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...

Haplosporidium nelsoni is a protistan pathogen of the eastern oyster Crassostrea virginica, and has contributed to the decline of the oyster population in the Chesapeake Bay. From comparison of the sequence data of the 16s-like rDNA of H. nelsoni with those of Minchinia teredinis and other related organisms, 2 oligonucleotides which were specific to H. nelsonj and suitable for use as PCR primers were identified. These primers amphfied a 564 base pair fragment of the small subunit (SSU) rRNA gene of H. nelsoni, but did not amplify genomic oyster DNA or the SSU rRNA genes of the haplosporidians Haplosporidium costale, Haplosporjdium louisiana, or M. teredinis. The PCR primers were able to detect the H. nelson1 SSU rDNA from 50 ng of infected oyster genomic DNA or from 10 fg of cloned H. nelsoni SSU rDNA. The ability of the PCR primers to diagnose H. nelsoni-infected oysters was compared to the established techniques of hemolymph settlement analysis in Farley chambers and histological examination from a sample of 20 oysters. Hemolymph settlement analysis detected infection in 10 oysters and histology revealed infections in 11 oysters. PCR amplification of DNA from hemolymph initially detected infections in 15 oysters and reamplification of the PCR products detected an additional 4 infections. PCR amphfication is a more sensitive diagnostic assay for H. nelsoni than traditional techniques.

The genus Bonamia (Haplosporidia) includes economically significant oyster parasites. Described species were thought to have fairly circumscribed host and geographic ranges: B. ostreae infecting Ostrea edulis in Europe and North America, B. exitiosa infecting O. chilensis in New Zealand, and B. roughleyi infecting Saccostrea glomerata in Australia. The discovery of B. exitiosa-like parasites in new locations and the observation of a novel species, B. perspora, in noncommercial O. stentina altered this perception and prompted our wider evaluation of the global diversity of Bonamia parasites. Samples of 13 oyster species from 21 locations were screened for Bonamia spp. by PCR, and small subunit and internal transcribed spacer regions of Bonamia sp. ribosomal DNA were sequenced from PCR-positive individuals. Infections were confirmed histologically. Phylogenetic analyses using parsimony and Bayesian methods revealed one species, B. exitiosa, to be widely distributed, infecting 7 oyster species from Australia, New Zealand, Argentina, eastern and western USA, and Tunisia. More limited host and geographic distributions of B. ostreae and B. perspora were confirmed, but nothing genetically identifiable as B. roughleyi was found in Australia or elsewhere. Newly discovered diversity included a Bonamia sp.

Previously, we described the pathology and ultrastructure of an apparently asporous haplosporidian-like parasite infecting the common shore crab Carcinus maenas from the European shoreline. In the current study, extraction of genomic DNA from the haemolymph, gill or hepatopancreas of infected C. maenas was carried out and the small subunit ribosomal DNA (SSU rDNA) of the pathogen was amplified by PCR before cloning and sequencing. All 4 crabs yielded an identical 1736 bp parasite sequence. BLAST analysis against the NCBI GenBank database identified the sequence as most similar to the protistan pathogen group comprising the order Haplosporida within the class Ascetosporea of the phylum Cercozoa Cavalier-Smith, 1998. Parsimony analysis placed the crab pathogen within the genus Haplosporidium, sister to the molluscan parasites H. montforti, H. pickfordi and H. lusitanicum. The parasite infecting C. maenas is hereby named as Haplosporidium littoralis sp. nov. The presence of a haplosporidian parasite infecting decapod crustaceans from the European shoreline with close phylogenetic affinity to previously described haplosporidians infecting molluscs is intriguing. The study provides important phylogenetic data for this relatively understudied, but commercially significant, pathogen group.