Identification of Heterotrophic Nanoflagellates by Restriction Fragment Length Polymorphism Analysis of Small Subunit Ribosomal DNA

Thirty clones derived from twenty isolates of heterotrophic nanoflagellates originating from a variety of marine and freshwater environments were examined by restriction fragment length polymorphism analysis of small subunit ribosomal RNA genes amplified by the polymerase chain reaction (riboprinting). The data were compared with light and electron microscopical identification of the isolates. On morphological criteria, sixteen of the thirty clones belonged to the genus Paraphysomonas De Saedeleer, seven to the genus Spumella Cienkowski, four to the genus Pteridomonas Penard and three to the genus Cafeteria Fenchel and Patterson. Among these taxa, eleven ribotypes were detected by analysis with the restriction enzymes Hinf I, Hae III, Sau3A I, and Msp I. Differentiation of nanoflagellate taxa by the riboprinting method supported taxonomic classification based on morphology at the generic and species level. The utility of the method for discriminating the 'naked' flagellates and for confirming the identity of polymorphic forms among species of Paraphysomonas is demonstrated.     

Phylogenetic Relationships Among Anuran Trypanosomes as Revealed by Riboprinting

Twenty trypanosome isolates from Anura (frogs and toads) assigned to several species were characterized by riboprinting–restriction enzyme digestion of polymerase chain reaction amplified small subunit ribosomal RNA genes. Restriction site polymorphisms allowed distinction of all the recognized species and no intraspecific variation in riboprint patterns was detected. Phylogenetic reconstruction using parsimony and distance estimates based on restriction fragment comigration showed Trypanosoma chattoni to be only distantly related to the other species, white T. ranarum and T. fallisi appear to be sister taxa despite showing non-overlapping host specificities.     

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.     

Multiple polymorphic sites at the ITS and ATAN loci in cultured isolates of Perkinsus marinus

Sequence analysis of genomic DNA from the protozoan parasite Perkinsus marinus at two loci revealed genetic polymorphisms within and among different cultured isolates. Genomic DNA from 12 Perkinsus marinus isolates was amplified at the internal transcribed spacer region and at an anonymous locus previously identified to contain polymorphisms by restriction fragment length polymorphism analysis. Fourteen polymorphic nucleotide positions were identified at the internal transcribed spacer region; eight in internal transcribed spacer 1 and six in internal transcribed spacer 2. Thirteen polymorphic nucleotide sites were identified within the anonymous locus. In some instances, more than three different sequences were observed at both the internal transcribed spacer region and at the anonymous locus from a single clonal isolate, suggesting the possibility of recombination in cultured cells and/or strand jumping during the polymerase chain reaction. Intra-isolate sequence variation (3.46% for the anonymous locus and 3.08% for internal transcribed spacer 1) was in several cases as high as inter-isolate sequence variation, even in one isolate where recombination was not evident. High intra- and inter-isolate variation detected at both loci demonstrates the importance of determining the genetic variation of each locus prior to development of sequence-based molecular diagnostics.     

Riboprinting: A Tool for the Study of Genetic Diversity in Microorganisms1

ABSTRACT. Classical morphology-based methods of taxonomic and phylogenetic analysis are inadequate in many groups of structurally simple eukaryotes. Molecular methods can generate data independently of the complexity of the organisms’ morphology. Riboprinting is one such technique, and involves restriction enzyme analysis of polymerase chain reaction amplified small subunit ribosomal RNA genes. The utility of the method is illustrated with examples from several genera of intestinal and bloodstream parasites. Among the applications of riboprinting are the detection of cryptic genetic variation within species, organism misidentifications and culture mix-ups, independent verification of DNA sequences, and the rapid generation of data useful in phylogenetic analyses.     

Intraspecific Variation and Phylogenetic Relationships in the Genus Entamoeba as Revealed by Riboprinting

ABSTRACT. Eighty-seven isolates of amebae assigned to the genus Entamoeba have been studied by riboprinting (restriction enzyme polymorphism analysis of polymerase chain reaction amplified small subunit ribosomal RNA genes). Twenty-four distinct patterns were obtained, most of which corresponded to previously described species. In three species ( Entamoeba coli, Entamoeba gingivalis and Entamoeba moshkovskii ) intraspecific variation was detected that led to the grouping of isolates into 'ribodemes' (populations of amebae that share the same riboprint pattern). The riboprint data were used to estimate genetic distances among and within species for the construction of phylogenetic trees based on parsimony and distance analyses. The trees obtained with the two methods are largely congruent. In some cases the estimated distances between species were greater than the upper limit recommended for the fragment comigration method of analysis indicating unusually deep branches within this genus. However, it appears that those species producing cysts with eight nuclei, those producing cysts with one nucleus, and those producing cysts with four nuclei form morphologically based groups that are supported by the riboprint data. The oral parasite Entamoeba gingivalis , which does not encyst, clusters with the third group indicating secondary loss of this ability.     

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.     

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).     

Natural and cultured populations of the mangrove oyster Saccostrea palmula from Sinaloa, Mexico, infected by Perkinsus marinus

The mangrove oyster Saccostrea palmula coexists with the pleasure oyster Crassostrea corteziensis in coastal lagoons of northwest Mexico. Recent discovery of Perkinsus marinus infecting the pleasure oyster in the region prompted evaluation of S. palmula as an alternative P. marinus host. An analysis to determine the possible presence of P. marinus in natural and cultured populations of S. palmula at four coastal lagoons in Sinaloa, Mexico was carried out during October-November 2010. Tissues from apparently healthy S. palmula were evaluated using Ray's fluid thioglycollate method (RFTM), which revealed a Perkinsus sp. to be present in all four locations at 6.7-20.0% prevalence. Histopathological analysis of these specimens showed tissue alterations and parasite forms consistent with moderate P. marinus infection, which was confirmed by ribosomal non-transcribed spacer (NTS)-based PCR assays on DNA samples from oysters positive by RFTM and histology. DNA sequencing of amplified NTS fragments (307 bp) produced a sequence 98-100% similar to GenBank-deposited sequences of the NTS from P. marinus. Fluorescent in situ hybridization for Perkinsus spp. and P. marinus corroborated the PCR results, showing clear hybridization of P. marinus in host tissues. This is the first record of P. marinus infecting a species from genus Saccostrea and the first record of the parasite from coastal lagoons in Sinaloa, Mexico.     

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.     

Monophyly of Endosymbiont Containing Trypanosomatids: Phylogeny versus Taxonomy

To obtain additional information on the phylogenetic relationships within the family Trypanosomatidae (order Kinetoplastida), we have sequenced the small subunit ribosomal RNA genes from the endosymbiont containing species Herpetomonas roitmani TCC080, Herpetomonas sp. TCC263, Crithidia oncopelti ATCC 12982 and a partial large subunit rRNA gene from H. roitmani. The small subunit sequences in the two isolates of Herpetomonas are very similar but not identical, and so are their restriction digest profiles of kinetoplast DNA. The size of minicircles in both isolates is 4.2 kilobases. The inferred ribosomal RNA phylogenetic trees shows the genera Herpetomonas and Crithidia as polyphyletic. Endosymbiont-bearing herpetomonads cluster with the endosymbiont-bearing crithidias and a blastocrithidia to form a monophyletic clade, whereas the endosymbiont-free members of these genera are found elsewhere in the tree. These data support the hypothesis of a monophyletic origin of endosymbiosis in trypanosomatid evolution and also suggest that a taxonomic revision is needed in order to better describe the natural affinities in this family.     

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.     

Application of a multiplex PCR for the detection of protozoan pathogens of the eastern oyster Crassostrea virginica in field samples

Populations of eastern oysters Crassostrea virginica along the east coast of North America have repeatedly experienced epizootic mass mortality due to infections by protozoan parasites, and molecular diagnostic methodologies are fast becoming more widely available for the diagnosis of protozoan diseases of oysters. In this study we applied a modified version of an existing multiplex polymerase chain reaction (PCR) for detection of the eastern oyster parasites Haplosporidium nelsoni, H. costale and Perkinsus marinus from field-collected samples. We incorporated primers for DNA quality control based on the large subunit ribosomal RNA (LSU rRNA) gene of C. virginica. The multiplex PCR (MPCR) simultaneously amplified genomic DNA of C. virginica, and cloned DNA of H. nelsoni, P. marinus and H. costale. In field trial applications, we compared the performance of the MPCR to that of the conventional diagnostic techniques of histopathological tissue examination and the Ray/Mackin fluid thioglycollate medium (RMFT) assay. A total of 530 oysters were sampled from 18 sites at 12 locations along the east coast of the United States from the Gulf of Mexico to southern New England. The modified MPCR detected 21% oysters with H. nelsoni, 2% oysters with H. costale, and 40% oysters with P. marinus infections. In comparison, histopathological examination detected H. nelsoni and H. costale infections in 6 and 0.8% oysters, respectively, and the RMFT assay detected P. marinus infection in 31% oysters. The MPCR is a more sensitive diagnostic assay for detection of H. nelsoni, H. costale, and P. marinus, and incorporation of an oyster quality control product limits false negative results.     

THE PHYLOGENETIC RELATIONSHIP OF PFIESTERIA PISCICIDA, CRYPTOPERIDINIOPSOID SP. AMYLOODINOUM OCELLATUM AND A PFIESTERIA-LIKE DINOFLAGELLATE TO OTHER DINOFLAGELLATES AND APICOMPLEXANS

The taxonomic relationship between heterotrophic and parasitic dinoflagellates has not been studied extensively at the molecular level. In order to investigate these taxonomic relationships, we sequenced the small subunit (SSU) ribosomal RNA gene of Pfiesteria piscicida (Steidinger et Burkholder), a Pfiesteria -like dinoflagellate, Cryptoperidiniopsoid sp., and Amyloodinium ocellatum (Brown) and submitted those sequences to GenBank. Pfiesteria piscicida and Cryptoperidiniopsoid sp. are heterotrophic dinoflagellates, purportedly pathogenic to fish, and A. ocellatum, a major fish pathogen, has caused extensive economic losses in both the aquarium and aquaculture industries. The pathogenicity of the Pfiesteria -like dinoflagellate is unknown at this time, but its growth characteristics and in vitro food preferences are similar to those of P. piscicda. The SSU sequences of these species were aligned with the other full-length dinoflagellate sequences, as well as those of representative apicomplexans and Perkinsus species, the groups most closely related to dinoflagellates. Phylogenetic analyses indicate that Cryptoperidiniopsoid sp., P. piscicida, and the Pfiesteria -like dinoflagellate are closely related and group into the class Blastodiniphyceae, as does A. ocellatum. None of the species examined were closely related to the apicomplexans or to Perkinsus marinus, the parasite that causes "Dermo disease" in oysters. The overall phylogenetic analyses largely supported the current class and subclass groupings within the dinoflagellates.     

Nucleotide sequence variability in the nontranscribed spacer of the rRNA locus in the oyster parasite Perkinsus marinus.

We examined the sequence variability of the nontranscribed spacer (NTS) and internal-transcribed spacer (ITS1 and ITS2) domains of the rRNA locus of Perkinsus marinus from Maryland, Florida, and Louisiana. The sequence of P. marinus DNA including the 5S rRNA, NTS, small subunit (SSU) rRNA, ITSI, and ITS2 regions confirmed their contiguity in the rRNA locus and revealed differences at 28 positions with the SSU rRNA sequences published earlier. The 307-bp polymerase chain reaction (PCR)-amplified fragments from the NTS domain of the various P. marinus isolates revealed the presence of 2 distinct sequences, designated as types I and II, that differed at 6 defined nucleotide positions. Based on these differences, nested PCR and restriction enzyme digests were used to distinguish between the 2 types. Sequences of the ITS1 and ITS2 domains of samples from either NTS type I (n = 3) or type II (n = 3) showed no variation and were identical to published sequences. Frequencies of the P. marinus NTS sequence types I and II in infected oysters varied with the geographic origin of the samples. All Maryland samples examined (n = 19) corresponded to the NTS type I sequence, the type II was the most frequent in the Florida samples (n = 17), and both types were about equally represented in the Louisiana samples (n = 19), with both sequence types found in individual oyster specimens. Although it has been suggested that P. marinus is diploid, it remains to be determined if both NTS sequence types can be present in a single P. marinus trophozoite.     

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.     

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).     

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.     

Identification and phylogenetic relationships of Vahlkampfia spp. (free-living amoebae) by riboprinting

Abstract The riboprinting technique (restriction fragment length polymorphism analysis of polymerase chain reaction amplified ribosomal DNA) was applied to 8 strains representing 7 species of amoebae from the Vahlkampfia genus (Family Vahlkampfiidae, Class Heterolobosea). The length of the 18S ribosomal gene was found to vary significantly between species. Restriction fragment length polymorphism analysis following digestion of the amplified ribosomal DNA with 18 restriction enzymes confirmed the separate identity of each species, originally based on morphological characteristics, and generated a phylogenetic tree. Two strains assigned to the same species yielded identical riboprints.