R. Wayne Litaker

Global distribution of ciguatera causing dinoflagellates in the genus Gambierdiscus

Dinoflagellates in the genus Gambierdiscus produce toxins that bioaccumulate in tropical and sub-tropical fishes causing ciguatera fish poisoning (CFP). Little is known about the diversity and distribution of Gambierdiscus species, the degree to which individual species vary in toxicity, and the role each plays in causing CFP. This paper presents the first global distribution of Gambierdiscus species. Phylogenetic analyses of the existing isolates indicate that five species are endemic to the Atlantic (including the Caribbean/West Indies and Gulf of Mexico), five are endemic to the tropical Pacific, and that two species, Gambierdiscus carpenteri and Gambierdiscus caribaeus are globally distributed. The differences in Gambierdiscus species composition in the Atlantic and Pacific correlated with structural differences in the ciguatoxins reported from Atlantic and Pacific fish. This correlation supports the hypothesis that Gambierdiscus species in each region produce different toxin suites. A literature survey indicated a >100-fold variation in toxicity among species compared with a 2 to 9-fold within species variation due to changing growth conditions. These observations suggest that CFP events are driven more by inherent differences in species toxicity than by environmental modulation. How variations in species toxicity may affect the development of an early warning system for CFP is discussed.

Taxonomy of Gambierdiscus including four new species, Gambierdiscus caribaeus, Gambierdiscus carolinianus, Gambierdiscus carpenteri and Gambierdiscus ruetzleri (Gonyaulacales, Dinophyceae)

Litaker R.W., Vandersea M.W., Faust M.A., Kibler S.R., Chinain M., Holmes M.J., Holland W.C. and Tester P.A. 2009. Taxonomy of Gambierdiscus including four new species, Gambierdiscus caribaeus, Gambierdiscus carolinianus, Gambierdiscus carpenteri and Gambierdiscus ruetzleri (Gonyaulacales, Dinophyceae). Phycologia 48: 344–390. DOI: 10.2216/07-15.1 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.

Rapid One-Step Quantitative Reverse Transcriptase PCR Assay with Competitive Internal Positive Control for Detection of Enteroviruses in Environmental Samples

ABSTRACT Human enteroviruses can serve as a more accurate indicator of human fecal contamination than conventional bacteriological fecal indicators. We describe here a quantitative reverse transcriptase PCR (qRT-PCR) assay specifically tailored to detect these viruses in environmental waters. The assay included a competitive internal positive control (CIPC) that allowed the inhibition of qRT-PCRs to be quantitatively assessed. Coamplification of the CIPC with enteroviral genetic material did not affect the sensitivity, specificity, or reproducibility of the enteroviral qRT-PCR assay. The assay is rapid (less than 5 h from sample to result), has a wide dynamic range (>3 logs), and is capable of detecting as few as 25 enteroviral genomes with an average amplification efficiency of 0.91. In samples with low or moderate inhibition, the delay in CIPC amplification was used to adjust enterovirus qRT-PCR concentrations to account for losses due to inhibition. Samples exhibiting significant inhibition were not corrected but instead diluted twofold and immediately assayed again. Using significantly inhibited samples, it was found that dilution relieved inhibition in 93% (25 of 27) of the samples. In addition, 15% (4 of 27) of these previously negative samples contained enteroviral genomes. The high-throughput format of the assay compared to conventional culture-based methods offers a fast, reliable, and specific method for detecting enteroviruses in environmental water samples. The ability of the assay to identify false negatives and provide improved quantitative assessments of enterovirus concentrations will facilitate the tracking of human fecal contamination and the assessment of potential public health risk due to enteroviruses in recreational and shellfish harvesting waters.

TRITRICHOMONAS FOETUS AND NOT PENTATRICHOMONAS HOMINIS IS THE ETIOLOGIC AGENT OF FELINE TRICHOMONAL DIARRHEA

Recently, several investigators have reported large-bowel diarrhea in cats associated with intestinal trichomonad parasites. These reports have presumptively identified the flagellates as Pentatrichomonas hominis, an organism putatively capable of infecting the intestinal tracts of a number of mammalian hosts, including cats, dogs, and man. The purpose of the present study was to determine the identity of this recently recognized flagellate by means of rRNA gene sequence analysis; restriction enzyme digest mapping; and light, transmission, and scanning electron microscopy (SEM).

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.

Late-summer phytoplankton in western Lake Erie (Laurentian Great Lakes): bloom distributions, toxicity, and environmental influences

Phytoplankton abundance and composition and the cyanotoxin, microcystin, were examined relative to environmental parameters in western Lake Erie during late-summer (2003–2005). Spatially explicit distributions of phytoplankton occurred on an annual basis, with the greatest chlorophyll (Chl) a concentrations occurring in waters impacted by Maumee River inflows and in Sandusky Bay. Chlorophytes, bacillariophytes, and cyanobacteria contributed the majority of phylogenetic-group Chl a basin-wide in 2003, 2004, and 2005, respectively. Water clarity, pH, and specific conductance delineated patterns of group Chl a, signifying that water mass movements and mixing were primary determinants of phytoplankton accumulations and distributions. Water temperature, irradiance, and phosphorus availability delineated patterns of cyanobacterial biovolumes, suggesting that biotic processes (most likely, resource-based competition) controlled cyanobacterial abundance and composition. Intracellular microcystin concentrations corresponded to Microcystis abundance and environmental parameters indicative of conditions coincident with biomass accumulations. It appears that environmental parameters regulate microcystin indirectly, via control of cyanobacterial abundance and distribution.

Microcystin Concentrations and Genetic Diversity of Microcystis in the Lower Great Lakes

The resurgence of Microcystis blooms in the lower Great Lakes region is of great concern to public and ecosystem health due to the potential for these colonial cyanobacteria to produce hepatotoxic microcystins. A survey of Microcystis cell densities and microcystin concentrations during August 2004 showed particularly high concentrations of both cells and toxin in the nearshore regions of Saginaw Bay (Lake Huron) and western Lake Erie, often exceeding the World Health Organizations recommended drinking water limit of 1 μg L−1. The dominant congener of microcystin in both basins was microcystin‐LR (MC‐LR), whereas the second most abundant congeners, accounting for up to 20–25% of the total microcystin concentrations, were MC‐LA in Saginaw Bay and MC‐RR in western Lake Erie. Multiplex PCR assays of Microcystis colonies isolated from these two regions showed that a much greater percentage of the Microcystis colonies from Saginaw Bay carried the mcyB gene necessary for microcystin production, in comparison with those from western Lake Erie. The mcyB genotypes sequenced separated into two distinct phylogenetic clusters, with Microcystis originating from Lake Erie predominantly in one branch and from Saginaw Bay present in both branches. These results indicate that the genetic composition of the bloom could impact the concentrations and congeners of microcystin produced and that the cell count methods currently being used to gauge public health threats posed by Microcystis blooms may not sufficiently assess actual bloom toxicity.

LIFE CYCLE OF THE HETEROTROPHIC DINOFLAGELLATE PFIESTERIA PISCICIDA (DINOPHYCEAE)1

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.

DEVELOPMENT OF SEMI-QUANTITATIVE PCR ASSAYS FOR THE DETECTION AND ENUMERATION OF GAMBIERDISCUS SPECIES (GONYAULACALES, DINOPHYCEAE)(1).

Ciguatera fish poisoning (CFP) is a serious health problem in tropical regions and is caused by the bioaccumulation of lipophilic toxins produced by dinoflagellates in the genus Gambierdiscus. Gambierdiscus species are morphologically similar and are difficult to distinguish from one another even when using scanning electron microscopy. Improved identification and detection methods that are sensitive and rapid are needed to identify toxic species and investigate potential distribution and abundance patterns in relation to incidences of CFP. This study presents the first species‐specific, semi‐quantitative polymerase chain reaction (qPCR) assays that can be used to address these questions. These assays are specific for five Gambierdiscus species and one undescribed ribotype. The assays utilized a SYBR green format and targeted unique sequences found within the SSU, ITS, and the D1/D3 LSU ribosomal domains. Standard curves were constructed using known concentrations of cultured cells and 10‐fold serial dilutions of rDNA PCR amplicons containing the target sequence for each specific assay. Assay sensitivity and accuracy were tested using DNA extracts purified from known concentrations of multiple Gambierdiscus species. The qPCR assays were used to assess Gambierdiscus species diversity and abundance in samples collected from nearshore areas adjacent to Ft. Pierce and Jupiter, Florida USA. The results indicated that the practical limit of detection for each assay was 10 cells per sample. Most interestingly, the qPCR analysis revealed that as many as four species of Gambierdiscus were present in a single macrophyte sample.

Molecular taxonomy of the suborder Bodonina (order Kinetoplastida), including the important fish parasite, Ichthyobodo necator

Abstract Ichthyobodo necator is an important fish ectoparasite with a broad host and ecological range. A novel method, involving the use of an anesthetic, allowed the collection of large numbers of parasites from the skin and gills of hybrid striped bass (Morone saxatilis male × M. chrysops female). Genomic DNA from these samples was used to amplify and clone the 18S rRNA gene. The 18S rRNA gene was similarly cloned from Bodo caudatus, Bodo edax, Bodo saltans, an unidentified Bodo species, and Dimastigella trypaniformis. The resulting sequences were aligned with other representative kinetoplastid species using pileup and similarities in secondary structure. Phylogenetic relationships within the suborder Bodonina and representatives of the suborder Trypanosomatina were determined using maximum-likelihood statistics. The phylogenetic analyses strongly supported the order Kinetoplastida as a monophyletic assemblage consisting of at least two major lineages. One lineage consisted exclusively of I. necator, indicating that it may represent a new suborder. The second lineage consisted of all other kinetoplastid species. This second lineage appeared to contain at least 8 bodonine sublineages, none of which correlated with currently recognized families. For three sublineages, there was a close correspondence between the 18S phylogeny and the classical taxonomy of Dimastigella, Rhynchobodo, and Rhynchomonas. In contrast, Bodo and Cryptobia were polyphyletic, containing species in two or more sublineages that may represent separate genera.