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.     

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.     

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.     

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.     

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.     

Minchinia mercenariae n. sp. (Haplosporidia) in the Hard Clam Mercenaria mercenaria: Implications of a Rare Parasite in a Commercially Important Host

ABSTRACT. During routine histopathology of 180 juvenile hard clams, Mercenaria mercenaria , from a site in Virginia, USA, in 2007, we discovered a single individual heavily infected with a parasite resembling a haplosporidian, some members of which cause lethal bivalve diseases. Scanning electron microscopy of spores and sequencing of small subunit ribosomal DNA confirmed a new species: Minchinia mercenariae n. sp. Further sampling of clams at the site found prevalences up to 38% using polymerase chain reaction (PCR). No parasites were found in routine histological screening of the same individuals, but re-examination of clams judged positive by in situ hybridization (ISH) revealed very faintly staining plasmodia. No unusual mortalities have occurred among the sampled groups. Analysis of clams from Massachusetts to Florida by PCR failed to detect the parasite, but a haplosporidian found in a clam from New Jersey in 2001 was subsequently identified by ISH as M. mercenariae . No other haplosporidians have been reported in thousands of hard clams from the US east coast examined histologically since the mid-1980s. The discovery underscores critical questions about how to assess the risks associated with parasites in groups known to be lethal, but that themselves are not considered a problem.     

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).     

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.     

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.     

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.     

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.     

A thraustochytrid protist isolated from Mercenaria mercenaria: molecular characterization and host defense responses

A previously undescribed thraustochytrid protist, designated C9G, was isolated from the gills of a clam, Mercenaria mercenaria, collected from the Bay of Fundy, Canada. Sequence data analysis showed C9G to be related to the clam pathogen QPX, quahog parasite unknown; however, it is not enveloped by secreted mucoid material as is the case for QPX. Clam hemocytes recognized and phagocytized C9G in vitro in the absence of plasma recognition factors. Hemocytes were also capable of killing ingested C9G, as shown by the use of a tetrazolium reduction viability assay. The mechanisms underlying intracellular antimicrobial activity are not yet established, but no detectable cytotoxic reactive oxygen species were generated during phagocytosis of C9G. Clam plasma proteins were shown to inhibit C9G growth at concentrations similar to those in unfractionated hemolymph.     

Prevalence of agglutinating antibodies to Toxoplasma gondii and Sarcocystis neurona in beavers (Castor canadensis) from Massachusetts

The present study examined the seroprevalence of Toxoplasma gondii and Sarcocystis neurona in a population of beavers (Castor canadensis) from Massachusetts. Sixty-two blood samples were collected during the field seasons over 3 consecutive years from different animals. Blood was collected onto filter paper and shipped to the Department of Biomedical Sciences, Virginia Tech, Blacksburg, Virginia, for parasite testing. The samples were tested at dilutions of 1:25, 1:50, and 1:100 against each parasite antigen by modified agglutination tests to determine whether antibodies to either parasite were present in the blood. Six of 62 samples (10%) were positive for T. gondii, with 2 samples having titers of 1:25 and 4 having titers of 1:50. Four of 62 samples (6%) were positive for S. neurona, with 2 samples having titers of 1:25 and 2 having titers of 1:50.     

The effect of benthic prey abundance and size on red knot (Calidris canutus) distribution at an alternative migratory stopover site on the US Atlantic Coast

A population decline of the western Atlantic red knot (Calidris canutus rufa) has been linked to food limitation during the spring migratory stopover in Delaware Bay, USA. The stopover ecology at potential alternative sites has received little attention. We studied factors affecting red knot habitat selection and flock size at a coastal stopover site in Virginia in 2006–2007. The most common potential prey items were coquina clams (Donax variabilis) and crustaceans. Red knot foraging sites had more clams and crustaceans than unused sites in 2006. Prey abundance increased during the 2007 stopover period and remained high after the red knot peak. Red knot flock size in 2007 increased with mean clam shell length, and probability of flock presence decreased with increasing distance from night use locations. Our results suggest that red knots preferred coquina clams and that these clams were not depleted during the stopover period in 2007. Thus prey abundance did not appear to be a population-limiting factor at this coastal stopover site in Virginia in that year. Protection of coastal sites outside of Delaware Bay, many of which have been altered by human development, would likely benefit red knot population recovery, as they can apparently provide abundant food resources during at least some years.     

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.     

Infectious diseases of marine molluscs and host responses as revealed by genomic tools

More and more infectious diseases affect marine molluscs. Some diseases have impacted commercial species including MSX and Dermo of the eastern oyster, QPX of hard clams, withering syndrome of abalone and ostreid herpesvirus 1 (OsHV-1) infections of many molluscs. Although the exact transmission mechanisms are not well understood, human activities and associated environmental changes often correlate with increased disease prevalence. For instance, hatcheries and large-scale aquaculture create high host densities, which, along with increasing ocean temperature, might have contributed to OsHV-1 epizootics in scallops and oysters. A key to understanding linkages between the environment and disease is to understand how the environment affects the host immune system. Although we might be tempted to downplay the role of immunity in invertebrates, recent advances in genomics have provided insights into host and parasite genomes and revealed surprisingly sophisticated innate immune systems in molluscs. All major innate immune pathways are found in molluscs with many immune receptors, regulators and effectors expanded. The expanded gene families provide great diversity and complexity in innate immune response, which may be key to mollusc''s defence against diverse pathogens in the absence of adaptive immunity. Further advances in host and parasite genomics should improve our understanding of genetic variation in parasite virulence and host disease resistance.     

Movement and aggregation in the fluted giant clam (Tridacna squamosa L.)

Aquaculture has been the traditional focus of tridacnid giant clam research whereas their ecology and behaviour have received much less attention. This study was based on the observation that juvenile fluted giant clams (Tridacna squamosa), when evenly distributed in a tank, will move and aggregate over time. We observed movement in clams ranging from 10 to 313 mm in shell length and ‘climbing’ up the sides of tanks was noted for clams with lengths between 10 and 22 mm. Locomotion also occurred after byssal attachment to the substrate; there was a highly significant association between type of movement (i.e. translation, rotation and no movement) and presence of attachment. Tests for phototaxis were negative. Aggregation was examined by placing clams in regular patterns on grids. After three days in the aquarium and 24 h in the field, their positions were analysed to obtain a statistical parameter for ‘clumpiness’. This was found to be greater in the live clam runs than both random walk and random distribution simulations, suggesting that clams were attracted to conspecifics. The latter was tested by recording clam movement with respect to five types of fixed ‘targets’ (i.e. live clam, fouled clam shell, foul-free clam shell, random inanimate object and none). The test clams moved, non-significantly, towards live clam targets and displayed higher mobility compared to tests with other target objects; a negative correlation between mobility and clam length was also observed. A choice experiment using bidirectional water inflow with clam effluent as one source resulted in clams moving toward the effluent, offering the first direct support for positive chemotaxis among conspecifics in Bivalvia. Together, our results indicate the presence of chemical signalling among clams, leading to movement toward one another and clumping. Aggregation could serve several ecological functions, such as defence against predation, physical stabilisation and facilitation of reproduction. With worldwide decline in natural giant clam densities, the opportunity for conspecific clumping is reduced, and local stocks could be facing increased vulnerability to Allee affects.