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.     

Cell types and hydrolytic enzymes of soft shell clam (Mya arenaria) hemocytes

Hemocytes of the soft shell clam, Mya arenaria, based on appearance after Romanowsky-type staining, can be shown to be either granulocytes or agranulocytes comprising 76.5 and 23.5% of the total cell population, respectively. Cytoplasmic granules are basophilic, eosinophilic, or refractile. Cytochemical studies indicate that these cells are markedly heterogeneous with respect to certain hydrolytic lysosomal enzymes and in cytoplasmic glycogen and lipofuscin. The overall activities of these enzymes in clam hemocytes, as estimated by the number of reactive sites, were unrelated to one another. Using consecutive double-staining techniques, individual cells were also found to vary in their enzyme content. These findings emphasize the biochemical individuality of circulating hemocytes and the variations noted probably reflect differences in number and composition of lysosomes.     

Seasonal gonad progesterone pattern in the soft-shell clam Mya arenaria

Steroidogenesis, which plays a major role in the reproductive cycle of vertebrates, is still for the most part, unknown in invertebrates. The aim of this study was to examine the link between progesterone and the reproductive cycle in Mya arenaria. The soft-shell clams, Mya arenaria were collected in Anse à l'Orignal (Parc Provincial du Bic, Québec, Canada) from July to November 1998. Histological data have shown that female gonads of M. arenaria were in the spawning stage in August and September, while the male gonads were in the ripe stage. This period of active gametogenesis was associated with a depletion of lipid reserves. These lipids could be used as a source of energy and as a substrate for steroidogenesis. Liquid Chromatography coupled to Mass Spectroscopy (LC-MS) and quantified by Enzyme Linked ImmunoSorbent Assay (ELISA) determined progesterone. Progesterone levels in the gonad were increased during the ripe stage in the male and during the spawning stage in the female. These results indicate, for the first time, that progesterone, as in vertebrates, may play a role in the reproductive cycle of M. arenaria.     

The effects of sediment type on growth rate and shell allometry in the soft shelled clam Mya arenaria L

Hatchery-reared juvenile Mya arenaria L. were grown for 11 wk in replicated gravel, sand, mud, and pearl net treatments under flow-through sea-water conditions in Maine. Analyses of variance showed significant differences between sediment treatments for final shell length, dry meat weight, chondrophore growth increment, and percent shell weight. Growth of juvenile M. arenaria was more rapid in fine sediments than in coarse sediments or nets.Regression slopes of shell length-shell height and shell length-shell depth varied significantly between sediment treatments. Slower-growing clams from nets and gravel were more globose than clams from sand or mud treatments. Clams grown in sand were longer and narrower than those from mud. Differences in growth rates and shell form were attributed primarily to the physical properties of the substrata, and their effects on the scope for growth of M. arenaria.     

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.     

Seasonal aspects of sarcomatous neoplasia in Mya arenaria (soft-shell clam) from Long Island Sound

Mya arenaria were collected monthly for 2.5 years from three populations in Long Island Sound. Histopathological examination revealed that 6.1% of the clams from Stonington, Connecticut, 12.9% of the clams from the Saugatuck River, Westport, Connecticut, and 12.7% of those from Old Mill Beach, Westport, also in Connecticut, had sarcomatous neoplasms. This is the first documented account of the occurrence of clam neoplasm in populations from this geographic area. Peak prevalences of 45, 59, and 60%, respectively, were found in clams from the three study sites. The prevalence of neoplasms in clams collected from three epizootic areas showed a pronounced seasonal pattern, with the highest incidences occurring in the late fall-winter of each year studied. The regular, seasonal occurrence of neoplasia in field populations does not support the hypothesis that pollution alone is the cause of the disorder.     

Bioaccumulation and depuration of enteroviruses by the soft-shelled clam, Mya arenaria.

Low levels of feces-associated natural virus, simulating virus numbers estimated to exist in moderately polluted shellfish-growing waters, were used to evaluate the effectiveness of depuration as a virus depletion procedure in soft-shell clams. Depuration effectiveness depended upon the numbers of virus bioaccumulated and whether virus was solids associated. Virus uptake was greatest when viruses were solids associated and pollution levels were equivalent or greater than those likely to be found in grossly polluted growing waters. Virtually all bioaccumulated feces-associated natural virus was deposited within either the hepatopancreas or siphon tissues. Viruses usually were eliminated within a 24- to 48-h depuration period. Dependence upon depuration of clams to elimate health hazards of virus etiology involved a risk factor not measureable in the study. The greatest reduction of health risks would come from the routine depuration of clams harvested from growing waters of good sanitary quality.     

Bioaccumulation and depuration of enteroviruses by the soft-shelled clam, Mya arenaria.

Low levels of feces-associated natural virus, simulating virus numbers estimated to exist in moderately polluted shellfish-growing waters, were used to evaluate the effectiveness of depuration as a virus depletion procedure in soft-shell clams. Depuration effectiveness depended upon the numbers of virus bioaccumulated and whether virus was solids associated. Virus uptake was greatest when viruses were solids associated and pollution levels were equivalent or greater than those likely to be found in grossly polluted growing waters. Virtually all bioaccumulated feces-associated natural virus was deposited within either the hepatopancreas or siphon tissues. Viruses usually were eliminated within a 24- to 48-h depuration period. Dependence upon depuration of clams to elimate health hazards of virus etiology involved a risk factor not measureable in the study. The greatest reduction of health risks would come from the routine depuration of clams harvested from growing waters of good sanitary quality.     

Lipase activity in the serum and hemolymph cells of the soft-shelled clam, Mya arenaria, during phagocytosis.

Quantitative determinations of lipase activity in the sera and hemolymph cell homogenates of Mya arenaria that had been challenged with heat-killed Bacillus megaterium and those of control clams have revealed that there is an increase in both fractions of the hemolymph during phagocytosis.The occurrence of lipase in serum and cells suggests that suitable substrates could be degraded by hydrolysis extracellularly as well as within phagocytes.     

Different proteins in the hemolymph sera from sarcomatous and healthy soft shell clams, Mya arenaria L.

1. Serum proteins showed quantitative and qualitative differences between sarcomatous and healthy soft shell clams, Mya arenaria L. 2. Total protein concentration was significantly higher in the serum of sarcomatous clams than of healthy clams. 3. According to SDS-PAGE, more serum proteins with more variability distinguished sarcomatous clams from healthy ones. 4. Sarcomatous clams had unique serum proteins of M(r)23,000, 45,000 and 54,000. Healthy clams had unique serum proteins of M(r) 24,000, 103,000 and 105,000. 5. During disease progression, sarcoma-specific proteins appeared while normal proteins disappeared. 6. We propose that some sarcoma-associated proteins may have tumor promoting and/or cytotoxic functions and that some normal proteins which disappear during disease progression may be involved in the humoral defense system.     

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.     

Aryl hydrocarbon receptor (AhR)-independent effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on softshell clam (Mya arenaria) reproductive tissue

A high prevalence of germinomas has been observed in certain populations of Mya arenaria from eastern Maine. The etiology of these tumors is unknown. We are investigating the hypothesis that exposure to environmental contaminants, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) contributes to gonadal carcinogenesis. Clams were exposed to TCDD with or without the initiating compound diethylnitrosamine (DEN) in an attempt to induce germinomas. A TCDD-dependent alteration in gametogenesis was observed in which 32.5+/-6.5% of individuals exhibited undifferentiated gonads. Analyses of AhR and p53 expression were carried out to identify similarities between naturally occurring neoplastic and TCDD (+/-DEN)-altered reproductive tissues. Neoplastic tissues had significantly less p53 protein than matched controls, whereas TCDD-induced undifferentiated samples exhibited no difference in p53 protein levels compared to controls. No gender-specific differences were observed in AhR mRNA, but there were significant differences in protein levels. AhR was undetectable in male gonadal tissue whereas females exhibited a significant positive relationship between AhR protein levels and stage of ovogenesis. Despite exhibiting some morphological similarity, we conclude the TCDD-induced pathology is not a germinoma. We further suggest the change in reproductive tissue is due to inhibition of cell differentiation and/or development by an AhR-independent mechanism.     

Long-term changes in cohort structure of the soft-shell clam Mya arenaria in the White Sea: growth rate affects lifespan and mortality

The population dynamics of a Mya arenaria cohort in the White Sea was studied throughout almost its entire duration (ca. 25 years). We aimed to study changes in the growth heterogeneity of the cohort in terms of individual growth rate and to find out whether there was a connection between the growth rate and both lifespan and mortality rate. Significant variation of the individual growth rate within the cohort of the soft-shell clams was detected. To a great extent it seemed to be determined by the characteristics of the growth achieved in the initial growth period (shell size by the start of the second growing season). Most of the cohort individuals (about 60%) had a similar growth rate during the study period. They belonged to the group of medium-growing clams and with a few exceptions represented the cohort at later stages of development (at the age of 16–24 years). In the most slow-growing group no individuals older than 15 years were found. Clams of this group had the highest mortality rate and the shortest lifespan. In all other groups, periods of low mortality alternated with periods of a much higher mortality. The survival of Mya arenaria seems to be affected mostly by the degree of protection (largely determined by burial depth), intraspecific relationships and ageing.     

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.     

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.