Perkinsus chesapeaki in stout razor clams Tagelus plebeius from Delaware Bay

Perkinsus chesapeaki is reported from stout razor clams Tagelus plebeius in Delaware Bay, extending the known range of P. chesapeaki north of Chesapeake Bay. P. marinus, which causes dermo disease, is prevalent in cultured and wild oysters at this site, but was not detected in T. plebeius. Evidence for the presence of disseminated neoplasia, also reported from Chesapeake Bay, was equivocal. Although P. chesapeaki infections were associated with mortality events, light infection intensities and a general lack of histopathological evidence of disease limit inferences about a causal relationship. A comparison of Ray's fluid thioglycollate medium (RFTM)-based and PCR-based detection assays highlight differences in detection capabilities related to the quantity and type of tissue processed rather than assay sensitivity per se, a point that should be considered when surveying populations for disease prevalence. Investigators are further cautioned to use care when applying and interpreting diagnostic assays when used with novel species.     

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.     

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.     

Selected enzyme activities in Mya arenaria hemolymph.

1. The activities of lysozyme, acid and alkaline phosphatases, beta-glucuronidase, amylase, lipase, glutamate-oxalacetate transaminase, and glutamate-pyruvate transaminase in the whole hemolymph and 4000 g pellets and supernatants of Mya arenaria were determined. 2. All of these enzymes, except for amylase, occurred in whole hemolymph as well as in the 4000 g pellet and supernatant. 3. Based on earlier observations, these enzymes are believed to be of cellular origin within hemolymph cells. 4. In the case of amylase, it only occurred in the whole hemolymph and/or serum and is believed to have originated from the crystalline style.     

Sex alteration in soft-shell clams (Mya arenaria) in an intertidal zone of the Saint Lawrence river (Quebec,Canada)

The purpose of this study was to verify whether any changes in sex ratio might occur in soft-shell clams (Mya arenaria) located in an intertidal harbor zone located at the mouth of the Saguenay Fjord in the Saint Lawrence estuary (Baie Sainte-Catherine (BSC), Québec, Canada) likely to be contaminated by organotin compounds. Bivalves were harvested at the BSC harbor site and from two reference sites. Condition index (weight to length ratio), gonado-somatic index, sex ratio, vitellin-like proteins, organotin concentrations in gonad tissue, maturation stages of the gonads, the number of estradiol-17beta binding sites and the capacity of female gonad extracts to produce estradiol-17beta were determined in collected animals. Results showed that sex ratio in clams was significantly skewed toward males. Moreover, the condition and gonad-somatic indices, vitellin-like proteins in female gonads and the capacity of female gonads to produce estradiol-17beta were significantly reduced at the harbor site with respect to the reference sites. Maturation status of male gonads was clearly delayed at the harbor site. Additionally, gonad tissue contained tributyltin (TBT) at an average level of 109+/-18 ngSn/gdry wt. at the harbor site while organotins were not detected from the reference sites. Finally, female gonads had a higher number of unoccupied estradiol binding sites at the harbor site indicating low levels of this steroid in this tissue. Overall, this paper is first to report that clams collected in the vicinity of a TBT contaminated harbor are subject to masculinizing effects which seems to be consistent with biological effects that organotins are known to exert toward some other marine invertebrates.     

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.     

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.     

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.     

Field and laboratory comparisons of mortality in normal and neoplastic Mya arenaria

The results of a 6-month mark and recapture experiment involving approximately 900 adult Mya arenaria demonstrated that under natural conditions, significantly higher (P much less than .001, chi 2 test) mortality occurred among animals with neoplasia than those diagnosed as normal. Using a blood screening technique, the clams were diagnosed and placed in one of three diagnostic groups based on the severity of the disease (the percentage neoplastic cells per total number of blood cells): Nonneoplastic (NN), 0%; low severity neoplastic (LSN), less than 50%; and high severity neoplastic (HSN), greater than 50%. Fifty-one percent of those clams initially diagnosed as HSN died by the end of the test period as compared to 8% of the LSN clams and only 3% of the normals. Both progression and remission of the disease were also evident. Approximately 10% of the clams in the NN and LSN groups progressed to a LSN or HSN condition, whereas 16% of those clams initially identified as LSN, and that were recovered alive, underwent complete remission during the test period. Comparison of the field results with those of an 18-week laboratory study suggests that studies of mortality done under laboratory conditions may not provide useful data for the interpretation of the quantitative effects of a disease process, such as molluscan neoplasia, on the natural population of the animal studied.     

Spatial and Temporal Distribution of Two Diazotrophic Bacteria in the Chesapeake Bay

The aim of this study was to initiate autecological studies on uncultivated natural populations of diazotrophic bacteria by examining the distribution of specific diazotrophs in the Chesapeake Bay. By use of quantitative PCR, the abundance of two nifH sequences (907h22 and 912h4) was quantified in water samples collected along a transect from the head to the mouth of the Chesapeake Bay during cruises in April and October 2001 and 2002. Standard curves for the quantitative PCR assays demonstrated that the relationship between gene copies and cycle threshold was linear and highly reproducible from 1 to 10⁷ gene copies. The maximum number of 907h22 gene copies detected was approximately 140 ml⁻¹ and the maximum number of 912h4 gene copies detected was approximately 340 ml⁻¹. Sequence 912h4 was most abundant at the mouth of the Chesapeake Bay, and in general, its abundance increased with increasing salinity, with the highest abundances observed in April 2002. Overall, the 907h22 phylotype was most abundant at the mid-bay station. Additionally, 907h22 was most abundant in the April samples from the mid-bay and mouth of the Chesapeake Bay. Despite the fact that the Chesapeake Bay is rarely nitrogen limited, our results show that individual nitrogen-fixing bacteria have distinct nonrandom spatial and seasonal distributions in the Chesapeake Bay and are either distributed by specific physical processes or adapted to different environmental niches.     

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.     

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.     

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.     

Soft-Shell Clam, Mya arenaria, a Convenient Laboratory Animal for Screening Pathogens of Bivalve Mollusks

Attempts to introduce infectious or foreign material into oysters and other bivalve mollusks usually involve force or trauma because of immediate, prolonged adduction of the tightly closing valves. The soft-shell clam, Mya arenaria, is unable to seal its valves completely and relaxes readily, exposing soft tissue and a large siphon. This species is free from fouling organisms and is readily available at all seasons in the New England and mid-Atlantic areas. Suspensions of five strains of Vibrio sp. that cause bacillary necrosis in larval and juvenile bivalve mollusks were injected into the heart, siphon tissue, and the incurrent and excurrent siphon lumina of soft-shell clams. All vibrio strains caused significant mortality, usually within 2 days. Heaviest losses resulted from heart and excurrent siphon injections. No mortality occurred in control clams injected with seawater, broth, Serratia sp., and Escherichia coli. The soft-shell clam appears to be a useful animal for testing the pathogenicity of marine microorganisms for bivalve mollusks.