Assessment of haemic neoplasia in different soft shell clam Mya arenaria populations from eastern Canada by flow cytometry

Diagnosis of haemic neoplasia (HN) in the soft shell clam, Mya arenaria, is often achieved by hematocytology and histology. Since neoplastic cells display tetraploid DNA contents, haemocyte cell cycle analysis was developed for use as a diagnosis tool. The aim of this study was to assess the application of a flow cytometry procedure of cell cycle analysis established for the common cockle, to clams and to evaluate different thresholds of value for the percentage of tetraploid cells for establishing HN disease status of individual clams and clam populations. HN status of six clam populations from eastern Canada was determined. Results of the present study demonstrate a flow cytometry procedure to be useful for HN diagnosis in clams. Individual clams were considered to be affected by HN when presenting at least 20% of haemocytes in S-4N phase; and negative when presenting less that 5% of haemocytes in S-4N phase. As discussed in this paper, intermediate cases represent uncertain diagnoses including either false-negative or false-positive clams, which are difficult to discriminate. At a population level, an additional threshold of 15% for the mean intensity of the disease is proposed, which means having in the population several individual clams presenting more than 20% of their haemocytes in S-4N phase. Based on these thresholds of value, only one population was considered as free of HN disease, and one population was unequivocally affected by HN. For the four other clam populations, further investigations are needed toward development and use of specific and objective biomarkers of HN.     

Behavioral plasticity of the soft-shell clam, Mya arenaria (L.), in the presence of predators increases survival in the field

Soft-shell clams, Mya arenaria, are sessile, suspension-feeding bivalves that are preyed upon by the exotic green crab, Carcinus maenas. Clams evade crab consumers by burrowing deeper into the sediment after perceiving a threat from a nearby predator. The purpose of this study was to determine the types of signals that M. arenaria use to detect predators and the types of behaviors clams use to avoid being eaten. In a field study, clams increased their burial depth in the presence of green crab predators consuming conspecifics that were caged nearby, and also increased burial depth after artificial tactile stimulation in the laboratory assay. These results indicate that clams can use chemical and mechanical cues to detect potential predatory threats. We performed a field study to examine the difference in survivability of clams that had burrowed deeper into the sediment in response to predators vs. control clams that were burrowed less deeply. Significantly higher survival rates were observed in clams that had initially burrowed more deeply, suggesting that increasing burial depth is a valid predator avoidance strategy. Some bivalves also alter their pumping rates in the presence of predators, making them less apparent and providing more structural defense by covering soft tissue, and we measured pumping time of soft-shell clams in the presence and absence of predators, when burrowing was not an option for escape. Soft-shell clams did not alter their pumping time in the presence of green crab predators, possibly because they employ a burrowing method called “hydraulic” or “jet-propelled” burrowing, where it is necessary for the clam to pump in order to burrow. Chemical signals and tactile cues instigated behavioral changes in M. arenaria, and this change in behavior (increasing burial depth) increased clam survival in the field.     

Differential in vivo response of soft-shell clam hemocytes against two strains of Vibrio splendidus: Changes in cell structure, numbers and adherence

Host-pathogen interaction models in aquatic species are useful tools for understanding the pathogenicity of diseases in cultured and wild populations. In this study we report the differential in vivo response of soft-shell clam (Mya arenaria) hemocytes against two strains of Vibrio splendidus. Responses were measured 24 h after injecting into the posterior adductor muscle either an endemic wild-type strain (7SHRW) or a strain associated with oyster mortalities (LGP32-GFP). Changes in hemocyte structure (percentage of rounded cells) were assessed microscopically. Changes in adherence and hemocyte numbers were analyzed by flow-cytometric cell counting. Increased percentages of rounded cells were found in response to both strains. However, values from the group infected with LGP32-GFP were significantly higher (p < 0.01) than with 7SHRW. The cell adherence was markedly diminished (p < 0.001) by LGP32-GFP whereas 7SHRW did not change it significantly. Increased numbers of hemocytes (p < 0.001) were induced by LGP32-GFP, while no significant changes were found after infection with 7SHRW. These results show the regulatory capacity of soft-shell clams hemocytes to perform specific responses against different strains of V. splendidus.     

Characterization of aspartate transcarbamylase activity from gonads of the soft shell clam,Mya arenaria

Aspartate transcarbamylase (ATCase, EC 2.1.3.2) has been shown to be a good index of the reproductive cycle in marine molluscs. However, this enzyme has never been studied in the soft shell clam Mya arenaria. The characteristics of gonadal ATCase of the soft shell clam, Mya arenaria were therefore determined since we need powerful tools to assess the degree of effects of endocrine disruptors in this species at risk. Enzyme kinetic values observed at pH 8.3 were significantly lower than those measured at pH 9.4. The optimal conditions for the enzyme assays were reached in the presence of a 10 mM of substrate concentration and at pH 9.2 for 60 min at 37 °C. We have found that the enzyme was heat sensitive, markedly activated by DMSO and DMF, but no effect was observed with ethanol, ATP or CTP. However, clam ATCase activity was partly inhibited by the addition of CuSO₄ and PHMB to the medium, an inhibition that could be attributed to the presence of SH sites in cysteine residues localized in the catalytic site of this enzyme. All these results will be very useful in the near future to study the gametogenetic process of Mya arenaria, since little is known about the factors that control the physiological process of reproduction in this bivalve of ecological and economic importance. Studies of variations of the activity of aspartate transcarbamylase will also be useful as a potential biomarker to evaluate the disruption of gametogenesis in clams exposed to endocrine disruptors in situ.     

Up-regulation of NFkappaB-responsive gene expression by DeltaNp73alpha in p53 null cells

Transactivation domain (TAD)-truncated p73, DeltaNp73, associates with p53, resulting in suppression of p53's functions. Using p53 null cell lines, we examined whether or not DeltaNp73 can regulate gene expression in a p53-independent manner. When DeltaNp73alpha was co-transfected with a luciferase reporter plasmid with various enhancer elements, NFkappaB-responsive luciferase gene expression was selectively up-regulated by DeltaNp73alpha, but not by other p73-isoforms with TAD and DeltaNp73beta. Deletion of the TAD endowed p73alpha with the ability to enhance the responsive gene's expression, but deletion of the N-terminal proline-rich domain (PRD) rendered the TAD-deleted p73alpha inactive. Considering the inability of DeltaNp73beta, which is the C-terminus-truncated form of DeltaNp73alpha, to function, these results indicate that both the PRD and C-terminus are necessary for DeltaNp73alpha to can activate NFkappaB-responsive luciferase expression. Over-expression of p53 suppressed the TAD-truncated p73alpha-mediated luciferase expression, suggesting that p53 interferes with the TAD-truncated p73alpha-mediated activation of NFkappaB. Inhibitors for NFkappaB activation reduced the TAD-truncated p73alpha-dependent NFkappaB-responsive gene expression, indicating that TAD-truncated p73alpha activates NFkappaB as does TNFalpha. In addition to the results obtained in the reporter gene assay, TAD-truncated p73alpha stimulated the translocation of NFkappaB to the nucleus and the expression of an endogenous NFkappaB-responsive gene, Bcl-XL. Taken together, these results demonstrate that TAD-truncated p73alpha can activate NFkappaB.     

Predation on soft-shell clams (Mya arenaria) by the nemertean Cerebratulus lacteus in Atlantic Canada: implications for control measures

The nemertean, Cerebratulus lacteus Verrill (Nemertinea: Heteronemertini), has been identified as an important threat to soft-shell clam (Mya arenariaL.) populations in Atlantic Canada. The biology of this species, however, is still largely unknown. Field and laboratory studies were undertaken in 1998 and 1999 in Prince Edward Island, Canada, to test certain control measures to reduce predation on soft-shell clam populations and to better describe the relationship between C. lacteus and M. arenaria. Field abundance of C. lacteus, M. arenaria and Nereis virens Sars were evaluated in relation to particular habitat modifications that were used as control measures. Sediment manipulations tested were: (1) addition of shells and (2) use of a hydraulic rake. No difference was observed on the abundance of C. lacteus, M. arenaria and N. virens after treatments were applied. In the laboratory, C. lacteus was shown to be an efficient predator of M. arenaria. Clam mortalities reached 100% in the presence of C. lacteus while 0% mortality was observed in its absence. A complementary set of experiments was carried out to see if the sympatric polychaetes N. virens and Glycera dibranchiata Ehlers had any impact on the relationship between C. lacteus and M. arenaria. N. virens showed no impact on C. lacteus predation on clams. The presence of G. dibranchiata significantly reduced the nemertean predation rate. Analysis of clam size selection revealed no significant preference by C. lacteus. Other experimental studies revealed that high predator densities did not impede predation on clams and that C. lacteus preferred soft-shell clams among other commercial bivalve species presented (Mercenaria mercenariaL., Crassostrea virginica Gmelin and Mytilus edulisL.). This study should provide a better understanding of the relationship between C. lacteus and M. arenaria and lead to the development of improved nemertean control measures.     

Relative importance of predation and intraspecific competition in regulating growth and survival of juveniles of the soft-shell clam, Mya arenaria L., at several spatial scales

Predation appears to be the single most important biotic factor regulating populations of bivalves in estuarine and coastal soft sediments. However, the relative roles of predation and intraspecific competition are rarely investigated simultaneously over different spatial scales, making generalities about these mechanisms difficult. Using juveniles of the soft-shell clam, Mya arenaria (initial mean shell length [SL] ± 95% CI = 12.4 ± 0.13 mm), I tested the interactive effects of predator deterrence and intraspecific density (660 vs. 1320 individuals m^− 2) on growth and survival responses over a 185-day period from May to November 2003 at spatial scales that spanned four orders of magnitude: embayments, sites within embayments, tidal gradients, and blocks that were 10,000's, 1000's, 100's, and 5 m apart, respectively. Replicate field experiments were conducted from May to November 2003 at the upper and lower tidal heights at each of two intertidal mud flats (sites) within each of two embayments (Passamaquoddy Bay [PB] and Cobscook Bay [CB]) in eastern Maine.Mean survival, relative growth, and the abundance of wild recruits each varied significantly over all spatial scales. Predation was the most important factor affecting clam survival, explaining 45% of the total variability, whereas embayment, sites within embayments, tidal gradient, and intraspecific density collectively accounted for less than 10% of the variation. At all four intertidal sites, clam survival in experimental units designed to deter predators averaged 72%, but the degree of enhancement varied between embayments (PB = 61%; CB = 267%). Average survival rate was higher (by 12-16%), but growth was slower (by ca. 50%) in upper vs. lower intertidal blocks; however, the patterns differed for both variables between sites within each embayment. The effect of increasing intraspecific clam density was to lower survival by ca. 17% (56% [660 m^− 2] vs. 48% [1320 m^− 2]) in both embayments, but growth was unaffected. Overall, clams doubled in SL, although mean relative growth was 15% greater in CB than PB. Tidal gradient, sites within embayments, and blocks were the three most important factors explaining 35%, 19%, and 22% of total variation in relative clam growth, respectively. In Maine and the northeast US, juveniles of Mya reach their highest abundance above mean low tide levels. Experimental evidence presented here suggests that differential predation along the tidal gradient is the dominant factor controlling clam abundance and distribution patterns in the intertidal zone.     

Limited genetic variation and structure in softshell clams (Mya arenaria) across their native and introduced range

To offset declines in commercial landings of the softshell clam, Mya arenaria, resource managers are engaged in extensive stocking of seed clams throughout its range in the northwest Atlantic. Because a mixture of native and introduced stocks can disrupt locally adapted genotypes, we investigated genetic structure in M. arenaria populations across its current distribution to test for patterns of regional differentiation. We sequenced mitochondrial cytochrome oxidase I for a total of 212 individuals from 12 sites in the northwest Atlantic (NW Atlantic), as well as two introduced sites, the northeast Pacific (NE Pacific), and the North Sea Europe (NS Europe). Populations exhibited extremely low genetic variation, with one haplotype dominating (65–100%) at all sites sampled. Despite being introduced in the last 150–400 years, both NE Pacific and NS Europe populations had higher diversity measures than those in the NW Atlantic and both contained private haplotypes at frequencies of 10–27% consistent with their geographic isolation. While significant genetic structure (F _ST = 0.159, P < 0.001) was observed between NW Atlantic and NS Europe, there was no evidence for genetic structure across the pronounced environmental clines of the NW Atlantic. Reduced genetic diversity in mtDNA combined with previous studies reporting reduced genetic diversity in nuclear markers strongly suggests a recent population expansion in the NW Atlantic, a pattern that may result from the retreat of ice sheets during Pleistocene glacial periods. Lack of genetic diversity and regional genetic differentiation suggests that present management strategies for the commercially important softshell clam are unlikely to have a significant impact on the regional distribution of genetic variation, although the possibility of disrupting locally adapted stocks cannot be excluded.     

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.     

Germline p53 single-base changes associated with Balkan endemic nephropathy

The Balkan endemic nephropathy (BEN) is a significant clinical and scientific problem in need of novel effective therapies. Though many genetic and environmental factors have been investigated the basis, cause, and predisposition to BEN are still unclear. In this study, based on the hypothesis that the genetic pathways leading to BEN might be associated with p53 dysfunction, we screened for p53 gene mutations 90 Bulgarian BEN patients using optimized PCR-SSCP-sequencing analysis. Germline p53 single-base changes were found in blood samples in 10% of BEN cases. Three of them caused amino acid substitutions (p.Arg283Cys, p.Gln317His, and p.Lys321Glu); the other six were either synonymous amino acid substitutions (p.Arg213Arg) or intron polymorphisms (T14766C). To the best of our knowledge, these are the first data investigating tumor suppressor gene mutations in patients with BEN. The obtained results are in support of our hypothesis that p53 gene alterations are possibly involved in BEN genetic pathways.     

Isolation of a viral agent causing hematopoietic neoplasia in the soft-shell clam,Mya arenaria

A virus from neoplastic soft-shell clams, Mya arenaria, has been isolated. Its morphological and physical properties are similar to those of B-type retroviruses. It is enveloped, pleomorphic, averaging 120 nm in size with an eccentric nucleoid. Possible A-type particles have also been observed. The bouyant density of the particle ranged from 1.17 to 1.18 g/cm³ and the ultraviolet absorption $\text{260}\text{280}\text{-}\text{nm}$ ratio was 1.23. The virus has been shown to cause a hematopoietic neoplasm in Mya. Purified virus from neoplastic clams was innoculated into nonneoplastic clams. Most of these clams developed tumors within 2 months. Virus isolated from inoculated clams which developed neoplasia was inoculated into nonneoplastic clams. Most of these clams also developed neoplasia thus completing Koch's postulates.     

The expression of phosphatidic acid phosphatase 2a, which hydrolyzes lipids to generate diacylglycerol, is regulated by p73, a member of the p53 family

p73, a p53-related gene, is essential for a development of animals, while p53 is important for tumor formation. And little is known about the target genes specifically regulated by p73. Identifying the specific targets of p73 is important to understand the physiological roles of p73. To identify the genes specifically regulated by p73, we conducted serial analysis of gene expression to quantitatively evaluate messenger RNA populations. We found that the gene for phosphatidic acid phosphatase 2a (PAP2a), an enzyme that hydrolyzes lipids to generate diacylglycerol, was specifically upregulated by ectopic production of p73beta. The promoter region of this gene contains an element that is functionally responsive to p73beta. And the quantity of PAP2a protein was upregulated by ectopic production of p73beta. These results suggest that the expression of PAP2a is directly regulated by p73.     

5-bromodeoxyuridine induction of hematopoietic neoplasia and retrovirus activation in the soft-shell clam, Mya arenaria

Hematopoietic neoplasia and virus replication were induced in shoft-shell clams, Mya arenaria, by 5-bromodeoxyuridine (5-BrdUrd). Eighty clams were found to be nonneoplastic by the in vivo bleeding method. These clams were randomly distributed into four aquaria and maintained in seawater at 1°C. 5-BrdUrd was added to aquaria in the following concentrations: 0, 20, 100, and 200 μg/ml. After 4 days of treatment, the aquaria were drained, rinsed, and fresh sea water without 5-BrdUrd was added. Sea water was changed on a weekly basis thereafter. The clams were diagnosed for neoplasia by the in vivo method at days 4, 9, 16, and 23 of the experiment. Results showed that on day 23, neoplasia was not found in the aquarium without 5-BrdUrd, but in the aquaria containing 20, 100, and 200 μg/ml of 5-BrdUrd the incidences of neoplasia were 37, 79, and 35%, respectively. 5-BrdUrd-induced neoplastic tissue was homogenized and subjected to differential ultracentrifugation. Retrovirus-like particles resembling ones previously shown to induce neoplasia were isolated from neoplastic clams which were induced by 5-BrdUrd. When these particles were inoculated into healthy clams, the clams developed neoplasia. Neoplastic hemocytes, cultured in sea water containing 50 μg/ml of 5-BrdUrd, formed pseudopodia after 4 days. Pseudopod formation is a trait of normal hemocytes. This indicates that differentiation of neoplastic hemocytes may be induced by the drug.