Disseminated neoplasia in cockles Cerastoderma edule: ultrastructural characterisation and effects on haemolymph cell parameters

Disseminated neoplasia (DN) has been detected in cockles from various beds in Galicia (NW Spain). A study was performed to characterise cockle neoplastic cell ultrastructure and to evaluate the effect of this disease at different severity stages on various haemolymph cell parameters. Examination of cockle neoplastic cells with transmission electron microscopy (TEM) showed round shapes and a lack of pseudopods, a high nucleus:cytoplasm diameter ratio, Golgi complexes, abundant mitochondria, ribosomes, and numerous endoplasmic reticulum tubes and electron-lucent vesicles. Various haemolymph cell parameters (cell mortality, non-specific esterase and lysosome biovolume, reactive oxygen intermediates [ROI] production, phagocytosis ability, intracellular Ca2+ and actin levels) were compared between DN severity categories by flow cytometry; haemocyte mortality, non-specific esterase activities and lysosome biovolume were found to be higher with increasing DN severity. The phagocytic ability of neoplastic cells was sharply reduced with regard to haemocytes. The cytoplasmic-free Ca2+ level was higher and actin content lower in haemolymph cells of diseased cockles compared to unaffected ones. A significant increase in ROI production was detected in later stages of disease progression.     

Prevalence, intensity, and aneuploidy patterns of disseminated neoplasia in cockles (Cerastoderma edule) from Arcachon Bay: Seasonal variation and position in sediment

The present report presents the first evidence of disseminated neoplasia (DN) in cockles Cerastoderma edule from Arcachon Bay (France). Aneuploidy of neoplastic cells allowed the use of flow cytometry (FCM) to diagnose and stage DN. A 1 year survey (2007) of the prevalence and intensity (% of aneuploid circulating cells in neoplastic cockles) was conducted. Prevalences ranged from 2.2% (June) to 13.6% (May), and disease intensity ranged from 18.7% (June) to 95.5% (September). These percentages were not correlated with seawater temperature, but rather showed unexplained oscillations over the year. Prevalence and intensity of DN were higher in cockles found at the surface of sediment compared to those buried normally (11.8% vs. 6.7% and 53.0% vs. 40.6%, respectively, p < 0.05). DN could thus be one mechanism leading to unexplained presence of cockles at the surface of the sediment in Arcachon Bay. Ploidy characteristics of neoplastic cells were also investigated using FCM, revealing an unusual, broad continuum of ploidy distribution from 1.6 to 9.6n. Ploidy values were not in whole numbers in contrast to the rounded values reported in other studies. Ploidy varied according to DN intensity, with the ploidy distribution of neoplastic cells from lightly-diseased cockles being unimodal (3.7n median). In contrast, highly-diseased cockles showed a bimodal ploidy distribution (3.0n and 4.7n medians). This suggests that, in cockles from Arcachon Bay, mechanisms leading to aneuploidy are complex, developing during disease progression.     

Influence of disseminated neoplasia, trematode infections and gametogenesis on surfacing and mortality in the cockle Cerastoderma edule

Cerastoderma edule is a widely distributed bivalve mollusc, commercially exploited throughout Europe and is also an important food source for birds and crustaceans. Recently, mass surfacing and mortalities of cockles have been observed and reported at sites in Ireland and elsewhere, particularly in the summer months. One such site is Flaxfort Strand, Courtmacsherry Bay, County Cork, Ireland, an important feeding area used by many seabirds during the summer months. For the past few years large numbers of surfaced cockles have been observed at the site in a moribund condition. Samples of cockles from this area were collected over the summer months and their health status assessed. Cockles that had surfaced (moribund) and those still buried in the sediment were quantified and screened: sex, gonadal maturity and size class of cockles were also determined. Disseminated neoplasia and trematodes were observed in screened cockles. The most significant finding during the study was that mortalities and surfacing of cockles was related to a greater incidence of disseminated neoplasia. No neoplasia was observed in the smallest and largest size classes. There was a significantly higher prevalence of neoplasia in moribund cockles than in buried cockles, whereas in both groups a similar concentration of trematode metacercariae was observed in the screened tissues. Also, most of the cockles that had surfaced were either in the process of spawning or were spent. Overall a much larger percentage of moribund cockles exhibited both trematode infections plus neoplasia compared with buried cockles. A combination of the presence of neoplasia and trematodes, along with stress related to spawning, may immunocompromise the cockless, causing the animals to surface and become moribund.     

The course and mortality of a hematopoietic neoplasm in the soft-shell clam,Mya arenaria

Results from two experiments containing approximately 280 Mya arenaria indicated that significantly higher (P < 0.05) mortality occurred within the neoplastic clam population than in the nonneoplastic clam population. Using an in vivo blood cytological technique, five levels of severity were established. The levels were based on the number of neoplastic cells in the circulation with level 1 as the lowest severity and level 5 as the highest severity. Neoplastic clams that were diagnosed as the lowest level had higher survival rates (60%) than those clams diagnosed as the highest level (0%). The hematopoietic neoplasm in M. arenaria followed one of three courses: (1) in approximately 50% of the neoplastic clams the disease progressed to a higher severity and resulted in death; (2) in approximately 40% of the neoplastic clams the disease was chronic, i.e., remained at a stable level; and (3) in approximately 10% of the neoplastic clams the disease diminished in severity or disappeared entirely. In addition, 10% of the clams diagnosed as nonneoplastic at the beginning of the experiments were neoplastic by the termination of the experiment. The contraction of the disease may have been de novo or by stimulation of latent infections.The prevalence of the neoplasm in clams collected from an epizootic area followed a biphasic seasonal pattern. The highest prevalences occurred in October, November, and in May. The hematopoietic neoplasm in M. arenaria was also age and species specific.     

Effects of digenean trematodes and heterotrophic bacteria on mortality and burying capability of the common cockle Cerastoderma edule (L.)

Population dynamics of marine invertebrates is controlled by a variety of abiotic and biotic factors. Among these, some have received lesser attention from marine ecologists because of their ‘discrete’ nature. This is the case of parasitism and bacterial load. In the present study, we focused on the role that both digenean trematodes and heterotrophic aerobic bacteria might play in the mortality and burying behaviour of cockles, Cerastoderma edule.The bivalves were sampled monthly during 1 year from two sites in Arcachon Bay (French Atlantic coast). Mortality rates were assessed after transferring in the laboratory normally buried and unburied (i.e. found lying at the sediment surface at low tide) cockles. Their digenean and bacterial loads were determined for both positions (normally buried and unburied). Mortality rate was significantly higher for cockles found out of the sediment at low tide, suggesting that this abnormal position was a prelude to cockles' death. Comparison of digenean load of cockles showed no significant difference between buried and unburied bivalves. In contrast, bacterial load was significantly higher in unburied cockles than in normally buried animals. The effect of high concentration of a marine bacterial strain (Pseudomonas fluorescens) on cockles' burying behaviour and mortality was tested in the laboratory. Results showed that these bacteria could trigger the emergence of animals from the sediment but did not cause cockles' death. These field observations and laboratory experiments suggest that bacteria, rather than digenean trematodes, could play a role in the emergence of cockles and, hence, affect their survival in the wild.     

PNA: a marker of neoplastic progression and differentiation in the gastro-intestinal tract

We examined 35 cases of stomach carcinoma and 40 cases of colonic carcinoma with PNA associated with peroxidase (peanut agglutinin, lectin which binds to the terminal disaccharide galactose beta (1,3)-N-acetil-galacto-samine). In this way evaluation of the functional aspects of the normal-neoplastic sequence was undertaken. This method was carried out for histological and ultrastructural investigations. The results obtained in both cases showed a different reactivity in the evolution of neoplastic disease: in fact, positivity in dysplasia is finely granular intracytoplasmic, whereas in well-differentiated neoplastic transformation such a reactivity is preferentially localized along the cellular membranes, with restoration of gross positivity in the cytoplasm for the poorly-differentiated neoplasm. We therefore believe PNA to be a marker not only of neoplastic progression but of differentiation as well: we also hypothesize it to reveal glycoprotein groups with possible antigenic power, involved in immunologic interactions between tumor and host.     

Parasite‐induced surfacing in the cockle Austrovenus stuchburyi: adaptation or not?

Parasite manipulation of host behaviour is a compelling example of the extended phenotype. However, in many cases, such manipulation may be incorrectly assumed. Previous work has demonstrated that Austrovenus stuchburyi cockles stranded on mud-flat surfaces due to an inability to re-burrow both contain significantly more metacercariae of the trematode Curtuteria australis and are predated by the definitive host of this parasite at a faster rate than burrowed cockles. These results have been interpreted as strong evidence for a manipulation of cockle behaviour by the trematode to facilitate transmission to the definitive host. The model presented here, however, indicates that the selective advantage to the parasite of the altered host behaviour is currently of a negligible level at our study site that is highly unlikely to have been realized as an adaptation over evolutionary time. Hence, there are no grounds on which the more parsimonious explanation, that the altered host behaviour observed is simply an incidental side-effect of infection, can be rejected. We thus maintain that for any change in the behaviour of infected hosts to be confirmed as potentially a parasite trait that has evolved in response to selection, the adaptive benefit taking into account the entire parasite life cycle may need to be considered.     

The decrement of carcinogenesis by dietary selenium and expression of placental form of glutathione-S-transferase in rat glioma

Supranutrition dietary levels of the element selenium (Se) that have been shown to reduce or retard tumor development resulting from transplantation. The rat placental form of glutathione-S-transferase (GST-p) has been reported to be a good marker for preneoplastic or neoplastic lesions. Four groups of rats with glioma were exposed to Se-free, 0.05, 2.0, and 4.0 ppm sodium selenite GST-p was investigated. Normal brain tissue did not differ significantly in all groups. In contrast, GST-p in tumor was significantly higher in Se-free and 4.0-ppm groups compared to 0.5- and 2.0-ppm groups. The concentration of Se in normal brain tissue did not differ significantly in Se-supplement groups. By contrast, Se in tumors was significantly higher in the 0.5- and 2.0-ppm groups compared to the Se-free and 4.0-ppm groups. Mean group survival at 30 d after treatment was determined and compared with previous dietary Se. Survival was significantly longer in the 0.5- and 2.0-ppm groups than in the Se-free and 4.0-ppm groups. The 2.0-ppm group had enhanced survival, similar to the 0.5-ppm group. The Se-free and 4.0-ppm groups might have no protection against carcinogenesis.     

Short-term adaptation of digestive processes in the cockle Cerastoderma edule exposed to different food quantity and quality

Feeding and digestive parameters were analysed in cockles Cerastoderma edule fed for 3 days on two foods of different qualities, both foods given in two different concentrations. With low quality food, gut content was found to increase with ingestion rate. Such increased capacity of the gut to allocate food precludes negative effects upon throughput time, and so absorption efficiency remained nearly constant at the two food concentrations. With high quality food, gut content remained at high constant values and consequently enhancement of food ingestion rate with a high food ration leads to a significant reduction in throughput time, resulting in lower absorption efficiencies. Significantly higher levels of amylases and cellulases have been found within the digestive gland of cockles fed high quality diets. Coincidentally, absorption of carbohydrates is increased and absorption of lipids decreased in such diets as compared to low quality diets. Implications of the positive correlation between digestive enzyme activity and food quality are discussed in relation to the role that both digestive investments and endogenous faecal losses play in digestive processes. Results obtained in this study indicate that investments in the form of digestive enzymes are a key factor in the functional response of cockles to short-term variations in the food regime. Accepted: 13 September 1997     

Cockle emergence at the sediment surface: 'favourization' mechanism by digenean parasites?

The aim of the present work was to assess the effect of digenean trematodes on indirect mortality of the cockle Cerastoderma edule, an infaunal bivalve. The tested hypothesis was that parasites altered the burrowing capacity of cockles and thus exposed them at the sediment surface, where they are more vulnerable to predators. If the predator is the final host, this mechanism, which drives the cockle out of the sediment, is considered as a 'favourization'. Cockle populations from 2 stations in Arcachon Bay (France)-Banc d'Arguin (oceanic situation) and La Canelette (lagoonal situation)--were sampled for 1 yr. At La Canelette, monitoring every 2 d showed that 50% of adult cockles regularly migrated to the sediment surface at a rate of 5 cockles m(-2) yr(-1) and disappeared in a few days. In the laboratory, 67% of these 'surface cockles' did not burrow again, suggesting that they would die in the field. Moreover, mortality measured after 7 d in the laboratory was 2 to 5 times higher than mortality of 'buried cockles', at both stations and particularly during summer. Species richness and abundance of digeneans from both stations were compared in 'buried' and 'surface' individuals to determine whether parasites played a role in cockle migration and mortality. Ten and 9 digeneans were found at Banc d'Arguin and La Canelette, respectively, with Himasthla quissetensis and Labratrema minimus being the most prevalent and abundant species at both stations. The abundance of H. quissetensis was slightly higher in surface cockles at Banc d'Arguin, but the difference fluctuated with station and cockle age (or size). L. minimus prevalence was only higher in surface cockles at La Canelette. In the latter station, we estimated that L. minimus and H. quissetensis were responsible for the emergence of 9 and 2%, respectively, of the buried cockles. Although this favourization mechanism may induce some mortality in cockles, it does not alone explain the magnitude of the observed mortalities (41 and 57% at La Canelette and Banc d'Arguin, respectively). A correspondence analysis did not show the presence of a particular parasite community in buried or surface cockles, which could explain these high surface cockle mortalities in association with the 2 dominant digeneans.     

Fetal antigens as tumor growth enhancing factors in Yoshida's tumor rats

Malignant neoplastic cells have been shown to have some antigenic features identical to those of embryonic cells. Since several antigens are likely to be shared by both embryonic cells and neoplastic tissue, we tried to understand the meaning of the appearance of such antigens and the type of effect that the immunization with embryonic antigens would have on the survival of Yoshida's tumor rats. Wistar rats were immunized with fetal antigens by fetal cells (1.5 x 10(6)) suspended in 0.5 ml of Hanks solution plus an equal volume of Freund adjuvant, were injected in hind footpads, i.p. and i.m., respectively, for active immunization. Rabbit antigen sera were used for passive immunization. All animals presented ascites and tumor growth. Animals immunized by means of fetal cell antigens showed a mean survival rate after neoplastic transplant of 14 days. Animals that received rabbit immune serum showed a mean survival rate after neoplastic transplant of 17 days. The immunization by means of fetal antigens elicited a scanty effect on the survival of Yoshida's tumor transplanted rats. It can be concluded that antibodies, which are able to cross react with neoplastic cells, do not have cytotoxic effect and do not interfere with the survival of the neoplastic transplanted animals. Therefore, fetal antigens are likely able to carry out an immunosuppressive action. The fact that they appear on neoplastic cells could be seen as a metabolic modification effect or as a growth enhancing factor.     

Parasites boosts biodiversity and changes animal community structure by trait-mediated indirect effects

Parasitism has long been emphasised as an important process structuring animal communities. However, empirical evidence documenting the impact of parasites in other than simple laboratory settings is lacking. Here we examine the trait-mediated indirect effects of echinostome trematodes on a New Zealand soft bottom intertidal community of macroinvertebrates. Curtuteria australis and a second related but undescribed trematode both utilise the cockle Austrovenus stutchburyi as second intermediate host in which the parasites infect the foot tissue. Heavily infected cockles are therefore more sessile than lightly infected individuals, and, unable to bury, often rest on the sediment surface. We utilised these behavioural changes in two long term field experiments, respectively manipulating the parasite load of buried cockle (i.e. bioturbation), and the density of surfaced cockles (i.e. surface structures and seabed hydrodynamics). Both high parasite loads in buried cockles and the presence of surfaced cockles increased species richness and generally also the density of certain species and of major systematic and functional groups of benthic macroinvertebrates. Species diversity (alpha) peaked under intermediate densities of surfaced cockles. Our results demonstrate that parasites, solely through their impact on the behaviour of a single community member, can be significant determinants of animal community structure and function.     

Prey depletion and foraging strategy in the Oystercatcher Haematopus ostralegus

Summary The responses of a population of Oystercatchers to their own depletion of their prey, the edible cockle Cerastoderma edule, have been examined in Strangford Lough, Northern Ireland., Cockle stocks were severely depleted each winter as a result of predation by Oystercatchers and about half the birds present in October in the main study area had left by March: the reduction was greater when the initial population of Oystercatchers was high than when it was low. Oystercatchers were initially widespread around the Lough but tended to aggregate by January into a few good sites. Within each site annual differences in the location of feeding effort were correlated with year to year variation in the location of second winter cockles.Within the feeding areas in a bay the birds apparently hunted in the short-term on the basis of expectation, ceasing to crop, the densest cockle beds once an average yield for the area has been removed; this yield was apparently estimated from the local density of the anvils on which captured cockles were broken open. This effect led to marginal areas being depleted of cockles more rapidly than high density areas, so that the birds gradually concentrated their hunting onto an increasingly restricted area of the bay. The birds initially took only second winter and larger cockles but by late winter cockle densities had fallen so much that smaller cockles were accepted: areas previously abandoned or unused for hunting were then economically viable because of the reduced expectations of the birds and were incorporated into the feeding areas.These results are seen as consistent with Royama's profitability theory.     

From first to second and back to first intermediate host: the unusual transmission route of Curtuteria australis (Digenea: Echinostomatidae)

The trematode Curtuteria australis uses the whelk Cominella glandiformis as first intermediate host and the cockle Austrovenus stutchburyi as second intermediate host before maturing in shorebirds. The whelk also happen to be an important predator of cockles on intertidal mudflats. In this study we show that whelks can act as temporary paratenic hosts for the trematode. A single whelk feeding on 1 cockle can ingest large numbers of metacercariae, which remain within the whelk for 1-3 days before passing out in feces. The viability of these metacercariae assessed as the percentage capable of successfully excysting under conditions simulating those inside a bird's digestive tract, is lower after passage through a whelk (48%) than before (59%). Still, given that shorebird definitive hosts prey on whelks as well as cockles, survival inside the whelk allows C. australis to complete its life cycle: overall, though, whelk predation is likely to be an important sink for the trematode population. To our knowledge, this is the first report of a trematode using a snail as both first intermediate host and paratenic host, offering an alternative transmission route for the parasite as a result of the unusual trophic relationships of its hosts.     

Ambient fauna impairs parasite transmission in a marine parasite-host system

To understand possible factors controlling transmission of trematode larvae between first and second intermediate hosts we examined the impact of ambient fauna on parasite transmission in a marine intertidal parasite-host association. Cockle hosts (Cerastoderma edule) kept together with selected co-occurring macrozoobenthic species in mesocosms acquired a lower parasite load compared to cockles kept alone, when targeted by cercariae of the trematode Himasthla elongata. The reduction of parasite load in the cockles differed between the 7 macrozoobenthic species tested and was between 35 and 91%. Three different types of reduction could be distinguished: (1) predators (Carcinus maenas, Crangon crangon) actively preying upon cercariae, (2) non-host filter feeders (Crepidula fornicata, Mya arenaria, Crassostrea gigas) filtering cercariae but not becoming infected and (3) alternative hosts (Mytilus edulis, Macoma balthica) becoming infected by the cercariae and thus distracting cercariae from the target hosts. In addition, interference competition may occur in the form of disturbance of cockles by ambient organisms resulting in lower filtration rates and subsequently lower parasite loads. Our results suggest that the species composition and relative abundance of the ambient fauna of parasite-host systems play an important role in controlling trematode transmission rates in benthic marine systems.     

Equal partnership: two trematode species, not one, manipulate the burrowing behaviour of the New Zealand cockle, Austrovenus stutchburyi

Metacercariae of the trematode Curtuteria australis (Echinostomatidae) accumulate in the foot of the New Zealand cockle Austrovenus stutchburyi, severely impairing the cockle's ability to burrow under the sediments. This results in increased predation by birds on cockles, and thus enhanced transmission rates of the parasite to its bird definitive hosts. This host manipulation by the trematode is costly: fish regularly crop the tip of the foot of cockles stranded on the sediment surface, killing any metacercariae they ingest. A second, previously undetected trematode species (characterized by 23 collar spines) co-existing with C. australis, has been found in the foot of cockles in the Otago Harbour, South Island, New Zealand. The relative abundance of the two species varies among localities, with the identity of the numerically dominant species also changing from one locality to the next. Both C. australis and the new species have a strong preference for encysting in the tip of the cockle's foot, where their impact on the burrowing ability of the host is greatest, and where they both face the risk of cropping by fish. Results indicate that these two species are ecological equivalents, and their combined numbers determine how the cockle population is affected.     

Historic discovery of natural thermodynamic cause of cancer

The essence of life is best manifested in cell, which, when brought to the edge of its existence in the actual environment may and sometimes must self-organise into an entirely different cell (neoplasm), but it must enhance dissipation of matter and energy in its closest environment. This phenomenon has been described before as self-organisation of dissipative structures in physics, chemistry and even sociology. Each neoplastic cell is such a dissipative system - with its clonal growth, the cell causes increasing disorganisation of the body, in consequence leading to neoplastic disease. The only adequate cause of formation of neoplasms is an internal dissipathogenic cellular state, which is clinically identify as preneoplastic ones at the level of morphology or molecular biology but also biophysics. Two general directions for therapy of neoplastic diseases arise from the thermodynamic essence of neogenesis: the direct one - targeting neoplasms, and the indirect one - leading to normalisation or sufficient alteration of their environment. The greatest disappointment in the fight against neoplasm was the discovery of its thermodynamic cause in a natural self-organisation of biological dissipative structures. It is this dissipation that causes the signs and symptoms of neoplastic diseases ending with destruction of the body if the treatment comes too late and/or is insufficient, limited only to removal of neoplastic lesions without the always necessary elimination and/or prevention of preneoplastic (dissipathogenic) states.     

Parasite-induced trophic facilitation exploited by a non-host predator: a manipulator's nightmare

Parasites with complex life cycles, relying on trophic transmission to a definitive host, very often induce changes in the behaviour or appearance of their intermediate hosts. Because this usually makes the intermediate host vulnerable to predation by the definitive host, it is generally assumed that the parasite's transmission rate is increased, and that the modification of the host is, therefore, of great adaptive significance to the parasite. However, in the ecological "real world" other predators unsuitable as hosts may just as well take advantage of the facilitation process and significantly erode the benefit of host manipulation. Here we show that the intertidal New Zealand cockle (Austrovenus stutchburyi), manipulated by its echinostome trematode (Curtuteria australis) to rest on the sediment surface fully exposed to predation from the avian definitive host, is also subject to sublethal predation from a benthic feeding fish (Notolabrus celidotus, Labridae). The fish is targeting only the cockle-foot, in which the parasite preferentially encysts, reducing the infection intensity of manipulated cockles to levels comparable with those in non-manipulated, buried cockles. Based on the frequency and intensity of the foot cropping and predation rates on surfaced cockles by avian hosts, it is estimated that 2.5% of the parasite population in manipulated cockles is transmitted successfully whereas 17.1% is lost to fish. We argue that the adaptive significance of manipulation in the present system depends critically on the feeding behaviour of the definitive host. If cockles constitute the majority of prey items, there will be selection against manipulation. If manipulated cockles are taken as an easily accessible supplement to a diet composed mostly of other prey organisms, behavioural manipulation of the cockle host appears a high risk, high profit transmission strategy. Both these feeding behaviours of birds are known to occur in the field.     

Towards a Case Definition for Devil Facial Tumour Disease: What Is It?

In the mid 1990s an emerging disease characterised by the development of proliferative lesions around the face of Tasmanian devils (Sarcophilus harrisii) was observed. A multi-disciplinary approach was adopted to define the condition. Histopathological and transmission electron microscopic examination combined with immunohistochemistry help define Devil Facial Tumour Disease (DFTD) as a neoplastic condition of cells of neuroendocrine origin. Cytogenetic analysis of neoplastic tissue revealed it to be markedly different from normal devil tissue and having a consistent karyotype across all tumours examined. Combined with evidence for Major histocompatability (MHC) gene analysis there is significant evidence to confirm the tumour is a transmissible neoplasm.