Effects of temperature,salinity and their interaction on growth of Japanese Gambierdiscus spp. (Dinophyceae)

Marine toxic dinoflagellates of the genus Gambierdiscus are the causative agents of ciguatera fish poisoning (CFP), a seafood poisoning that is widespread in tropical, subtropical and temperate regions of the world. In the main island of Japan, distributions of Gambierdiscus australes, Gambierdiscus scabrosus and two phylotypes of Gambierdiscus spp. type 2 and type 3, have been reported. To discuss the bloom dynamics of these Japanese species/phylotypes of Gambierdiscus, first we tested six culture media to optimize growth conditions and then clarified the effects of temperature and salinity and temperature–salinity interactions on growth. All strains of the species/phylotypes tested showed the highest cell yields when they were cultivated in IMK/2 medium. G. australes, G. scabrosus and Gambierdiscus sp. type 2 grew in the range 17.5–30 °C, whereas Gambierdiscus sp. type 3 grew in 15–25 °C. The semi-optimal temperature ranges (≥80% of the maximal growth rate) of the former three species/phylotypes were 19–28 °C, 24–31 °C and 21–28 °C, respectively, whereas that of the latter phylotype was 22–25 °C. Hence, Gambierdiscus sp. type 3 may be adapted to relatively lower water temperatures of ≤25 °C. In contrast, G. australes, G. scabrosus and Gambierdiscus sp. type 2 presumably possess adaptability to relatively high water temperatures. The optimal temperature for G. scabrosus was 30 °C, whereas the optimal temperature for the others was 25 °C. G. australes and Gambierdiscus sp. type 3 grew in a salinity range of 25–40 whereas G. scabrosus and Gambierdiscus sp. type 2 grew in salinity 20–40. Furthermore, the semi-optimal salinity range of G. australes, G. scabrosus, Gambierdiscus spp. type 2 and type 3 were salinity 27–38, 24–36, 22–36 and 29–37, respectively. Among the species/phylotypes, G. scabrosus and Gambierdiscus sp. type 2 grew even at salinity 20 where the others did not grow, thus possessing adaptability to low salinity waters. Our results clearly demonstrate that the optimal and tolerable temperature–salinity conditions differ among Japanese Gambierdiscus species/phylotypes. Considering these results, temperature–salinity interactions may play an important role in bloom dynamics and the distribution of the Gambierdiscus species/phylotypes in Japanese coastal waters.     

First report of three benthic dinoflagellates,Gambierdiscus pacificus,G. australes and G. caribaeus (Dinophyceae), from Hainan Island,South China Sea

Species of the marine benthic dinoflagellate genus Gambierdiscus are the principal cause of Ciguatera fish poisoning. This genus has been recorded from tropical to temperate oceans, although Gambierdiscus species have rarely been found in Chinese waters. Our work revealed the morphological and genetic characteristics of three potentially toxic Gambierdiscus species observed in the temperate to subtropical waters of China. The fine thecal morphology was determined based on light microscopy and scanning electron microscopy analyses, and these species were also characterized by sequencing the D1–D3 and D8–D10 regions of the LSU rDNA. The morphological and genetic data indicated that these three Gambierdiscus species were G. pacificus, G. australes and G. caribaeus. This work provides the first report of these species in Chinese waters, which increases the known species distribution of this genus.     

The effects of light intensity on the growth of Japanese Gambierdiscus spp. (Dinophyceae)

Marine toxic dinoflagellates of the genus Gambierdiscus are the causative agents of ciguatera fish poisoning (CFP), a form of seafood poisoning that is widespread in tropical, subtropical and temperate regions worldwide. The distributions of Gambierdiscus australes, Gambierdiscus scabrosus and two phylotypes of Gambierdiscus spp. type 2 and type 3 have been reported for the waters surrounding the main island of Japan. To explore the bloom dynamics and the vertical distribution of these Japanese species and phylotypes of Gambierdiscus, the effects of light intensity on their growth were tested, using a photoirradiation-culture system. The relationship between the observed growth rates and light intensity conditions for the four species/phylotypes were formulated at R > 0.92 (p < 0.01) using regression analysis and photosynthesis-light intensity (P-L) model. Based on this equation, the optimum light intensity (L_max) and the semi-optimum light intensity range (L_s-opt) that resulted in the maximum growth rate (μ_max) and ≥80% μ _max values of the four species/phylotypes, respectively, were as follows: (1) the L_max and L_s-opt of G. australes were 208 μmol photons m⁻² s⁻¹ and 91–422 μmol photons m⁻² s⁻¹, respectively; (2) those of G. scabrosus were 252 and 120–421 μmol photons m⁻² s⁻¹, respectively; (3) those of Gambierdiscus sp. type 2 were 192 and 75–430 μmol photons m⁻² s⁻¹, respectively; and (4) those of Gambierdiscus sp. type 3 were ≥427 and 73–427 μmol photons m⁻² s⁻¹, respectively. All four Gambierdiscus species/phylotypes required approximately 10 μmol photons m⁻² s⁻¹ to maintain growth. The light intensities in coastal waters at a site in Tosa Bay were measured vertically at 1 m intervals once per season. The relationships between the observed light intensity and depth were formulated using Beer’s Law. Based on these equations, the range of the attenuation coefficients at Tosa Bay site was determined to be 0.058–0.119 m⁻¹. The values 1700 μmol photons m⁻² s⁻¹, 500 μmol photons m⁻² s⁻¹, and 200 μmol photons m⁻² s⁻¹ were substituted into the equations to estimate the vertical profiles of light intensity at sunny midday, cloudy midday and rainy midday, respectively. Based on the regression equations coupled with the empirically determined attenuation coefficients for each of the four seasons, the ranges of the projected depths of L_max and L_s-opt for the four Gambierdiscus species/phylotypes under sunny midday conditions, cloudy midday conditions, and rainy midday conditions were 12–38 m and 12–54 m, 1–16 m and 1–33 m, and 0 m and 0–16 m, respectively. These results suggest that light intensity plays an important role in the bloom dynamics and vertical distribution of Gambierdiscus species/phylotypes in Japanese coastal waters.     

MORPHOLOGY AND MOLECULAR ANALYSES OF THREE TOXIC SPECIES OF GAMBIERDISCUS (DINOPHYCEAE): G. PACIFICUS, SP. NOV., G. AUSTRALES, SP. NOV., AND G. POLYNESIENSIS, SP. NOV.

Three new dinoflagellate species, Gambierdiscus polynesiensis, sp. nov., Gambierdiscus australes, sp. nov., and Gambierdiscus pacificus, sp. nov., are described from scanning electron micrographs. The morphology of the three new Gambierdiscus species is compared with the type species Gambierdiscus toxicus Adachi et Fukuyo 1979, and two other species: Gambierdiscus belizeanus Faust 1995 and Gambierdiscus yasumotoi Holmes 1998. The plate formula is: Po, 3′, 7", 6C, 8S, 5‴, 1p, 2". Culture extracts of these three new species displayed both ciguatoxin- and maitotoxin-like toxicities. The following morphological characteristics differentiated each species. 1) Cells of G. polynesiensis are 68–85 μm long and 64–75 μm wide, and the cell’s surface is smooth. They are identified by a large triangular apical pore plate (Po), a narrow fish-hook opening surrounded by 38 round pores, and a large, broad posterior intercalary plate (1p) wedged between narrow postcingular plates 2‴ and 4‴. Plate 1p occupies 60% of the width of the hypotheca. 2) Cells of G. australes also have a smooth surface and are 76–93 μm long and 65–85 μm wide in dorsoventral depth. They are identified by the broad ellipsoid apical pore plate (Po) surrounded by 31 round pores and a long and narrow 1p plate wedged between postcingular plates 2‴ and 4‴. Plate 1p occupies 30% of the width of the hypotheca. 3) Cells of G. pacificus are 67–77 μm long and 60–76 μm wide in dorsoventral depth, and its surface is smooth. They are identified by the four-sided apical pore plate (Po) surrounded by 30 round pores. A short narrow 1p plate is wedged between the wide postcingular plates 2‴ and 4‴. Plate 1p occupies 20% of the width of the hypotheca. These three newly described species were also characterized by isozyme electrophoresis and DNA sequencing of the D8–D10 region of their large subunit (LSU) rRNA genes. The consistency between species designations based on SEM microscopy and classification inferred from biochemical and genetic heterogeneities was examined among seven isolates of Gambierdiscus. Their classification into four morphospecies was not consistent with groupings inferred from isozyme patterns. Three molecular types could be distinguished based on the comparison of their LSU rDNA sequences. Although G. toxicus TUR was found to be more closely related to G. pacificus, sp. nov. than to other G. toxicus strains, the molecular classification was able to discriminate G. polynesiensis, sp. nov. and G. australes, sp. nov. from G. toxicus. These results suggest the usefulness of the D8–D10 portion of the Gambierdiscus LSU rDNA as a valuable taxonomic marker.     

Taxonomic assignment of the benthic toxigenic dinoflagellate Gambierdiscus sp. type 6 as Gambierdiscus balechii (Dinophyceae), including its distribution and ciguatoxicity

Recent molecular phylogenetic studies of Gambierdiscus species flagged several new species and genotypes, thus leading to revitalizing its systematics. The inter-relationships of clades revealed by the primary sequence information of nuclear ribosomal genes (rDNA), however, can sometimes be equivocal, and therefore, in this study, the taxonomic status of a ribotype, Gambierdiscus sp. type 6, was evaluated using specimens collected from the original locality, Marakei Island, Republic of Kiribati; and specimens found in Rawa Island, Peninsular Malaysia, were further used for comparison. Morphologically, the ribotype cells resembled G. scabrosus, G. belizeanus, G. balechii, G. cheloniae and G. lapillus in thecal ornamentation, where the thecal surfaces are reticulate-foveated, but differed from G. scabrosus by its hatchet-shaped Plate 2′, and G. belizeanus by the asymmetrical Plate 3′. To identify the phylogenetic relationship of this ribotype, a large dataset of the large subunit (LSU) and small subunit (SSU) rDNAs were compiled, and performed comprehensive analyses, using Bayesian-inference, maximum-parsimony, and maximum-likelihood, for the latter two incorporating the sequence-structure information of the SSU rDNA. Both the LSU and SSU rDNA phylogenetic trees displayed an identical topology and supported the hypothesis that the relationship between Gambierdiscus sp. type 6 and G. balechii was monophyletic. As a result, the taxonomic status of Gambierdiscus sp. type 6 was revised, and assigned as Gambierdiscus balechii. Toxicity analysis using neuroblastoma N2A assay confirmed that the Central Pacific strains were toxic, ranging from 1.1 to 19.9 fg P-CTX-1 eq cell⁻¹, but no toxicity was detected in a Western Pacific strain. This suggested that the species might be one of the species contributing to the high incidence rate of ciguatera fish poisoning in Marakei Island.     

Detection of Gambierdiscus and Fukuyoa single cells using recombinase polymerase amplification combined with a sandwich hybridization assay

Dinoflagellates of the genera Gambierdiscus and Fukuyoa are known to produce several bioactive compounds including the potent neurotoxic ciguatoxins (CTXs) which are able to accumulate in fish and through the food web. When humans ingest fish contaminated with CTXs, it can result in an intoxication named ciguatera. Although not all the currently recognized species are able to produce toxins, G. australes and G. excentricus have been highlighted to be the most abundant and toxic among the species present in the Atlantic. Even though the genera Gambierdiscus and Fukuyoa are endemic to tropical areas, recently their presence was recorded in subtropical and temperate regions. In this work, the development of three molecular assays for the detection of the Gambierdiscus and Fukuyoa genera and for G. australes and G. excentricus species, based on the combination of recombinase polymerase amplification with detection via hybridization, is successfully described. Furthermore, a remarkable limit of detection of a single cell was achieved. Additionally, six different species have been used to check the ability of each primer set to give an amplified product, even in presence of potentially interfering non-target DNAs. Therefore, these developments provide a rapid and cost-effective strategy for detection of both genera and two of the most toxic species, which will undoubtedly contribute to reliable screening of samples and ciguatera risk assessment, guaranteeing seafood safety and protection of human health.

     

Characterization of Gambierdiscus and Coolia (Dinophyceae) isolates from Thailand based on morphology and phylogeny

The benthic dinoflagellates in the genus Gambierdiscus produce toxins that bioaccumulate in tropical and sub‐tropical fish causing ciguatera fish poisoning (CFP). Other co‐occurring genera such as Coolia have also been implicated in causing CFP. Little is known about the diversity of the two genera Gambierdiscus and Coolia along the Thai coasts. The results of morphological analyses based on observation under light microscopy and scanning electron microcopy showed that strains of Gambierdiscus from Thailand displayed the typical Gambierdiscus plate formula: Po, 4′, 0a, 6″, 6c,?s, 5′′′, 0p and 2′′′′. Morphological examination of Thai Gambierdiscus enabled it to be identified as Gambierdiscus caribaeus: round and anterior‐posteriorly compressed cell shape, broad 2′′′′ plate, rectangular 2′ plate, and symmetrical 3″ plate. The phylogenetic analyses based on the large subunit (LSU) rDNA D8/D10 sequences of Gambierdiscus from Thailand confirmed the morphological identification. The thecal plate formula for all of the Coolia isolates from Thailand was Po, 4′, 0a, 6″,?c,?s, 5′′′, 0p and 2′′′′. Most, but not all, of these isolates could be identified morphologically as Coolia malayensis. An LSU rDNA D1/D2 phylogenetic analysis confirmed identity of C. malayensis isolates identified morphologically. The remaining unidentified isolates fell in the C. tropicalis clade.     

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.     

Gambierdiscus (Gonyaulacales,Dinophyceae) diversity in Vietnamese waters with description of G. vietnamensis sp. nov.

Viet Nam has a coastline of 3200 km with thousands of islands providing diverse habitats for benthic harmful algal species including species of Gambierdiscus. Some of these species produce ciguatera toxins, which may accumulate in large carnivore fish potentially posing major threats to public health. This study reports five species of Gambierdiscus from Vietnamese waters, notably G. australes, G. caribaeus, G. carpenteri, G. pacificus, and G. vietnamensis sp. nov. All species are identified morphologically by LM and SEM, and identifications are supported by molecular analyses of nuclear rDNA (D1–D3 and D8–D10 domains of LSU, SSU, and ITS1-5.8S-ITS2 region) based on cultured material collected during 2010–2021. Statistical analyses of morphometric measurements may be used to differentiate some species if a sufficiently large number of cells are examined. Gambierdiscus vietnamensis sp. nov. is morphologically similar to other strongly reticulated species, such as G. belizeanus and possibly G. pacificus; the latter species is morphologically indistinguishable from G. vietnamensis sp. nov., but they are genetically distinct, and molecular analysis is deemed necessary for proper identification of the new species. This study also revealed that strains denoted G. pacificus from Hainan Island (China) should be included in G. vietnamensis sp. nov.     

Production of ciguatoxin and maitotoxin by strains of Gambierdiscus australes,G. pacificus and G. polynesiensis (Dinophyceae) isolated from Rarotonga,Cook Islands

The isolation and culturing of toxic Gambierdiscus species is desirable for obtaining reliable sources of toxins responsible for Ciguatera Fish Poisoning (CFP) in order to provide reference materials and purified standards for chemical analyses and bioassays. Gambierdiscus polynesiensis CAWD212, G. pacificus CAWD213 and G. australes CAWD149 and 216 have been isolated from samples of sea water from the Cook Islands. G. polynesiensis produced ciguatoxins (CTX; 18.2 pg per cell) and G. australes CAWD149 produced maitotoxin (MTX; 8.3 pg per cell) and in both extracts putative MTX-3 was detected. G. pacificus also produced putative MTX-3. The toxicities of extracts of G. polynesiensis and G. pacificus were similar by i.p. injection (LD_50s of 1.0 mg/kg and 0.8 mg/kg respectively), but when administered by gavage, the MTX-3 analogue producing G. pacificus (LD₅₀ 251 mg/kg) was 30-times less toxic than the CTX-producing G. polynesiensis (LD₅₀ 7.9 mg/kg). Consumption of fish contaminated by CFP has caused illness and even death in the Cook Islands and throughout the Pacific region and is now considered a globally neglected disease. Large-scale cultures of these organisms are now available, and isolation of the toxins they produce is in progress.     

Fukuyoa paulensis gen. et sp. nov., a New Genus for the Globular Species of the Dinoflagellate Gambierdiscus (Dinophyceae)

The marine epiphytic dinoflagellate Gambierdiscus is a toxicologically important genus responsible for ciguatera fish poisoning, the principal cause of non-bacterial illness associated with fish consumption. The genus currently contains species exhibiting either globular or anterior-posteriorly compressed morphologies with marked differences in cell shape and plate arrangement. Here we report a third globular, epiphytic and tychoplanktonic species from the coasts of Ubatuba, Brazil. The new species can be distinguished from G. yasumotoi and G. ruetzleri by its broader first apical plate that occupies a larger portion of the epitheca. Accordingly, phylogenetic trees from small subunit (SSU) and large subunit (LSU) ribosomal DNA sequences also showed strongly supported separation of the new species from the G. yasumotoi / G. ruetzleri group albeit with short distance. The molecular phylogenies, which included new sequences of the planktonic species Goniodoma polyedricum, further indicated that the globular species of Gambierdiscus formed a tight clade, clearly separated (with strong bootstrap support) from the clade of lenticular species including the type for Gambierdiscus. The morphological and molecular data in concert support the split of Gambierdiscus sensu lato into two genera. Gambierdiscus sensu stricto should be reserved for the species with lenticular shapes, highly compressed anterioposteriorly, with short-shank fishhook apical pore plate, large 2'' plate, low and ascending cingular displacement, and pouch-like sulcal morphology. The new genus name Fukuyoa gen. nov. should be applied to the globular species, slightly laterally compressed, with long-shank fishhook apical pore plate, large 1'' plate, greater and descending cingular displacement, and not pouch-like vertically-oriented sulcal morphology. Fukuyoa contains the new species Fukuyoa paulensis gen. et sp. nov., and F. yasumotoi comb. nov. and F. ruetzleri comb. nov.     

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.     

Gambierdiscus species exhibit different epiphytic behaviors toward a variety of macroalgal hosts

Ciguatera fish poisoning is a common form of seafood poisoning caused by toxins (ciguatoxins) that accumulate in demersal (reef) food webs. The precursors of ciguatoxins are produced by dinoflagellates of the genus Gambierdiscus, and enter the food web via herbivory and detritivory. The Gambierdiscus genus was recently revised and new research on the physiology and ecology of the revised species is needed. While it has been demonstrated that Gambierdiscus spp. are predominately epiphytic, the variability in epiphytic behavior among the various Gambierdiscus species is not known. Five Gambierdiscus species isolated from the Greater Caribbean Region were the focus of this study (G. belizeanus, G. caribaeus, G. carolinianus, G. carpenteri, and G. yasumotoi). Cells of Gambierdiscus were grown in wells with algae fragments from eight different macroalgal host genera (Acanthophora, Caulerpa, Dasya, Derbesia, Dictyota, Laurencia, Polysiphonia, and Ulva) where the epiphytic behavior and growth of the different Gambierdiscus species were monitored over 29 days. The results of this experiment demonstrate that epiphytic behavior (growth and attachment) differs among the Gambierdiscus species toward the various macroalgal hosts. Results tended to be specific to Gambierdiscus – host pairings with few commonalities in the way a particular Gambierdiscus species interacted across hosts or how the various Gambierdiscus species responded to a particular host. The Gambierdiscus – host pairings that resulted in the highest growth and attachment combinations were examined in terms of known cellular toxicity and host palatability to determine which pairings could represent the most likely vectors for the transfer of ciguatoxins (or precursors) into the demersal food web. Two pairings, Gambierdiscus belizeanus – Polysiphonia and G. belizeanus – Dictyota, best met these criteria, providing a hypothetical approach to better focus sampling and monitoring efforts on such potential vectors in the benthic environment.     

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.     

Gambierdiscus balechii sp. nov (Dinophyceae), a new benthic toxic dinoflagellate from the Celebes Sea (SW Pacific Ocean)

A new benthic toxic dinoflagellate is described from the Celebes Sea. Gambierdiscus balechii sp. nov. was isolated from seaweeds growing in tidal ponds. Its morphology was studied by means of LM and SEM; G. balechii has a very ornamented theca, a hatchet shaped second apical plate, a narrow second antapical plate and an asymmetrical third precigular plate, a unique combination of characters among Gambierdiscus species. It has a very wide size range with widths from 36 to 88 μm. Phylogenetic analyses of two G. balechii strains, based on LSU rRNA (D8–D10) and partial SSUrRNA sequences confirmed that these clustererd in its’ own group, separated from the rest of Gambierdiscus species and with G. pacificus, G. belizeanus and G. scabrosus as its closest relatives. Thecate cysts are described from culture as non motile vegetative-like cells which germinated after being isolated and transferred to fresh medium. Mouse tests showed that this species is toxic and hence it is a potential cause of ciguatera in the Celebes Sea.     

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.     

Toxicity of the dinoflagellate Gambierdiscus sp. toward the marine copepod Tigriopus japonicus

The toxic dinoflagellate Gambierdiscus sp. has recently been observed in temperate areas in the Southern Sea of the Republic of Korea. The purpose of this study was to examine the toxicity of Gambierdiscus sp. toward the marine copepod Tigriopus japonicus. T. japonicus adult females and nauplii were exposed to various concentrations of algae or culture medium to assess toxicity and analyze gene expression patterns in the copepod. Based on the toxicity tests, Gambierdiscus sp. increased the mortality of nauplii and the immobility of adult females. The survival and mobility of T. japonicus were not affected by culture medium lacking Gambierdiscus sp. cells in the toxicity test. However, based on the analysis of gene expression in the copepod, exposure of the copepod to culture medium affected the expression of stress or detoxification-related genes. Further studies to identify toxins in Gambierdiscus sp. are required to increase our understanding of dinoflagellate toxicity.     

Genetic diversity of soybean-nodulating rhizobia in Nepal in relation to climate and soil properties

Backgroud and aims

This study was conducted to reveal the genetic diversity of soybean-nodulating rhizobia in Nepal in relation to climate and soil properties.

Method

A total of 102 bradyrhizobial strains were isolated from the root nodules of soybeans cultivated in 12 locations in Nepal varying in climate and soil properties, and their genetic diversity was examined based on 16S rDNA, ITS regions of 16S–23S rDNA, nodC and nifH. In vitro growth properties of some representative strains were examined to elucidate their characteristic distribution in Nepal.

Results

Four species of the genus Bradyrhizobium were isolated, and B. japonicum dominated at temperate locations, while in subtropical locations, B. elkanii, B. yuanmingense, and B. liaoningense dominated at acidic, moderately acidic, and slightly alkaline soils, respectively. The relative nodule occupancies could not be fully explained by their in vitro growth properties. Similar nodC and nifH genes among the strains suggested co-evolution of these genes also in Nepal, probably through horizontal gene transfer.

Conclusions

The influence of climate and soil pH on diversity at the sub-species level was revealed. It is concluded that the highly diverse climate and soils in Nepal might be conducive for the existence of diverse soybean rhizobial strains.