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The identification of sea bass (Centropristis) larvae to species is difficult because of similar morphological characters, spawning times, and overlapping species ranges. Black sea bass (Centropristis striata) is an important [...]

Sensitive PCR and fluorescent peptide nuclei acid in situ hybridization (FISH) assays were developed to detect and identify Aphanomyces invadans and to use the assays to screen populations of infected fish. Neither the FISH assay nor the PCR assay cross-reacted with other closely related oomycetes providing strong evidence that the pathogenic oomycete is the primary etiological agent in ulcerative mycosis, an ulcerative skin disease caused by a fungus-like agent of wild and cultured fish.

The two most toxic Gambierdiscus species identified from the Caribbean are G. excentricus and G. silvae. These species are the primary causes of ciguatera fish poisoning and likely contribute disproportionately to the toxicity of marine food webs. While Gambierdiscus species are difficult to distinguish using light or scanning electron microscopy, reliable species-specific molecular identification methods have been developed and used successfully to identify a number of other Gambierdiscus species. Corresponding speciesspecific assays are not yet available for G. excentricus and G. silvae, which imposes limitations on species identification and related ecological studies. The following note describes species-specific polymerase chain reaction assays for G. excentricus and G. silvae that can be used for these purposes. [ABSTRACT FROM AUTHOR]

A voided urine sample, obtained from a 13-year-old intact male dog residing in a laboratory animal research facility, was observed to contain biflagellate protozoa 5 days following an episode of gross hematuria. The protozoa were identified as belonging to the class Kinetoplastea on the basis of light microscopic observation of Wright–Giemsa-stained urine sediment in which the kinetoplast was observed basal to 2 anterior flagella. A polymerase chain reaction (PCR) assay using primers corresponding with conserved regions within the 18S ribosomal RNA gene of representative kinetoplastid species identified nucleotide sequences with 100% identity to Parabodo caudatus. Parabodo caudatus organisms were unable to be demonstrated cytologically or by means of PCR in samples collected from the dog’s environment. The dog had a history of 50 complete urinalyses performed over the 12-year period preceding detection of P. caudatus, and none of these were noted to contain protozoa. Moreover, the gross hematuria that was documented 5 days prior to detection of P. caudatus had never before been observed in this dog. Over the ensuing 2.5 years of the dog’s life, 16 additional complete urinalyses were performed, none of which revealed the presence of protozoa. Bodonids are commonly found in soil as well as in freshwater and marine environments. However, P. caudatus, in particular, has a 150-year-long, interesting, and largely unresolved history in people as either an inhabitant or contaminant of urine. This historical conundrum is revisited in the current description of P. caudatus as recovered from the urine of a dog. [ABSTRACT FROM PUBLISHER]

The article presents a study which investigated Aphanomyces invadans pathogens which cause epizootic ulcerative syndrome (EUS) in captive bullseye snakehead Channa marulfus from freshwater canals in Miami-Dade County, Florida. It is stated that histological examination revealed presence into the musculature and internal organs. The pathogens can gain entry through surface wounds or an otherwise compromised mucus barrier. The findings of therapeutic treatments with nitrofurazone and furazolidone are discussed.

Gambierdiscus species produce toxins that cause ciguatera fish poisoning (CFP), the most common nonbacterial illness associated with fish consumption worldwide. Understanding the role that individual Gambierdiscus species play in causing CFP is hampered because the morphologically similar species in the genus are difficult to distinguish. Ambiguities in the description of the type species Gambierdiscus toxicus also exist. This paper presents detailed line drawings along with additional scanning electron microscopy (SEM) and light micrographs to more fully characterize the six known Gambierdiscus species in addition to describing four new species, Gambierdiscus caribaeus sp. nov., Gambierdiscus carolinianus sp. nov., Gambierdiscus carpenteri sp. nov., and Gambierdiscus ruetzleri sp. nov. All four of the new species are photosynthetic and epibenthic and have a Kofoidian plate formula of Po, 3′, 7″, 6C, 6 or 7S, 5′″, 1p, and 2‴′. Establishment of these species is supported by both morphological differences and three independent phylogenetic analyses utilizing small-subunit, as well as D1-D3 and D8-D10 large-subunit rDNA gene sequences. During the course of this study it became apparent that the description of the type species, G. toxicus, included more than one species. Since no type material exists we propose that Fig. 1 in Adachi and Fukuyo (1979) be designated as the lectotype for this species and that the epithecal view of isolate GTT-91 shown in Fig. 1 from Chinain et al. (1999) be designated as the epitype. The GTT-91 isolate has been well characterized genetically and the original SEM stub of this isolate archived at the Smithsonian Institution has been re-examined to provide a more detailed morphological analysis of the epitype. [ABSTRACT FROM AUTHOR]

A new genus of Pfiesteria-like heterotrophic dinoflagellate, Luciella gen. nov., and two new species, Luciella masanensis sp. nov. and Luciella atlantis sp. nov., are described. These species commonly occur with other small (<20 μm) heterotrophic and mixotrophic dinoflagellates in estuaries from Florida to Maryland and the southern coast of Korea, suggesting a possible global distribution. An SEM analysis indicates that members of the genus Luciella have the enhanced Kofoidian plate formula of Po, cp, X, 4′, 2a, 6″, 6c, PC, 5+s, 5‴, 0p, and 2″″. The two four-sided anterior intercalary plates are diamond shaped. The genus Luciella differs from the other genera in the Pfiesteriaceae by a least one plate in the plate tabulation and in the configuration of the two anterior intercalary plates. An SSU rDNA phylogenetic analysis confirmed the genus as monophyletic and distinct from the other genera in the Pfiesteriaceae. The morphology of Luciella masanensis closely resembles Pfiesteria piscicida Steid. et J. M. Burkh. and other Pfiesteria-like dinoflagellates in size and shape, making it easily misidentified using LM. Luciella atlantis, in contrast, has a more distinctive morphology. It can be distinguished from L. masanensis and other Pfiesteria-like organisms by a larger cell size, a more conical-shaped epitheca and hypotheca, larger rhombic-shaped intercalary plates, and an asymmetrical hypotheca. The genus Luciella is assigned to the order Peridiniales and the family Pfiesteriaceae based on plate tabulation, plate pattern, general morphology, and phylogenetic analysis. [ABSTRACT FROM AUTHOR]

Dinoflagellate taxonomy is based primarily on morphology and morphometric data that can be difficult to obtain. In contrast, molecular data can be rapidly and cost-effectively acquired, which has led to a rapid accumulation of sequence data in GenBank. Currently there are no systematic criteria for utilizing taxonomically unassigned sequence data to identify putative species that could in turn serve as a basis for testable hypotheses concerning the taxonomy, diversity, distribution, and toxicity of these organisms. The goal of this research was to evaluate whether simple, uncorrected genetic distances ( p) calculated using ITS1/5.8S/ITS2 (ITS region) rDNA sequences could be used to develop criteria for recognizing putative species before formal morphological evaluation and classification. The current analysis used sequences from 81 dinoflagellate species belonging to 14 genera. For this diverse assemblage of dinoflagellate species, the within-species genetic distances between ITS region copies ( p=0.000–0.021 substitutions per site) were consistently less than those observed between species ( p=0.042–0.580). Our results indicate that a between-species uncorrected genetic distance of p≥0.04 could be used to delineate most free-living dinoflagellate species. Recently evolved species, however, may have ITS p values <0.04 and would require more extensive morphological and genetic analyses to resolve. For most species, the sequence of the dominant ITS region allele has the potential to serve as a unique species-specific “DNA barcode” that could be used for the rapid identification of dinoflagellates in field and laboratory studies. [ABSTRACT FROM AUTHOR]

Pfiesteria shumwayaeGlasgow et Burkholder is assigned to a new genusPseudopfiesteriagen. nov. Plate tabulation differences betweenPfiesteriaandPseudopfiesteriagen. nov. as well as a maximum likelihood phylogenetic analysis based on rDNA sequence data warrant creation of this new genus. The Kofoidian thecal plate formula for the new genus is Po, cp, X, 4′, 1a, 6′′, 6c, PC, 5+s, 5′′′, 0p, 2′′′′. In addition to having six precingular plates,P. shumwayaecomb. nov. also has a distinctive diamond or rectangular-shaped anterior intercalary plate. BothPfiesteriaandPseudopfiesteriagen. nov. are reassigned to the order Peridiniales based on an apical pore complex (APC) with a canal (X) plate that contacts a symmetrical 1′, four to five sulcal plates, and the conservative hypothecal tabulation of 5′′′, 0p, and 2′′′′. These morphological characters and the life histories ofPfiesteriaandPseudopfiesteriaare consistent with placement of both genera in the Peridiniales. Based on the plate tabulations forP.shumwayae, P. piscicida, and the closely related“cryptoperidiniopsoid” and“lucy” groups, the family Pfiesteriaceae is amended to include species with the following tabulation: 4-5′, 0-2a, 5-6′′, 6c, PC, 5+s, 5′′′, 0p, and 2′′′′ as well as an APC containing a pore plate (Po), a closing plate (cp), and an X plate; the tabulation is expanded to increase the number of sulcal plates and to include a new plate, the peduncle cover (PC) plate. Members of the family have typical dinoflagellate life cycles characterized by a biflagellated free-living motile stage, a varying number of cyst stages, and the absence of multiple amoeboid stages. [ABSTRACT FROM AUTHOR]

The putative harmful algal bloom dinoflagellate, Pfiesteria piscicida (Steidinger et Burkholder), frequently co-occurs with other morphologically similar species collectively known as Pfiesteria -like organisms (PLOs). This study specifically evaluated whether unique sequences in the internal transcribed spacer (ITS) regions, ITS1 and ITS2, could be used to develop PCR assays capable of detecting PLOs in natural assemblages. ITS regions were selected because they are more variable than the flanking small subunit or large subunit rRNA genes and more likely to contain species-specific sequences. Sequencing of the ITS regions revealed unique oligonucleotide primer binding sites for Pfiesteria piscicida , Pfiesteria shumwayae (Glasgow et Burkholder), Florida “Lucy” species, two cryptoperidiniopsoid species, “H/V14” and “PLO21,” and the estuarine mixotroph, Karlodinium micrum (Leadbetter et Dodge). These PCR assays had a minimum sensitivity of 100 cells in a 100-mL sample (1 cell·mL-1 ) and were successfully used to detect PLOs in the St. Johns River system in Florida, USA. DNA purification and aspects of PCR assay development, PCR optimization, PCR assay controls, and collection of field samples are discussed. [ABSTRACT FROM AUTHOR]

The putative harmful algal bloom dinoflagellate, Pfiesteria piscicida (Steidinger et Burkholder), frequently co-occurs with other morphologically similar species collectively known as Pfiesteria -like organisms (PLOs). This study specifically evaluated whether unique sequences in the internal transcribed spacer (ITS) regions, ITS1 and ITS2, could be used to develop PCR assays capable of detecting PLOs in natural assemblages. ITS regions were selected because they are more variable than the flanking small subunit or large subunit rRNA genes and more likely to contain species-specific sequences. Sequencing of the ITS regions revealed unique oligonucleotide primer binding sites for Pfiesteria piscicida , Pfiesteria shumwayae (Glasgow et Burkholder), Florida “Lucy” species, two cryptoperidiniopsoid species, “H/V14” and “PLO21,” and the estuarine mixotroph, Karlodinium micrum (Leadbetter et Dodge). These PCR assays had a minimum sensitivity of 100 cells in a 100-mL sample (1 cell·mL-1 ) and were successfully used to detect PLOs in the St. Johns River system in Florida, USA. DNA purification and aspects of PCR assay development, PCR optimization, PCR assay controls, and collection of field samples are discussed. [ABSTRACT FROM AUTHOR]

Free-living, marine dinoflagellates are typified by a well-defined, haplontic life cycle with relatively few stages. The most unusual departure from this life cycle is one reported for the heterotrophic dinoflagellate Pfiesteria piscicida Steidinger et Burkholder. This species is alleged to have at least 24 life cycle stages including amoebae and a chrysophyte-like cyst form (Burkholder et al. 1992, Burkholder and Glasgow 1997a) not previously known in free-living marine dinoflagellates. Litaker et al. (2002) redescribed the life cycle of P. piscicida from single-cell isolates and found only life cycle stages typical of free-living marine dinoflagellates. The discrepancy between these observations and the life cycle reported in the literature prompted a rigorous study to resolve the life cycle of P. piscicida. Burkholder and Glasgow (2002) took exception to this study, arguing that Litaker et al. (2002) misunderstood the life cycle of P. piscicida and ignored recent publications. We present a rebuttal of their criticisms and suggest a simple way to resolve the discrepancies in the P. piscicida life cycle. [ABSTRACT FROM AUTHOR]

Free-living, marine dinoflagellates are typified by a well-defined, haplontic life cycle with relatively few stages. The most unusual departure from this life cycle is one reported for the heterotrophic dinoflagellate Pfiesteria piscicida Steidinger et Burkholder. This species is alleged to have at least 24 life cycle stages including amoebae and a chrysophyte-like cyst form (Burkholder et al. 1992, Burkholder and Glasgow 1997a) not previously known in free-living marine dinoflagellates. Litaker et al. (2002) redescribed the life cycle of P. piscicida from single-cell isolates and found only life cycle stages typical of free-living marine dinoflagellates. The discrepancy between these observations and the life cycle reported in the literature prompted a rigorous study to resolve the life cycle of P. piscicida. Burkholder and Glasgow (2002) took exception to this study, arguing that Litaker et al. (2002) misunderstood the life cycle of P. piscicida and ignored recent publications. We present a rebuttal of their criticisms and suggest a simple way to resolve the discrepancies in the P. piscicida life cycle. [ABSTRACT FROM AUTHOR]

The putatively toxic dinoflagellate Pfiesteria piscicida (Steidinger et Burkholder) has been reported to have an unusual life cycle for a free-living marine dinoflagellate. As many as 24 life cycle stages were originally described for this species. During a recent phylogenetic study in which we used clonal cultures of P. piscicida , we were unable to confirm many reported life cycle stages. To resolve this discrepancy, we undertook a rigorous examination of the life cycle of P. piscicida using nuclear staining techniques combined with traditional light microscopy, high-resolution video microscopy, EM, and in situ hybridization with a suite of fluorescently labeled peptide nucleic acid (PNA) probes. The results showed that P. piscicida had a typical haplontic dinoflagellate life cycle. Asexual division occurred within a division cyst and not by binary fission of motile cells. Sexual reproduction of this homothallic species occurred via the fusion of isogamous gametes. Examination of tanks where P. piscicida was actively feeding on fish showed that amoebae were present; however, they were contaminants introduced with the fish. Whole cell probing using in situ hybridization techniques confirmed that these amoebae were hybridization negative for a P. piscicida -specific PNA probe. Direct observations of clonal P. piscicida cultures revealed no unusual life cycle stages. Furthermore, the results of this study provided no evidence for transformations to amoebae. We therefore conclude that P. piscicida has a life cycle typical of free-living marine dinoflagellates and lacks any amoeboid or other specious stages. [ABSTRACT FROM AUTHOR]

The putatively toxic dinoflagellate Pfiesteria piscicida (Steidinger et Burkholder) has been reported to have an unusual life cycle for a free-living marine dinoflagellate. As many as 24 life cycle stages were originally described for this species. During a recent phylogenetic study in which we used clonal cultures of P. piscicida , we were unable to confirm many reported life cycle stages. To resolve this discrepancy, we undertook a rigorous examination of the life cycle of P. piscicida using nuclear staining techniques combined with traditional light microscopy, high-resolution video microscopy, EM, and in situ hybridization with a suite of fluorescently labeled peptide nucleic acid (PNA) probes. The results showed that P. piscicida had a typical haplontic dinoflagellate life cycle. Asexual division occurred within a division cyst and not by binary fission of motile cells. Sexual reproduction of this homothallic species occurred via the fusion of isogamous gametes. Examination of tanks where P. piscicida was actively feeding on fish showed that amoebae were present; however, they were contaminants introduced with the fish. Whole cell probing using in situ hybridization techniques confirmed that these amoebae were hybridization negative for a P. piscicida -specific PNA probe. Direct observations of clonal P. piscicida cultures revealed no unusual life cycle stages. Furthermore, the results of this study provided no evidence for transformations to amoebae. We therefore conclude that P. piscicida has a life cycle typical of free-living marine dinoflagellates and lacks any amoeboid or other specious stages. [ABSTRACT FROM AUTHOR]