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

Abstract

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 paraffinembedded 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. KEY WORDS: Hard clams · Disease · QPX · Molecular techniques · PCR · ProbeResale or republication not permitted without written consent of the publisher Dis Aquat Org 52: [233][234][235][236][237][238][239][240][241][242][243][244][245][246][247] 2002 observed in the late 1950s in New Brunswick, Canada, where it caused high mortalities of wild hard clams (Drinnan & Henderson 1963), it did not become a recognized threat to hard clam aquaculture until the late 1980s and early 1990s. Morphological and molecular evidence suggests that the organism is a member of the phylum Labyrinthulomycota (Whyte et al. 1994, Maas et al. 1...

Environmental distribution and persistence of Quahog Parasite Unknown (QPX)

“…Negative controls for contamination without added template DNA were run with every PCR experiment. The primers used range from selective for the thraustochytrid group (Mo et al 2002) to specific for the QPX organism (Stokes et al 2002). Products from the DGGE amplification were detected on 1% agarose gels stained with ethidium bromide prior to confirmation as QPX on denaturing gels.…”

“…Using the protocols presented here, however, these primers enhanced the detection level 10-fold. The QPX primers of Stokes et al (2002) may also not be as specific for the QPX organism as originally thought, although they do primarily amplify DNA sequences from organisms that are closely related to the parasite. We have not observed the same level of amplification diversity in the MA samples as was found in VA, which may suggest that populations of related thraustochytrids in VA are more diverse, or simply different, from those in MA.…”

“…QPX epizootics have decimated clam-growing areas, resulting in cessation of local aquaculture development in some regions. In other locales, following detection of QPX disease in selected clam beds, shellfish growers voluntarily destroyed potentially infected seed clams, worth hundreds of thousands of dollars in marketable clams, in an attempt to minimize the impact of the disease.Recent studies of the parasite have reported on the development of molecular methods to detect its presence (Ragone Calvo et al 2001, Stokes et al 2002, Lyons et al 2005a, Gast et al 2006, the mechanisms of transmission (Smolowitz et al 2001, Ford et al 2002, Dahl & Allam 2007, the general lack of genetic variation between isolates (Qian et al 2007) and the potential for macroalgae to support growth of the parasite (Lyons et al 2005b, Bugge & Allam 2007. Currently, there are also studies in progress to elucidate aspects of the parasite physiology, pathogenicity and disease development in clams.…”

“…Molecular detection tools are appropriate for searching for QPX in various environments and other species, since these tools are specific to all potential QPX life stages, regardless of host or reservoir species. We used an improved method for the molecular detection of the protist (Gast et al 2006), along with in situ hybridization (Stokes et al 2002), to investigate the distribution of the QPX organism in environmental samples from impacted regions in Massachusetts and Virginia. …”

“…Alternately, fixed tissues from macrophytes and snails were decalcified using formic acid, and then, if needed, processed in paraffin, sectioned at 6 µm, deparaffinated (Howard et al 2004) and stained using the ISH method. Resulting slides were examined and photographed using a Zeiss Axioskop2 with attached digital camera.The ISH staining procedure used to stain QPX organisms was previously described by Stokes et al (2002). Briefly, the method uses a cocktail of 2 digoxigenin-labeled DNA oligonucleotide probes (4 ng µl -1 each of QPX 641 and QPX1318) that target the small subunit ribosomal RNA of the QPX organism.…”