Effects of temperature on hard clam (Mercenaria mercenaria) immunity and QPX (Quahog Parasite Unknown) disease development: II. Defense parameters

Quahog Parasite Unknown (QPX) is a protistan parasite affecting hard clams Mercenaria mercenaria along the Northeastern coast of the United States. The geographic distribution and occurrence of disease epizootics suggests a primary role of temperature in disease development. This study was designed to investigate the effect of temperature on constitutive and QPX-induced defense factors in M. mercenaria. Control and QPX-challenged (both experimentally and naturally) clams were maintained at 13, 21 and 27 °C for 4 months. Control and experimentally-infected clams originated from a southern broodstock (Florida, no prior reports of disease outbreak) while naturally-infected clams originated from a northern broodstock (Massachusetts, enzootic area). Standard and QPX-specific cellular and humoral defense parameters were assessed after 2 and 4 months. Measured parameters included total and differential hemocyte counts, reactive oxygen species production, phagocytic activity of hemocytes, lysozyme concentration in plasma, anti-QPX activity in plasma and resistance of hemocytes to cytotoxic QPX extracellular products. Results demonstrated a strong influence of temperature on constitutive clam defense factors with significant modulation of cellular and humoral parameters of control clams maintained at 13 °C compared to 21 and 27 °C. Similarly, clam response to QPX challenge was also affected by temperature. Challenged clams exhibited no difference from controls at 27 °C whereas different responses were observed at 21 °C and 13 °C compared to controls. Despite differences in infection mode (experimentally or naturally infected) and clam origin (northern and southern broodstocks), similarities were observed at 13 °C and 21 °C between QPX infected clams from Florida and Massachusetts. Clam response to temperature and to QPX exhibited interesting relationship with QPX disease development highlighting major influence of temperature on disease development.     

Modulatory effects of hard clam (Mercenaria mercenaria) tissue extracts on the in vitro growth of its pathogen QPX

Quahog parasite unknown (QPX) is a fatal protistan parasite affecting cultured and wild hard clams Mercenaria mercenaria along the northeastern coasts of the USA and maritime Canada. Field investigations and laboratory transmission studies revealed some variations in the susceptibility of different hard clam stocks to QPX infection. In this study, we used in vitro QPX cultures to investigate the effect of plasma and tissue extracts from two different clam stocks on parasite survival and growth. Results demonstrated the presence of factors in clams that significantly modulate QPX growth. Extracts from gills and mantle tissues as well as plasma inhibited in vitro QPX growth, whereas foot and adductor muscle extracts enhanced parasite growth. Investigations of anti-QPX activities in plasma from two clam stocks displaying different susceptibility toward QPX disease in vivo demonstrated higher inhibition of QPX growth by plasma from New York (resistant) clams compared to Florida (susceptible) clams. Some clams appeared to be deficient in inhibitory factors, suggesting that such animals may become more easily infected by the parasite.     

Molecular diagnostics,field validation,and phylogenetic analysis of Quahog Parasite Unknown (QPX), a pathogen of the hard clam Mercenaria mercenaria

Quahog Parasite Unknown (QPX) is a protistan parasite that causes disease and mortality in the hard clam Mercenaria mercenaria. PCR primers and DNA oligonucleotide probes were designed and evaluated for sensitivity and specificity for the QPX organism specifically and for the phylum Labyrinthulomycota in general. The best performing QPX-specific primer pair amplified a 665 bp region of the QPX small-subunit ribosomal DNA (SSU rDNA) and detected as little as 1 fg cloned QPX SSU rDNA and 20 fg QPX genomic DNA. The primers did not amplify DNA of uninfected hard clams M. mercenaria or of the thraustochytrids Schizochytrium aggregatum, Thraustochytrium aureum, and T. striatum. The general labyrinthulomycete primers, which were designed to offer broader specificity than the QPX primers, amplified a 435 bp region of SSU rDNA from QPX, and a 436 to 437 bp region of SSU rDNA from S. aggregatum, T. aureum, and T. striatum, but did not amplify that of the clam M. mercenaria. Field validation of the QPX-specific primer pair, through comparative sampling of 224 clams collected over a 16 mo period from a QPX endemic site in Virginia, USA, indicated that the PCR assay is equivalent to histological diagnosis if initially negative PCR products are reamplified. Oligonucleotide DNA probes specific for QPX and the phylum Labyrinthulomycota were evaluated for in situ hybridization assays of cell smears or paraffin-embedded tissues. Two DNA probes for QPX offered limited sensitivity when used independently; however, when used together as a probe cocktail, sensitivity was greatly enhanced. The probe cocktail hybridized to putative QPX organisms in tissues of hard clams collected from Virginia, New Jersey, Massachusetts and Canada, suggesting that the QPX organisms in these areas are either very closely related or the same species. The QPX probe cocktail did not hybridize with clam tissue or with the thraustochytrids S. aggregatum, T. aureum, and T. striatum. The labyrinthulomycete DNA probe hybridized with QPX and the 3 thraustochytrids, with no background hybridization to clam tissue. SSU rDNA sequences were obtained for the putative QPX organisms from geographically distinct sites. Phylogenetic analyses based on the QPX and Labyrinthulomycota sequences confirmed earlier reports that QPX is a member of this phylum, but could not definitively demonstrate that all of the QPX organisms were the same species.     

Molecular genetic variation within and among isolates of QPX (Thraustochytridae), a parasite of the hard clam Mercenaria mercenaria

The thraustochytrid known as QPX (Quahog Parasite Unknown) has sporadically caused disease in the hard clam Mercenaria mercenaria along the east coast of North America since the 1960s. We hypothesized that genetically distinct QPX strains might be responsible for outbreaks of QPX disease in different areas and tested this hypothesis by comparing several QPX isolates recovered from the recent outbreak in Raritan Bay, New York with QPX strains isolated from 2 outbreaks in Massachusetts, USA. There was no variation in small subunit rDNA (SSU rDNA), 5.8S rDNA, or 4 mitochondrial gene sequences. In contrast, both of the ribosomal ribonucleic acid (rRNA) operon intergenic spacers, internal transcribed spacers 1 and 2 (ITS1 and ITS2), revealed substantial sequence variation. However, strain-specific sequences were not detected because the ITS sequence variation within QPX isolates was comparable to the variation between isolates. ITS1 sequences recovered from an infected clam by amplification with a QPX ITS2-specific primer were identical to those recovered from the QPX isolates.     

Effects of salinity on hard clam (Mercenaria mercenaria) defense parameters and QPX disease dynamics

QPX (Quahog Parasite Unknown) is a protistan parasite affecting hard clams (Mercenaria mercenaria) along the Northeast coast of the United States. The fact that QPX disease epizootics are usually observed in field sites with high salinities led to the general assumption that salinity represents an important factor for disease distribution. This study was designed to investigate the effect of salinity on QPX disease development as well as constitutive and QPX-induced defense factors in M. mercenaria. Naïve and QPX-infected (both experimentally and naturally) clams were submitted to 17 and 30 psu for 4 months. Standard and QPX-specific cellular and humoral defense parameters were assessed after 2 and 4 months. These included total and differential hemocyte counts, reactive oxygen species production, phagocytic activity of hemocytes, lysozyme concentration in plasma, anti-QPX activity in plasma and resistance of hemocytes to cytotoxic QPX extracellular products. Results demonstrated higher QPX-associated mortality in naturally infected clams maintained at high salinity compared to those held at 17 psu. Our findings also showed an increase in mortality following experimental challenge with QPX in clams submitted to 30 psu but not in those held at 17 psu. Constitutive clam defense factors and the response to QPX challenge were also affected by salinity. QPX challenge caused significant but transitory changes in hemolymph parameters that were obvious at 2 months but disappeared at 4 months. Overall, our results show that salinity modulates clam immunity and the progress of QPX disease although its impact appears secondary as compared to findings we reported earlier for temperature.     

Development of a real time quantitative PCR assay for the hard clam pathogen Quahog Parasite Unknown (QPX)

Quahog Parasite Unknown (QPX) is a thraustochytrid pathogen responsible for catastrophic mortalities of the northern quahog (hard clam) Mercenaria mercenaria. A real-time quantitative polymerase chain reaction (qPCR) assay was developed to assist research efforts on QPX ecology and pathology. Sensitivity of the assay was evaluated with serial dilutions of QPX-cultured cells to determine the lowest concentration of DNA that remained detectable in both the presence and absence of extraneous environmental substances. QPX cells were quantified before DNA extraction to calibrate standard curves to cell counts. Based on our results, the qPCR assay is able to quantify QPX within the range of 1 to several thousand organisms per reaction. Specificity of the assay was assessed by testing 29 thraustochytrid-like protists isolated from suspension-feeding bivalves from China, Oregon, Maryland, and Virginia. Application of the assay was demonstrated with positive qPCR results from naturally contaminated environmental samples including marine aggregates (i.e. marine snow), clam pseudofeces, and inflammatory nodules from infected clams. This quantitative assay for QPX will provide a valuable tool for characterizing QPX parasite abundances in coastal environments and for improving clam disease diagnostics.     

Effects of temperature on hard clam (Mercenaria mercenaria) immunity and QPX (Quahog Parasite Unknown) disease development: I. Dynamics of QPX disease

Quahog Parasite Unknown (QPX) causes disease and mortality in hard clams, Mercenaria mercenaria. Seasonality of QPX disease prevalence in the field and changes in QPX growth and survival in vitro suggest a role of temperature in the hard clam-QPX interaction and disease development. This study specifically examined the effect of temperature on QPX disease development and dynamics. Naturally and experimentally infected clams were separately maintained in the laboratory at 13 °C, 21 °C, or 27 °C for 4 months. Following this initial treatment, temperature was adjusted to 21 °C for 5 additional months to simulate seasonal changes of temperature in the field and to investigate the effect of temperature variations on QPX disease dynamics. Mortality was continuously monitored during the experiment and clams were sampled at 2, 4 and 9 months for the assessment of QPX disease prevalence and intensity using our standard histological and quantitative PCR techniques. Results demonstrated significantly higher QPX disease prevalence and intensity, as well as higher mortality, in naturally-infected clams maintained at 13 °C as compared to those held at 21 °C or 27 °C. Similarly, disease development was significantly higher in experimentally infected clams maintained at the colder temperature (70% prevalence after 4 months) as compared to those maintained under warmer conditions (<10%). Additionally, our results demonstrated an improvement in the condition of clams initially maintained at 13 °C for 4 months after transfer to 21 °C for 5 additional months, with a significant reduction of QPX prevalence (down to 19%). Interestingly, disease development or healing in clams maintained at different temperatures exhibited a strong relationship with clam defense status (jointly submitted paper) and highlighted the impact of temperature on clam activity and QPX disease dynamics. These findings should be taken into account for the timing of activities involving the monitoring, movement (e.g. relays, transplants) or grow out (e.g. commercial culture, municipal enhancement) of hard clams in enzootic areas.     

DGGE-based detection method for Quahog Parasite Unknown (QPX)

Quahog Parasite Unknown (QPX) is a significant cause of hard clam Mercenaria mercenaria mortality along the northeast coast of the United States. It infects both wild and cultured clams, often annually in plots that are heavily farmed. Subclinically infected clams can be identified by histological examination of the mantle tissue, but there is currently no method available to monitor the presence of QPX in the environment. Here, we report on a polymerase chain reaction (PCR)-based method that will facilitate the detection of QPX in natural samples and seed clams. With our method, between 10 and 100 QPX cells can be detected in 1 l of water, 1 g of sediment and 100 mg of clam tissue. Denaturing gradient gel electrophoresis (DGGE) is used to establish whether the PCR products are the same as those in the control QPX culture. We used the method to screen 100 seed clams of 15 mm, and found that 10 to 12% of the clams were positive for the presence of the QPX organism. This method represents a reliable and sensitive procedure for screening both environmental samples and potentially contaminated small clams.     

Protistan parasite QPX of hard-shell clam Mercenaria mercenaria is a member of Labyrinthulomycota

Biomass of the protistan parasite QPX (quahaug parasite X) of hard-shell clam Mercenaria mercenaria was enriched from in vitro culture. The nuclear gene encoding the 18S RNA of the small-subunit ribosomal (ssu-rDNA) was recovered using the polymerase chain reaction (PCR) and sequenced. Phylogenetic analysis clearly showed that QPX is a member of phylum Labyrinthulomycota, within which it appears as a specific relative of Thraustochytrium pachydermum. These results confirm the provisional assignment of QPX to the Labyrinthulomycota made previously on the basis of morphological and ultrastructural characters found in some, but not all, geographic isolates.     

Effect of environmental factors on survival and growth of quahog parasite unknown (QPX) in vitro

Quahog parasite unknown (QPX) is a protistan microorganism associated with mass mortalities of hard clams (Mercenaria mercenaria) along the northeastern coasts of the United States and maritime Canada. Because several studies indicate modulatory effects of prevailing environmental parameters on disease outbreaks, this study tested the effect of major environmental parameters (temperature, salinity and oxygen concentration; individually or combined) on QPX survival in artificial seawater and parasite growth in culture media in vitro. Three QPX isolates from two different geographic locations were compared. Results indicated that in vitro growth of QPX was optimal in standard culture medium at 34 ppt between 20 °C and 23 °C. Additionally, significant differences in temperature optima were observed for geographically distinct QPX isolates (p < 0.001) confirming previous studies suggesting the existence of different QPX strains (or ecotypes). When tested in seawater, QPX exhibited opposite trends with higher survival at 15 °C and 15 ppt. Results also demonstrated limited survival and growth of QPX under anoxic conditions. Additionally, results showed that the parasite is able to survive extreme temperatures (−12 °C to 32 °C) suggesting that QPX could overcome short periods of extreme conditions in the field. These results contribute to a better understanding of interactions between QPX and its environment, but potential impacts of environmental conditions on QPX disease development need further work as it also involves clam response to these factors.     

Environmental distribution and persistence of Quahog Parasite Unknown (QPX)

Quahog Parasite Unknown (QPX) is the cause of mass mortality events of hard clams Mercenaria mercenaria from Virginia, USA, to New Brunswick, Canada. Aquaculture areas in Massachusetts, USA, have been particularly hard hit. The parasite has been shown to be a directly infective organism, but it is unclear whether it could exist or persist outside of its clam host. We used molecular methods to examine water, sediment, seaweeds, seagrass and various invertebrates for the presence of QPX. Sites in Virginia and Massachusetts were selected based upon the incidence of QPX-induced clam die-offs, and they were monitored seasonally. QPX was detectable in almost all of our different sample types from Massachusetts, indicating that the parasite was widely distributed in the environment. Significantly more samples from Massachusetts were positive than from Virginia, and there was a seasonal pattern to the types of samples positive from Massachusetts. The data suggest that, although it may be difficult to completely eradicate QPX from the environment, it may be possible to keep the incidence of disease under control through good plot husbandry and the removal of infected and dying clams.     

Quantitative Real-Time PCR Assay for QPX (Thraustochytriidae), a Parasite of the Hard Clam (Mercenaria mercenaria)

We developed a real-time quantitative PCR (qPCR) assay targeting the rRNA internal transcribed spacer region of the hard clam pathogen QPX. The qPCR assay was more sensitive than was histology in detecting clams with light QPX infections. QPX was detected in 4 of 43 sediment samples but in none of 40 seawater samples.The thraustochytrid called QPX (for quahog parasite unknown) has caused high mortalities in hatchery-reared and wild hard clams (Mercenaria mercenaria, also known as quahogs) from Prince Edward Island (Canada) to Virginia (United States) since the late 1950s (17, 22, 25, 29). In the summer of 2002, QPX infections appeared in the previously healthy Raritan Bay (off the coast of Staten Island in New York) M. mercenaria population, causing significant clam mortality and closure of the fishery (6). Management of hard clam populations affected by QPX disease is hampered by an incomplete understanding of factors controlling the occurrence and severity of QPX infections. Environmental factors, such as salinity and temperature, appear to be important (22), as do clam population density and the planting of seed from nonlocal sources (7). More quantitative information about the occurrence and progression of QPX disease in relation to these and other variables would support better prediction of, and response to, QPX outbreaks. QPX is thought to be an opportunistic pathogen (4, 7, 11), capable of growing outside its host. However, there is very little known about substrates that might support QPX organisms outside of hard clams (4). The abilities to detect and enumerate QPX cells in potential reservoirs would allow the dynamics of the QPX organism in the environment to be related to the occurrence of QPX disease, offering new insight into fundamental questions about the natural transmission mechanisms of the infection.The 18S ribosomal DNA (rDNA) primer pair QPX-F and QPX-R2 can be used in a standard PCR assay to detect the presence of QPX DNA in clam tissue samples (26). Unfortunately, the products are too long (∼650 bp), and often include too much primer dimer, for use in a SYBR green real-time quantitative PCR (qPCR) assay. The low sequence variability in rRNA genes made it difficult to design other primers specific for QPX 18S rDNA. Instead, we used our previously reported rRNA internal transcribed spacer (ITS) region (including ITS1, the 5.8S rRNA gene, and ITS2) sequences for QPX isolates from Massachusetts and New York (20) to develop a qPCR assay targeting the more variable ITS region (1).     

Blood clam (Anadara inflata) red cells. partition in aqueoues two-polymer phase systems

Anadara inflata is a clam which has red blood cells in its hemolymph. Furthermore, the nucleated red blood cells contain two structurally distinct hemoglobins. Clam red cells were subjected to partition in aqueous dextran-polyethylene glycol two-phase systems with the following results:

1.

1. Clam red cells are the largest cells (about 20 μm in diameter) so far studied in two-polymer phases. It is shown that not only can such cells be partitioned in dextran-polyethylene glycol phase systems, but that countercurrent distribution resolves the clam red cell population into more and less metabolically active cells. The distribution of these cells in relation to the whole population is similar to that of young and old red cells from mammals.     

Prevalence of Perkinsus marinus (dermo), Haplosporidium nelsoni (MSX), and QPX in bivalves of Delaware's inland bays and quantitative, high-throughput diagnosis of dermo by QPCR

Restoration of oyster reef habitat in the Inland Bays of Delaware was accompanied by an effort to detect and determine relative abundance of the bivalve pathogens Perkinsus marinus, Haplosporidium nelsoni, and QPX. Both the oyster Crassostrea virginica and the clam Mercenaria mercenaria were sampled from the bays. In addition, oysters were deployed at eight sites around the bays as sentinels for the three parasites. Perkinsus marinus prevalence was measured with a real-time, quantitative polymerase chain reaction (PCR) methodology that enabled high-throughput detection of as few as 31 copies of the ribosomal non-transcribed spacer region in 500 ng oyster DNA. The other pathogens were assayed using PCR with species-specific primers. Perkinsus marinus was identified in Indian River Bay at moderate prevalence ( approximately 40%) in both an artificial reef and a wild oyster population whereas sentinel oysters were PCR-negative after 3-months exposure during summer and early fall. Haplosporidium nelsoni was restricted to one oyster deployed in Little Assawoman Bay. QPX and P. marinus were not detected among wild clams. While oysters in these bays have historically been under the greatest threat by MSX, it is apparent that P. marinus currently poses a greater threat to recovery of oyster aquaculture in Delaware's Inland Bays.     

Cdc2 protein kinase is complexed with both cyclin A and B: evidence for proteolytic inactivation of MPF

In the clam, Spisula, two previously described proteins known as cyclin A and B display the unusual property of selective proteolytic degradation at the end of each mitosis. We show here that clam oocytes and embryos contain a cdc2 protein kinase. This protein kinase is a component of the M phase promoting factor (MPF) in frog eggs and the M phase-specific histone H1 kinase in starfish. Clam cdc2 is found in association with both cyclin A and B, probably not as a trimolecular association, but as separate cdc2/cyclin A and cdc2/cyclin B complexes. Clam cdc2 and the associated cyclins bind to p13suc1-Sepharose. The p13-bound complex, and also anti-cyclin A or B immunoprecipitates, each display cell cycle-dependent histone H1 kinase activity. We suggest that in addition to the cdc2 protein kinase, the cyclins are further components of the M phase promoting factor and that cyclin proteolysis provides the mechanism of MPF inactivation and thus exit from mitosis.     

Effects of biological behaviors of benthic bivalve (Corbicula japonica) on its passive transport

Hydrobiologia - The bivalve Corbicula japonica is a popular food in Japan. The abundance of the clam in a lake changes over time. Clam transport is assumed to be passive even after the clam’s...     

A naturally occurring cancer with molecular connectivity to human diseases

As Jessani et al. 1 point out development of cell and animal models that accurately depict human tumorigenesis remains a major goal of cancer research. Clam cancer offers significant advantages over traditional models for genotoxic and non-genotoxic preclinical analysis of treatments for human cancers with a similar molecular basis. The naturally occurring clam model closely resembles an out-breeding, human clinical population and provides both in vitro and in vivo alternatives to those generated from inbred mouse strains or by intentional exposure to known tumor viruses. Fly and worm in vivo models for adult human somatic cell cancers do not exist because their adult somatic cells do not divide. Clam cancer is the best characterized, naturally occurring malignancy with a known molecular basis remarkably similar to those observed in several unrelated human cancers where both genotoxic and non-genotoxic strategies can restore the function of wild-type p53. To further emphasize this point of view, we here demonstrate a p53-induced, mitochondrial-directed mechanism for promoting apoptosis in the clam cancer model that is similar to one recently identified in mammals. Discerning the molecular basis for naturally occurring diseases in non-traditional models and correlating these with related molecular mechanisms responsible for human diseases is a virtually unexplored aspect of toxico-proteomics and genomics and related drug discovery.     

基于大叶藻成苗率的新型海草播种技术评价

蛤蜊播种技术是一种新型海草播种技术,该技术中,种子通过糯米糊粘在蛤蜊贝壳上,随蛤蜊穴居被埋入底质。为评价蛤蜊和糯米糊对种子成苗率的影响,以菲律宾蛤仔(Ruditapes philippinarum)和大叶藻(Zostera marina)种子为试验对象,设置了直接播撒种子(A1组)、直接埋种(A2组)、包埋糯米后播撒(B1组)、包埋糯米后埋入底质(B2组)、蛤蜊播种(C)5种处理,每种处理设置3个重复,在实验室的水槽中对其进行试验。结果表明:糯米糊对种子萌发有负面影响,但由于蛤蜊的行为使底质具有透气性,减轻了糯米糊对种子萌发的影响,种子成苗率可达到23.2%;海区试验中利用蛤蜊播种的两个样方中的成苗率分别为19.1%和9.9%。试验表明,蛤蜊播种技术适合作为一种经济、有效的播种技术用于海区海草床的建立和修复。