Allelochemical interactions and short-term effects of the dinoflagellate Alexandrium on selected photoautotrophic and heterotrophic protists

The marine dinoflagellate genus Alexandrium (Halim) Balech contains members that produce highly potent phycotoxins (PSP toxins or spirolides) as well as lytic substances and other allelochemicals of unknown structure and ecological significance. One isolate each of six Alexandrium species (A. tamarense, A. ostenfeldii, A. lusitanicum, A. minutum, A. catenella, A. taylori), of the closely related gonyaulacoid dinoflagellate Fragilidium subglobosum, and of the peridinioid Scrippsiella trochoidea were tested in 24 h co-incubation experiments for their short-term deleterious effects on a diversity of marine protists. Both autotrophs (Rhodomonas salina, Dunaliella salina, Thalassiosira weissflogii) and heterotrophs (Oxyrrhis marina, Amphidinium crassum, Rimostrombidium caudatum) were included as target species. All donor isolates except S. trochoidea exhibited lytic effects on at least some target species. Lytic effects were observed with all Alexandrium species, for both whole cell samples and culture filtrate (<10 μm and <0.2 μm). Antibiotic treated cultures with drastically reduced bacterial numbers did not show any general reduction in lytic capacity, therefore direct involvement of extracellular bacteria in allelochemical production is unlikely. Values of EC₅₀, defined as the Alexandrium cell concentration causing lysis of 50% of target cells, differed by two orders of magnitude depending on the donor/target combination, from 3.1 × 10³ cells ml⁻¹ (A. minutum/O. marina) down to 0.02 × 10³ cells ml⁻¹ (A. catenella/D. salina). Within the array of nine donor Alexandrium/target combinations, variable ratios in EC₅₀ values between donor/target combination cannot be explained by quantitative differences in allelochemical production, but rather indicate qualitative differences in the composition of compounds produced by different Alexandrium strains. In conclusion, our study confirms the widespread lytic capacity within the genus Alexandrium, although allelochemical effects are not restricted to this genus. Allelochemical interactions mediated by such lytic substances may be significant in explaining the formation and maintenance of Alexandrium blooms through direct destructive effects on competing algae or unicellular grazers.     

Alexandrium (Dinophyceae) species in Malaysian waters

A study was carried out to determine the presence of paralytic shellfish poisoning (PSP) toxin-producing dinoflagellates in the coastal waters of Peninsula Malaysia. This followed first ever occurrences of PSP in the Straits of Malacca and the northeast coast of the peninsula. The toxic tropical dinoflagellate Pyrodinium bahamense var. compressum was never encountered in any of the plankton samples. On the other hand, five species of Alexandrium were found. They were Alexandrium affine, Alexandrium leei, Alexandrium minutum, Alexandrium tamarense and Alexandrium tamiyavanichii. Not all species were present at all sites. A. tamiyavanichii was present only in the central to southern parts of the Straits of Malacca. A. tamarense was found in the northern part of the straits, while A. minutum was only found in samples from the northeast coast of the peninsula. A. leei and A. affine were found in both the north and south of the straits. Cultured isolates of A. minutum and A. tamiyavanichii were proven toxic by the receptor binding assay for PSP toxins but A. tamarense clones were not toxic. Mean toxin content for the A. tamiyavanichii and A. minutum clones were 26 and 15 fmol per cell STX equivalent, respectively. This study has provided evidence on the presence of PSP toxin-producing Alexandrium species in Malaysian waters which suggests that PSP could increase in importance in the future.     

Morphogenetic diversity and biotoxin composition of Alexandrium (Dinophyceae) in Irish coastal waters

The diversity of Alexandrium spp. in Irish coastal waters was investigated through the morphological examination of resting cysts and vegetative cells, the determination of PSP toxin and spirolide profiles and the sequence analysis of rDNA genes. Six morphospecies were characterised: A. tamarense, A. minutum, A. ostenfeldii, A. peruvianum, A. tamutum and A. andersoni. Both PSP toxin producing and non-toxic strains of A. tamarense and A. minutum were observed. The average toxicities of toxic strains for both cultured species were respectively 11.3 (8.6 S.D.) and 2.3 (0.5 S.D.) pg STX equiv. cell⁻¹. Alexandrium ostenfeldii and A. peruvianum did not synthesise PSP toxins but HPLC–MS analysis of two strains showed distinct spirolide profiles. A cyst-derived culture of A. peruvianum from Lough Swilly mainly produced spirolides 13 desmethyl-C and 13 desmethyl-D whereas one of A. ostenfeldii, from Bantry Bay, produced spirolides C and D. Species identification was confirmed through the analyses of SSU, ITS1-5.8S-ITS2 and LSU rDNA genes. Some nucleotide variability was observed among clones of toxic strains of A. tamarense, which all clustered within the North American clade. However, rDNA sequencing did not allow discrimination between the toxic and non-toxic forms of A. minutum. Phylogenetic analysis also permitted the differentiation of A. ostenfeldii from A. peruvianum. Resting cysts of PSP toxin producing Alexandrium species were found in Cork Harbour and Belfast Lough, locations where shellfish contamination events have occurred in the past, highlighting the potential for the initiation of harmful blooms from cyst beds. The finding of supposedly non-toxic and biotoxin-producing Alexandrium species near aquaculture production sites will necessitate the use of reliable discriminative methods in phytoplankton monitoring.     

Experimental study on the impact of dinoflagellate Alexandrium species on populations of the rotifer Brachionus plicatilis

To investigate harmful effects of the dinoflagellate Alexandrium species on microzooplankton, the rotifer Brachionus plicatilis was chosen as an assay species, and tested with 10 strains of Alexandrium including one known non-PSP-producer (Alexandrium tamarense, AT-6). HPLC analysis confirmed the PSP-content of the various strains: Alexandrium lusitanicum, Alexandrium minutum and Alexandrium tamarense (ATHK, AT5-1, AT5-3, ATCI02, ATCI03) used in the experiment were PSP-producers. No PSP toxins were detected in the strains Alexandrium sp1, Alexandrium sp2.Exposing rotifer populations to the densities of 2000 cells ml⁻¹ of each of these 10 Alexandrium strains revealed that the (non-PSP) A. tamarense (AT-6) and two other PSP-producing algae: A. lusitanicum, A. minutum, did not appear to adversely impact rotifer populations. Rotifers exposed to these three strains were able to maintain their population numbers, and in some cases, increase them. Although some increases in rotifer population growth following exposures to these three algal species were noted, the rate was less than for the non-exposed control rotifer groups.In contrast, the remaining seven algal strains (A. tamarense ATHK, AT5-1, AT5-3, ATCI02, ATCI03; also Alexandrium sp1 and Alexandrium sp2) all have adverse effects on the rotifers. Dosing rotifers with respective algal cell densities of 2000 cells ml⁻¹ each, for Alexandrium sp1, Alexandrium sp2, and A. tamarense strains ATHK and ATCI03 showed mean lethal time (LT₅₀) on rotifer populations of 21, 28, 29, and 36h, respectively. The remaining three species (A. tamarense strains AT5-1, AT5-3, ATCI02) caused respective mean rotifer LT₅₀s of 56, 56, and 71 h, compared to 160 h for the unexposed “starved control” rotifers. Experiments to determine ingestion rates for the rotifers, based on changes in their Chlorophyll a content, showed that the rotifers could feed on A. lusitanicum, A. minutum and A. tamarense strain AT-6, but could graze to little or no extent upon algal cells of the other seven strains. The effects on rotifers exposed to different cell densities, fractions, and growth phases of A. tamarense algal culture were respectively compared. It was found that only the whole algal cells had lethal effects, with strongest impact being shown by the early exponential growth phase of A. tamarense. The results indicate that some toxic mechanism(s), other than PSP and present in whole algal cells, might be responsible for the adverse effects on the exposed rotifers.     

Toxin composition variations in cultures of Alexandrium species isolated from the coastal waters of southern China

The composition of the paralytic shellfish toxins (PSTs) of five Alexandrium tamarense strains isolated from the coastal waters of southern China and one Alexandrium minutum strain from Taiwan Island were investigated. A. tamarense CI01 and A. tamarense Dapeng predominantly produced C2 toxin (over 90%) with trace amounts of C1 toxin (C1), gonyautoxin-2 (GTX2) and GTX3; two strains of A. tamarense HK9301 maintained in different locations produced C1-4 toxins and GTX1, 4, 5 and 6; no PSTs were found in A. tamarense NEW, while A. minutum TW produced only GTX1-4. The toxin compositions of cultured A. tamarense strains did not vary as much during different growth phases as did the toxin composition of A. minutum TW. The toxin compositions of A. tamarense HK9301-1 did not change significantly under different salinity, light intensity, and nitrate and phosphate levels in the culture medium, although the toxin productivity varied expectably. Another strain HK9301-2 maintained in a different location produced much less toxins with a considerably different toxin composition. Under similar culture maintenance conditions for 3 years, the toxin profiles of A. tamarense HK9301-1 did not change as much as did A. tamarense CI01. Our results indicate that toxin compositions of the dinoflagellate strains are strain-specific and are subject to influence by nutritional and environmental conditions but not as much by the growth phase. Use of toxin composition in identifying a toxigenic strain requires special caution.     

Toxin profiles of natural populations and cultures ofAlexandrium minutum Halim from Galician (Spain) coastal waters

The toxin profiles of three isolates and natural populations of the PSP agentAlexandrium minutum from several Galician rías (NW Spain) was obtained by HPLC. The toxin content of cultures ofA. minutum is dominated by GTX4 (80–90%) and GTX4 (10–15%) with small amounts of GTX3 and GTX2 (less than 3% of each); similar results were obtained for natural populations ofAlexandrium from three different Galician rías, where a mixture ofA. lusitanicum Balech andA. minutum can occur. Important quantitative differences were found between the three isolates, one being highly and two weakly toxic. The results obtained from these isolates and natural populations ofAlexandrium were very similar to those obtained from HPLC analyses of mussels intoxicated during a PSP outbreak in Ría de Ares (Rías Altas) in 1984, confirming thatA. minutum (previously identified asGonyaulax tamarensis Lebour andAlexandrium lusitanicum) was the PSP agent during the toxic outbreak in May 1984. Toxin profiles obtained from natural populations during different PSP outbreaks in different rías and from cultures are fairly consistent and suggest that at least from the toxin point of view,A. lusitanicum andA. minutum are identical, and that the toxin profile ofA. minutum from Galicia can be used as a biochemical marker.     

Comparative studies of four protein preparation methods for proteomic study of the dinoflagellate Alexandrium sp. using two-dimensional electrophoresis

Alexandrium is a wide-spread genus of dinoflagellate causing harmful algal blooms and paralytic shellfish poisoning around the world. Proteomics has been introduced to the study of Alexandrium, but the protein preparation method is still unsatisfactory with respect to protein spot number, separation and resolution, and this has limited the application of a proteomic approach to the study of dinoflagellates. In this study we compared four protein preparation methods for the two-dimensional electrophoresis (2DE) analysis of A. tamarense: (1) urea/Triton X-100 buffer extraction with trichloroacetic acid (TCA)/acetone precipitation; (2) direct precipitation with TCA/acetone; (3) 40 mM Tris (hydroxymethyl) aminomethane (Tris) buffer extraction; and (4) 50 mM Tris/5% glycerol buffer extraction. The results showed that, among the four protein preparation methods, the method combining the urea/Triton X-100 buffer extraction and TCA/acetone precipitation allowed detection of the highest number and quality of protein spots with a clear background. Although the direct TCA/acetone precipitation method also detected a high number of protein spots with a clear background, the spot number, separation and intensity were not as good as those obtained from the urea/Triton X-100 buffer extraction with TCA/acetone precipitation method. The 40 mM Tris buffer and 50 mM Tris/5% glycerol buffer methods allowed the detection of fewer protein spots and a pH range only from 4 to 7. Subsequently, the urea/Triton X-100 buffer extraction with TCA/acetone precipitation method was successfully applied to profiling protein expression in A. catenella under light stress conditions and the differential expression proteins were identified using MALDI TOF–TOF mass spectrometry. The method developed here appears to be promising for further proteomic studies of this organism and related species.     

Pfiesteria piscicida and Pfiesteria shumwayae in coastal waters of Long Island, New York, USA

Water and sediment samples were collected during summer and early fall 1999–2004 from coastal waters of New York State, USA, to test for the presence of Pfiesteria piscicida and Pfiesteria shumwayae. Physical and chemical conditions were characterized, and real-time polymerase chain reaction assays were conducted. Both species were relatively common and found at most sites at least once, and the frequency of positive assays was higher in sediments than in the water column. In a subset of the data from Suffolk County, Long Island, the presence of Pfiesteria was related to high chlorophyll a and relatively high nutrient concentrations. Partial SSU rDNA sequences of four PCR amplicons generated using P. shumwayae primers indicated two sequences: three were identical to GenBank P. shumwayae entries, but one showed enough sequence difference (15 positions in a 454 bp amplicon) to suggest a possible new species. Three isolates were tested for toxicity, and one was found to kill fish in bioassays. Despite the widespread presence of both Pfiesteria species and demonstration of potential to harm fish, no blooms of these dinoflagellates have been observed, nor has there been evidence of Pfiesteria-related fish or human health problems in these waters, likely related to colder temperatures than optimal for Pfiesteria species.     

Hypocrella panamensis sp. nov. (Clavicipitaceae, Hypocreales): a new species infecting scale insects on Piper carrilloanum in Panama

A new species, Hypocrella panamensis, is described from collections and cultures obtained on Barro Colorado Island, Panama. In order to aid in placement of this fungus, phylogenetic analyses were conducted using LSU (rDNA) sequences. Hypocrella panamensis is characterized by possessing pulvinate stromata with a Lecanicillium-like anamorphic state and superficial perithecia. Hypocrella panamensis consistently grouped in a clade containing Hypocrella nectrioides, H. phyllogena, and H. africana (100 % PP). Most species of Hypocrella possess Aschersonia or Hirsutella anamorphs. Hypocrella panamensis is unique in the genus Hypocrella in possession of a Lecanicillium-like anamorphic state. In its biological habit Hypocrella panamensis is similar to other species in Hypocrella in that it infects and degrades the scale insect, then grows superficially on nutrients that emerge to the plant surface through the stylet wound.     

Cystobasidiopsis nirenbergiae, a new agaricostilbomycete (Pucciniomycotina)

A new genus, Cystobasidiopsis, and a new species, Cystobasidiopsis nirenbergiae, are described for a fungus isolated from an arable loess soil in Ahlum near Braunschweig, Niedersachsen, Germany. An integrated analysis of morphological, ecological, ultrastructural and molecular data indicates that the new species belongs to the Chionosphaeraceae within the Agaricostilbales. Relevant characteristics of the new species are discussed and compared with those of related taxa.     

Latitudinal differentiation in the effects of the toxic dinoflagellate Alexandrium spp. on the feeding and reproduction of populations of the copepod Acartia hudsonica

Blooms of the dinoflagellate Alexandrium spp. increase in their frequency, toxicity and historical presence with increasing latitude from New Jersey (USA) to the Gaspé peninsula (Canada). Biogeographic variation in these blooms results in differential exposure of geographically separate copepod populations to toxic Alexandrium. We hypothesize that the ability of copepods to feed and reproduce on toxic Alexandrium should be higher in copepods from regions that are frequently exposed to toxic Alexandrium blooms. We tested this hypothesis with factorial common environment experiments in which female adults of the copepod Acartia hudsonica from five separate populations ranging from New Jersey to New Brunswick were fed toxic and non-toxic strains of Alexandrium, and the non-toxic flagellate Tetraselmis sp. Consistent with the hypothesis, when fed toxic Alexandrium we observed significantly higher ingestion and egg production rates in the copepods historically exposed to toxic Alexandrium blooms relative to copepods from regions in which Alexandrium is rare or absent. Such differences among copepod populations were not observed when copepods were fed non-toxic Alexandrium or Tetraselmis sp. These results were also supported by assays in which copepods from populations both historically exposed and naïve to toxic Alexandrium blooms were fed mixtures of toxic Alexandrium and Tetraselmis sp. Two-week long experiments demonstrated that when copepods from populations naïve to toxic Alexandrium were fed a toxic strain of Alexandrium they failed to acclimate, such that their ingestion rates remained low throughout the entire two-week period. The differences observed among populations suggest that local adaptation of populations of A. hudsonica from Massachusetts (USA) to New Brunswick (Canada) has occurred, such that some populations are resistant to toxic Alexandrium.     

Life cycle of the ichthyotoxic dinoflagellate Cochlodinium polykrikoides in Korean coastal waters

Since 1995, blooms of the harmful dinoflagellate, Cochlodinium polykrikoides, have caused considerable mortality of aquatic organisms and economic loss in Korea. However, little is known about the life cycle of the species, except for the planktonic vegetative stage; therefore, the aim of this paper was to elucidate the life cycle of C. polykrikoides. Its life cycle has two morphologically different stages: an armored and an unarmored vegetative stage. Armored vegetative cells were found in seawater samples collected in late-November and developed into four-cell chained, unarmored vegetative cells under laboratory culture. In samples collected in late-May, both the armored and unarmored types (vegetative swimming stage) occurred; the former easily developed into an unarmored vegetative cell type, suggesting that the armoured–unarmored transition occurs as early as May. A presumptive resting cyst, round but folded at one side, was produced from armored type cells in laboratory conditions. It was also collected from natural bottom sediments, which suggests it is the dormant resting cyst of C. polykrikoides.     

Improved phylogenetic resolution of toxic and non-toxic Alexandrium strains using a concatenated rDNA approach

Dinoflagellates of the genus Alexandrium are known producers of paralytic shellfish toxins. Species within the genus have similar phenotypes making morphological identification problematical. The use of Alexandrium rDNA sequence data is therefore increasing, resulting in the improved resolution of evolutionary relationships by phylogenetic inferences. However, the true branching pattern within Alexandrium remains unresolved, with minimal support shown for the main phylogentic branch. The aim of this study is to improve phylogenetic resolution via a concatenated rDNA approach with a broad sample of taxa, allowing inference of the evolutionary pattern between species and toxins. 27 Alexandrium strains from 10 species were tested with HPLC for PSP toxin presence and additionally sequenced for 18S, ITS1, 5.8S, ITS2 and 28S rDNA before being phylogenetically inferred together with all available orthologous sequences from NCBI. The resulting alignment is the largest to date for the genus, in terms of both inferred characters and taxa, thus allowing for the improved phylogenetic resolution of evolutionary patterns there in. No phylogenetic pattern between PSP producing and non-producing strains could be established, however the terminal tamarense complex was shown to produce more PSP analogues than basal clades. Additionally, we distinguish a high number of polymorphic regions between the two copies of A. fundyense rDNA, thus allowing us to demonstrate the presence of chimeric sequences within GenBank, as well as a possible over estimation of diversification within the tamarense complex.     

Toxin production in migrating dinoflagellates: a modelling study of PSP producing Alexandrium

A mechanistic model of dinoflagellate physiology, previously developed and parameterised to simulate paralytic shellfish poison (PSP) content and cell growth for Alexandrium fundyense in response to N and P nutrition, was operated within a vertical water structure in which the organism migrated. Simulations showed the expected development of vertical migration behaviour in response to light and mineral nutrient interactions. Growth in a N-limited water column resulted in a continual, though low level, PSP production with a large population biomass. A sequence of P-stress and nutrient re-feeding during vertical migration stimulated an enhancement of PSP content even with only moderately elevated supply of N:P ratios. This was exacerbated by low absolute P concentrations below the nutricline as well as by the N:P ratio. Although the final biomass was lower in these P-limited simulations, the total toxin production was much higher. The simulations suggest that vertical migration in stratified waters in even moderately high N:P waters could result in the formation of highly toxic populations of Alexandrium. One may expect a similar enhancement of toxicity in other harmful algal species that are engaged in vertical migration, where nutrient supply ratios affect toxin production.     

A new fungal genus, Teracosphaeria, with a phialophora-like anamorph (Sordariomycetes, Ascomycota)

The new genus and species Teracosphaeria petroica is described for a perithecial ascomycete and its anamorph occurring on decayed wood collected in New Zealand. The fungus produces immersed, non-stromatic ceratosphaeria-like perithecia in nature, with hyaline, septate ascospores produced in unitunicate, non-amyloid asci. The anamorph produced in vitro is phialophora-like with lightly pigmented phialides terminating in flaring, deep collarettes that are often noticeably brown with conspicuous periclinal thickening. Phylogenetic analysis of LSU rDNA sequence data indicates that this fungus is distinct from morphologically similar fungi classified in the Chaetosphaeriales, the Trichosphaeriales or the Magnaporthaceae. It forms a monophyletic group with recently described, chaetosphaeria-like ascomycetes, such as the pyrenomycete genus Mirannulata, and shows affinity with the anamorphic species Dictyochaeta cylindrospora. The usefulness of describing anamorph genera for morphologically reduced anamorphs, when anamorph characteristics are actually part of the holomorph diagnosis, is discussed. An apparently contradictory example of the so-called Cordana and Pseudobotrytis anamorphs of Porosphaerella spp. is also discussed.     

Corylomyces: a new genus of Sordariales from plant debris in France

The new genus Corylomyces, isolated from the surface of a hazelnut (Corylus avellana) in the French Pyrenees, is described, illustrated and compared with morphologically similar taxa. It is characterised by tomentose, ostiolate ascomata possessing long necks composed of erect to sinuose hairs, and one- or two-celled, opaque, lunate to reniform ascospores. Analyses of the SSU and LSU fragments rDNA gene sequences support its placement in the Lasiosphaeriaceae (Sordariales).     

Morphological differences and molecular phylogeny of freshwater blooming species, Peridiniopsis spp. (Dinophyceae) from China

In 2008–2010, several freshwater dinoflagellate blooms caused by Peridiniopsis spp. were observed in China. P. penardii and P. niei sampled from various geographical localities were examined by means of light and scanning electron microscopy. After comparing morphological and molecular differences, the new freshwater variety Peridiniopsis penardii var. robusta var. nov. (Peridiniales, Dinophyceae) found in Manwan Reservoir, Yunnan Province was described. The new variety differed from P. penardii since it possessed numerous robust antapical spines and a conspicuous apical spine. Molecular phylogenetic analyses based on SSU, LSU and ITS indicated P. niei, P. penardii and P. penardii var. robusta were closely related with P. kevei, and clustered into a monophyletic clade. The new variety possessed an endosymbiotic diatom which was similar to P. penardii and P. kevei, whereas the endosymbiont was not present in cells of P. niei. The endosymbiont SSU and ITS phylogenies showed that the endosymbionts of these three dinoflagellates were closely related to members of Thalassiosirales. Furthermore it was concluded that the endosymbionts might originate from Discostella-like species.     

Intra-population clonal variability in allelochemical potency of the toxigenic dinoflagellate Alexandrium tamarense

Clonal variability in exponential growth rate and production of secondary metabolites was determined from clonal isolates of Alexandrium tamarense originating from a single geographical population from the east coast of Scotland. To assess variability in the selected phenotypic characteristics over a wide spectrum, 10 clones were chosen for experimentation from 67 clonal isolates pre-screened for their lytic capacity in a standardized bioassay with the cryptophyte Rhodomonas salina. Specific growth rates (μ) of the 10 clonal isolates ranged from 0.28 to 0.46 d⁻¹ and were significantly different among clones. Cell content (fmol cell⁻¹) and composition (mol%) of paralytic shellfish toxins (PSTs), analyzed by liquid chromatography with fluorescence detection (LC–FD), varied widely among these isolates, with total PST quotas ranging from 20 to 89 fmol cell⁻¹. Except for strain 3, the toxins C1/C2, neosaxitoxin (NEO), saxitoxin (STX), and gonyautoxins-1 and -4 (GTX1/GTX4), were consistently the most relatively abundant, with lesser amounts of GTX2/GTX3 evident among all isolates. Only clone 3 contained >20 mol% of toxin B1, with C1/C2, GTX2/GTX3 and NEO in almost equimolar ratios.Eight of the 10 clones caused cell lysis of both R. salina and the heterotrophic dinoflagellate Oxyrrhis marina, as quantified from the dose–response curves from short-term (24 h) co-incubation bioassays. For two clones, no significant mortality even at high Alexandrium cell concentrations (ca. 10⁴ mL⁻¹) was observed. Allelochemical activity expressed as EC₅₀ values, defined as the Alexandrium cell concentration causing lysis of 50% of target cells, varied by about an order of magnitude and was significantly different among clones. No correlation was observed between growth rate und allelochemical potency (as EC₅₀) indicating that at least under non-limiting growth conditions no obvious growth reducing costs are associated with the production of allelochemically active secondary metabolites.