Perkinsus Species Research Articles

Description of Perkinsus beihaiensis n. sp., a new Perkinsus sp. Parasite in Oysters of Southern China

Oysters were collected from coastal locations in China from 1999-2006 for parasite analyses by molecular, culture, and histological techniques. Polymerase chain reaction-based assays targeting the internal transcribed spacer (ITS) region of the ribosomal RNA gene complex were performed to detect the presence of Perkinsus species. Sequencing and phylogenetic analysis of amplified Perkinsus sp. DNAs indicated that a novel Perkinsus sp. infects Crassostrea hongkongensis, Crassostrea ariakensis, and other bivalve hosts from Fujian to Guangxi provinces in southern China. Prevalence of this Perkinsus sp. reaches as high as 60% in affected oyster populations. Analyses of nucleotide sequences of the rRNA ITS region and of large subunit rRNA and actin genes, consistently confirmed the genus affiliation of this Perkinsus sp., but distinguished it from currently accepted Perkinsus species. Parasite cell types, such as signet ring trophozoites of 2-8 microm diameter, were observed by histology, and application of both genus Perkinsus and Perkinsus species-specific in situ hybridization probes consistently labelled the same Perkinsus sp. cells in histological sections from infected oyster tissues. Combined phylogenetic and histological results support the identity of a new parasite species, Perkinsus beihaiensis n. sp.

Determination of the effects of temperature on viability, metabolic activity and proliferation of two Perkinsus species, and its significance to understanding seasonal cycles of perkinsosis

The range of water temperatures in which Perkinsus species can survive and proliferate remains ill-defined, particularly at lower temperatures. The in vitro viability, metabolic activity, and proliferation of 3 isolates each of P. marinus and P. olseni trophozoites at 28 degrees C, and at 15 and 4 degrees C, after transfer from 28 degrees C, were compared. Both species showed declines in metabolic activity and proliferation from 28 degrees C to 15 degrees C. At 4 degrees C, both species had viability after 30 days incubation time (P. marinus 49%, P. olseni 58%), but limited metabolic activity and no proliferation. Perkinsus marinus viability was further compared when transferred directly from 28 degrees C, 18 degrees C and progressively from 18 degrees C (0.5 degrees C/day) to 2, 4 and 6 degrees C and maintained for up to 4 months. Viability was highest under progressive transfer (77% and 54% after 30 and 60 days exposure to test temperatures). The decrease in P. marinus viability at the lower temperatures in vitro only partially explains decreasing parasite infection intensities in eastern oysters in the colder months of the year. Moreover, the significant decrease in parasite infection intensities in late winter and early spring, as temperatures increase, is likely due to an active process of elimination by oyster host defences.

Continuous Culture of Perkinsus mediterraneus, a Parasite of the European Flat Oyster Ostrea edulis, and Characterization of Its Morphology, Propagation, and Extracellular Proteins in Vitro

Continuous in vitro cultures of Perkinsus mediterraneus were established from tissues of infected European flat oysters, Ostrea edulis. The parasite proliferated in protein-free medium and divided by schizogony in vitro. Cell morphology was similar to that observed for P. mediterraneus in tissues of naturally infected O. edulis and for other Perkinsus spp. cultured in vitro. Parasite cells enlarged approximately 8-fold when placed in alternative Ray's fluid thioglycollate medium, and stained black with Lugol's iodine solution, a response characteristic of Perkinsus spp. DNA sequences matched those determined previously for P. mediterraneus, and phylogenetic analyses on three different data sets indicated that this was a Perkinsus species with a close relationship to another recently described species, Perkinsus honshuensis. Parasite viability was high (>90%) in vitro, but the proliferation rate was low, with densities generally increasing 2-to-6-fold between subcultures at 6-wk intervals. Enzyme analysis of cell-free culture supernatants revealed protease-, esterase-, glycosidase-, lipase-, and phosphatase-like activities. Incubation with class-specific protease inhibitors showed that P. mediterraneus produced serine proteases, and eight proteolytic bands with molecular weights ranging from 34 to 79 kDa were detected in the supernatants by gelatin sodium dodecylsulfate-polyacrylamide gel electrophoresis.

Noble tandem-repeat galectin of Manila clam Ruditapes philippinarum is induced upon infection with the protozoan parasite Perkinsus olseni

The galectin family of lectins plays crucial roles in the innate immunity systems of vertebrates and invertebrates. Noble galectin (MCGal) was cloned from the marine invertebrate Ruditapes philippinarum and characterized. This protein has an open reading frame of 918 nucleotides, with 309 amino acid residues, and a predicted molecular weight of 33.9kDa. Similar to other galectins, MCGal has neither a signal peptide nor a transmembrane domain, but it contains tandemly repeated carbohydrate recognition domains (CRDs), with typical conserved motifs that are important for carbohydrate recognition. Carbohydrate recognition by the recombinant MCGal (rMCGal), as determined by hapten inhibition of hemagglutination, revealed that rMCGal has features common to the galectin family, i.e., significant affinity for galactose and N-acetylgalactosamine. MCGal mRNA expression was detected mainly in the heart, mantle, foot, adductor, palp, and siphon tissues. Immunohistochemistry (IHC) using an anti-MCGal antibody confirmed MCGal expression in these tissues and in hemocytes. Temporal expression of MCGal mRNA in Manila clams challenged with Perkinsus or Vibrio species was up-regulated as compared with non-challenged healthy clams. rMCGal agglutinated Vibrio tapetis, and agglutination was inhibited by incubation with alpha-lactose. rMCGal also bound to the surface of Perkinsus olseni. MCGal plays a crucial role in Manila clam defense, particularly with respect to pathogen recognition.

Pathogens in Crassostrea ariakensis and other Asian oyster species: implications for non-native oyster introduction to Chesapeake Bay

With the drastic decline of eastern oyster Crassostrea virginica populations in the Chesapeake Bay due to over-fishing, diseases and habitat destruction, there is interest in Maryland and Virginia in utilizing the non-native oyster species Crassostrea ariakensis for aquaculture, fishery resource enhancement, and ecological restoration. The International Council for the Exploration of the Sea (ICES) recommends that non-native species be examined for ecological, genetic and disease relationships in the native range prior to a deliberate introduction to a new region. Therefore, a pathogen survey of C. ariakensis and other sympatric oyster species was conducted on samples collected in the PR China, Japan and Korea using molecular diagnostics and histopathology. Molecular assays focused on 2 types of pathogens: protistan parasites in the genus Perkinsus and herpesviruses, both with known impacts on commercially important molluscan species around the world, including Asia. PCR amplification and DNA sequence data from the internal transcribed spacer region of the rRNA gene complex revealed the presence of 2 Perkinsus species not currently found in USA waters: P. olseni and an undescribed species. In addition, 3 genetic strains of molluscan herpesviruses were detected in oysters from several potential C. ariakensis broodstock acquisition sites in Asia. Viral gametocytic hypertrophy, Chlamydia-like organisms, a Steinhausia-like microsporidian, Perkinsus sp., Nematopsis sp., ciliates, and cestodes were also detected by histopathology.

Experimental cross-infections by Perkinsus marinus and P. chesapeaki in three sympatric species of Chesapeake Bay oysters and clams

In controlled laboratory transmission experiments, uniform doses of axenic in vitro isolate cultures of Perkinsus marinus from a Crassostrea virginica oyster, and of independent P. chesapeaki isolates from Chesapeake Bay Mya arenaria and Macoma balthica clams, were used to reciprocally challenge Perkinsus sp.-free C. virginica, M. arenaria, and M. balthica experimental hosts. Following mantle cavity inoculations, all 3 experimental hosts acquired high incidences (30 to 100%) of infections by each of the 3 Perkinsus sp. isolates, based on PCR assays of DNAs from experimental host tissues that were collected through 60 d post-inoculation. Lesions containing proliferating pathogen cells were documented histologically in tissues of all experimental host species challenged with all isolates of both Perkinsus species. Experimental Perkinsus sp. challenge isolates were re-isolated and propagated in vitro from infected tissues of host molluscs from most (5 of 9) experimental treatment groups. Koch's postulates were generally satisfied to confirm experimental infections in all bivalve molluscs that were challenged with 3 isolates of 2 Perkinsus spp. These results suggest potential broad and overlapping host specificities for the 2 current Chesapeake Bay-endemic Perkinsus species: P. marinus and P. chesapeaki.

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.

Differential diagnosis of Perkinsus species by polymerase chain reaction-restriction fragment length polymorphism assay

Perkinsosis is an infection of marine molluscs caused by the protistan parasites of the genus Perkinsus, which has been classified by the OIE as a disease that warrants notification. In the present study, we have applied a molecular genetic approach to develop an optional method for the specific identification of Perkinsus species. A species-specific polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay of the rRNA ITS region was developed to identify and distinguish among Perkinsus species. A taxonomic key was established that allows successful identification of Perkinsus species using a single restriction enzyme (Rsa I) to discriminate P. chesapeaki and P. marinus or by a combination of two endonucleases (Rsa I plus Hinf I) to discriminate P. olseni and P. mediterraneus. In order to validate the RFLP assay, the PCR products were cloned and sequenced, and its phylogenetic affinity was determined. Phylogenetic analysis confirmed the specific identification carried out by RFLPs. Herein is the first report of P. olseni in Manila clams from the NW Adriatic Sea (Italy), which we identified by employing this method. The PCR-RFLP assay herein described may be useful to provide accurate, rapid and inexpensive identification of Perkinsus species, and may aid in ongoing epizooetiological studies and diseases control programmes.

Salinity effects on viability, metabolic activity and proliferation of three Perkinsus species

Little is known regarding the range of conditions in which many Perkinsus species may proliferate, making it difficult to predict conditions favorable for their expansion, to identify conditions inducing mortality, or to identify instances of potential cross-infectivity among sympatric host species. In this study, the effects of salinity on viability, metabolic activity and proliferation of P. marinus, P. olseni and P. chesapeaki were determined. Specifically, this research examined the effects of 5 salinities (7, 11, 15, 25, 35 per thousand), (1) without acclimation, on the viability and metabolic activity of 2 isolates of each Perkinsus species, and (2) with acclimation, on the viability, metabolic activity, size and number of 1 isolate of each species. P. chesapeaki showed the widest range of salinity tolerance of the 3 species, with high viability and cell proliferation at all salinities tested. Although P. chesapeaki originated from low salinity areas (i.e. <15 per thousand), several measures (i.e. cell number and metabolic activity) indicated that higher salinities (15, 25 per thousand) were more favorable for its growth. P. olseni, originating from high salinity areas, had better viability and proliferation at the higher salinities (15, 25, 35 per thousand). Distinct differences in acute salinity response of the 2 P. olseni isolates at lower salinities (7, 11 per thousand), however, suggest the need for a more expansive comparison of isolates to better define the lower salinity tolerance. Lastly, P. marinus was more tolerant of the lower salinities (7 and 11 per thousand) than P. olseni, but exhibited reduced viability at 7 per thousand, even after acclimation.

A histopathological survey of some commercially exploited bivalve molluscs in northern Patagonia, Argentina

A survey of pathological conditions affecting natural beds of the most economically important bivalve species was performed for first time in the Argentinean Sea. The bivalves studied were Aequipecten tehuelchus (Pectinidae), Mytilus edulis, Aulacomya atra (Mytilidae), Protothaca (= Venus) antiqua (Veneridae), Ostrea puelchana (Ostreidae) and Pododesmus rudis (Anomiidae), all from San José gulf (42°20′S, 64°20′W), in northern Patagonia, Southwest Atlantic Ocean. Samples of about 30 adult individuals from each molluscan species were collected and processed by standard histological techniques. Rickettsia-like organisms were the commonest parasites found, followed by ciliates, Nematopsis-like spores, sporocysts and metacercariae of Trematoda, larval Cestoda, Turbellaria and two crustaceans (an isopod and a pea crab). A protozoan of uncertain taxonomic affinities, similar to that formerly reported as Perkinsus karlssoni in Argopecten irradians, was found in A. tehuelchus. DNA analysis demonstrated that it does not belong to a Perkinsus species. The commercially exploited bivalves here studied seem to be devoid of serious pathogens. None of the parasites appear to be a problem to future farming due either to low infestation levels or low pathological effect. Moreover, none of these parasites is OIE notifiable, although to confirm these initial results it is necessary to perform a more exhaustive survey in several beds from different seasons. In order to manage Argentinean stocks, a continuous, organized survey, satisfying international standards, is strongly recommended.

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).

Ultrastructural localization of antigenic determinants conserved during Perkinsus atlanticus trophozoite to prezoosporangium differentiation

Trophozoite, prezoosporangium and zoospore are the 3 main developmental stages that form the life cycle of protozoa of the genus Perkinsus. Several studies have shown that the differentiation of Perkinsus species from the trophozoite to the prezoosporangium stage involves a substantial modification of the antigenic characteristics of these molluscan parasites. With the aim of determining the presence and distribution of antigenic determinants conserved during trophozoite to prezoosporangium differentiation, a polyclonal serum was raised against trophozoites of P. atlanticus purified from parasitized gills of the clam Tapes semidecussatus. Immunocytochemical analyses showed that the serum generated against P. atlanticus trophozoites strongly cross-reacted with the prezoosporangium stage. Immunogold electron microscopy studies revealed that the granular component of the nucleolus, chromatin, cell wall, plasmalemma, lomasomes and vacuolar membrane are the main subcellular structures where the immunodominant epitopes consistently expressed by trophozoites and prezoosporangia are located. Furthermore, analysis of the immunogold staining pattern revealed that the labelling density obtained for prezoosporangia in the nucleolus, cell wall, plasmalemma and lomasomes was significantly higher than that obtained for trophozoites. The most immunoreactive structure in trophozoites was the granular component of the nucleolus, whereas in prezoosporangia it was the lomasome. Interestingly, the main antigenic compartment of P. atlanticus, considering both developmental stages, was the lomasome of the prezoosporangium. These findings show that P. atlanticus trophozoite to prezoosporangium differentiation is accompanied by significant qualitative and quantitative changes in the ultrastructural distribution of the immunodominant antigens shared by these 2 developmental stages.

Identification of a second rRNA gene unit in the Perkinsus andrewsi genome.

Perkinsus species are parasitic protozoa of mollusks, currently classified within the Perkinsozoa, a recently established phylum that is basal to the Apicomplexa and Dinozoa. Ribosomal RNA (rRNA) genes and their intergenic spacers have been used to support the taxonomy of Perkinsus species, the description of new species, and to develop molecular probes for their detection and identification. We previously described ultrastructure, behavior in culture, and partial sequence of the rRNA locus of a Perkinsus species isolated from the baltic clam Macoma balthica. The rRNA genes and intergenic spacers of this Perkinsus isolate differed from those described in the currently accepted species to a degree that led to its designation as a new species, Perkinsus andrewsi. In this study, we identify an additional rRNA gene unit (rRNA-B) in the P. andrewsi holotype, and report the complete sequences of both rRNA gene units. Except for the 5.8S, all regions of the rRNA-B gene unit exhibited sequence differences from that initially described (rRNA-A). Each rRNA gene unit is arranged in a "head-to-tail" tandem repeat. This is the first report demonstrating two distinct rRNA units in a Perkinsus species.

Development of a PCR-ELISA assay for diagnosis of Perkinsus marinus and Perkinsus atlanticus infections in bivalve molluscs

Perkinsus atlanticus and P. marinus have been associated with mass mortality of bivalve molluscs. Perkinsus infections are routinely diagnosed by histology or the fluid thioglycollate medium (FTM) assay. In this study, we describe the development of a PCR-enzyme-linked immunosorbent assay (ELISA) for amplification and rapid detection of Perkinsus species. The PCR reactions were selected to either amplify an IGS sequence region shared by currently accepted Perkinsus species or to simultaneously amplify IGS regions specific to either P. atlanticus or P. marinus. The specific hybridisation of DIG-labelled amplified products to species-specific capture probes was detected colorimetrically. This assay is able to specifically detect P. atlanticus and P. marinus, and the intensity of the colorimetric signal is dependent upon the amount of amplified product. The PCR-ELISA assay format is 100-fold more sensitive than visualisation of PCR products on ethidium bromide (EtdBr)-stained agarose gels, and as sensitive as Southern hybridisation. The sensitivity limit of PCR-ELISA was 1 pg of DNA from P. atlanticus. No cross-reactivity of the assay was observed against the host DNA. When applied to the detection of P. atlanticus in clams, 39 samples out of 45 yielded concordant results for FTM assay and PCR-ELISA detection.

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.

Continuous in vitro culture of the carpet shell clam Tapes decussatus protozoan parasite Perkinsus atlanticus.

Continuous in vitro cultures of the clam Tapes decussatus parasite Perkinsus atlanticus were established from infected gill fragments, infected haemolymph and parasite hypnospores isolated from infected gill fragments following incubation in Ray's fluid thioglycollate medium (RFTM). No continuous cultures could be initiated from P. atlanticus zoospores. Cultures initiated from hypnospores yielded the highest percentage of continuous cultures (100%, 6/6), followed by cultures initiated from gill fragments (93%, 43/46) and from haemolymph (30%, 3/10). Failures to establish continuous cultures were due to microbial contamination. The source of parasite influenced the success rate, the time taken to establish cultures and the size of cultured cells. In vitro proliferation of parasite cells was mainly by vegetative multiplication. Zoosporulation, yielding motile biflagellated zoospores, was observed at a low frequency (< 1% of dividing cells) in every culture. Morphology of cultured cells examined with light and transmission electron microscopy corresponded to that of P. atlanticus found in clam tissues. Cultured cells enlarged in RFTM and stained blue-black with Lugol's solution, which are characteristics of the Perkinsus species cells. DNA sequences of the internal transcribed spacer (ITS) region of the ribosomal RNA gene complex matched those of P. atlanticus. All cultures were established in a medium designated JL-ODRP-2A that was similar in composition to the culture medium JL-ODRP-1 originally used to propagate Perkinsus marinus in vitro. Proliferation of P. atlanticus in vitro could be supported by the commercial culture medium (1:2 v/v) DME:Ham's F-12 with fetuin.

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.

Fine structure of clonally propagated in vitro life stages of a Perkinsus sp. isolated from the Baltic clam Macoma balthica.

We established monoclonal in vitro cultures of a Perkinsus sp. isolated from the baltic clam Macoma balthica and compared morphological features of various life stages by light and transmission electron microscopy to those of the currently accepted Perkinsus species: Perkinsus marinus, Perkinsus olseni, Perkinsus atlanticus, and Perkinsus qugwadi. Except that trophozoites were slightly larger than those of P. marinus, and that they underwent zoosporulation in culture, observation of our isolate under light microscopy did not reveal striking differences from any Perkinsus species. Perkinsus sp. from M. balthica shared fine structural characteristics with other Perkinsus species that clearly place it within this genus. Although zoospores of Perkinsus sp. from M. balthica were slightly smaller than those from other species, the ultrastructural arrangement and appearance of the apical complex and flagella seem to be identical to those of P. marinus and P. atlanticus. Our isolate also appeared, in some sections, to have cortical alveolar expansions of the plasmalemma at regions other than the anterior end and lobulated mitochondria that were reported as unique for P. qugwadi. Little consensus exists among authors in the assignment of taxonomic weight to any particular morphological feature to designate Perkinsus species. The present study of gross morphology and ultrastructure was complemented with molecular studies reported elsewhere, which propose that Perkinsus sp. from Macoma balthica is a distinct species.

Molecular Determination of the Phylogenetic Position of a Species in the Genus Colpodella (Alveolata)

The phylogenetic position of a species of the free-living protist genus Colpodella is assessed using its SSU rDNA gene sequence in the context of a wide array of other alveolate taxa. Phylogenetic analyses indicates that this species is not related to species of Bodo as has previously been suggested. However, SSU rDNA data alone are insufficient to provide a wholly stable hypothesis of relationships for Colpodella sp. among the various alveolate phyla. Much of this instability can be attributed to alignment procedure sensitivity. Analyses of SSU rDNA and actin gene sequences in combination provide strong support for a species of Colpodella as sister group to the ciliates, irrespective of alignment procedure. Moreover, use of SSU rDNA data alone strongly support a recent common ancestry of the genera Perkinsus and Parvilucifera with the dinoflagellates, and not with the apicomplexans. These findings, and the nearly identical ultrastructural characteristics of Colpodella, Perkinsus, and Parvilucifera species, suggest that this morphology is the ancestral condition for all of Alveolata.

CHARACTERIZATION OF THE RIBOSOMAL RNA LOCUS OFPERKINSUS ATLANTICUSAND 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.