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Ciguatera poisoning is caused by the consumption of reef fish or shellfish that have accumulated ciguatoxins, neurotoxins produced by benthic dinoflagellates of the genera Gambierdiscus or Fukuyoa. Although ciguatera constitutes the primary cause of seafood intoxication in Cuba, very little information is available on the occurrence of ciguatoxins in the marine food web and the causative benthic dinoflagellate species. This study conducted on the south-central coast of Cuba reports the occurrence of Gambierdiscus and Fukuyoa genera and the associated benthic genera Ostreopsis and Prorocentrum. Gambierdiscus/Fukuyoa cells were present at low to moderate abundances depending on the site and month of sampling. This genus was notably higher on Dictyotaceae than on other macrophytes. PCR analysis of field-collected samples revealed the presence of six different Gambierdiscus and one Fukuyoa species, including G. caribaeus, G. carolinianus, G. carpenteri, G. belizeanus, F. ruetzleri, G. silvae, and Gambierdiscus sp. ribotype 2. Only Gambierdiscus excentricus was absent from the eight Gambierdiscus/Fukuyoa species known in the wider Caribbean region. Eleven clonal cultures were established and confirmed by PCR and SEM as being either G. carolinianus or G. caribaeus. Toxin production in each isolate was assessed by a radioligand receptor binding assay and found to be below the assay quantification limit. These novel findings augment the knowledge of the ciguatoxin-source dinoflagellates that are present in Cuba, however further studies are needed to better understand the correlation between their abundance, species-specific toxin production in the environment, and the risk for fish contamination, in order to develop better informed ciguatera risk management strategies.

Despite the long history of paralytic shellfish poisoning (PSP) events in Alaska, little is known about the seasonal distribution and abundance of the causative organism, Alexandrium, or the environmental factors that govern toxic bloom development. To address this issue, a five year study (2012–2017) was undertaken in Kachemak Bay and lower Cook Inlet Alaska to determine how the occurrence of Alexandrium catenella, the dominant PSP-causing Alexandrium species, was influenced by temperature, salinity, nutrient concentrations, and other environmental factors. Cell concentrations from 572 surface water samples were estimated using quantitative PCR. Monthly sampling revealed a seasonal pattern of A. catenella bloom development that was positively correlated with water temperature. Prevailing salinity conditions did not significantly affect abundance, nor was nutrient limitation a direct factor. Elevated cell concentrations were detected in 35 samples from Kachemak Bay (100-3050 cell eq. L−1) while a maximum abundance of 67 cell eq. L−1 was detected in samples from lower Cook Inlet sites. Monitoring data showed average water temperatures in Kachemak Bay increased by ∼2 °C over the course of the study and were accompanied by an increase in Alexandrium abundance. Based on these findings, 7–8 °C appears to represent a temperature threshold for significant bloom development in Kachemak Bay, with the greatest risk of shellfish toxicity occurring when temperatures exceed 10–12 °C. The role of temperature is further supported by time series data from the Alaska Coastal Current (station GAK1), which showed that summertime shellfish toxicity events in Kachemak Bay generally followed periods of anomalously high winter water temperatures. These data indicate monitoring changes in water temperatures may be used as an early warning signal for subsequent development of shellfish toxicity in Kachemak Bay.

A brown tide bloom of Aureoumbra lagunensis developed in Guantanamo Bay, Cuba during a period of drought in 2013 that followed heavy winds and rainfall from Hurricane Sandy in late October 2012. Based on satellite images and water turbidity measurements, the bloom appeared to initiate in January 2013. The causative species (A. lagunensis) was confirmed by microscopic observation, and pigment and genetic analyses of bloom samples collected on May 28 of that year. During that time, A. lagunensis reached concentrations of 900,000 cells ml−1 (28 ppm by biovolume) in the middle portion of the Bay. Samples could not be collected from the northern (Cuban) half of the Bay because of political considerations. Subsequent sampling of the southern half of the Bay in November 2013, April 2014, and October 2014 showed persistent lower concentrations of A. lagunensis, with dominance shifting to the cyanobacterium Synechococcus (up to 33 ppm in April), an algal group that comprised a minor bloom component on May 28. Thus, unlike the brown tide bloom in Laguna Madre, which lasted 8 years, the bloom in Guantanamo Bay was short-lived, much like recent blooms in the Indian River, Florida. Although hypersaline conditions have been linked to brown tide development in the lagoons of Texas and Florida, observed euhaline conditions in Guantanamo Bay (salinity 35–36) indicate that strong hypersalinity is not a requirement for A. lagunensis bloom formation. Microzooplankton biomass dominated by ciliates was high during the observed peak of the brown tide, and ciliate abundance was high compared to other systems not impacted by brown tide. Preferential grazing by zooplankton on non-brown tide species, as shown in A. lagunensis blooms in Texas and Florida, may have been a factor in the development of the Cuban brown tide bloom. However, subsequent selection of microzooplankton capable of utilizing A. lagunensis as a primary food source may have contributed to the short-lived duration of the brown tide bloom in Guantanamo Bay.

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 species-specific 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.

Paralytic shellfish poisoning (PSP) poses a serious health threat in Alaska and prevents effective utilization of shellfish resources by subsistence and recreational harvesters. Substantial economic losses also affect shellfish growers during PSP events. The toxins responsible for PSP are produced by dinoflagellates in the genus Alexandrium. Despite the persistent threat posed by PSP and the long history of shellfish toxicity research, there is still confusion concerning the Alexandrium species that cause PSP in Alaska. The primary objective of this study was to identify the toxic Alexandrium species present in Alaska and to develop polymerase chain reaction (PCR) assays for use in screening phytoplankton and sediment samples. Before developing the PCR assays for this study, we evaluated published assays and many were not adequate because of primer dimer formation or because of cross-reactivity. Rather than continue to grapple with the uncertainty and inadequacy of published assays, we developed new assays for the Alexandrium species most likely to be present in Alaska. Only Alexandrium fundyense Group I and A. ostenfeldii were identified from four sampling regions from southeast Alaska to Kodiak Island, indicating that these two species are widely distributed. PCR assays for these two species were converted to quantitative (q)PCR format for use in monitoring programs. During the course of this study, we realized that a systematic evaluation of all published (~150) Alexandrium species-specific assays would be of benefit. Toward this objective, we collated published Alexandrium PCR, qPCR, and in situ hybridization assay primers and probes that targeted the small-subunit (SSU), internal transcribed spacer (ITS/5.8S), or D1-D3 large-subunit (LSU) (SSU/ITS/LSU) ribosomal DNA genes. Each individual primer or probe was screened against the GenBank database and Alexandrium gene sequence alignments constructed as part of this study. These data were used to identify a suite of species-specific Alexandrium assays that can be recommended for evaluation by the global harmful algal bloom community.

In autumn of 2013 an immense dinoflagellate bloom developed in Kachemak Bay, AK, USA. Much of the Bay was discolored a dark amber color and raised public concerns as small scale fish kills were reported in a few locations. Light microscopy revealed a monospecific bloom of gymnodinoid dinoflagellates that were previously unknown from the Bay. Gene sequencing of SSU rDNA from cells collected from the bloom confirmed the causative species to be Karenia mikimotoi. This represents the first report of a K. mikimotoi bloom in Alaska. After the bloom organism was confirmed, a K. mikimotoi species-specific qPCR assay was developed and used to assess K. mikimotoi abundances in DNA extracted from phytoplankton samples from Kachemak Bay and Lower Cook Inlet (LCI) obtained over a six-year period. The K. mikimotoi abundances were compared with corresponding time series of environmental variables (water temperature, salinity, water column stability, nutrients, precipitation and wind speed) to assess the factors contributing to the development of the bloom. The results showed early bloom development occurred in August when snow melt reduced salinities and increased water column stability during a period of calm winds. Peak bloom concentrations occurred in late September (107 cell eq. L-1) even as water temperatures were decreasing. The bloom gradually declined over the winter but persisted until April of 2014. Karenia mikimotoi cells were not detected two years prior or three years following the bloom, suggesting cells were introduced to Kachemak Bay at a time when conditions allowed K. mikimotoi to thrive.

In Cuba, ciguatera poisoning associated with fish consumption is the most commonly occurring non-bacterial seafood-borne illness. Risk management through fish market regulation has existed in Cuba for decades and consists of bans on selected species above a certain weight, however, the actual occurrence of ciguatoxins (CTXs) in seafood has never been verified. From this food safety risk management perspective, a study site locally known to be at risk for ciguatera was selected. Analysis of the epiphytic dinoflagellate community identified the microalga Gambierdiscus. Gambierdiscus species included six of the seven species known to be present in Cuba (G. caribaeus, G. belizeanus, G. carpenteri, G. carolinianus, G. silvae, and F. ruetzleri). CTX-like activity in invertebrates, herbivorous and carnivorous fishes were analyzed with a radioligand receptor-binding assay and, for selected samples, with the N2A cell cytotoxicity assay. CTX activity was found in 80% of the organisms sampled, with toxin values ranging from 2 to 8 ng CTX3C equivalents g&minus, 1 tissue. Data analysis further confirmed CTXs trophic magnification. This study constitutes the first finding of CTX-like activity in marine organisms in Cuba and in herbivorous fish in the Caribbean. Elucidating the structure&ndash, activity relationship and toxicology of CTX from the Caribbean is needed before conclusions may be drawn about risk exposure in Cuba and the wider Caribbean.

The sea urchin Tripneustes gratilla (Toxopneustidae, Echinoids) is a source of protein for many islanders in the Indo-West Pacific. It was previously reported to occasionally cause ciguatera-like poisoning; however, the exact nature of the causative agent was not confirmed. In April and July 2015, ciguatera poisonings were reported following the consumption of T. gratilla in Anaho Bay (Nuku Hiva Island, Marquesas archipelago, French Polynesia). Patient symptomatology was recorded and sea urchin samples were collected from Anaho Bay in July 2015 and November 2016. Toxicity analysis using the neuroblastoma cell–based assay (CBA-N2a) detected the presence of ciguatoxins (CTXs) in T. gratilla samples. Gambierdiscus species were predominant in the benthic assemblages of Anaho Bay, and G. polynesiensis was highly prevalent in in vitro cultures according to qPCR results. Liquid chromatography–tandem mass spectrometry (LC-MS/MS) analyses revealed that P-CTX-3B was the major ciguatoxin congener in toxic sea urchin samples, followed by 51-OH-P-CTX-3C, P-CTX-3C, P-CTX-4A, and P-CTX-4B. Between July 2015 and November 2016, the toxin content in T. gratilla decreased, but was consistently above the safety limit allowed for human consumption. This study provides evidence of CTX bioaccumulation in T. gratilla as a cause of ciguatera-like poisoning associated with a documented symptomatology.

Ciguatera fish poisoning (CFP) is a foodborne disease caused by the consumption of seafood (fish and marine invertebrates) contaminated with ciguatoxins (CTXs) produced by dinoflagellates in the genus Gambierdiscus. The report of a CFP-like mass-poisoning outbreak following the consumption of Tectus niloticus (Tegulidae, Gastropod) from Anaho Bay on Nuku Hiva Island (Marquesas archipelago, French Polynesia) prompted field investigations to assess the presence of CTXs in T. niloticus. Samples were collected from Anaho Bay, 1, 6 and 28 months after this poisoning outbreak, as well as in Taiohae and Taipivai bays. Toxicity analysis using the neuroblastoma cell-based assay (CBA-N2a) detected the presence of CTXs only in Anaho Bay T. niloticus samples. This is consistent with qPCR results on window screen samples indicating the presence of Gambierdiscus communities dominated by the species G. polynesiensis in Anaho Bay. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses revealed that P-CTX-3B was the major congener, followed by P-CTX-3C, P-CTX-4A and P-CTX-4B in toxic samples. Between July 2014 and November 2016, toxin content in T. niloticus progressively decreased, but was consistently above the safety limit recommended for human consumption. This study confirms for the first time T. niloticus as a novel vector of CFP in French Polynesia.

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.

Globally, ciguatera fish poisoning (CFP) is the principal cause of non-bacterial illness associated with seafood consumption. The toxins (ciguatoxins) responsible for CFP are produced by dinoflagellates in the genus Gambierdiscus, which are endemic to tropical and sub-tropical areas. Ciguatoxins are lipophilic and bioaccumulate in marine food webs, typically reaching their highest concentrations in fish. Following a CFP event in 2008, the U.S. Food and Drug Administration (USFDA) issued a ciguatera toxin alert that included fish harvested in the northern Gulf of Mexico in and near the Flower Garden Banks National Marine Sanctuary (FGBNMS). The East Flower Garden Bank (EFGB) and West Flower Garden Bank (WFGB) are characterized by thriving coral communities that support Gambierdiscus growth. This study was undertaken specifically to document the diversity of Gambierdiscus species present in the sanctuary that may be sources of ciguatoxins entering the food web. Samples collected from the FGBNMS over a three year period were screened using species-specific polymerase chain reaction assays. A diverse assemblage of Gambierdiscus species was distributed to depths of >45 m, a new depth record for Gambierdiscus. Gambierdiscus belizeanus, Gambierdiscus caribaeus, Gambierdiscus carolinianus, Gambierdiscus carpenteri and Gambierdiscus ribotype 2 were all found on both East and West FGB with Gambierdiscus ruetzleri also recorded from the WFGB. The most common species was G. carolinianus, originally identified from samples collected between 35 and 40 m off the coast of NC, USA. Our findings are consistent with recent physiological studies showing that some Gambierdiscus species can grow year round at the temperatures and salinities at the FGBNMS and at light levels as low as 10 μmol photons m−2 s−1. Such irradiances are estimated to occur in the FGBNMS at depths of ∼70–80 m. The consistent recovery of Gambierdiscus species from deep sampling sites in areas known to produce ciguatoxic fish signals a substantial change in our concept of suitable habitats for Gambierdiscus to include depths greater than 50 m.

As part of a long-term study of benthic dinoflagellates from the Belizean barrier reef system, we report a new species: Prorocentrum levis M. A. Faust, Kibler, Vandersea, P. A. Tester et Litaker sp. nov. P. levis cells are oval in valve view and range in size from 40 to 44 μm long and 37 to 40 μm wide. Each valve surface is smooth, with 221-238 valve pores and 99-130 marginal pores. These pores are uniformly small and range in diameter from 0.13 to 0.19 μm. Asexual reproduction in P. levis is atypical, occurring within a hyaline envelope, and produces long branching chains of adherent cells. A phylogenetic analysis of SSU rDNA indicated that of the Prorocentrum species sequenced so far, P. levis was most closely related to P. concavum. P. levis produces okadaic acid and dinophysis toxin-2 (DTX2). Further, SEM observations and SSU rDNA sequence for P. belizeanum M. A. Faust, which was isolated at the same time, are also presented.

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.

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.

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.

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, Gambie...

As part of a long-term study of benthic dinoflagellates from the Belizean barrier reef system, we report a new species: Prorocentrum levis M. A. Faust, Kibler, Vandersea, P. A. Tester et Litaker sp. nov. P. levis cells are oval in valve view and range in size from 40 to 44 lm long and 37 to 40 lm wide. Each valve surface is smooth, with 221–238 valve pores and 99–130 marginal pores. These pores are uniformly small and range in diameter from 0.13 to 0.19 lm. Asexual reproduction in P. levis is atypical, occurring within a hyaline envelope, and produces long branching chains of adherent cells. A phylogenetic analysis of SSU rDNA indicated that of the Prorocentrum species sequenced so far, P. levis was most closely related to P. concavum. P. levis produces okadaic acid and dinophysis toxin-2 (DTX2). Further, SEM observations and SSU rDNA sequence for P. belizeanum M. A. Faust, which was isolated at the same time, are also presented

All dinoflagellates that infest the skin and gills of fish have traditionally been placed within the class Blastodiniphyceae. Their relatedness was primarily based upon a similar mode of attachment to the host, i.e., attachment disc with holdfasts. Results of recent molecular genetic analyses have transferred these parasites, including Amyloodinium, to the class Dinophyceae, subclass Peridiniphycidae. In our study, a small subunit rDNA gene from a parasitic dinoflagellate that has features diagnostic for species in the genus Piscinoodinium, i.e., typical trophont with attachment disc having rhizocysts, infesting the skin of freshwater tropical fish, places this organism within the dinophycean subclass Gymnodiniphycidae. This suggests a close relationship of Piscinoodinium spp. to dinoflagellates that include symbionts, e.g., species of Symbiodinium, and free-living algae, e.g., Gymnodinium spp. These molecular and morphological data suggest that evolution of this mode of fish ectoparasitism occurred independently in 2 distantly related groups of dinoflagellates, and they further suggest that the taxonomic status of parasites grouped as members of Piscinoodinium requires major revision.

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

In this study, inter- and intraspecific genetic diversity within the marine harmful dinoflagellate genus Coolia Meunier was evaluated using isolates obtained from the tropics to subtropics in both Pacific and Atlantic Ocean basins. The aim was to assess the phylogeographic history of the genus and to clarify the validity of established species including Coolia malayensis. Phylogenetic analysis of the D1-D2 LSU rDNA sequences identified six major lineages (L1–L6) corresponding to the morphospecies Coolia malayensis (L1), C. monotis (L2), C. santacroce (L3), C. palmyrensis (L4), C. tropicalis (L5), and C. canariensis (L6). A median joining network (MJN) of C. malayensis ITS2 rDNA sequences revealed a total of 16 haplotypes; however, no spatial genetic differentiation among populations was observed. These MJN results in conjunction with CBC analysis, rDNA phylogenies and geographical distribution analyses confirm C. malayensis as a distinct species which is globally distributed in the tropical to warm-temperate regions. A molecular clock analysis using ITS2 rDNA revealed the evolutionary history of Coolia dated back to the Mesozoic, and supports the hypothesis that historical vicariant events in the early Cenozoic drove the allopatric differentiation of C. malayensis and C. monotis.

Pfiesteria shumwayae Glasgow et Burkholder is assigned to a new genus Pseudopfiesteria gen. nov. Plate tabulation differences between Pfiesteria and Pseudopfiesteria gen. 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. shumwayae comb. nov. also has a distinctive diamond or rectangular-shaped anterior intercalary plate. Both Pfiesteria and Pseudopfiesteria gen. 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 of Pfiesteria and Pseudopfiesteria are consistent with placement of both genera in the Peridiniales. Based on the plate tabulations for P. 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.

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.

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.

In the spring of 1998, the Florida Fish and Wildlife Research Institute received numerous reports of lesioned or ulcerated fish primarily from the St. Lucie Estuary on the southeast coast of Florida, an area known since the late 1970s for lesions of the ulcerative mycosis (UM) type. From these and archived reports, as well as others received from different areas of Florida, we documented that diseased specimens had randomly distributed skin ulcers (usually reddened or hemorrhagic) with raised irregular margins and, in some cases, deeply penetrating hyphae in the surrounding muscle tissue. Since 1998, 256 fish (comprising 18 species) with ulcerative lesions (from 15 different locations) were confirmed with hyphae in fresh squash preparation or by histological evaluation. Squash preparations revealed nonseptate, sparsely branching, thick-walled hyphae; histological sections revealed mycotic granulomas in the dermis that occasionally penetrated into the skeletal muscle. These pathological characteristics were consistent with UM caused by the oomycete Aphanomyces invadans in Southeast Asia, Japan, Australia, and the United States. For specific identification, six isolates from ulcerated fish were cultured and prepared for molecular characterization using established diagnostic methods. Ribosomal RNA gene sequence analysis identified three isolates as Aphanomyces invadans, one as the oomycete Achlya bisexualis, and two as the ascomycete Phialemonium dimorphosporum. A more extensive survey of 67 ulcerated skin samples from fish collected between 1998 and 2003 was performed using a polymerase chain reaction assay specific for Aphanomyces invadans. Of these, 26 (38.8%) samples from seven fish species and nine collection locations were positive. Confirmation of UM associated with Aphanomyces invadans represents new host records in Florida for the sheepshead Archosargus probatocephalus, striped mullet Mugil cephalus, white mullet Mugil curema, silver perch Bairdiella chrysoura, black drum Pogonias cromis, largemouth bass Micropterus salmoides, and American shad Alosa sapidissima.

Based on isolations from naturally infected fish in Florida, we investigated the role of the fungi Aphanomyces invadans, Achlya bisexualis, and Phialemonium dimorphosporum in the etiology of ulcerative mycosis (UM) in striped mullet Mugil cephalus. We injected healthy striped mullet subcutaneously with secondary zoospores of four oomycete isolates: two concentrations (50 and 115 zoospores/mL) of SJR (an endemic isolate of Aphanomyces invadans in American shad Alosa sapidissima from the St. Johns River); two concentrations each of CAL (25 and 65 zoospores/mL) and ACH (1,400 and 2,000 zoospores/mL; endemic isolates of Aphanomyces invadans and Achlyva bisexualis, respectively, in striped mullet from the Caloosahatchee River); and two concentrations of the ascomycete culture MTZ (2,500 and 3,500 zoospores/mL; endemic isolate of P. dimorphosporum from whirligig mullet M. gyrans in the Matanzas Inlet). All fish injected with either concentration of SJR developed granulomatous ulcers after 8 d and died within 21 d. Eighty percent (8/10) of fish injected with the high dose of CAL developed ulcers after 13 d and died within 28 d, but only 30% (3/10) of fish injected with the low dose of CAL developed ulcers. Four of the ulcerated fish died within 28 d, and the remaining fish were terminated after 32 d. Fish injected with zoospores of Aphanomyces invadans developed ulcers that were grossly and histologically similar to those observed in naturally infected striped mullet with UM from several estuaries or rivers in Florida. These hemorrhagic skin ulcers were characterized by myonecrosis and the presence of mycotic granulomas. None of the fish injected with ACH, MTZ, or sterile water developed ulcers. This study fulfilled Koch's postulates and demonstrated that ulcers could be experimentally induced in striped mullet after exposure via injection to secondary zoospores of an endemic Florida strain of Aphanomyces invadans.

Amyloodiniosis, caused by the dinoflagellate ectoparasite Amyloodinium ocellatum, is one of the most serious diseases affecting marine fish in warm and temperate waters. Current diagnostic methods rely entirely on the microscopic identification of parasites on the skin or gills of infested fish. However, subclinical infestations usually go undetected, while no method of detecting the free-swimming, infective (dinospore) stage has been devised. Targeting the parasite's ribosomal DNA region, we have developed a sensitive and specific PCR assay that can detect as little as a single cell from any of the 3 stages of the parasite's life cycle (trophont, tomont, dinospore). This assay performs equally well in a simple artificial seawater medium and in natural seawater containing a plankton community assemblage. The assay is also not inhibited by gill tissue. Sequence analysis of the internal transcribed spacer region of 5 A. ocellatum isolates, obtained from fish in the Red Sea (Israel), eastern Mediterranean Sea (Israel), Adriatic Sea (Italy), Gulf of Mexico (Florida), and from an unknown origin, revealed insignificant variation, indicating that all isolates were the same species. However, 3 of these isolates propagated in cell culture varied in behavior and morphology, and these differences were consistent during at least 2 yr in culture. Thus, our findings do not eliminate the possibility that different strains are in fact 'subspecies' or lower taxa, which may also differ in pathogenic and immunogenic characteristics, environmental tolerance, and other features.

The pathogenic oomyceteAphanomyces invadansis the primary etiological agent in ulcerative mycosis, an ulcerative skin disease caused by a fungus-like agent of wild and cultured fish. We developed sensitive PCR and fluorescent peptide nucleic acid in situ hybridization (FISH) assays to detectA. invadans. Laboratory-challenged killifish (Fundulus heteroclitus) were first tested to optimize and validate the assays. Skin ulcers of Atlantic menhaden (Brevoortia tyrannus) from populations found in the Pamlico and Neuse River estuaries in North Carolina were then surveyed. Results from both assays indicated that all of the lesioned menhaden (n= 50) collected in September 2004 were positive forA. invadans. Neither the FISH assay nor the PCR assay cross-reacted with other closely related oomycetes. These results provided strong evidence thatA. invadansis the primary oomycete pathogen in ulcerative mycosis and demonstrated the utility of the assays. The FISH assay is the first molecular assay to provide unambiguous visual confirmation that hyphae in the ulcerated lesions were exclusivelyA. invadans.

• Patricia A. Testera, , , , • Steven R. Kiblera, • William C. Hollanda, • Gires Usupb, • Mark W. Vanderseaa, • Chui Pin Leawc, • Po Teen Limc, • Jacob Larsend, • Normawaty Mohammad-Noore, • Maria A. Faustf, • R. Wayne Litakera Show more DOI: 10.1016/j.hal.2014.09.004 Refers To • Patricia A. Tester, Steven R. Kibler, William C. Holland, Gires Usup, Mark W. Vandersea, Chui Pin Leaw, Lim Po Teen, Jacob Larsen, Normawaty Mohammad-Noor, Maria A. Faust, R. Wayne Litaker • Sampling harmful benthic dinoflagellates: Comparison of artificial and natural substrate methods • Harmful Algae, Volume 39, October 2014, Pages 8-25 •

Retinoic acid (RA), a derivative of vitamin A, plays a critical role as a signaling molecule in axial patterning of vertebrates. Here we report that RA exposure of zebrafish (Danio rerio) and mummichog (Fundulus heteroclitus) embryos during gastrulation results in homeotic duplications of the pectoral fins in up to 94% of fish. We have observed three to four pairs of fins in an individual fish. Although some duplications are partial, many represent complete axial duplications of the pectoral girdle and fin and include coracoscapulae, proximal radials, and dermal fin elements. Fin duplications are observed only at a defined dose of RA. Inhibition of RA synthesis by exposure to citral during a narrow developmental window leads to fish which lack pectoral fins but can be rescued by addition of exogenous RA, suggesting that RA signaling is critical to fin specification during early development. The ability to consistently induce multiple fins in a large number of vertebrate embryos should contribute to the understanding of genetic regulation of the normal positioning of limbs during embryogenesis.

The putative harmful algal bloom dinoflagellate, Pfiesteria piscicida, frequently co-occurs with other morphologically similar species collectively known as Pfiesteria-like organisms (PLOs). This study specifically evaluated whether unique sequences in the ribosomal internal transcribed spacer 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 (SSU) or large subunit (LSU) ribosomal RNA 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, Florida “Lucy” species, two cryptoperidiniopsoid species, “H/V14” and “PLO21,” and the estuarine mixotroph, Karlodinium micrum. 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.