Microcystins (MCs) are secondary metabolites produced by cyanobacteria and have been well-documented in temperate and tropical regions. However, knowledge of the production of MCs in extremely cold environments is still in its infancy. Recently, examination of 100-year-old Antarctic cyanobacterial mats collected from Ross Island and the McMurdo Ice Shelf during Captain R.F. Scott's Discovery Expedition revealed that the presence of MCs in Antarctica is not a new phenomenon. Here, morphological and molecular phylogenetic analyses are used to identify a new microcystin-producing freshwater cyanobacterium, Anagnostidinema pseudacutissimum. The strain was isolated from a deep-frozen (-15 °C) sample collected from a red-brown cyanobacterial mat in a frozen pond at Cape Crozier (Ross Island, continental Antarctica) in 1984-1985. Amplification of the mcyE gene fragment involved in microcystin biosynthesis from A. pseudacutissimum confirmed that it is identical to the sequence from other known microcystin-producing cyanobacteria. Analysis of extracts from this A. pseudacutissimum strain by HPLC-MS/MS confirmed the presence of MC-LR and -YR at concentrations of 0.60 μg/L and MC-RR at concentrations of 0.20 μg/L. This is the first report of microcystin production from a species of Anagnostidinema from Antarctica.
The genus Amphidinium is an important group of athecated dinoflagellates because of its high abundance in marine habitats, its member's ability to live in a variety of environmental conditions and ability to produce toxins. Furthermore, the genus is of particular interest in the biotechnology field for its potential in the pharmaceutical arena. Taxonomically the there is a history of complication and confusion over the proper identities and placements of Amphidinium species due to high genetic variability coupled with high morphological conservation. Thirteen years has passed since the most recent review of the group, and while many issues were resolved, some remain. The present study used microscopy, phylogenetics of the 28S region of rDNA, secondary structure of the ITS2 region of rDNA, compensatory base change data, and cytotoxicity data from Amphidinium strains collected world-wide to elucidate remaining confusion. This holistic approach using multiple lines of evidence resulted in a more comprehensive understanding of the morphological, ecological, and genetic characteristics that are attributed to organisms belonging to Amphidinium, including six novel species: A. fijiensis, A. magnum, A. paucianulatum, A. pseudomassartii, A. theodori, and A. tomasii.
A new species of toxic benthic dinoflagellate is described based on laboratory cultures isolated from two locations from Brazil, Rio de Janeiro and Bahia. The morphology was studied with SEM and LM. Cells are elliptical in right thecal view and flat. They are 37-44μm long and 29-36μm wide. The right thecal plate has a V shaped indentation where six platelets can be identified. The thecal surface of both thecal plates is smooth and has round or kidney shaped and uniformly distributed pores except in the central area of the cell, and a line of marginal pores. Some cells present an elongated depression on the central area of the apical part of the right thecal plate. Prorocentrum caipirignum is similar to Prorocentrum lima in its morphology, but can be differentiated by the general cell shape, being elliptical while P. lima is ovoid. In the phylogenetic trees based on ITS and LSU rDNA sequences, the P. caipirignum clade appears close to the clades of P. lima and Prorocentrum hoffmannianum. The Brazilian strains of P. caipirignum formed a clade with strains from Cuba, Hainan Island and Malaysia and it is therefore likely that this new species has a broad tropical distribution. Prorocentrum caipirignum is a toxic species that produces okadaic acid and the fast acting toxin prorocentrolide.
In 2015, a remarkably high density bloom of Alexandrium minutum occurred in Sungai Geting, a semi-enclosed lagoon situated in the northeast of Peninsular Malaysia, causing severe discoloration and contaminated the benthic clams (Polymesoda). Plankton and water samples were collected to investigate the mechanisms of bloom development of this toxic species. Analysis of bloom samples using flow cytometry indicated that the bloom was initiated by the process of active excystment, as planomycetes (>4C cells) were observed in the early stage of the bloom. Increase in planozygotes (2C cells) was evident during the middle stage of the bloom, coinciding with an abrupt decrease in salinity and increase of temperature. The bloom was sustained through the combination of binary division of vegetative cells, division of planozygotes, and cyst germination through continuous excystment. Nutrient depletion followed by precipitation subsequently caused the bloom to terminate. This study provides the first continuous record of in situ life-cycle stages of a natural bloom population of A. minutum through a complete bloom cycle. The event has provided a fundamental understanding of the pelagic life-cycle stages of this tropical dinoflagellate, and demonstrated a unique bloom development characteristic shared among toxic Alexandrium species in coastal embayments.
Strains of a dinoflagellate from the Salton Sea, previously identified as Protoceratium reticulatum and yessotoxin producing, have been reexamined morphologically and genetically and Pentaplacodinium saltonense n. gen. et sp. is erected to accommodate this species. Pentaplacodinium saltonense differs from Protoceratium reticulatum (Claparède et Lachmann 1859) Bütschli 1885 in the number of precingular plates (five vs. six), cingular displacement (two widths vs. one), and distinct cyst morphology. Incubation experiments (excystment and encystment) show that the resting cyst of Pentaplacodinium saltonense is morphologically most similar to the cyst-defined species Operculodinium israelianum (Rossignol, 1962) Wall (1967) and O. psilatum Wall (1967). Collections of comparative material from around the globe (including Protoceratium reticulatum and the genus Ceratocorys) and single cell PCR were used to clarify molecular phylogenies. Variable regions in the LSU (three new sequences), SSU (12 new sequences) and intergenic ITS 1-2 (14 new sequences) were obtained. These show that Pentaplacodinium saltonense and Protoceratium reticulatum form two distinct clades. Pentaplacodinium saltonense forms a monophyletic clade with several unidentified strains from Malaysia. LSU and SSU rDNA sequences of three species of Ceratocorys (C. armata, C. gourreti, C. horrida) from the Mediterranean and several other unidentified strains from Malaysia form a well-supported sister clade. The unique phylogenetic position of an unidentified strain from Hawaii is also documented and requires further examination. In addition, based on the V9 SSU topology (bootstrap values >80%), specimens from Elands Bay (South Africa), originally described as Gonyaulax grindleyi by Reinecke (1967), cluster with Protoceratium reticulatum. The known range of Pentaplacodinium saltonense is tropical to subtropical, and its cyst is recorded as a fossil in upper Cenozoic sediments. Protoceratium reticulatum and Pentaplacodinium saltonense seem to inhabit different niches: motile stages of these dinoflagellates have not been found in the same plankton sample.
Thirty-four strains of Heterocapsa were established from Malaysian waters and their morphologies were examined by light, scanning, and transmission electron microscopy. Three species, H. bohaiensis, H. huensis, and H. rotundata, and three new species, H. borneoensis sp. nov., H. limii sp. nov., and H. iwatakii sp. nov. were described in this study. The three species were differentiated morphologically by unique characteristics of cell size, shape, displacement of the cingulum, shape and position of nucleus, the number and position of pyrenoids, and body scale ultrastructure. The species delimitations were robustly supported by the molecular data. A light-microscopy-based key to species of Heterocapsa is established, with two major groups, i.e., species with a single pyrenoid, and species with multiple pyrenoids. Bioassays were conducted by exposing Artemia nauplii to Heterocapsa densities of 1-5 × 105 cells mL-1, and treatments exposed to H. borneoensis showed naupliar mortality, while no naupliar death was observed in the treatments exposed to cells of H. bohaiensis, H. huensis, H. limii, and H. iwatakii. Naupliar death was observed during the initial 24 h for both tested H. borneoensis strains, and mortality rates increased up to 50% after 72-h exposure. This study documented for the first time the diversity and cytotoxic potency of Heterocapsa species from Malaysian waters.
Twenty cyanobacterial strains of eight morphospecies isolated from deep-frozen (-15 °C) mat samples originally collected on Ross Island, in Victoria Land, and on the McMurdo Ice Shelf were screened for the presence of genes encoding for production of anatoxins, cylindrospermopsin, microcystin/nodularin and saxitoxin. One strain of each of Microcoleus autumnalis and Phormidesmis priestleyi and two strains of Wilmottia murrayi were found to produce microcystin. No toxin production was detected in the other 16 strains representing five species. The four toxin-producing strains were characterised using both morphological and molecular approaches. Phylogenetic analyses using partial 16S rRNA sequences were consistent with the morphological identification of all four strains. They were all found to contain a fragment of the mcyE gene, which is involved in microcystin biosynthesis. ELISA analysis of extracts from cultures of these strains confirmed the presence of low concentrations of microcystin: 0.35 μg/L in M. autumnalis, <0.15 μg/L in P. priestleyi, 1.60 μg/L in W. murrayi strain 1 and 0.9 μg/L in W. murrayi strain 2. This study includes the first report of microcystin synthesis by W. murrayi.
Pseudo-nitzschia pungens is a widely distributed marine pennate diatom. Hybrid zones, regions in which two different genotypes may interbreed, are important areas for speciation and ecology, and have been reported across the globe for this species. However, sexual reproduction between differing clades in the natural environment is yet to be observed and is difficult to predict. Here we carried out experiments using two mono-clonal cultures of P. pungens from different genotypes to measure the frequency and timing of sexual reproduction across varying biotic (growth phases and cell activity potential) and abiotic conditions (nutrients, light, turbulence). We found the mating rates and number of zygotes gradually decreased from exponential to late stationary growth phases. The maximum zygote abundance observed was 1,390 cells mL-1 and the maximum mating rate was 7.1%, both which occurred during the exponential growth phase. Conversely, only 9 cells mL-1 and a maximum mating rate of 0.1% was observed during the late stationary phase. We also found the higher the relative potential cell activity (rPCA) in parent cells, as determined by the concentration of chlorophyll a per cell and the ratio of colony formation during parent cultivations, revealed higher mating rates. Furthermore, sexual events were reduced under nutrient enrichment conditions, and mating pairs and zygotes were not formed under aphotic (dark) or shaking culture conditions (150 rpm). In order to understand the sexual reproduction of Pseudo-nitzschia in the natural environment, our results highlight that it is most likely the combination of both biotic (growth phase, Chl. a content) and abiotic factors (nutrients, light, turbulence) that will determine the successful union of intraspecific populations of P. pungens in any given region.
A large-scale sampling was undertaken during a research cruise across the South China Sea in August 2016, covering an area of about 100,000 km2 to investigate the molecular diversity and distributions of micro-eukaryotic protists, with a focus on the potentially harmful microalgal (HAB) species along the east coast of Peninsular Malaysia. Environmental DNAs from 30 stations were extracted and DNA metabarcoding targeting the V4 and V9 markers in the 18S rDNA was performed. Many protistan molecular units, including previously unreported HAB taxa, were discovered for the first time in the water. Our findings also revealed interesting spatial distribution patterns, with a marked signal of compositional turnover between latitudinal regimes of water masses, where dinophytes and diatom compositions were among the most strongly enhanced at the fronts, leading to distinct niches. Our results further confirmed the widespread distribution of HAB species, such as the toxigenic Alexandrium tamiyavaichii and Pseudo-nitzschia species, and the fish-killing Margalefidinium polykrikoides and Karlodinium veneficum. The molecular information obtained from this study provides an updated HAB species inventory and a toolset that could facilitate existing HAB monitoring schemes in the region to better inform management decisions.
This study describes two novel species of marine dinophytes in the genus Alexandrium. Morphological characteristics and phylogenetic analyses support the placement of the new taxa, herein designated as Alexandrium limii sp. nov. and A. ogatae sp. nov. Alexandrium limii, a species closely related to A. taylorii, is distinguished by having a shorter 2'/4' suture length, narrower plates 1' and 6'', with larger length: width ratios, and by the position of the ventral pore (Vp). Alexandrium ogatae is distinguishable with its metasert plate 1' having almost parallel lateral margins, and by lacking a Vp. Production of paralytic shellfish toxins (PSTs), cycloimines, and goniodomins (GDs) in clonal cultures of A. ogatae, A. limii, and A. taylorii were examined analytically and the results showed that all strains contained GDs, with GDA as major variants (6-14 pg cell-1) for all strains except the Japanese strain of A. limii, which exclusively had a desmethyl variant of GDA (1.4-7.3 pg cell-1). None of the strains contained detectable levels of PSTs and cycloimines.
Pseudo-nitzschia is a cosmopolitan phytoplankton genus of which some species can form blooms and produce the neurotoxin domoic acid (DA). Identification of Pseudo-nitzschia is generally based on field material or strains followed by morphological and/or molecular characterization. However, this process is time-consuming and laborious, and can not obtain a relatively complete and reliable profile of the Pseudo-nitzschia community, because species with low abundance in the field or potentially unavailable for culturing may easily be overlooked. In the present study, specific ITS primer sets were designed and evaluated using in silico matching. The primer set ITS-84F/456R involving the complete ITS1 region was found optimal. Based on matching with a Pseudo-nitzschia ITS1 reference sequence database carefully-calibrated in this study, a metabarcoding approach using annotated amplicon sequence variants (ASV) was applied in the Taiwan Strait of the East China Sea during two cruises in the spring and summer of 2019. In total, 48 Pseudo-nitzschia species/phylotypes including 36 known and 12 novel were uncovered, and verified by haplotype networks, ITS2 secondary structure comparisons and divergence analyses. Correlation analyses revealed that temperature was a key factor affecting the seasonal variation of the Pseudo-nitzschia community. This study provides an overview of the Pseudo-nitzschia community in the Taiwan Strait, with new insights into the diversity. The developed metabarcoding approach may be used elsewhere as a standard reference for accurate annotation of Pseudo-nitzschia.