Molecular, morphological, and experimental evidence support the synonymy of Perkinsus chesapeaki and Perkinsus andrewsi

Diverse analytical and experimental results confirm that two protistan parasites, Perkinsus chesapeaki and Perkinsus andrewsi, described separately as parasites of Mya arenaria and Macoma balthica clams sympatric in Chesapeake Bay, USA, represent a single species. Ribosomal RNA (rRNA) internal transcribed spacer (ITS) regions, rRNA large subunit (LSU) gene, and actin gene sequences were obtained from clonal Perkinsus spp. cultured in vitro. Although multiple polymorphic sequences were found in DNA from clonal cultures at each locus, identical ITS region and actin gene sequences were found in the P. andrewsi holotype culture and in Perkinsus sp. clonal cultures from M. arenaria and Tagelus plebius. All sequences determined from cultures of P. chesapeaki and P. andrewsi at each locus grouped together in monophyletic clades with high support values in phylogenetic analyses. In vitro isolates of Perkinsus spp. from M. arenaria and M. balthica were reciprocally infective for each other's cognate host. Lesions and histozoic parasite cell morphologies were consistent with those described for the original host/parasite interactions. In vitro isolate cell cycles and cell types of both parasites were indistinguishable. In accordance with the International Code of Zoological Nomenclature rules of priority, P. andrewsi is declared a junior synonym of P. chesapeaki.     

Perkinsus chesapeaki in stout razor clams Tagelus plebeius from Delaware Bay

Perkinsus chesapeaki is reported from stout razor clams Tagelus plebeius in Delaware Bay, extending the known range of P. chesapeaki north of Chesapeake Bay. P. marinus, which causes dermo disease, is prevalent in cultured and wild oysters at this site, but was not detected in T. plebeius. Evidence for the presence of disseminated neoplasia, also reported from Chesapeake Bay, was equivocal. Although P. chesapeaki infections were associated with mortality events, light infection intensities and a general lack of histopathological evidence of disease limit inferences about a causal relationship. A comparison of Ray's fluid thioglycollate medium (RFTM)-based and PCR-based detection assays highlight differences in detection capabilities related to the quantity and type of tissue processed rather than assay sensitivity per se, a point that should be considered when surveying populations for disease prevalence. Investigators are further cautioned to use care when applying and interpreting diagnostic assays when used with novel species.     

Assessment of the northern distribution range of selected Perkinsus species in eastern oysters (Crassostrea virginica) and hard clams (Mercenaria mercenaria) with the use of PCR-based detection assays

Perkinsus species are protistan parasites of molluscs. In Chesapeake Bay, Perkinsus marinus, Perkinsus chesapeaki, and Perkinsus andrewsi are sympatric, infecting oysters and clams. Although P. marinus is a pathogen for Crassostrea virginica, it remains unknown whether P. andrewsi and P. chesapeaki are equally pathogenic. Perkinsus species have been reported in C. virginica as far north as Maine, sometimes associated with high prevalence, but low mortality. Thus, we hypothesized that, in addition to P. marinus, Perkinsus species with little or no pathogenicity for C. virginica may be present. Accordingly, we investigated the distribution of Perkinsus species in C. virginica and Mercenaria mercenaria, collected from Maine to Virginia, by applying PCR-based assays specific for P. marinus, P. andrewsi, and a Perkinsus sp. isolated from M. mercenaria. DNA samples of M. mercenaria possessed potent PCR inhibitory activity, which was overcome by the addition of 1 mg/ml BSA and 5% (v/v) DMSO to the PCR reaction mixture. All 3 Perkinsus species were found in both host species throughout the study area. Interestingly, the prevalence of P. marinus in M. mercenaria was significantly lower than in C. virginica, suggesting that M. mercenaria is not an optimal host for P. marinus.     

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.     

Description of Perkinsus andrewsi n. sp. isolated from the Baltic clam (Macoma balthica) by characterization of the ribosomal RNA locus, and development of a species-specific PCR-based diagnostic assay

A Perkinsus species was isolated from the baltic clam Macoma balthica and an in vitro culture established under conditions described for P. marinus. As reported previously, morphological features remarkable enough to clearly indicate that this isolate is a distinct Perkinsus species were lacking. In this study, regions of the rRNA locus (NTS, 18S, ITS1, 5.8S, and ITS2) of this isolate were cloned, sequenced, and compared by alignment with those available for other Perkinsus species and isolates. Sequence data from the rRNA locus and species-specific PCR assays indicated not only that Perkinsus sp. from M. balthica was not P. marinus, but it was different from P. atlanticus and P. olseni. The degree of difference was comparable to or greater than differences between accepted Perkinsus species. In particular, NTS sequence and length were dramatically different from that of P. marinus and P. atlanticus. Therefore, we formally propose to designate the Perkinsus sp. from M. balthica as a separate species, P. andrewsi n. sp. Primers based on P. andrewsi NTS sequence were used to develop a PCR-based diagnostic assay that was validated for species-specificity and sensitivity. PCR-based assays specific for either P. andrewsi or P. marinus were used to test for their presence in bivalve species sympatric to M. balthica. Although isolated from M. balthica, P. andrewsi was also detected in the oyster Crassostrea virginica and clams Macoma mitchelli and Mercenaria mercenaria, and could coexist with P. marinus in all four bivalve species tested.     

In vitro propagation of two Perkinsus spp. parasites from Japanese Manila clams Venerupis philippinarum and description of Perkinsus honshuensis n. sp

Perkinsus species are destructive parasites of commercial Manila clams, Venerupis philippinarum, in Japan, Korea, and Spain. However, in vitro parasite cultures from this important host clam are not available. Tissues of Manila clams collected during April 2002 in Gokasho Bay, Japan harbored Perkinsus sp. parasites at a 97% prevalence (28/29) of moderate- and high-intensity infections. Perkinsus sp. cells in tissue samples were enlarged in alternative Ray's fluid thioglycollate medium, before propagation in DME:Ham's F-12 Perkinsus sp. culture medium. Enlarged parasite hypnospores zoosporulated at high frequencies to release motile zoospores, which gave rise to continuous schizogonic cell lines that also zoosporulated continuously at low frequencies. Four Perkinsus sp. in vitro isolates comprising two distinct morphotypes were cryopreserved, cloned, and archived for public distribution. For three isolates of one morphotype, nucleotide sequences of the ribosomal DNA internal transcribed spacer region, of the large subunit rRNA gene, and of actin genes, were consistent with those reported for P. olseni. Similar sequences from one morphologically unique isolate differed from those of all described Perkinsus species. These results show that at least two Perkinsus spp. infect Japanese Manila clams, and that one represents a new species, Perkinsus honshuensis n. sp.     

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.     

Development of an in vitro clonal culture and characterization of the rRNA gene cluster of Perkinsus atlanticus,a protistan parasite of the clam Tapes decussatus

Perkinsus atlanticus cultures were established either with trophozoites isolated from fresh gills, with hypnospores isolated from tissues incubated in fluid thioglycollate medium, or directly from infected hemocytes of carpet shell clams Tapes decussatus from Algarve (Southern Portugal), using a culture medium and conditions optimized for Perkinsus marinus. Perkinsus atlanticus isolates were cloned by limiting dilution, and their identity unequivocally established by PCR-based species-specific diagnostic assays, and by sequencing the complete rRNA gene cluster. The rRNA gene cluster is 7.5-kb in length including 5S, IGS, SSU, ITS1, 5.8S, ITS2, LSU, and an inter-cluster spacer. rDNA sequences of the P. atlanticus clone were between 98.3-100% identical to P. atlanticus sequences previously obtained from clam tissue (non-clonal) isolates. Based on the IGS sequences available from Perkinsus species, a set of primers was designed to amplify P. atlanticus and the two clonally cultured Perkinsus species (P. marinus and P. andrewsi) currently available from a recognized repository. This Perkinsus "genus-specific" PCR-based assay complements the species-specific assays developed earlier and strengthen the detection of Perkinsus species for which specific detection assays are not yet available.     

Infection prevalence and phylogenetic analysis of Perkinsus olseni in Ruditapes philippinarum from East China

The prevalence of Perkinsus sp. infection in Manila clam Ruditapes philippinarum was investigated in the coastal areas of east China. Thirteen groups of clams were collected from 5 sites: Dandong and Qingdao Bays (Yellow Sea), Weifang Bay (Bohai Sea), and Ningbo and Fuzhou Bays (East China Sea). The clams were tested for perkinsosis infection using Ray's fluid thioglycollate medium culture assay. Perkinsus sp. was found in samples from all 5 sites from May 2008 to May 2009. Infection prevalence ranged from 43.75 to 95.83%, and was significantly higher in October than in May. The only 3 uninfected groups of clams were collected from Weifang Bay, the site farthest from the ocean. There was no difference in the prevalence of infection among the remaining 4 sites. The conserved internal transcribed spacer regions of the ribosomal RNA gene complex in each of the Perkinsus sp. isolates were amplified by PCR. The resulting amplicons were sequenced and phylogenetically analyzed. All the Perkinsus isolates were identified as Perkinsus olseni.     

A HECT E3 ubiquitin-protein ligase with sequence similarity to E6AP does not target p53 for degradation in the softshell clam (Mya arenaria)

Numerous reports have raised the level of national concern that chemicals found in the environment may have adverse effects on the health of humans and wildlife. Environmental exposure to pollutants, such as dioxin, has been implicated in gonadal tumor formation in Maine softshell clams (Mya arenaria). Prevalence of these tumors is as high as 40% in some populations. Although their etiology is still unknown, investigations into the mechanisms of tumor formation have revolved around a hypothesis of dioxin-induced toxicity. The aryl hydrocarbon receptor (AHR) was initially investigated, but was later determined to not bind the prototypical ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), suggesting that dioxin toxicity is mediated through an AHR-independent pathway. An alternative mechanism of tumor formation has been investigated, involving a protein with significant sequence similarity to mammalian E6AP, a HECT (homologous to E6AP carboxy terminus) E3 ubiquitin-protein ligase. E6AP, in association with the high-risk human papillomavirus (HPV) E6 protein, is involved in the abnormal degradation of the p53 tumor suppressor protein in human cervical cancer. Tumorigenic clam reproductive tissue revealed higher M. arenaria E3 (MaE3) protein levels concomitant with lower M. arenaria p53 (Map53) levels. While the function of MaE3 as a HECT E3 was verified, results from three methods agree that MaE3 does not associate with Map53. However, alteration in Map53 levels may still play a role in clam gonadal tumorigenesis. Due to upregulation of MaE3 in neoplastic reproductive tissue, further investigations will focus on determining the proteolytic targets of MaE3. In conjunction with our previous findings that dioxin toxicity in the softshell clam is not mediated by AHR, the results from our current investigation suggest a complex etiology for the clam germinomas.     

Experimental challenges of wild Manila clams with Perkinsus species isolated from naturally infected wild Manila clams

Manila clams, Ruditapes philippinarum, are widely harvested in the coastal waters in Japan. However, there have been significant decreases in the populations of Manila clams since the 1980s. It is thought that infection with the protozoan Perkinsus species has contributed to these decreases. A previous study demonstrated that high infection levels of a pure strain of Perkinsus olseni (ATCC PRA-181) were lethal to hatchery-raised small Manila clams, however, the pathogenicity of wild strain Perkinsus species to wild Manila clam is unclear. To address this, we challenged large (30-40mm in shell length) and small (3-15mm in shell length) wild Manila clams with Perkinsus species isolated from naturally infected wild Manila clams. We report high mortalities among the small clams, but not among the large ones. This is the first report to confirm the pathogenicity of wild isolate of Perkinsus species to wild Manila clams.     

Perkinsus olseni in vitro isolates from the New Zealand clam Austrovenus stutchburyi

Perkinsus olseni infections are reported at 10%-84% prevalences among Austrovenus stutchburyi clams (cockles) in northern New Zealand coastal waters. However, P. olseni has not yet been propagated in vitro from New Zealand clams. In our sample of A. stutchburyi clams from Mangemangaroa Stream, New Zealand, 24% (8/34) showed low-intensity Perkinsus sp. infections among mantle and gill tissues incubated in alternative Ray's fluid thioglycollate medium (ARFTM), and 5% (4/79) showed Perkinsus sp. lesions by histological analyses. Among clams that were screened using a polymerase chain reaction (PCR) assay, 16% (3/19) were positive for Perkinsus sp. DNA. Alternative Ray's fluid thioglycollate medium-enlarged hypnospores from tissues of five infected clams yielded three in vitro Perkinsus sp. isolate cultures that were cloned before sequencing internal transcribed spacer (ITS) regions of their rRNA gene complex. For one isolate, ATCC PRA-205, large subunit (LSU) rRNA and actin genes were also sequenced. All nucleotide sequences from all isolates consistently identified them as P. olseni, as did their in vitro cell cycles and zoosporulation characteristics. All in vitro isolate cultures and their respective monoclonal derivative strains were cryopreserved and deposited for archiving and distribution by the American Type Culture Collection (http://www.atcc.org).     

Use of molecular markers for species identification of Korean Perkinsus sp. isolated from Manila clams Ruditapes philippinarum

Perkinsus is the pathogen responsible for mass mortality of the Manila clam Ruditapes philippinarum. Perkinsus sp. isolated from Manila clams collected in Korean waters was assayed by polymerase chain reaction (PCR) to determine its phylogenetic affinity with other congeneric species. Regions of rRNA of Perkinsus sp. isolated from clam haemolymph were cloned and sequenced. Sequences of a non-transcribed spacer (NTS), internal transcribed spacers (ITS 1, 2) and 5.8S rRNA genes were compared to those available from other Perkinsus species. The NTS sequence of Korean Perkinsus was approximately 99.9 to 100% similar to that of P. atlanticus and 98.06 to 98.15% and 73.05 to 73.14% similar to those of P. olseni and P. marinus, respectively. The ITS 1, 5.8S rRNA and ITS 2 sequences of Korean Perkinsus showed 100% similarity to P. atlanticus and Perkinsus sp. reported from Japan. The ITS-5.8S rRNA sequences of Korean Perkinsus were 99.86 and 93.73% similar to those of P. olseni and P. marinus, respectively. The sporulation pattern and morphology of the Korean Perkinsus were very similar to those of P. atlanticus. Our data suggest that the Perkinsus sp. isolated from clams in Korean waters is P. atlanticus, which is currently synonymous with P. olseni reported from Australia. By considering that P. olseni has taxonomic priority, Korean Perkinsus sp. is accepted as P. olseni (atlanticus).     

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.     

CONSPECIFICITY AND INDO-PACIFIC DISTRIBUTION OF SYMBIODINIUM GENOTYPES (DINOPHYCEAE) FROM GIANT CLAMS

We previously reported the occurrence of genetically‐diverse symbiotic dinoflagellates (zooxanthellae) within and between 7 giant clam species (Tridacnidae) from the Philippines based on the algal isolates' allozyme and random amplified polymorphic DNA (RAPD) patterns. We also reported that these isolates all belong to clade A of the Symbiodinium phylogeny with identical 18S rDNA sequences. Here we extend the genetic characterization of Symbiodinium isolates from giant clams and propose that they are conspecific. We used the combined DNA sequences of the internal transcribed spacer (ITS)1, 5.8S rDNA, and ITS2 regions (rDNA‐ITS region) because the ITS1 and ITS2 regions evolve faster than 18S rDNA and have been shown to be useful in distinguishing strains of other dinoflagellates. DGGE of the most variable segment of the rDNA‐ITS region, ITS1, from clonal representatives of clades A, B, and C showed minimal intragenomic variation. The rDNA‐ITS region shows similar phylogenetic relationships between Symbiodinium isolates from symbiotic bivalves and some cnidarians as does 18S rDNA, and that there are not many different clade A species or strains among cultured zooxanthellae (CZ) from giant clams. The CZ from giant clams had virtually identical sequences, with only a single nucleotide difference in the ITS2 region separating two groups of isolates. These data suggest that there is one CZ species and perhaps two CZ strains, each CZ strain containing individuals that have diverse allozyme and RAPD genotypes. The CZ isolated from giant clams from different areas in the Philippines (21 isolates, 7 clam species), the Australian Great Barrier Reef (1 isolate, 1 clam species), Palau (8 isolates, 7 clam species), and Okinawa, Japan (1 isolate, 1 clam species) shared the same rDNA‐ITS sequences. Furthermore, analysis of fresh isolates from giant clams collected from these geographical areas shows that these bivalves also host indistinguishable clade C symbionts. These data demonstrate that conspecific Symbiodinium genotypes, particularly clade A symbionts, are distributed in giant clams throughout the Indo‐Pacific.     

Occurrence of Perkinsus olseni in the Venus clam Protothaca jedoensis in Korean waters

This is the first report of the occurrence of Perkinsus olseni in the Venus clam Protothaca jedoensis off the western and southern coasts of South Korea. Histological observations revealed Perkinsus-like organisms in the mantle, gills, digestive tubules, and gonad. Haemocytic infiltration and tissue necrosis were also observed in heavily infected clams. Hypnospore formation of the Perkinsus-like organism was confirmed with Ray's fluid thioglycollate medium assay (RFTM). When incubated in filtered and aerated seawater, the hypnospore gave rise to cell division and subsequently discharged hundreds of motile zoospores. Genus- and species-specific polymerase chain reaction (PCR) assays and the DNA sequences of the internal transcribed spacer region (ITS) of the Perkinsus sp. isolated from the Venus clam were identical to those of P. olseni reported from the Manila clam Venerupis (=Ruditapes)philippinarum. Based on the DNA sequences and microscopic data, the Perkinsus-like pathogen isolated from P. jedoensis was identified as P. olseni, which parasitizes the Manila clam in European and Asian waters and Haliotis rubra (abalone) in Australian waters. The prevalence and infection intensity of a clam population collected from Yosu, Korea, was determined using RFTM and Choi's 2M NaOH digestion technique. The intensities averaged 10,768 and 7438 Perkinsus cells per gram tissue in 2003 and 2004, and the prevalence ranged from 37.0 to 53.9%, respectively.     

Characterization of the ribosomal RNA locus of Perkinsus atlanticus and development of a polymerase chain reaction-based diagnostic assay

The rRNA locus of Perkinsus atlanticus from the clam Ruditapes decussatus cultivated on the Atlantic coast of Spain was cloned and sequenced. Sequences of the internal transcribed spacer (ITS) from the rRNA locus were compared to sequences reported earlier for a P. atlanticus isolate from Portugal and to those from other Perkinsus species. The ITS I sequence of the Spanish P. atlanticus isolate was identical to the Portuguese P. atlanticus sequence and had 76.6% identity to the ITS1 of Perkinsus marinus. The ITS2 sequence had 99.7% identity to the Portuguese P. atlanticus ITS2, 92.5% identity to the P. marinus ITS2, and 99.5% identity to the Perkinsus olseni ITS2. We report for first the time the small subunit (SSU) and nontranscribed spacer (NTS) of P. atlanticus. The P. atlanticus SSU sequence was 99.6% identical to that of an unidentified Perkinsus species from the Australian clam Anadara trapezia and 98.0% identical to that of P. marinus. Further, our results support the proposal that P. atlanticus, P. olseni, and the Perkinsus sp. from A. trapezia constitute a subgroup of Perkinsus species distributed in the Pacific and eastern Atlantic, different from P. marinus that is distributed along the western edge of the Atlantic. Based on the NTS sequence of P. atlanticus from Spain and the differences with P. marinus NTS (62.2% identity), we developed a polymerase chain reaction (PCR)-based diagnostic assay with a lowest limit of detection of 0.01 amol of cloned NTS DNA as assessed on ethidium bromide-stained agarose gels. Specificity of the PCR-based assay was tested with samples from the clams R. decussatus, Ruditapes philippinarum, and Venerupis pullastra collected in P. atlanticus-enzootic areas of Spain. The specificity and sensitivity demonstrated for this NTS-based PCR assay validate its use as a tool for assessment of P. atlanticus in molluscs.     

Development of real-time PCR assays for discrimination and quantification of two Perkinsus spp. in the Manila clam Ruditapes philippinarum

The Manila clam Ruditapes philippinarum is infected with 2 Perkinsus species, Perkinsus olseni and P. honshuensis, in Japan. The latter was described as a new species in Mie Prefecture, Japan, in 2006. Ray's Fluid Thioglycollate Medium (RFTM) assay has been most commonly used to quantify Perkinsus infection. However, this assay cannot discriminate between species that resemble one another morphologically. We developed real-time PCR assays for the specific quantification of P. olseni and P. honshuensis. DNA was extracted using Chelex resin. Cultured P. olseni and P. honshuensis cells were counted and spiked into uninfected clam gill tissue prior to DNA extraction to generate standard curves, which allowed quantification based on the PCR cycle threshold values. We compared the RFTM assay with both real-time PCR assays by quantifying Perkinsus spp. in gill tissue samples from the same individual clams obtained from various localities in Japan. Infection intensities estimated by both assays were significantly correlated (r2 = 0.70). Our results suggest that the prevalence and infection intensity of P. honshuensis are much lower than for P. olseni in Manila clams.     

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. marinus isolate of the eastern oyster.