The clammy grip of parasitic tumors.

An epidemic of leukemia among bivalve molluscs is spreading along the Atlantic coast of North America, with a serious population decline of soft-shelled clams. In this issue of Cell, Metzger et al. use forensic DNA markers to demonstrate that the leukemia cells have a clonal origin and appear to be transmitted through sea water.

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.

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.

Gill epithelial cell nuclear virus disease is prevalent and widespread in Mya arenaria clams of Chesapeake Bay, USA.

Several historic investigations have reported intranuclear virus infections of Mya arenaria soft-shell clams from the Atlantic coast of North America, but their descriptive details are limited. Among numerous multi-clam samples of Chesapeake Bay M. arenaria that were analyzed histopathologically during clam population surveys from 2000-2009, virus replication apparently caused extreme hypertrophy among the infected nuclei of gill epithelial cells. Infected cells were often abundant within the gill epithelia of affected clams, where their nuclear abnormalities suggested compromised genetic controls of critical cellular physiological functions. Infection prevalences were generally elevated, reaching 90% in 25% of samples. A grand mean prevalence of 67% resulted for all (69) M. arenaria samples of the decadal investigation, which included 1934 individual clams. Infected nuclei of gill epithelial cells were microscopically conspicuous by their extreme hypertrophic diameters of 10 µm or more and their prominent DNA-inclusion bodies. Cells with abnormal, hypertrophic nuclei were often abundant in the epithelia of M. arenaria gills. Transmission electron microscopy revealed abundant, replicating, icosahedral viral particles of 65-85 nm diameter within such hypertrophic nuclei. Viruses frequently occurred in paracrystalline nuclear arrays, showed granular internal contents, and had radial structures that suggested capsid surface ornamentation. Normal heterochromatin of infected nuclei appeared emarginated by dense central masses of replicating virions. Large, electron-dense DNA inclusion bodies routinely occurred at the internal margins of virus-infected nuclei. These may be virus replication centers based on their ultrastructural features and close proximity to replicated viral particles.

Effects of variable oxygen regimes on mitochondrial bioenergetics and reactive oxygen species production in a marine bivalve, Mya arenaria.

Estuarine and coastal benthic organisms often experience fluctuations in oxygen levels that can negatively impact their mitochondrial function and aerobic metabolism. To study these impacts, we exposed a common sediment-dwelling bivalve, the soft-shell clam Mya arenaria, for 21 days to chronic hypoxia (PO2  âˆ¼4.1 kPa), cyclic hypoxia (PO2  âˆ¼12.7-1.9 kPa, mean 5.7 kPa) or normoxia (PO2  âˆ¼21.1 kPa). pH was manipulated to mimic the covariation in CO2/pH and oxygen levels in coastal hypoxic zones. Mitochondrial respiration, including proton leak, the capacity for oxidative phosphorylation (OXPHOS), the maximum activity of the electron transport system (ETS), reactive oxygen species (ROS) production, and activity and oxygen affinity of cytochrome c oxidase (CCO) were assessed. Acclimation to constant hypoxia did not affect the studied mitochondrial traits except for a modest decrease in the OXPHOS coupling efficiency. Cyclic hypoxia had no effect on OXPHOS or ETS capacity, but increased proton leak and lowered mitochondrial OXPHOS coupling efficiency. Furthermore, mitochondria of clams acclimated to cyclic hypoxia had higher rates of ROS generation compared with the clams acclimated to normoxia or chronic hypoxia. CCO activity was upregulated under cyclic hypoxia, but oxygen affinity of CCO did not change. These findings indicate that long-term cyclic hypoxia has a stronger impact on the mitochondria of M. arenaria than chronic hypoxia and might lead to impaired ATP synthesis, higher costs of mitochondrial maintenance and oxidative stress. These changes might negatively affect populations of M. arenaria in the coastal Baltic Sea under increasing hypoxia pressure.

Effects of prolonged food limitation on energy metabolism and burrowing activity of an infaunal marine bivalve, Mya arenaria.

Benthic organisms are subject to prolonged seasonal food limitation in the temperate shallow coastal waters that can cause energetic stress and affect their performance. Sediment-dwelling marine bivalves cope with prolonged food limitation by adjusting different physiological processes that might cause trade-offs between maintenance and other fitness-related functions. We investigated the effects of prolonged (42 days) food deprivation on bioenergetics, burrowing performance and amino acid profiles in a common marine bivalve, Mya arenaria collected in winter and spring. Food limitation of >15 days decreased respiration of the clams by 80%. Total tissue energy content was higher in spring-collected clams (reflecting higher lipid content) than in their winter counterparts. Prolonged food deprivation decreased the tissue energy content of clams, especially in winter. The levels of free amino acids transiently increased during the early phase of food deprivation possibly reflecting suppression of the protein synthesis or enhanced protein degradation. The levels of amino acids considered essential for bivalves were more tightly conserved than those of non-essential amino acids during starvation. The burrowing capacity of clams was negatively affected by food deprivation so that the time required for a burial cycle increased by 35-50% after 22-42 days of starvation. During the early phase of starvation, clams preferentially used lipids as fuel for burrowing, whereas carbohydrates were used at the later phase. These findings suggest that although M. arenaria can withstand prolonged food deprivation by lowering their basal maintenance costs and switching their fuel usage, their ecological functions (e.g. bioturbation and the energy transferable to the next trophic level) could be negatively impacted by starvation.

Transmissible Tumors: Breaking the Cancer Paradigm.

Transmissible tumors are those that have transcended the bounds of their incipient hosts by evolving the ability to infect another individual through direct transfer of cancer cells, thus becoming parasitic cancer clones. Coitus, biting, and scratching are transfer mechanisms for the two primary species studied, the domestic dog (Canis lupus familiaris) and the Tasmanian devil (Sarcophilus harrisii). Canine transmissible venereal tumors (CTVT) are likely thousands of years old, and have successfully travelled from host to host around the world, while the Tasmanian devil facial tumor disease (DFTD) is much younger and geographically localized. The dog tumor is not necessarily lethal, while the devil tumor has driven the population to near extinction. Transmissible tumors are uniform in that they have complex immunologic profiles, which allow them to escape immune detection by their hosts, sometimes for long periods of time. In this review, we explore how transmissible tumors in CTVT, DFTD, and as well as the soft-shell clam and Syrian hamster, can advance studies of tumor biology.

Effects of mechanical disturbance and salinity stress on bioenergetics and burrowing behavior of the soft-shell clam Mya arenaria.

Bioturbation of sediments by burrowing organisms plays a key role in the functioning of coastal ecosystems. Burrowing is considered an energetically expensive activity, yet the energy costs of burrowing and the potential impacts of multiple stressors (such as salinity stress and wave action) on bioenergetics and burrowing performance of marine bioturbators are not well understood. We investigated the effects of mechanical disturbance and salinity stress on the burrowing behavior, aerobic capacity and energy expense of digging in a common marine bioturbator, the soft-shell clam Mya arenaria from the Baltic Sea (control salinity 15). Mya arenaria showed large individual variability in the burrowing efficiency, with an average of ∼7% of the body energy reserves used per burial. Clams with higher mitochondrial capacity and lower energy expenditure per burial showed higher endurance. Acclimation for 3-4 weeks to low (5) or fluctuating (5-15) salinity reduced the burrowing speed and the number of times the clams can rebury but did not affect the mitochondrial capacity of the whole body or the gill. Acclimation to the fluctuating salinity shifted the predominant fuel use for burrowing from proteins to lipids. Our data indicate that the reduced burrowing performance of clams under the salinity stress is not due to the limitations of energy availability or aerobic capacity but must involve other mechanisms (such as impaired muscle performance). The reduction in the burrowing capacity of clams due to salinity stress may have important implications for survival, activity and ecological functions of the clams in shallow coastal ecosystems.

Insights into CNS ageing from animal models of senescence.

In recent years, novel model systems have made significant contributions to our understanding of the processes that control the ageing of whole organisms. However, there are limited data to show that the mechanisms that gerontologists have identified as having a role in organismal ageing contribute significantly to the ageing of the central nervous system. Two recent discoveries illustrate this particularly well. The first is the consistent failure of researchers to demonstrate a simple relationship between organismal ageing and oxidative stress--a mechanism often assumed to have a primary role in brain ageing. The second is the demonstration that senescent cells play a causal part in organismal ageing but remain essentially unstudied in a CNS context. We argue that the animal models now available (including rodents, flies, molluscs and worms), if properly applied, will allow a paradigm shift in our current understanding of the normal processes of brain ageing.

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.

Field and laboratory transmission studies of haemic neoplasia in the soft-shell clam, Mya arenaria, from Atlantic Canada.

A two-year laboratory and field study was initiated in 2001 in response to mass mortalities associated with haemic neoplasia (HN) in 1999 in Prince Edward Island (PEI) soft-shell clams, Mya arenaria. A laboratory proximity experiment (cohabitation) and an inoculation challenge were conducted with clams and mussels (Mytilus edulis). Three field exposure experiments were also conducted, in which naive clams were held in sediment (in trays) or out of sediment (in mesh bags) at three high HN prevalence sites on PEI. There was a conversion to HN positive in clams in the proximity experiment and in clams injected with whole blood and cell-free homogenate, but not at statistically significant levels. No mussels or control clams became HN positive. There was a significant conversion to HN positive in as little as 24 and 58 days after transfer with clams held out of sediment and in sediment, respectively. The laboratory and field experiments' results suggest that HN-infected clams are spreading the disease through water from infected clams to naïve individuals and via transplantation from affected to unaffected sites. Some environmental conditions (e.g. abnormally high water temperature and hypoxia-induced sea lettuce [Ulva lacteus] invasion) may make clams susceptible to infections or exacerbate the proliferation of HN.

Distribution of haemic neoplasia of soft-shelled clams in Prince Edward Island: an examination of anthropogenic factors and effects of experimental fungicide exposure.

Haemic neoplasia was first considered a disease of concern for soft-shell clams in Prince Edward Island (PEI) when it was diagnosed as the cause of mass mortalities in 1999. The aetiology of the disease remains elusive, but has been associated with environmental degradation. In this study, a 2-year (2001-2002) geographic and seasonal survey was conducted for haemic neoplasia, using histology, in soft-shell clams from PEI. In addition, using geographic information system, the association between anthropogenic factors in the watersheds at sites affected by haemic neoplasia and the prevalence of the disease was investigated. Finally, histopathological changes were assessed in soft-shell clams experimentally exposed to four concentrations of chlorothalonil for 27 days. Haemic neoplasia could not be induced at any concentration of chlorothalonil. Clams exposed to a concentration of 1000 µg L(-1) of the fungicide, however, exhibited an LC50 of 17 days. Although this information provides additional toxicity information (LC50) for soft-shell clams, further experiments are required to assess longer term exposure to the fungicide. The highest prevalences of haemic neoplasia in PEI were found in North River and Miscouche (28.3-50.9% and 33.0-77.8%, respectively). No clear seasonal patterns were found. There was a correlation between haemic neoplasia prevalence and watersheds with a high percentage of potato acreage and forest coverage (P = 0.026 and P = 0.045, respectively), suggesting a link between anthropogenic activity and the prevalence of the disease.

Tracking larvae with molecular markers reveals high relatedness and early seasonal recruitment success in a partially spawning marine bivalve.

The partial synchronized spawning strategy adopted by some marine invertebrate broadcast-spawners can lead to the production of many distinct pools of larvae within a single reproductive cycle. Following the fate of these larval groups from birth to settlement with molecular markers might shed light on mechanisms regulating their population recruitment. Larvae and recruits of Mya arenaria, a partially spawning marine bivalve, were monitored and collected over 13 consecutive weeks during an entire reproductive cycle. Each sampled individual (n = 218) was sorted according to size (early veligers, late veligers, post-larval recruits) and genotyped at seven microsatellite loci for comparisons among samples and with adult reference samples (n = 270). While traditional differentiation statistics (e.g., pairwise Δ(ST), allelic richness) suggested the absence of sweepstakes reproductive success, the level of relatedness found within and among larvae and recruit samples suggested otherwise. Four samples out of ten were observed to have higher within-sample relatedness values than randomly expected, including the very first group of early veligers produced in the season (E1) and the last group of post-larvae who survived recruitment (P10). E1 individuals were also found to be more related than randomly expected to individuals of more than 80% of all other samples including the last surviving recruits (P8 and P10). These results suggest that the first larvae produced in the season were the most successful to survive recruitment. Results also show direct evidence for larval retention and demonstrate for the first time larval and post-larval kin aggregation in a marine bivalve.

[Recent advances in transmissible tumors].

Transmissible tumors are a class of tumor that can be transmitted between individuals through living cells. So far, four types of transmissible tumors including canine transmissible venereal tumor (CTVT),Tasmanian devil facial tumor disease (DFTD), soft-shell clams leukemia (SSCL), and hamsters reticulum cell sarcoma (HRCS)have been discovered and identified. In the last decades, these transmissible tumors have been proved to be transmitted through living cells by cytological, histological and genetic studies. CTVT, the oldest mammalian somatic cell line, and DFTD originated from Schwann cell have been reported to avoid immunological recognition by down-regulating MHC expression, while a high copy number of Steamer retrotransposon is commonly exist in SSCL. In recent years, the whole-genome sequencing of CTVT and DFTD have been completed which facilitates studies on the mechanisms of tumorigenesis, transmission and evolution of transmissible tumors at the whole-genome level. In this review, we summarize the recent advances in transmissible tumors and discuss the research focus in next decade.

Using a metabolomics approach to investigate the sensitivity of a potential Arctic-invader and its Arctic sister-species to marine heatwaves and traditional harvesting disturbances.

Coastal species are threatened by fishing practices and changing environmental conditions, such as marine heatwaves (MHW). The mechanisms that confer tolerance to such stressors in marine invertebrates are poorly understood. However, differences in tolerance among different species may be attributed to their geographical distribution. To test the tolerance of species occupying different thermal ranges, we used two closely related bivalves the softshell clam Mya arenaria (Linnaeus, 1758), a cold-temperate invader with demonstrated potential for establishment in the Arctic, and the blunt gaper Mya truncata (Linnaeus, 1758), a native polar species. Clams were subjected to a thermal stress, mimicking a MHW, and harvesting stress in a controlled environment. Seven acute temperature changes (2, 7, 12, 17, 22, 27, and 32 °C) were tested at two harvesting disturbance intensities (with, without). Survival was measured after 12 days and three tissues (gills, mantle, and posterior adductor muscle) collected from surviving individuals for targeted metabolomic profiling. MHW tolerance differed significantly between species: 26.9 °C for M. arenaria and 17.8 °C for M. truncata, with a negligeable effect of harvesting. At the upper thermal limit, M. arenaria displayed a more profound metabolomic remodelling when compared to M. truncata, and this varied greatly between tissue types. Network analysis revealed differences in pathway utilization at the upper MHW limit, with M. arenaria displaying a greater reliance on multiple DNA repair and expression and cell signalling pathways, while M. truncata was limited to fewer pathways. This suggests that M. truncata is ill equipped to cope with warming environments. MHW patterning in the Northwest Atlantic may be a strong predictor of population survival and future range shifts in these two clam species. As polar environments undergo faster rates of warming compared to the global average, M. truncata may be outcompeted by M. arenaria expanding into its native range.

Selective initiation and transmission of disseminated neoplasia in the soft shell clam Mya arenaria dependent on natural disease prevalence and animal size.

Disseminated neoplasia, a diffuse tumor of the hemolymph system, is one of the six most destructive diseases among bivalve mollusk populations, characterized by the development of abnormal, rounded blood cells that actively proliferate. Though the specific etiology of disseminated neoplasia in Mya arenaria remains undetermined, the involvement of viral pathogens and/or environmental pollutants has been suggested and considered. The current study used 5-bromodeoxyuridine (BrDU) known to induce the murine leukemia virus and filtered neoplastic hemolymph to initiate disseminated neoplasia in clams from different populations and size classes respectively. M. arenaria from three locations of different natural neoplasia occurrences were divided into a control and three experimental treatments and injected with 200 µl of sterile filtered seawater or 50-200 µg/ml BrDU respectively. In a concurrent experiment, animals from different size classes were injected with 2.5% total blood volume of 0.2 µm filtered blood from a fully neoplastic animal. Animals were biopsied weekly and cell neoplasia development was counted and scored as 0-25, 26-50, 51-75 and 76-100% neoplastic hemocytes (stages 1-4) in 50 µl samples. BrDU injection demonstrated that neoplasia development in M. arenaria was dose dependent on BrDU concentration. In addition, natural disease prevalence at the source location determined initiation of neoplasia induction, with animals from the area of the highest natural disease occurrence displaying fastest neoplasia development (p=0.0037). This could imply that depending on the natural disease occurrence, a potential infectious agent may remain dormant in normal (stage 1) individuals in higher concentrations until activated, i.e. through chemical injection or potentially stress. The size experiment demonstrated that only M. arenaria between 40 and 80 mm developed 26-100% neoplastic hemocytes when injected with filtered neoplastic hemolymph, indicating that individuals smaller than 20mm or larger than 80 mm were not or no longer susceptible to disease development. So far neoplasia studies have not considered natural disease prevalence or size involvement in neoplasia development and our results indicate that these should be future considerations in neoplasia examinations.

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.

Evidence of polystyrene nanoplastic contamination and potential impacts in Mya arenaria clams in the Saint-Lawrence estuary (Canada).

Plastic materials found in the environment are expected to degrade into smaller plastic nanoparticles (NPs) posing a greater toxic risk because they sorb contaminants and pass physiological barriers. Moreover the presence and effects of NPs is difficult to tease out from the contamination background at polluted sites. The purpose of this study was to examine for the presence of polystyrene NPs in feral Mya arenaria clam population near anthropogenic sources of pollution and potential toxic effects. Polystyrene NPs were determined by a newly developed fluorescence-based and size exclusion chromatography methodologies. Clam health status was determined by following changes in air survival time, condition factor, growth, alcohol/aldehyde dehydrogenase (AADH), protein aggregation and lactate dehydrogenase (LDH). In addition, multi-elemental analysis in tissues was also determined. The results revealed that clams collected at 2 polluted sites contained elevated amounts of polystyrene-like NPs between 10 and 110 nm in size based on size exclusion chromatography. Elevated levels of AADH suggest the presence of hydroxylated products and were correlated with plastic NPs in tissues. Moreover, principal component analysis revealed that As, Ca, Cu, Sn and V were closely related to either polystyrene-like NPs in tissues or AADH activity. Although we cannot rule out other pollutants, clams contaminated by polystyrene-like NPs had lower condition, growth rate, air survival time and LDH activity. Increased metal/element contamination reported to sorb onto plastic polymers were also related to NPs in tissues. In conclusion, clams populations close to anthropogenic sources of pollution show evidence of polystyrene-like NPs contamination and could contribute to decreased clam health status.

Effects of different oxygen regimes on ecological performance and bioenergetics of a coastal marine bioturbator, the soft shell clam Mya arenaria.

Benthic species are exposed to oxygen fluctuations that can affect their performance and survival. Physiological effects and ecological consequences of fluctuating oxygen are not well understood in marine bioturbators such as the soft-shell clam Mya arenaria. We explored the effects of different oxygen regimes (21 days of exposure to constant hypoxia (~4.1 kPa PO2), cyclic hypoxia (~2.1-~10.4 kPa PO2) or normoxia (~21 kPa PO2)) on energy metabolism, oxidative stress and ecological behaviors (bioirrigation and bioturbation) of M. arenaria. Constant hypoxia and post-hypoxic recovery in cyclic hypoxia led to oxidative injury of proteins and lipids, respectively. Clams acclimated to constant hypoxia maintained aerobic capacity similar to the normoxic clams. In contrast, clams acclimated to cyclic hypoxia suppressed aerobic metabolism and activated anaerobiosis during hypoxia, and strongly upregulated aerobic metabolism during recovery. Constant hypoxia led to decreased lipid content, whereas in cyclic hypoxia proteins and glycogen accumulated during recovery and were broken down during the hypoxic phase. Digging of clams was impaired by constant and cyclic hypoxia, and bioirrigation was also suppressed under constant hypoxia. Overall, cyclic hypoxia appears less stressful for M. arenaria due to the metabolic flexibility that ensures recovery during reoxygenation and mitigates the negative effects of hypoxia, whereas constant hypoxia leads to depletion of energy reserves and impairs ecological functions of M. arenaria potentially leading to negative ecological consequences in benthic ecosystems.

Comparative toxicity of micro and nanopolystyrene particles in Mya arenaria clams.

The contamination of coastal marine environments by plastics of sizes ranging from mm down to the nanoscale (nm) could pose a threat to aquatic organisms. The purpose of this study was to examine the toxicity of polystyrene nanoparticles (PsNP) of various sizes (50, 100 and 1000 nm) to the marine clams Mya arenaria. Clams were exposed to concentrations of PsPP for 7 days at 15 °C and analyzed for uptake/transformation, changes in energy metabolism, oxidative stress, genotoxicity and circadian neural activity. The results revealed that PsNP accumulated in the digestive gland was 50 nm > 100 nm > 1000 nm. All sized increased oxidative stress as follows: 50 nm (peroxidase, antioxidant potential and LPO), 100 nm (LPO and antioxidant potential) and 1000 nm (LPO). Tissue damage was also size dependent by increasing genotoxicity. The 100 nm PsPP altered the levels of the circadian metabolite melatonin. We conclude that the toxicity of plastics is size dependent in clams.