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A gill-associated Perkinsus sp. isolated from the softshell clam [Mya arenaria) is described as a new species, P. chesapeaki sp. nov. Examination of the parasite in seawater cultures revealed life cycle stages and zoosporulation processes similar to those described for other species of the genus Perkinsus. Prezoosporangia developed thickened cell walls upon contraction of the cytoplasm and development of a distinctive clear area between the cell wall and the protoplast. Successive bipartition of the protoplast led to the formation of hundred's of zoospores within mature sporangia. Zoospores were released into seawater through one or more discharge tubes. Ultrastructural studies revealed an oblong zoospore possessing two flagella that arose from a concave side located in the upper third of the zoospore body. The anterior flagellum possessed a unilateral array of hair-like structures. A large anterior vacuole and basolateral nucleus dominated the cytoplasm of the zoospore body. The presence of a rudimentary apical complex including on open-sided conoid, rhoptries, micronemes, and subpellicular microtubules were also discerned. Differences in zoospore morphology, and sequence analyses of two genes previously reported, support the designation of the gillassociated Perkinsus from the softshell clam as a new species. KEYWORDS :

The internal transcribed spacer (ITS-1 and ITS-2) regions and the 5.8S ribosomal RNA gene of 2 Perkinsus spp. (G117 and H49) originating from the softshell clam, Mya arenaria, of the Chesapeake Bay were cloned and sequenced to obtain evidence for their genetic divergence. A high level of heterogeneity in both regions, probably resulting from deletions, insertions, and base substitutions, was evident from alignments of the sequences of the 2 isolates with published sequences of other Perkinsus spp. The isolate G117 and other Perkinsus spp. were highly divergent (13-26% and 19-20% sequence divergence in ITS-1 and ITS-2, respectively). These regions in the isolate H49 and Perkinsus marinus were similar (99.07% and 99% for ITS-1 and ITS-2, respectively). Evidence obtained from a phylogenetic analysis using the aligned sequences suggests that G117 and H49 belong to 2 distinct species of Perkinsus. The isolate G117 possibly belongs to an as yet undescribed species of Perkinsus, and H49 belongs to the species P. marinus. The conclusions drawn from the genetic analysis of H49 and G117 are supported by previously reported morphological characteristics (McLaughlin & Faisal, 1998b). Isolates H49 and G117 originated from the same molluscan species demonstrating that at least 2 different species of Perkinsus can co-exist in 1 host.

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An 11-kbp DNA element of unknown function interrupts the nifD gene in vegetative cells of Anabaena sp. strain PCC 7120. In developing heterocysts the niJD element excises from the chromosome via site-specific recombination between short repeat sequences that flank the element. The nucleotide sequence of the niJp-proximal half of the element was determined to elucidate the genetic potential of the element. Four open reading frames with the same relative orientation as the nijD element-encoded xisA gene were identified in the sequenced region. Each of the open reading frames was preceded by a reasonable ribosome-binding site and had biased codon utilization preferences consistent with low levels of expression. Open reading frame 3 was highly homologous with three cytochrome P-450 w-hydroxylase proteins and showed regional homology to functionally significant domains common to the cytochrome P-450 superfamily. The sequence encoding open reading frame 2 was the most highly conserved portion of the sequenced region based on heterologous hybridization experiments with three genera of heterocystous cyanobacteria.Anabaena sp. strain PCC 7120 is an oxygen-evolving photoautotroph that is capable of simultaneous photosynthesis and nitrogen fixation. This strain is a well-studied representative of a class of filamentous cyanobacteria that protect nitrogenase from oxygen inactivation by sequestering the enzyme in specialized nitrogen-fixing cells called heterocysts.

Sequence analysis and riboprinting of the small subunit ribosomal RNA genes were used to characterize two morphologically different Perkinsus species isolates from the gill (G117) and the hemolymph (H49) of the softshell clam, Mya arenaria. Sequence data of the polymerase chain reaction amplified ribosomal RNA loci of G117 and H49 indicated that these genes are 1803 and 1806 base-pair long, respectively. A sequence similarity of > 98.9% was calculated among ribosomal RNA sequences of the two isolates of this study and the published sequences of Perkinsus marinus from the American eastern oyster, Crassostrea virginica, and Perkinsus sp. from the blood cockle of the Australian mollusc, Anadara trapezia. From a phylogenetic tree obtained from Jukes-Cantor distances of the aligned ribosomal RNA gene sequences of 13 eukaryotic taxa using the Neighbor-Joining method, we showed that G117 and H49 clustered within the genus Perkinsus. Guided by the sequence data of Perkinsus marinus (accession # X75762) and Perkinsus sp. (accession # L07375), restriction endonucleases were selected for restriction fragment analysis of polymerase chain reaction products of the small subunit ribosomal RNA genes (riboprinting). Riboprinting was used to distinguish the four members of the genus Perkinsus from each other.

Summary :Softshell clams [Mya arenaria) collected from the Chester River in the upper Chesapeake Bay showed the presence of Perkinsus spp. in -12% (28/240) of clams examined. The infection seems to run a mild course with the host prevailing in encapsulating invading para sites. The gills appear to be the major site of infection; however, the parasite was also found in the digestive gland, gonads, and kidneys and occasionally in the tissue and sinuses of adductor muscles. Typi cally, clusters of protozoal cells were embedded in an amorphous PAS-positive substrate and were surrounded by one or more layers of granulocytes. In heavy infections, both free and encapsulated Per kinsus spp. cells were observed in affected tissue forming aggrega tions of different sizes. Within the tissues of M. arenaria, the para site propagated by schizogony. The presence of large encapsulations in vital organs such as the gills and gonads may adversely affect growth and fertility of affected clams. KEY WORDS