Analysis of extracellular proteins of two Perkinsus spp. isolated from the softshell clam Mya arenaria in vitro.

Biochemical characterization of the extracellular proteins (ECP) of two softshell clam Perkinsus spp. cloned isolates, Perkinsus chesapeaki isolate G-117 and Perkinsus marinus H-49, was performed and compared to that of the oyster-derived P. marinus isolate P-1. G-117 and H-49 demonstrated distinct differences in enzyme activities; however, all three isolates shared common bands. Substrate-impregnated gels showed H-49 to possess proteolytic activities while G-117 did not. Inhibition studies revealed that H-49 ECP contain serine proteases similar to those described for P-1. The G-117 ECP lacked proteolytic activity but showed a higher production of lipolytic enzymes than H-49 or P-1. Optimal in vitro growth temperatures for the two clam isolates were generally lower than those for P-1. G-117 showed faster growth at lower salinities than either H-49 or P-1. Clam Perkinsus spp. isolates appear to be better adapted to lower salinities and temperatures than the P. murinus isolate of the eastern oyster.

Zoosporulation of a new Perkinsus species isolated from the gills of the softshell clam Mya arenaria.

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 an 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 gill-associated Perkinsus from the softshell clam as a new species.

Discrimination between two Perkinsus spp. isolated from the softshell clam, Mya arenaria, by sequence analysis of two internal transcribed spacer regions and the 5.8S ribosomal RNA gene.

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.

Characterization of two Perkinsus spp. from the softshell clam, Mya arenaria using the small subunit ribosomal RNA gene.

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.

Acanthamoeba pearcei n. sp. (Protozoa: Amoebida) from sewage contaminated sediments.

Seabottom sediments from a discontinued Philadelphia-Camden 40-Mile ocean sewage disposal site were cultured for cyst-forming free-living amoebae. Barge delivered wastes were discharged at the site from 1973 until 1980 when the site was closed. One station at the southeast margin of the site was sampled at a depth of approximately 50 m, twice in 1978 and once in 1982, 1983 and 1984. Sediment from the 1978 collection yielded Acanthamoeba polyphaga, Vahlkampfia sp., and an unknown amoeba with stellate endocysts similar to those of A. astronyxis. Trophozoites and cysts of the isolate were typical of those described for the genus Acanthamoeba. Biochemical tests employing enzyme electrophoresis and morphological studies on live and stained specimens showed that the isolate was distinct from other well-described species within the family Acanthamoebidae Sawyer & Griffin, 1975.

Transmission studies of sarcoma in the soft-shell clam, Mya arenaria.

Transmission experiments with adult soft-shell clams (Mya arenaria) demonstrated that clam sarcomas are transmissible with hemolymph from neoplastic animals but not with cell-free ultrafiltrates. Non-neoplastic clams were injected with either hemolymph from neoplastic clams or a cell-free ultrafiltrate prepared from a subsample of the same hemolymph. Injected clams were held in separate flow-through aquaria and examined for sarcomas by histocytology and histology. Data at 17 weeks showed a 44% prevalence of sarcomas in clams injected with neoplastic inoculum. No sarcomas were observed either in clams injected with a cell-free ultrafiltrate or in the control animals. The lack of sarcomas in clams injected with the ultrafiltrate argues against a viral etiology for the disease.

Cause of changes in the thin filament-associated reflexions on activation of frog muscle--myosin binding or conformational change of actin.

Although the intensity increase of the 5.9 nm layer-line (5.9 L.L.) on activation can potentially be informative, systematic investigation is left to be done. The present study is aimed to assign the cause of the intensity increase. Using the synchrotron radiation, X-ray diffraction patterns were recorded from live sartorius or semitendinosus muscle of the frog (mostly at a temperature of 3-4 degrees C) either on the "Imaging Plates", a newly developed X-ray image recording system, or with the fast linear detector. The Imaging Plates enabled us to measure the disorientation of the thin filaments in the contracting muscle, so that the intensity profiles of the layer-lines were corrected for the disorientation. After the correction, a slight inward shift of the intensity peak of 5.9 L.L. was measured which is associated with the intensity increase on activation at 4 degrees C. At higher temperatures, 5.9 L.L. increases less. The extent of the intensity increase is linearly related to the sarcomere overlap, whereas the outer part of the 2nd actin layer-line (2nd L.L.) is as high as 60% at 10% overlap. The rise of the intensity of 5.9 L.L. is as fast as that of 2nd L.L., but much faster than the changes of the equatorial reflexions and the tension rise. Although these results do not let us assign the cause, some properties are elucidated of the structural change which causes the intensity increase.

Improvement in the ultrasonic evaluation of the gall bladder by using the left lateral decubitus position.

The authors discuss the value of routinely using the left lateral decubitus position for ultrasound evaluation of the gall bladder. This technique often affords improved visualization, clarifies abnormalities suspected in the supine position, and in some cases demonstrates a gall bladder that cannot be seen in the supine position.