrotransposon among ecological cohort bivalve species.

Bivalve specimens from legacy frozen tissue collections, and others freshly obtained, were surveyed for the presence of the Steamer long terminal repeat (LTR)-retrotransposon associated with disseminated hemic neoplasia of the soft-shelled clam Mya areneria. Of 22 species investigated using primers for the pol region, only Atlantic M. arenaria, Atlantic and North Sea razor clams Ensis directus, and Baltic clams Macoma balthica from the North Sea were found to possess copies of Steamer in their genomes. Notably, close relatives like Mya truncata and Siliqua patula did not exhibit evidence of Steamer. Amplified Steamer sequences were uniformly identical in all M. areneria specimens, and were highly variable across specimens of E. directus. Variation in the latter included nucleotide polymorphisms among and within individuals as well as length variation in 2 specimens corresponding to the deletion of a predicted stable hairpin structure. Results implicate Atlantic razor clams as the proximal source for horizontal transmission of Steamer among ecologically similar yet markedly distantly related bivalves. The consequences of cross-species transmission of the Steamer retrotransposon are unknown, and the finding of Steamer in 3 bivalve species suggests that further spread is possible.

Widespread transmission of independent cancer lineages within multiple bivalve species.

Most cancers arise from oncogenic changes in the genomes of somatic cells, and while the cells may migrate by metastasis, they remain within that single individual. Natural transmission of cancer cells from one individual to another has been observed in two distinct cases in mammals (Tasmanian devils and dogs), but these are generally considered to be rare exceptions in nature. The discovery of transmissible cancer in soft-shell clams (Mya arenaria) suggested that this phenomenon might be more widespread. Here we analyse disseminated neoplasia in mussels (Mytilus trossulus), cockles (Cerastoderma edule), and golden carpet shell clams (Polititapes aureus) and find that neoplasias in all three species are attributable to independent transmissible cancer lineages. In mussels and cockles, the cancer lineages are derived from their respective host species; however, unexpectedly, cancer cells in P. aureus are all derived from Venerupis corrugata, a different species living in the same geographical area. No cases of disseminated neoplasia have thus far been found in V. corrugata from the same region. These findings show that transmission of cancer cells in the marine environment is common in multiple species, that it has originated many times, and that while most transmissible cancers are found spreading within the species of origin, cross-species transmission of cancer cells can occur.

Horizontal transmission of clonal cancer cells causes leukemia in soft-shell clams.

Outbreaks of fatal leukemia-like cancers of marine bivalves throughout the world have led to massive population loss. The cause of the disease is unknown. We recently identified a retrotransposon, Steamer, that is highly expressed and amplified to high copy number in neoplastic cells of soft-shell clams (Mya arenaria). Through analysis of Steamer integration sites, mitochondrial DNA single-nucleotide polymorphisms (SNPs), and polymorphic microsatellite alleles, we show that the genotypes of neoplastic cells do not match those of the host animal. Instead, neoplastic cells from dispersed locations in New York, Maine, and Prince Edward Island (PEI), Canada, all have nearly identical genotypes that differ from those of the host. These results indicate that the cancer is spreading between animals in the marine environment as a clonal transmissible cell derived from a single original clam. Our findings suggest that horizontal transmission of cancer cells is more widespread in nature than previously supposed.

Activation of transcription and retrotransposition of a novel retroelement, Steamer, in neoplastic hemocytes of the mollusk Mya arenaria.

Bivalve mollusks of the North Atlantic, most prominently the soft shell clam Mya arenaria, are afflicted with an epidemic transmissible disease of the circulatory system closely resembling leukemia. The disease is characterized by a dramatic expansion of blast-like cells in the hemolymph with high mitotic index. Examination of hemolymph of diseased clams revealed high levels of reverse transcriptase activity, the hallmark of retroviruses and retroelements. By deep sequencing of RNAs from hemolymph, we identified transcripts of a novel retroelement, here named Steamer. The DNA of the element is marked by long terminal repeats and encodes a single large protein with similarity to mammalian retroviral Gag-Pol proteins. Steamer mRNA levels were specifically elevated in diseased hemocytes, and high expression was correlated with disease status. DNA copy number per genome was present at enormously high levels in diseased hemocytes, indicative of extensive reverse transcription and retrotransposition. Steamer activation in M. arenaria is an example of a catastrophic induction of genetic instability that may initiate or advance the course of leukemia.

Design and analysis of site-specific single-strand nicking endonucleases for gene correction.

Single-strand nicking endonucleases ("nickases") have been shown to induce homology-mediated gene correction with reduced toxicity of DNA double-strand break-producing enzymes, and nickases have been engineered from both homing endonuclease and FokI-based scaffolds. We describe the strategies used to engineer these site-specific nickases as well as the in vitro methods used to confirm their activity and specificity. Additionally, we describe the Traffic Light Reporter system, which uses a flow cytometric assay to simultaneously detect both gene repair and mutagenic nonhomologous end-joining outcomes at a single targeted site in mammalian cells. With these methods, novel nickases can be designed and tested for use in gene correction with novel target sites.

PARP-mediated repair, homologous recombination, and back-up non-homologous end joining-like repair of single-strand nicks.

Double-strand breaks (DSBs) in chromosomal DNA can induce both homologous recombination (HR) and non-homologous end-joining (NHEJ). Recently we showed that single-strand nicks induce HR with a significant reduction in toxicity and mutagenic effects associated with NHEJ. To further investigate the differences and similarities of DSB- and nick-induced repair, we used an integrated reporter system in human cells to measure HR and NHEJ produced by the homing endonuclease I-AniI and a designed 'nickase' variant that nicks the same target site, focusing on the PARP and HR repair pathways. PARP inhibitors, which block single-strand break repair, increased the rate of nick-induced HR up to 1.7-fold but did not affect DSB-induced HR or mutNHEJ. Additionally, expression of the PALB2 WD40 domain in trans acted as a dominant-negative inhibitor of both DSB- and nick-induced HR, sensitized cells to PARP inhibition, and revealed an alternative mutagenic repair pathway for nicks. Thus, while both DSB- and nick-induced HR use a common pathway, their substrates are differentially processed by cellular factors. These results also suggest that the synthetic lethality of PARP and BRCA may be due to repair of nicks through an error prone, NHEJ-like mechanism that is active when both PARP and HR pathways are blocked.

Association of maternal smoking during pregnancy with infant hospitalization and mortality due to infectious diseases.

BACKGROUND: Maternal smoking is associated with infant respiratory infections and with increased risk of low birth weight infants and preterm birth. This study assesses the association of maternal smoking during pregnancy with both respiratory and nonrespiratory infectious disease (ID) morbidity and mortality in infants. METHODS: We conducted 2 retrospective case-control analyses of infants born in Washington State from 1987 to 2004 using linked birth certificate, death certificate and hospital discharge records. One assessed morbidity--infants hospitalized due to IDs within 1 year of birth (47,404 cases/48,233 controls). The second assessed mortality--infants who died within 1 year due to IDs (627 cases/2730 controls). RESULTS: Maternal smoking was associated with both hospitalization (adjusted odds ratio [AOR] = 1.52; 95% confidence interval [CI]: 1.46, 1.58) and mortality (AOR = 1.51; 95% CI: 1.17, 1.96) due to any ID. In subgroup analyses, maternal smoking was associated with hospitalization due to a broad range of IDs including both respiratory (AOR = 1.69; 95% CI: 1.63, 1.76) and nonrespiratory IDs (AOR = 1.27; 95% CI: 1.20, 1.34). Further stratification by birth weight and gestational age did not appreciably change these estimates. In contrast, there was no association of maternal smoking with ID infant mortality when only low birth weight infants were considered. CONCLUSIONS: Maternal smoking was associated with a broad range of both respiratory and nonrespiratory ID outcomes. Despite attenuation of the mortality association among low birth weight infants, ID hospitalization was found to be independent of both birth weight and gestational age. These findings suggest that full-term infants of normal weight whose mothers smoked may suffer an increased risk of serious ID morbidity and mortality.

Single-strand nicks induce homologous recombination with less toxicity than double-strand breaks using an AAV vector template.

Gene targeting by homologous recombination (HR) can be induced by double-strand breaks (DSBs), however these breaks can be toxic and potentially mutagenic. We investigated the I-AniI homing endonuclease engineered to produce only nicks, and found that nicks induce HR with both plasmid and adeno-associated virus (AAV) vector templates. The rates of nick-induced HR were lower than with DSBs (24-fold lower for plasmid transfection and 4- to 6-fold lower for AAV vector infection), but they still represented a significant increase over background (240- and 30-fold, respectively). We observed severe toxicity with the I-AniI 'cleavase', but no evidence of toxicity with the I-AniI 'nickase.' Additionally, the frequency of nickase-induced mutations at the I-AniI site was at least 150-fold lower than that induced by the cleavase. These results, and the observation that the surrounding sequence context of a target site affects nick-induced HR but not DSB-induced HR, strongly argue that nicks induce HR through a different mechanism than DSBs, allowing for gene correction without the toxicity and mutagenic activity of DSBs.

Island biogeography reveals the deep history of SIV.

Simian immunodeficiency virus (SIV) lineages have been identified that are endemic to Bioko Island. The time the island formed offers a geological time scale calibration point for dating the most recent common ancestor of SIV. The Bioko viruses cover the whole range of SIV genetic diversity, and each Bioko SIV clade is most closely related to viruses circulating in hosts of the same genus on the African mainland rather than to SIVs of other Bioko species. Our phylogeographic approach establishes that SIV is ancient and at least 32,000 years old. Our conservative calibration point and analyses of gene sequence saturation and dating bias suggest it may be much older.