Distribution of Pfiesteria piscicida cyst populations in sediments of the Delaware Inland Bays, USA

The toxic dinoflagellate, Pfiesteria piscicida, is a common constituent of the phytoplankton community in the Delaware Inland Bays, USA. In this study, molecular methods were used to investigate the distributions of benthic stages (cysts) of P. piscicida in sediment cores from the Delaware Inland Bays. Cores from 35 sites were partitioned into nephloid and anoxic layers and analyzed for P. piscicida by nested amplification of the 18S rDNA gene using P. piscicida-specific primers. The presence of inhibitory substances in the PCR reaction was evaluated by inclusion of an exogenous control DNA in the extraction buffer, thus eliminating samples that may yield false-negative results. Our results indicate a patchy distribution of P. piscicida in sediments of the Delaware Inland Bays, with distinct differences between each of the three bays. Overall, P. piscicida was found more frequently in sediments from Rehoboth Bay compared to Indian River and Little Assawoman Bays. These differences suggest (i) that populations of P. piscicida may be more widely distributed in Rehoboth Bay, (ii) that populations of P. piscicida may have been introduced to Rehoboth Bay at an earlier time, (iii) that past blooms of P. piscicida in Rehoboth Bay estuaries may have seeded the sediments with higher numbers of cysts, and/or (iv) that Rehoboth Bay sediments may be more resistant to clearing due to storm turbulence.     

Efficient 5' ETS walking from conserved 18S rDNA sequences of the dinoflagellates <Emphasis Type="Italic">Alexandrium</Emphasis> and <Emphasis Type="Italic">Akashiwo sanguinea</Emphasis> (Dinophyceae)

An external transcribed spacer (ETS) walking PCR technique was developed for the isolation of unknown sequences adjacent to the 18S rDNA. This strategy relied on four "walking primers", which were designed to bind unknown sequences upstream from the 18S rDNA, and a specially programmed series of thermocycles. This method was successful in the isolation of the 5' ETS regions from harmful dinoflagellates, including Alexandrium affine, A. catenella, A. minutum, A. tamarense, and Akashiwo sanguinea. Mono-directional sequencing reactions revealed the PCR products to be 392–962 nucleotides in length, and the 5' ETS in these products were longer than 362 bp. These are the first such sequences available for A. sanguinea and the Alexandrium. In comparisons of the ETS sequences, genetic distance was considerably high within the Alexandrium. Furthermore, the sequences were significantly variable among the different strains of identical species: genetic distance was recorded at 0.0420 for A. tamarense strains and as less than 0.7841 within strains of A. sanguinea. The 5'-start nucleotide of 18S rDNA was variable between the two genera: the five species of Alexandrium contained a T base, and A. sanguinea contained an A base. These results demonstrate the effectiveness of the ETS walking PCR method. This method will be valuable in directional ETS walking from known regions to unknown regions, particularly concerning the boundary sequences of rRNA genes.     

Morphological and molecular genetic characterization of Cryptoperidiniopsis brodyi (Dinophyceae) from Australia-wide isolates

Cryptoperidiniopsis brodyi is a common heterotrophic dinoflagellate known to often co-occur with Pfiesteria species in eastern U.S. estuaries. In this study, C. brodyi from Australia and Pfiesteria piscicida from ballast water from Indonesia were characterized by morphological and genetic analyses. Two P. piscicida strains originating from ballast water samples showed little genetic differences compared to P. piscicida from other countries and their morphology was identical. This finding indicates a potential inflow of P. piscicida into Australian estuaries via ballast water. Nine cultures of C. brodyi were established from Tasmania, South Australia and Western Australia. All C. brodyi cultures exhibited identical thecal plate patterns and could not be discriminated from other non-Australian strains. In contrast, two distinct genotypes could be identified by rDNA sequence analyses which were distinct from the U.S. genotype of C. brodyi. A previous survey using PCR-based methods reported a wide distribution of Pfiesteria shumwayae in Australia. However, the present study demonstrated that SSU rDNA-based P. shumwayae-specific primers produce false-positive PCR reactions with Australian C. brodyi. These results suggest that genetic variants of C. brodyi are widely distributed in Australia and Australian genotypes of C. brodyi had previously been misidentified as P. shumwayae. This finding also indicates that previous Australian distribution studies of P. shumwayae using SSU rDNA-based primers are potentially erroneous and need to be revisited.     

Predator/Prey Interaction between <Emphasis Type="Italic">Pfiesteria piscicida</Emphasis> and <Emphasis Type="Italic">Rhodomonas</Emphasis> Mediated by a Marine Alpha Proteobacterium

The dinoflagellate Pfiesteria piscicida coexists with bacteria in aquatic environments and as such, may interact with them at the physiological level. This study was designed to investigate the influence of bacteria, present in a clonal culture of Pfiesteria piscicida, on the predator/prey relationship of this dinoflagellate with the alga Rhodomonas. A series of replenishment experiments with bacteria isolated from P. piscicida clonal culture and the bacteria-free P. piscicida derived from the same culture were carried out. In the presence of bacteria, the number of P. piscicida increased significantly when incubated with alga Rhodomonas. This enhanced growth was almost entirely due to the increased consumption rate of Rhodomonas by P. piscicida since in bacteria-free (axenic) cultures Rhodomonas were consumed at significantly reduced rates relative to cultures with bacteria. Subsequent replenishment experiments with individual bacterial isolates showed that a single isolate was responsible for the increased predation rate of P. piscicida. The presence or absence of this specific bacterium determined the outcome of the interaction between P. piscicida and Rhodomonas. Partial sequence analysis of the 16S rDNA of this isolate indicated that it was a novel marine alpha proteobacterium with sequence similarities to a Roseobacter sp. and a bacterium recently isolated from a toxic dinoflagellate Alexandrium sp.     

Assessing the Biodiversity of Monoraphidium Using 18S rDNA Sequences

The taxonomy of the genus Monoraphidium is unclear due in part to the absence of morphological features to clearly distinguish one species from another. Phytoplankton samples collected from lakes in the Arrowwood National Refuge in eastern North Dakota were found to contain several morphological species of Monoraphidium. Eighteen Monoraphidium isolates were examined with light microscopy and six morphological species were identified. PCR–RFLP of the 18S rDNA was used to type the isolates. Following digestion by Hae III and Taq I, the 18S rDNA PCR–RFLP patterns indicated 10 different types. Presently, the 18S rDNA product is being sequenced for each of the 10 types. By examining morphological characters and 18S rDNA sequences, congruence between morphology and sequence data may be compared. Also, because there is a lack of morphological characters defining Monoraphidium species, diversity within the 18S rDNA sequences may aid in the taxonomy of the genus and its place within the Chlorococcales. Supported by National Science Foundation Grants MCB‐0084188 and DBI‐0070387.     

Identification of Two Species of Yeast-like Symbiotes in the Brown Planthopper, <Emphasis Type="Italic">Nilaparvata lugens</Emphasis>

To determine the species of the yeast-like symbionts (YLS) in the brown planthoppers (BPH), Nilaparvata lugens, YLS were first isolated and purified by ultracentrifugation from the fat bodies of BPH, and then 18S rDNA and internal transcribed spacer (ITS)–5.8S rDNA sequences of YLS were amplified with the different general primers for fungi. The results showed that the two different 18S and ITS–5.8S rDNA sequences of YLS were obtained. One 2291-bp DNA sequence, which contained 18S and ITS–5.8S rDNA, showed the high similarity to Cryptococcus and was named Cryp-Like symbiotes. Another 1248-bp DNA sequence, which contained a part of 18S and ITS–5.8S rDNA, showed the high similarity to Pichia guilliermondii and was named Pichia-Like symbiotes. It was further proved that Cryp- and Pichia-Like symbiotes existed in BPH through nested PCR with specific primers for two symbiotes and in situ hybridization analysis using digoxigenin-labeled probes. Our results showed that BPH harbored more than one species of eukaryotic YLS, which suggested that diversity of fungal endosymbiotes may be occurred in planthoppers, just like bacterial endosymbiotes.     

Molecular detection of Pectobacterium species causing soft rot of <Emphasis Type="Italic">Amorphophallus konjac</Emphasis>

Soft rot disease of Amorphophallus konjac is caused by Pectobacterium species. Infected corms are considered a primary and important source of inocula. Based on the 16S rDNA sequences of the soft rot pathogens, one pair of specific primers was designed to identify the soft rot disease by real-time PCR and the other two were used to identify the pathogens of Pectobacterium carotovorum subsp. carotovorum. and P. chrysanthemi respectively. According to the results, a single cell of Pectobacterium could be detected by real-time PCR with the designed primer pair, while at least 100 bacteria were required for conventional PCR. Moreover, the two special primers can directly and accurately authenticate to Pectobacterium carotovorum subsp. carotovorum and P. chrysanthemi by the conventional PCR system without testing the pathogenicity, biochemical and phenotypic characterizations and so on. In conclusion, the PCR-based techniques showed several significant advantages in identifying the soft rot pathogens from konjac, such as higher sensitivity, rapidness and precision, and it could be widely used in seed quarantine.     

Detection of Pfiesteria spp. by PCR in surface sediments collected from Chesapeake Bay tributaries (Maryland)

In 1997 blooms of Pfiesteria piscicida occurred in association with fish kills and human health problems in tributaries of the Chesapeake Bay (Maryland) and the scientific and media response resulted in large economic losses in seafood sales and tourism. These events prompted the Maryland Department of Natural Resources (MDNR) to begin monitoring for Pfiesteria spp. in water column samples. Real-time PCR assays targeted to the 18S rRNA gene were developed by our laboratories and utilized in conjunction with traditional microscopy and fish kill bioassays for detection of these organisms in estuarine water samples. This monitoring strategy aided in determining temporal and spatial distribution of motile forms of Pfiesteria spp. (i.e. zoospores), but did not assess resting stages of the dinoflagellates’ life cycle. To address this area, a 3-year study was designed using real-time PCR assays for analysis of surface sediment samples collected from several Chesapeake Bay tributaries. These samples were tested with the real-time PCR assays previously developed by our laboratories. The data reported herein suggest a strong positive association between presence of Pfiesteria spp. in the sediment and water column, based on long-term water column monitoring data. P. piscicida is detected more commonly in Maryland's estuarine waters than Pfiesteria shumwayae and sediment ‘cyst beds’ may exist for these organisms.     

Phylogenetic and genomic relationships in Setaria italica and its close relatives based on the molecular diversity and chromosomal organization of 5S and 18S-5.8S-25S rDNA genes

We have analyzed the phylogenetic and genomic relationships in the genus Setaria Beauv. including diploid and tetraploid species, by means of the molecular diversity of the 5S rDNA spacer and chromosomal organization of the 5S and 18S-5.8S-25S rDNA genes. PCR amplification of the 5S rDNA sequences gave specific patterns. All the species studied here share a common band of about 340 bp. An additional band of an approximately 300-bp repeat unit was found for Setaria verticillata and the Chinese accessions of Setaria italica and Setaria viridis. An additional band of 450 bp was found in the sole species Setaria faberii. Fluorescent in situ hybridization was used for physical mapping of the 5S and 18S-5.8S-25S rDNA genes and showed that they are localized at two separate loci with no polymorphism of chromosome location among species. Two chromosome pairs carrying the 5S and 18S-5.8S-25S rDNA clusters can now be unambiguously identified using FISH. Phylogenetic trees based on the variation of the amplified 5S rDNA sequences showed a clear separation into four groups. The clustering was dependent on the genomic composition (genome A versus genome B) and confirmed the closest relationship of S. italica and S. viridis accessions from the same geographical region. Our results confirm previous hypotheses on the domestication centers of S. italica. They also show the wide difference between the A and B genomes, and even clarify the taxonomic position of S. verticillata. Received: 28 August 2000 / Accepted: 27 January 2001     

Genetic Diversity and Molecular Markers of the Tropical Abalone (<Emphasis Type="Italic">Haliotis asinina</Emphasis>) in Thailand

Genetic diversity of abalone in Thailand, Haliotis asinina, H. ovina, and H. varia, was analyzed by polymerase chain reaction (PCR) of 18S and 16S rDNAs, with randomly amplified polymorphic DNA (RAPD) and restriction fragment length polymorphism (RFLP). Species-specific RAPD markers were found in each abalone species. Restriction analysis of 18S (nuclear) ribosomal DNA with Alu I, Taq I, and Hae III and 16S (mitochondrial) rDNA with Bam HI, Eco RI, Hae III, and Alu I gave 12 and 13 digestion patterns, respectively. A total of 49 composite haplotypes were found. A dendogram obtained by the unweighted pair-group method with arithmetic mean, constructed from divergence between pairs of composite haplotypes, revealed reproductively isolated gene pools of these abalone and indicated that H. asinina and H. ovina are genetically closer than H. varia. When H. varia was discovered owing to small sample sizes, geographic heterogeneity analysis and F_ST estimate indicated clear genetic differentiation between H. ovina originating from the Andaman Sea (west) and the Gulf of Thailand (east, P < 0.0001), whereas partial differentiation was observed between the Philippines and the remaining H. asinina samples (P < 0.0021). The amplified 16S rDNAs of individuals representing composite haplotypes found in this study were cloned and sequenced. A neighbor-joining tree constructed from sequence divergence of 16S rDNA accurately allocated those sequences according to species origins of abalone. Species-specific PCR based on 16S rDNA polymorphism was successfully developed in H. asinina and H. varia but not in H. ovina.     

Development of a Real-Time PCR Probe for Quantification of the Heterotrophic Dinoflagellate Cryptoperidiniopsis brodyi (Dinophyceae) in Environmental Samples

A TaqMan format real-time PCR probe was developed against the internal transcribed spacer 2 ribosomal DNA region for the specific detection and quantification of Cryptoperidiniopsis brodyi in environmental samples. The assay specificity was confirmed by testing against related dinoflagellates and verified by sequencing PCR amplicons from natural water samples. Phylogenetic analysis of the sequenced environmental samples also showed that this assay is specific to C. brodyi. The C. brodyi-specific assay was used in conjunction with Pfiesteria piscicida- and Pfiesteria shumwayae-specific real-time PCR assays to investigate the temporal variations of C. brodyi, P. piscicida, and P. shumwayae abundance in the Derwent estuary, Tasmania. The 18-month field survey from November 2004 to April 2006 revealed that C. brodyi occurred in all seasons at very low densities, mostly below 25 cells liter⁻¹, with higher abundance (maximum, 112 cells liter⁻¹) in April and May. P. piscicida was detected only once, in May 2005 at 60 cells liter⁻¹. P. shumwayae was not detected during the survey.     

New species in the genus <Emphasis Type="Italic">Francisella</Emphasis> (Gammaproteobacteria; Francisellaceae); <Emphasis Type="Italic">Francisella piscicida</Emphasis> sp. nov. isolated from cod (<Emphasis Type="Italic">Gadus morhua</Emphasis>)

A Francisella strain, GM2212, previously isolated from moribund farmed Atlantic cod (Gadus morhua) in Norway, is closely related to Francisella philomiragia among Francisella spp. according to its complete 16S rDNA, 16S-23S intergenic spacer, 23S rDNA, 23S–5S intergenic spacer, 5S rDNA, FopA, lipoprotein TUL4 (LpnA), malate dehydrogenase and hypothetical lipoprotein (LpnB) sequences. A comparison between GM2212 and the type strain of Francisella philomiragia were performed by DNA–DNA hybridization and fatty acid analysis. The DNA–DNA hybridization showed a 70% similarity. The fatty acid analysis showed only minor differences between the Francisella isolates. Due to the inconclusive result from the DNA–DNA hybridisation, major emphasis concerning the status of this isolate is made on previously published molecular, phenotypic and biochemical characters. All characteristics taken together support the establishment of GM2212 as a novel species, for which the name Francisella piscicida sp. nov. is proposed (=CNCM I-3511^T = DSM 18777^T = LMG registration number not yet available).     

GENETIC VARIATION AMONG STRAINS OF PSEUDOPFIESTERIA SHUMWAYAE AND PFIESTERIA PISCICIDA (DINOPHYCEAE)1

The putatively toxic dinoflagellates Pseudopfiesteria shumwayae (Glasgow et J. M. Burkh.) Litaker, Steid., P. L. Mason, Shields et P. A. Tester and Pfiesteria piscicida Steid. et J. M. Burkh. have been implicated in massive fish kills and of having negative impacts on human health along the mid‐Atlantic seaboard of the USA. Considerable debate still remains as to the mechanisms responsible for fish mortality (toxicity vs. micropredation) caused by these dinoflagellates. Genetic differences among these cultures have not been adequately investigated and may account for or correlate with phenotypic variability among strains within each species. Genetic variation among strains of Ps. shumwayae and P. piscicida was examined by PCR–RFLP analysis using cultures obtained from the Provasoli‐Guillard National Center for Culture of Marine Phytoplankton (CCMP), as well as those from our own and other colleagues’ collection efforts. Examination of restriction digest banding profiles for 22 strains of Ps. shumwayae revealed the presence of 10 polymorphic restriction endonuclease sites within the first and second internal transcribed spacers (ITS1 and ITS2) and the 5.8S gene of the rDNA complex, and the cytochrome oxidase subunit I (COI) gene. Three compound genotypes were represented within the 22 Ps. shumwayae strains. Conversely, PCR–RFLP examination of 14 strains of P. piscicida at the same ITS1, 5.8S, and ITS2 regions revealed only one variable restriction endonuclease site, located in the ITS1 region. In addition, a dinoflagellate culture listed as P. piscicida (CCMP 1928) and analyzed as part of this study was identified as closely related to Luciella masanensis P. L. Mason, H. J. Jeong, Litaker, Reece et Steid.     

Characterization of the rRNA Locus of Pfiesteria piscicida and Development of Standard and Quantitative PCR-Based Detection Assays Targeted to the Nontranscribed Spacer

Pfiesteria piscicida is a heterotrophic dinoflagellate widely distributed along the middle Atlantic shore of the United States and associated with fish kills in the Neuse River (North Carolina) and the Chesapeake Bay (Maryland and Virginia). We constructed a genomic DNA library from clonally cultured P. piscicida and characterized the nontranscribed spacer (NTS), small subunit, internal transcribed spacer 1 (ITS1), 5.8S region, ITS2, and large subunit of the rRNA gene cluster. Based on the P. piscicida ribosomal DNA sequence, we developed a PCR-based detection assay that targets the NTS. The assay specificity was assessed by testing clonal P. piscicida and Pfiesteria shumwayae, 35 additional dinoflagellate species, and algal prey (Rhodomonas sp.). Only P. piscicida and nine presumptive P. piscicida isolates tested positive. All PCR-positive products yielded identical sequences for P. piscicida, suggesting that the PCR-based assay is species specific. The assay can detect a single P. piscicida zoospore in 1 ml of water, 10 resting cysts in 1 g of sediment, or 10 fg of P. piscicida DNA in 1 μg of heterologous DNA. An internal standard for the PCR assay was constructed to identify potential false-negative results in testing of environmental sediment and water samples and as a competitor for the development of a quantitative competitive PCR assay format. The specificities of both qualitative and quantitative PCR assay formats were validated with >200 environmental samples, and the assays provide simple, rapid, and accurate methods for the assessment of P. piscicida in water and sediments.     

DISCRIMINATIVE POWER OF NUCLEAR rDNA SEQUENCES FOR THE DNA TAXONOMY OF THE DINOFLAGELLATE GENUS PERIDINIUM (DINOPHYCEAE)1

The genus Peridinium Ehrenb. comprises a group of highly diversified dinoflagellates. Their morphological taxonomy has been established over the last century. Here, we examined relationships within the genus Peridinium, including Peridinium bipes F. Stein sensu lato, based on a molecular phylogeny derived from nuclear rDNA sequences. Extensive rDNA analyses of nine selected Peridinium species showed that intraspecies genetic variation was considerably low, but interspecies genetic divergence was high (>1.5% dissimilarity in the nearly complete 18S sequence; >4.4% in the 28S rDNA D1/D2). The 18S and 28S rDNA Bayesian tree topologies showed that Peridinium species grouped according to their taxonomic positions and certain morphological characters (e.g., epithecal plate formula). Of these groups, the quinquecorne group (plate formula of 3′, 2a, 7″) diverged first, followed by the umbonatum group (4′, 2a, 7″) and polonicum group (4′, 1a, 7″). Peridinium species with a plate formula of 4′, 3a, 7″ diverged last. Thus, 18S and 28S rDNA D1/D2 sequences are informative about relationships among Peridinium species. Statistical analyses revealed that the 28S rDNA D1/D2 region had a significantly higher genetic divergence than the 18S rDNA region, suggesting that the former as DNA markers may be more suitable for sequence‐based delimitation of Peridinium. The rDNA sequences had sufficient discriminative power to separate P. bipes f. occultaum (Er. Lindem.) M. Lefèvre and P. bipes f. globosum Er. Lindem. into two distinct species, even though these taxa are morphologically only marginally discriminated by spines on antapical plates and the shape of red bodies during the generation of cysts. Our results suggest that 28S rDNA can be used for all Peridinium species to make species‐level taxonomic distinctions, allowing improved taxonomic classification of Peridinium.     

Dinoflagellate community analysis of a fish kill using denaturing gradient gel electrophoresis

A molecular method using the polymerase chain reaction (PCR) amplification of small subunit gene sequences (18S rDNA) and denaturing gradient gel electrophoresis (DGGE) was used to determine both the population complexity and species identification of organisms in harmful algal blooms. Eighteen laboratory cultures of dinoflagellates, including Akashiwo, Gymnodinium, Heterocapsa, Karenia, Karlodinium, Pfiesteria, and Pfiesteria-like species were analyzed using dinoflagellate-specific oligonucleotide primers and DGGE. The method is sensitive and able to determine the number of species in a sample, as well as the taxonomic identity of each species, and is particularly useful in detecting differences between species of the same genus, as well as differences between morphologically similar species. Using this method, each of eight Pfiesteria-like species was verified as being clonal isolates of Pfiesteria piscicida. The sensitivity of dinoflagellate DGGE is approximately 1000 cells/ml, which is 100-fold less sensitive than real-time PCR. However, the advantage of DGGE lies in its ability to analyze dinoflagellate community structure without needing to know what is there, while real-time PCR provides much higher sensitivity and detection levels, if probes exist for the species of interest, attributes that complement DGGE analysis. In a blinded test, dinoflagellate DGGE was used to analyze two environmental fish kill samples whose species composition had been previously determined by other analyses. DGGE correctly identified the dominant species in these samples as Karlodinium micrum and Heterocapsa rotundata, proving the efficacy of this method on environmental samples. Toxin analysis of a clonal isolate obtained from the fish kill samples confirmed the presence of KmTx2, corroborating the earlier genetic identification of toxic K. micrum in the fish kill water sample.     

Phenotypic and genotypic identification and phylogenetic characterisation of <Emphasis Type="Italic">Taphrina</Emphasis> fungi on alder

All Taphrina species are dimorphic with a mycelium stage biotrophic on vascular plants and a saprophytic yeast stage. European species of Taphrina on Alnus species (Betulaceae) were identified using morphological, physiological and molecular characteristics, the latter including determination of PCR fingerprints and of nucleotide sequences from selected nuclear ribosomal DNA regions. PCR fingerprinting gives a good overview of species identification, as do nucleotide sequences, which in addition, help to clarify phylogenetic relationships. Taphrina alni is a homogeneous species that exhibited more than 50% similarity in PCR fingerprinting with three different primers. Morphologically, it produces tongue-like outgrowths from female catkins of Alnus incana. Taphrina robinsoniana from A. rugosa and A. serrulata in North America is phylogenetically closely related to T. alni, but the two species could be separated by their PCR fingerprints, partial sequences of 26S rDNA (D1/D2) and ITS1/ITS2 sequences. T. epiphylla and T. sadebeckii are two phylogenetically closely related species. T. epiphylla causes witches brooms in crowns of A. incana. In addition, T. epiphylla forms slightly yellow white-grey leaf spots in midsummer on A. incana. Yellow white-grey leaf spots up to 10 mm on A. glutinosa are characteristic for T. sadebeckii. Both species can be separated well by PCR fingerprinting. Different from T. epiphylla, T. sadebeckii is genotypically more heterogeneous. Only two out of three different primers showed similarity values above 50% in different European strains of T. sadebeckii. Although genetic variability was not detected in complete sequences of the 18S ribosomal DNA of T. sadebeckii, ITS1/ITS2 sequences appeared to be more heterogeneous, too. Taphrina tosquinetii is a genotypically homogeneous species causing leaf curl on Alnus glutinosa. It was not possible to distinguish the yeast phases from different Taphrina species on Alnus using morphological and physiological characteristics only. Dedicated to Prof. Dr. Hanns Kreisel on the occasion of his 70^th birthday     

Rapid molecular identification of the harmful freshwater dinoflagellate <Emphasis Type="Italic">Peridinium</Emphasis> in various life stages using genus-specific single-cell PCR

Cysts of the freshwater dinoflagellate Peridinium are typically different from vegetative cells in shape and remain largely undescribed. Molecular discrimination of such cysts would be useful to a number of research disciplines. A reliable method for the amplification of ribosomal DNA (rDNA) from Peridinium at different life stages is described. This genotyping strategy relies on whole cell PCR using Peridinium-specific primers designed from available 18S rDNA sequences. Here, we demonstrate the effectiveness of Peridinium-specific PCR for the rapid molecular identification of Peridinium cells in various life stages such as vegetative, planozygote, hypnozygote and cyst.     

<Emphasis Type="Italic">Dasytricha</Emphasis> Dominance in Surti Buffalo Rumen Revealed by 18S rRNA Sequences and Real-Time PCR Assay

The genetic diversity of protozoa in Surti buffalo rumen was studied by amplified ribosomal DNA restriction analysis, 18S rDNA sequence homology and phylogenetic and Real-time PCR analysis methods. Three animals were fed diet comprised green fodder Napier bajra 21 (Pennisetum purpureum), mature pasture grass (Dicanthium annulatum) and concentrate mixture (20% crude protein, 65% total digestible nutrients). A protozoa-specific primer (P-SSU-342f) and a eukarya-specific primer (Medlin B) were used to amplify a 1,360 bp fragment of DNA encoding protozoal small subunit (SSU) ribosomal RNA from rumen fluid. A total of 91 clones were examined and identified 14 different 18S RNA sequences based on PCR–RFLP pattern. These 14 phylotypes were distributed into four genera-based 18S rDNA database sequences and identified as Dasytricha (57 clones), Isotricha (14 clones), Ostracodinium (11 clones) and Polyplastron (9 clones). Phylogenetic analyses were also used to infer the makeup of protozoa communities in the rumen of Surti buffalo. Out of 14 sequences, 8 sequences (69 clones) clustered with the Dasytricha ruminantium-like clone and 4 sequences (13 clones) were also phylogenetically placed with the Isotricha prostoma-like clone. Moreover, 2 phylotypes (9 clones) were related to Polyplastron multivesiculatum-like clone. In addition, the number of 18S rDNA gene copies of Dasytricha ruminantium (0.05% to ciliate protozoa) was higher than Entodinium sp. (2.0 × 10⁵ vs. 1.3 × 10⁴) in per ml ruminal fluid.     

Pfiesteria piscicida and Pfiesteria shumwayae in coastal waters of Long Island, New York, USA

Water and sediment samples were collected during summer and early fall 1999–2004 from coastal waters of New York State, USA, to test for the presence of Pfiesteria piscicida and Pfiesteria shumwayae. Physical and chemical conditions were characterized, and real-time polymerase chain reaction assays were conducted. Both species were relatively common and found at most sites at least once, and the frequency of positive assays was higher in sediments than in the water column. In a subset of the data from Suffolk County, Long Island, the presence of Pfiesteria was related to high chlorophyll a and relatively high nutrient concentrations. Partial SSU rDNA sequences of four PCR amplicons generated using P. shumwayae primers indicated two sequences: three were identical to GenBank P. shumwayae entries, but one showed enough sequence difference (15 positions in a 454 bp amplicon) to suggest a possible new species. Three isolates were tested for toxicity, and one was found to kill fish in bioassays. Despite the widespread presence of both Pfiesteria species and demonstration of potential to harm fish, no blooms of these dinoflagellates have been observed, nor has there been evidence of Pfiesteria-related fish or human health problems in these waters, likely related to colder temperatures than optimal for Pfiesteria species.