First report of the benthic dinoflagellate,Gambierdiscus belizeanus (Gonyaulacales: Dinophyceae) for the east coast of Sabah,Malaysian Borneo

Species of the genus Gambierdiscus Adachi & Fukuyo, in particular G. toxicus Adachi & Fukuyo are known producers of neurotoxins associated with ciguatera fish poisoning (CFP). In this study live samples were collected from seaweed beds of the east coast of Sabah, Malaysian Borneo and a strain of Gambierdiscus was isolated and cultured. Examination of the thecal fine morphology was undertaken using light, epifluorescence, and scanning electron microscopy. Observed morphological features and their associated morphometric information enabled identification to Gambierdiscus belizeanus Faust. This represents the first report for the occurrence of G. belizeanus in the Asia Pacific region.     

Cellular and nuclear morphological variability within a single species of the toxigenic dinoflagellate genus Gambierdiscus: Relationship to life-cycle processes

Dinoflagellates belonging to the genus Gambierdiscus are the causative agent of ciguatera fish poisoning (CFP). This syndrome, which is widespread in tropical and subtropical regions, has recently been reported also in temperate latitudes. Taxonomic studies of Gambierdiscus have yet to completely couple the morphological features of member species with their genetics. In this study, the cellular and nuclear morphology of a single strain of one species of Gambierdiscus was determined in cells grown under different culture conditions. The results showed a wide-ranging variability of cell sizes, together with a clear relationship between cell size and nuclear morphology. Thus, small cells were associated with round to oval or slightly U-shaped nuclei and large cells with obviously U-shaped nuclei. Most cells exhibited the typical anterio-posteriorly compressed lenticular, shape of Gambierdiscus, with the exception of a few small globular-shaped specimens. In all cells, regardless of their size, the arrangement of the thecal plates was typical of lenticular Gambierdiscus. Dividing cells were consistently the largest. In these cells, nuclear morphology, karyokinesis, and cytokinesis were characterized. Cells underwent division only during the dark period, thus demonstrating their spontaneous synchronized division. Cellular forms related to the sexual cycle were also present in the cultures and included gamete pairs and putative meiotic planozygotes. The effect of the culture medium was studied by means of principal component analyses, which showed a positive correlation between the medium used and nuclear size and shape but not cell size.     

OBSERVATION OF SAND-DWELLING TOXIC DINOFLAGELLATES (DINOPHYCEAE) FROM WIDELY DIFFERING SITES, INCLUDING TWO NEW SPECIES

Dinoflagellate associations, including toxic and potentially toxic benthic species, were examined in sand from South Water Cay and Carrie Bow Cay, Belize. The inshore sand habitat in localized areas of warm shallow lagoonal waters supported blooms of toxic assemblages of dinoflagellates. In the sand, the dominant microalgae were dinoflagellates; cyanobacteria were a minor component and diatoms were absent. Ciliates and nematodes were present. Assemblages of microorganisms in colored sand were examined for 4 consecutive days after which a storm washed away the patch. The sand-dwelling dinoflagellate assemblage included 16 species where densities ranged from as low as 1.3% to 15% of total cell densities. The dominant species was Scrippsiella subsalsa, having 1.8 × 10⁵ to 2.6 × 10⁵ cells g^-1 sand. Toxic dinoflagellates identified in the sand were Gambierdiscus toxicus, Ostreopsis lenticularis, Prorocentrum lima, Prorocentrum mexicanum, and Amphidinium carteri. The potentially toxic Ostreopsis labens, Gambierdiscus belizeanussp. nov., and Coolia tropicalis sp. nov. were also identified. Toxic and potentially toxic species represented 36% to 60% of total microalgal cell assemblage. The morphology of a new sand-dwelling species, Gambierdiscus belizeanus sp. nov., was examined with the scanning electron microscope. The plate formula was P_o, 3′, 7″, 6c, s?, 5?, 1p, and 2″″.Dimensions of G. belizeanus cells were 53–67 pm long, 54–63 μm wide, and 92–98 μm in dorsoventral depth. Cells were deeply areolated, ellipsoid in apical view, and compressed anteroposteriorly. The cells of G. belizeanus were identified by the cell's long, narrow, pentagonal, posterior intercalary plate (1p) wedged between the wide postcingular plates 2″’and 4″; 1p occupied 20% of the width of the hypotheca. The plate formula for Coolia tropicalis sp. nov. was P_o, 3′, 7″, 7c, 8s?, 5″″, and 2″″, Cell size ranges were 23–40 μm long, 25–39 μm wide, and 35–65 μm in dorsoventral diameter. Cells were spherical, smooth, and covered with scattered round pores. The epitheca was smaller than the hypotheca. Precingular plates 1″ and 7″ were small and narrow, and the first apical plate 1″ and precingular plate 6″ were the largest plates on the epitheca. The apical pore was straight and 7 μm long, and was situated in the apical plate complex. Cells of C. tropicalis were distinguished from C. monotis by the wedge-shaped plate 1′, a four-sided 3’plate, and a short apical pore.     

GAMBIERDISCUS YASUMOTOI SP. NOV. (DINOPHYCEAE), A TOXIC BENTHIC DINOFLAGELLATE FROM SOUTHEASTERN ASIA

A new, toxin-producing, benthic dinoflagellate named Gambierdiscus yasumotoi sp. nov. was isolated from macroalgae from the fringing coral reef surrounding the Singapore island of Pulau Hantu. The plate formula of G. yasumotoi is P_o , 3', 7", 6c, 6s, 5"', and 2"". Gambierdiscus yasumotoi has a globular shape and is not anterior-posteriorly compressed as are the two other known species in this genus, G. toxicus Adachi et Fukuyo 1979 and G. belizeanus Faust 1995. The girdle descends one to two girdle widths without overhang in contrast to the ascending girdles of G. toxicus and G. belizeanus. The dimensions of cultured G. yasumotoi were 45–63 μm in length, 38–50 μm in transdiameter and 43–61 μm in dorsoventral diameter. The thecae are smooth without areolae. The apical pore plate has the characteristic fishhook shape of Gambierdiscus, but it is significantly longer than G. toxicus. Cells from young cultures of G. yasumotoi are positively phototropic.     

First Report of the Epiphytic Dinoflagellate Gambierdiscus caribaeus in the Temperate Waters off Jeju Island,Korea: Morphology and Molecular Characterization

Gambierdiscus spp. are epiphytic, benthic dinoflagellates. Some species have been shown to be toxic and cause ciguatera fish poisoning. We report, for the first time, the occurrence of Gambierdiscus caribaeus isolated from the waters off Jeju Island in Korea. Its morphology was similar to that of the original Belize strains of G. caribaeus. Gambierdiscus caribaeus has been reported in the tropical and subtropical waters of the Pacific, Gulf of Mexico, Caribbean Sea, and Floridian coast. Our report extends its range to the North Pacific Ocean. The plates of the Korean strain were arranged in a Kofoidian series of Po, 3′, 7′′, 6c, 6s, 5′′′, 1p, and 2′′′′, morphologically closer to other strains of G. caribaeus than to G. carpenteri. When properly aligned, its small subunit (SSU) rDNA was 0.5% different from those of Gambierdiscus sp. C‐1, a strain that was isolated from the waters off eastern Japan, but was 2.4–4.0% different from those of the NOAA strains of G. caribaeus and 3.1–3.4% different from those of the NOAA strains of G. carpenteri. Additionally, the D1–D3 large subunit (LSU) rDNA sequence of the Korean strain of G. caribaeus was 4.7–5.3% different from those of the NOAA strains of G. caribaeus and 7.1–7.5% different from those of all reported G. carpenteri strains, including the NOAA strains. In phylogenetic trees based on SSU and LSU rDNA sequences, our Korean strain was basal to the clade consisting of the NOAA strains of G. caribaeus, which in turn was sister clade to all reported G. carpenteri strains.     

Characterization of Gambierdiscus lapillus sp. nov. (Gonyaulacales,Dinophyceae): a new toxic dinoflagellate from the Great Barrier Reef (Australia)

Gambierdiscus is a genus of benthic dinoflagellates found worldwide. Some species produce neurotoxins (maitotoxins and ciguatoxins) that bioaccumulate and cause ciguatera fish poisoning (CFP), a potentially fatal food‐borne illness that is common worldwide in tropical regions. The investigation of toxigenic species of Gambierdiscus in CFP endemic regions in Australia is necessary as a first step to determine which species of Gambierdiscus are related to CFP cases occurring in this region. In this study, we characterized five strains of Gambierdiscus collected from Heron Island, Australia, a region in which ciguatera is endemic. Clonal cultures were assessed using (i) light microscopy; (ii) scanning electron microscopy; (iii) DNA sequencing based on the nuclear encoded ribosomal 18S and D8‐D10 28S regions; (iv) toxicity via mouse bioassay; and (v) toxin profile as determined by Liquid Chromatography‐Mass Spectrometry. Both the morphological and phylogenetic data indicated that these strains represent a new species of Gambierdiscus, G. lapillus sp. nov. (plate formula Po, 3′, 0a, 7″, 6c, 7‐8s, 5?, 0p, 2″″ and distinctive by size and hatchet‐shaped 2′ plate). Culture extracts were found to be toxic using the mouse bioassay. Using chemical analysis, it was determined that they did not contain maitotoxin (MTX1) or known algal‐derived ciguatoxin analogs (CTX3B, 3C, CTX4A, 4B), but that they contained putative MTX3, and likely other unknown compounds.     

ISOLATION OF A SULFOQUINOVOSYL MONOACYLGLYCEROL FROM BRYOPSIS SP. (CHLOROPHYTA): IDENTIFICATION OF A FACTOR CAUSING A POSSIBLE SPECIES-SPECIFIC ECDYSIS RESPONSE IN GAMBIERDISCUS TOXICUS (DINOPHYCEAE)1,

A bioactive compound that induced ecdysis (thecal loss) in Gambierdiscus toxicus Adachi et Fukuyo (Dinophyceae) cultures was isolated from a host macroalga, Bryopsis sp. (Chlorophyta). The ecdysis factor was identified by spectroscopic methods as 1- O -palmitoyl-3- O -(6'-sulfo-α- d -quinovopyranosyl)- sn -glycerol (PSQG). From our results, PSQG induced ecdysis at a high frequency and appeared not to inhibit the growth of G. toxicus cultures. The induction of ecdysis followed a dose-dependent saturation curve from 4 to 8 μM PSQG. To determine specificity of PSQG, the effects of palmitoyl- l -α-lysophosphatidylcholine (PLPC) were observed. Because PLPC contains a lipophilic palmitoyl moiety and hydrophilic phosphatidyl choline group, the compound possesses a detergent-like amphiphathic property similar to PSQG. Our results demonstrate that PLPC induced ecdysis at a high frequency in G. toxicus cultures and generated a similar dose–response curve as PSQG. The ecdysis activity observed in PSQG and PLPC may correlate with the detergent-like amphiphathic property of both compounds. Although PLPC induced a similar ecdysis response as PSQG, PLPC appeared to inhibit the growth of G. toxicus cultures. Preliminary results on the effects of PSQG on the dinoflagellates Prorocentrum lima (Ehrenberg) Dodge and Coolia monotis Meunier did not parallel the results observed in G. toxicus. This study demonstrated the existence of a factor from Bryopsis sp. that elicited a possible species-specific ecdysis response in G. toxicus cultures. This is the first report of a compound that induced ecdysis in G. toxicus or in any dinoflagellate.     

Genetic diversity of Gambierdiscus spp. (Gonyaulacales, Dinophyceae) in Japanese coastal areas

The genetic diversity of the ciguatera fish poisoning-related dinoflagellate distributed in Japanese coastal areas was investigated. The entire sequence of the 5.8S rRNA gene and two internal transcribed (ITS) regions were determined, which included putative pseudogenes, from 19 strains of dinoflagellates assigned to the genus Gambierdiscus Adachi et Fukuyo collected from Japanese subtropical and temperate coastal areas. The sequences obtained from the 19 strains were divided into two types based on sequence similarity. Here we designate the two types as type 1 and type 2. For the relationship between the genotypes and origins of the strains used, the strains collected from subtropical areas possessed the type 1 sequence; whereas those from temperate areas possessed the type 2. This observation led us to question former reputations that Gambierdiscus cells observed in Japanese temperate areas are immigrants from Japanese subtropical areas. Subsequently, we sequenced a part of the 18S rRNA gene from two strains from subtropical areas and two from temperate areas. Unfortunately, phylogenetic analysis including the sequences obtained from various gonyaulacales dinoflagellates failed to determine the species phylogenetically closely related to and possible origin(s) of the Gambierdiscus sp. from the Japanese coastal areas.     

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.