Population genetic structure and connectivity of the harmful dinoflagellate Alexandrium minutum in the Mediterranean Sea

The toxin-producing microbial species Alexandrium minutum has a wide distribution in the Mediterranean Sea and causes high biomass blooms with consequences on the environment, human health and coastal-related economic activities. Comprehension of algal genetic differences and associated connectivity is fundamental to understand the geographical scale of adaptation and dispersal pathways of harmful microalgal species. In the present study, we combine A. minutum population genetic analyses based on microsatellites with indirect connectivity (C(i)) estimations derived from a general circulation model of the Mediterranean sea. Our results show that four major clusters of genetically homogeneous groups can be identified, loosely corresponding to four regional seas: Adriatic, Ionian, Tyrrhenian and Catalan. Each of the four clusters included a small fraction of mixed and allochthonous genotypes from other Mediterranean areas, but the assignment to one of the four clusters was sufficiently robust as proved by the high ancestry coefficient values displayed by most of the individuals (>84%). The population structure of A. minutum on this scale can be explained by microalgal dispersion following the main regional circulation patterns over successive generations. We hypothesize that limited connectivity among the A. minutum populations results in low gene flow but not in the erosion of variability within the population, as indicated by the high gene diversity values. This study represents a first and new integrated approach, combining both genetic and numerical methods, to characterize and interpret the population structure of a toxic microalgal species. This approach of characterizing genetic population structure and connectivity at a regional scale holds promise for the control and management of the harmful algal bloom events in the Mediterranean Sea.     

Comparison of Suitability of Great Bay, New Jersey, and Parsonage Creek, New York, for Alexandrium tamarense

ABSTRACT. Alexandrium tamarense (Lebour) Balech (= Gonyaulax tamarensis Lebour) has been widely distributed and occasionally abundant in coastal waters of Long Island, New York in recent years. However, the distribution on the New Jersey coast has been sparse and this fact cannot be explained by this dinoflagellate's ability to migrate, or by its nutritional and physiological characteristics, or by the region's general suitability for phytoplankton. Therefore, the possibility that New Jersey coastal waters might be chemically exclusionary for A. tamarense seemed worth exploring. In a limited approach, we tested for water quality detrimental to the species in one New Jersey site (Great Bay) with a series of annual assays. Parsonage Creek, Long Island, New York, was assayed for comparison; this creek is assumed to have had at least marginal suitablity for A. tamarense , based on its reported long-term presence. Results provide tentative support for our working hypothesis, i.e. Great Bay chemical water quality is generally unfavorable for A. tamarense. Inhibition of A. tamarense growth, or culture decline, occurred in both assay series, but was substantially greater in Great Bay water. Inimical water quality was the most important factor distinguishing the two sites. Chelation with EDTA had greatest overall benefit in Great Bay assays, suggesting that lower availability of a natural chelator in the bay could be a secondary factor. Assay metal response is problematic, but we believe it permits speculation that essential metals could be partially limiting to A. tamarense in Great Bay, but would not be a critical regulator.     

Detecting the bloom-forming dinoflagellate Alexandrium tamarense using the absorption signature

Pulsed N inputs were simulated by exposing Alexandrium tamarense to sudden increase in concentration of three different N sources to evaluate an approach of using absorption coefficient (a _ph) ratio at three wavelengths to detect toxic dinoflagellates such as A. tamarense. The present study examined the absorption properties of A. tamarense for a range of concentrations under three different nitrogen sources and concluded that using absorption such as chlorophyll-specific absorption coefficient (a _ph ^* ) at a single wavelength is not appropriate for detecting bloom forming species due to variability of a _ph ^* caused by varying physiological states. With appropriate wavelengths established, the a _ph ratio approach with three wavelengths has the capability of detecting A. tamarense. Variable environmental conditions such as nutrient species, light quantity and light quality did not affect the a _ph ratios significantly, particularly the ratio at 510 and 675 relative to 555 nm. This ratio allows differentiation of A. tamarense from other phytoplankton species including other bloom-forming dinoflagellates. In addition, this ratio could also detect the presence of A. tamarense in mixed cultures from diluted samples of as low as 5% in cell abundance (c. 30% in Chl a concentration). The present study suggests that this ratio could be an adequate biomarker for dinoflagellates such as A. tamarense. This absorption ratio technique could provide means of interpreting field populations and enhancing the capability for detecting harmful species such as A. tamarense.     

Six new microsatellite markers for the toxic marine dinoflagellate Alexandrium tamarense

We report the characterization of six new microsatellite loci for the toxic marine dinoflagellate Alexandrium tamarense (North American ribotype), using 56 isolates from a range of locations. The numbers of alleles per locus ranged from five to nine and gene diversities ranged from 0.041 to 0.722. We tested primers for these six loci on other A. tamarense ribotypes and on other Alexandrium species; the results suggest that the primers are specific to A. tamarense isolates belonging to the North American ribotype.     

A review of the global ecology,genomics, and biogeography of the toxic cyanobacterium,Microcystis spp.

This review summarizes the present state of knowledge regarding the toxic, bloom-forming cyanobacterium, Microcystis, with a specific focus on its geographic distribution, toxins, genomics, phylogeny, and ecology. A global analysis found documentation suggesting geographic expansion of Microcystis, with recorded blooms in at least 108 countries, 79 of which have also reported the hepatatoxin microcystin. The production of microcystins (originally “Fast-Death Factor”) by Microcystis and factors that control synthesis of this toxin are reviewed, as well as the putative ecophysiological roles of this metabolite. Molecular biological analyses have provided significant insight into the ecology and physiology of Microcystis, as well as revealed the highly dynamic, and potentially unstable, nature of its genome. A genetic sequence analysis of 27 Microcystis species, including 15 complete/draft genomes are presented. Using the strictest biological definition of what constitutes a bacterial species, these analyses indicate that all Microcystis species warrant placement into the same species complex since the average nucleotide identity values were above 95%, 16S rRNA nucleotide identity scores exceeded 99%, and DNA–DNA hybridization was consistently greater than 70%. The review further provides evidence from around the globe for the key role that both nitrogen and phosphorus play in controlling Microcystis bloom dynamics, and the effect of elevated temperature on bloom intensification. Finally, highlighted is the ability of Microcystis assemblages to minimize their mortality losses by resisting grazing by zooplankton and bivalves, as well as viral lysis, and discuss factors facilitating assemblage resilience.     

沿岸海域富营养化与赤潮发生的关系

综述了赤潮的发生与沿岸海域富营养化的关系。近几十年来,人类活动使得天然水体的富营养化进程大大加速。营养负荷的增加与高生物量水华的增多相联系。控制营养输入后,浮游植物生物量或有害藻类水华事件也相应减少。营养的组成与浮游植物的种类组成及水华的形成有密切联系。有机营养对有害藻类水华的促进作用受到关注。营养输入时机影响浮游植物种间竞争的结果,因而对浮游植物的群落演替具有深远影响。由于浮游植物存在生理差异,因而对营养加富的反应因种而异。营养在调控某些有毒藻类的毒素产量方面也发挥着重要作用。此外,营养输入与藻类水华之间存在复杂的间接联系。当然,营养状况并非浮游植物群落演替的唯一决定因素。研究结果提示,控制营养输入、减缓水域富营养化是减少有害藻类水华发生的有效途径,而深入研究典型有害藻类的营养生理对策则为防治并最终消除有害藻类水华提供了理论基础。     

First reports of Pseudo‐nitzschia micropora and P. hasleana (Bacillariaceae) from the Southern Hemisphere: Morphological,molecular and toxicological characterization

Pseudo‐nitzschia H. Peragallo is a marine diatom genus found worldwide in polar, temperate, subtropical and tropical waters. It includes toxigenic representatives that produce domoic acid (DA), a neurotoxin responsible for Amnesic Shellfish Poisoning. In this study we characterized two species of Pseudo‐nitzschia collected from Port Stephens and the Hawkesbury River (south eastern Australia) previously unreported from Australian waters. Clonal isolates were sub‐sampled for (i) light and transmission electron microscopy; (ii) DNA sequencing, based on the nuclear‐encoded partial large subunit ribosomal RNA gene and internal transcribed spacer (ITS)‐ITS1, 5.8S and ITS2 rDNA regions and, (iii) DA production as measured by liquid chromatography‐mass spectrometry. Morphological and molecular data unambiguously revealed the species to be Pseudo‐nitzschia micropora Priisholm, Moestrup & Lundholm (Port Stephens) and Pseudo‐nitzschia hasleana Lundholm (Hawkesbury River). This is the first report of the occurrence of these species from the Southern Hemisphere and the first report of P. micropora in warm‐temperate waters. Cultures of P. micropora, tested for DA production for the first time, proved to be non‐toxic. Similarly, no detectable toxin concentrations were observed for P. hasleana. Species resolution and knowledge on the toxicity of local Pseudo‐nitzschia species has important implications for harmful algal bloom monitoring and management.     

中国赤潮的发生趋势和研究进展

通过对中国沿海赤潮发生历史的回顾以及主要赤潮事件的分析,阐明了中国沿海赤潮发生所呈现的趋势,即频率增加,规模扩大,新的赤潮藻种不断出现,有毒赤潮种比例上升,以及有害赤潮危害程度日益增加,且初步分析了赤潮频发的内因和外因,综述了我国科学家在赤潮生消过程监测,赤潮灌的培养生物学和分类学,赤潮藻类的营养动力学及生理生态学特性,赤潮藻类的生活史,赤潮藻类毒素,赤潮的模型和赤潮防治及国际合作等方面工作的进展,指出了研究还存在的不足之处,并对未来赤潮研究和管理提出了建议。     

Short‐term variations in development of a recurrent toxic Alexandrium minutum–dominated dinoflagellate bloom induced by meteorological conditions1

Development of an Alexandrium minutum Halim bloom affecting a Mediterranean harbor was monitored in detail using a multidisciplinary approach. A. minutum was by far the most abundant species at and near the bloom maximum, but always coexisted with members of three additional dinoflagellate genera and prasinophytes. Bloom initiation (early February) occurred during prolonged influences of sunny weather conditions, when day length exceeded 10.5 h and water temperatures reached 10.2°C. Subsequent development toward its maximum (end of March) also relied on good weather conditions, with specific wind directions favoring accumulation of cells. Arrival of rainy weather, associated with frontal boundaries of large‐scale low‐atmospheric‐pressure systems and characterized by reduced solar irradiance (heavy cloud coverage), opposite wind directions, and enhanced wind speeds, always caused temporal declines of the bloom. These declines were attributed to dispersal or displacement of algae, but a vertical migration of A. minutum cells toward the sediment was not excluded. Delayed inflows of excess terrestrial rainwater along the inner harbor wall strongly reduced salinity and prolonged a temporal decline far beyond influences of bad weather. The associated nutrient supply favored development of the phytoplankton population but reduced the toxin production of A. minutum cells. The HPLC‐determined Gonyautoxin (GTX) 1 + 4/GTX 2 + 3 ratio strongly increased toward the bloom maximum. This ratio was influenced by nutrient status and cell density and has a potential value for monitoring developmental stages of blooms. Prolonged bad weather conditions eventually hindered continuation of bloom development, and subsequent declines of algal biomass were attributed to grazing.     

Alteration of plankton communities and biogeochemical cycles by harmful Cochlodinium polykrikoides (Dinophyceae) blooms

Cochlodinium polykrikoides is a globally distributed, ichthyotoxic, bloom-forming dinoflagellate. Blooms of C. polykrikoides manifest themselves as large (many km²) and distinct patches with cell densities exceeding 10³ ml⁻¹ while water adjacent to these patches can have low cell densities (<100 cells ml⁻¹). While the effect of these blooms on fish and shellfish is well-known, their impacts on microbial communities and biogeochemical cycles are poorly understood. Here, we investigated plankton communities and the cycling of carbon, nitrogen, and B-vitamins within blooms of C. polykrikoides and compared them to areas in close proximity (<100 m) with low C. polykrikoides densities. Within blooms, C. polykrikoides represented more than 90% of microplankton (>20 μm) cells, and there were significantly more heterotrophic bacteria and picoeukaryotic phytoplankton but fewer Synechococcus. Terminal restriction fragment length polymorphism analysis of 16S and 18S rRNA genes revealed significant differences in community composition between bloom and non-bloom samples. Inside the bloom patches, concentrations of vitamin B₁₂ were significantly lower while concentrations of dissolved oxygen were significantly higher. Carbon fixation and nitrogen uptake rates were up to ten times higher within C. polykrikoides bloom patches. Ammonium was a more important source of nitrogen, relative to nitrate and urea, for microplankton within bloom patches compared to non-bloom communities. While uptake rates of vitamin B₁ were similar in bloom and non-bloom samples, vitamin B₁₂ was taken up at rates five-fold higher (>100 pmol⁻¹ L⁻¹ d⁻¹) in bloom samples, resulting in turn-over times of hours during blooms. This high vitamin demand likely led to the vitamin B₁₂ limitation of C. polykrikoides observed during nutrient amendment experiments conducted with bloom water. Collectively, this study revealed that C. polykrikoides blooms fundamentally change microbial communities and accelerate the cycling of carbon, some nutrients, and vitamin B₁₂.     

Characterization of the Parasitic Dinoflagellate Amoebophrya sp. Infecting Akashiwo sanguinea in Coastal Waters of China

The endoparasitic dinoflagellate Amoebophrya infects a number of free‐living marine dinoflagellates, including harmful algal bloom species. The parasitoid eventually kills its host and has been proposed to be a significant loss factor for dinoflagellate blooms in restricted coastal waters. For several decades, the difficulties of culturing host‐parasitoid systems have been a great obstacle for further research on the biology of Amoebophrya. Here, we established an Akashiwo sanguinea‐Amoebophrya sp. coculture from Chinese coastal waters and studied the parasitoid's generation time, dinospore survival and infectivity, as well as its host specificity. The lifespan of Amoebophrya sp. ex. A. sanguinea was approximately 58 h. The infective dinospores can survive up to 78 h in ambient waters but gradually lose their infectivity. The parasitoid was unable to infect other dinoflagellate species, its infection rate reached as high as 91% when the ratio of dinospores to host cells was 20:1. The high infectivity of dinospores suggests that the Amoebophrya strain was capable of removing a considerable fraction of host biomass within a short period, but that it is probably unable to maintain high infection levels under nonbloom conditions of its host, due to limited survival and time constraints in encountering host cells.     

First report of the photosynthetic dinoflagellate genus Azadinium in the Pacific Ocean: morphology and molecular characterization of Azadinium cf. poporum

A strain of a dinoflagellate belonging to the genus Azadinium was obtained by the incubation of sediments collected from Shiwha Bay, Korea. This report of the genus Azadinium is the first outside of northern Europe and furthermore from the Pacific Ocean. The diagnostic morphological features of the isolate very closely resemble the recently described species Azadinium poporum isolated from the North Sea. However, the shape of the 3' apical plate and the occasional morphological variations unreported from A. poporum bring minor distinctions between strains from different locations. The DNA sequences of small subunit, ITS, and large subunit (LSU) rDNA differed by 0.2%, 2.6%, and 3.6%, respectively, from those of A. poporum, whereas the COI gene was identical to those found in all strains of Azadinium. Phylogenetic analyses of the ribosomal DNA regions generally positioned the Korean strain as a sister taxon of A. poporum. However, the Korean isolate tends to occupy a basal position within Azadinium species with ITS rDNA and LSU rDNA. Using liquid chromatography coupled with tandem mass spectrometry, no known azaspiracids were detected. The slight but discernible morphological differences, the distinct rDNA sequences, and the tendency of the Korean strain to diverge phylogenetically based on ITS rDNA and LSU rDNA from A. poporum do not enable us to clearly assign the isolate to A. poporum. However, these characteristics do not allow us to classify it as a distinct species, and it is therefore designated as Azadinium cf. poporum. The examination of more strains to find more diagnostic characteristics might enable the attribution of this material to a well-defined taxonomic position.     

Genome evolution and host‐microbiome shifts correspond with intraspecific niche divergence within harmful algal bloom‐forming Microcystis aeruginosa

Intraspecific niche divergence is an important driver of species range, population abundance and impacts on ecosystem functions. Genetic changes are the primary focus when studying intraspecific divergence; however, the role of ecological interactions, particularly host‐microbiome symbioses, is receiving increased attention. The relative importance of these evolutionary and ecological mechanisms has seen only limited evaluation. To address this question, we used Microcystis aeruginosa, the globally distributed cyanobacterium that dominates freshwater harmful algal blooms. These blooms have been increasing in occurrence and intensity worldwide, causing major economic and ecological damages. We evaluated 46 isolates of M. aeruginosa and their microbiomes, collected from 14 lakes in Michigan, USA, that vary over 20‐fold in phosphorus levels, the primary limiting nutrient in freshwater systems. Genomes of M. aeruginosa diverged along this phosphorus gradient in genomic architecture and protein functions. Fitness in low‐phosphorus lakes corresponded with additional shifts within M. aeruginosa including genome‐wide reductions in nitrogen use, an expansion of phosphorus assimilation genes and an alternative life history strategy of nonclonal colony formation. In addition to host shifts, despite culturing in common‐garden conditions, host‐microbiomes diverged along the gradient in taxonomy, but converged in function with evidence of metabolic interdependence between the host and its microbiome. Divergence corresponded with a physiological trade‐off between fitness in low‐phosphorus environments and growth rate in phosphorus‐rich conditions. Co‐occurrence of genotypes adapted to different nutrient environments in phosphorus‐rich lakes may have critical implications for understanding how M. aeruginosa blooms persist after initial nutrient depletion. Ultimately, we demonstrate that the intertwined effects of genome evolution, host life history strategy and ecological interactions between a host and its microbiome correspond with an intraspecific niche shift with important implications for whole ecosystem function.     

Haemolytic activity and reactive oxygen species production of four harmful algal bloom species

Based on haemolytic activity and reactive oxygen species (ROS) production of Chattonella marina, Chattonella antiqua, Heterocapsa circularisquama, Alexandrium tamiyavanichii and Karenia mikimotoi, the species were categorized into four types. (1) H. circularisquama: haemolytic activity was detected in both cell suspension and cell-free culture supernatant, but with greater activity in cell suspension than in the supernatant suggesting the presence of both cell surface and secreted haemolytic agents. (2) A. tamiyavanichii: equal haemolytic activities were detected in both the cell suspension and cell-free culture supernatant suggesting the presence of only secreted haemolytic agents. (3) K. mikimotoi: haemolytic activity was detected only in the cell suspension, indicating haemolytic agents occur only on the cell surface. (4) C. marina and C. antiqua: no significant haemolytic activity was detected in either cell suspension or cell-free culture supernatant, but high ROS were detected in the cell suspensions. Heterocapsa circularisquama and K. mikimotoi showed lethal effects on rotifers (Brachionus plicatilis), whereas A. tamiyavanichii, C. marina and C. antiqua had no effect. Our results suggest that H. circularisquama, K. mikimotoi and A. tamiyavanichii produce haemolytic agents with distinct characteristics, whereas C. marina and C. antiqua have an extremely potent ability to produce ROS.     

The application of a molecular clock based on molecular sequences and the fossil record to explain biogeographic distributions within the Alexandrium tamarense "species complex" (Dinophyceae)

The cosmopolitan dinoflagellate genus Alexandrium, and especially the A. tamarense species complex, contain both toxic and nontoxic strains. An understanding of their evolution and paleogeography is a necessary precursor to unraveling the development and spread of toxic forms. The inclusion of more strains into the existing phylogenetic trees of the Alexandrium tamarense species complex from large subunit rDNA sequences has confirmed that geographic distribution is consistent with the molecular clades but not with the three morphologically defined species that constitute the complex. In addition, a new clade has been discovered, representing Mediterranean nontoxic strains. The dinoflagellates fossil record was used to calibrate a molecular clock: key dates used in this calibration are the origins of the Peridiniales (estimated at 190 MYA), Gonyaulacaceae (180 MYA), and Ceratiaceae (145 MYA). Based on the data set analyzed, the origin of the genus Alexandrium was estimated to be around late Cretaceous (77 MYA), with its earliest possible origination in the mid Cretaceous (119 MYA). The A. tamarense species complex potentially diverged around the early Neogene (23 MYA), with a possible first appearance in the late Paleogene (45 MYA). A paleobiogeographic scenario for Alexandrium is based on (1) the calculated possible ages of origination for the genus and its constituent groups; (2) paleogeographic events determined by plate movements, changing ocean configurations and currents, as well as climatic fluctuations; and (3) the present geographic distribution of the various clades of the Alexandrium tamarense species complex.     

Characterization of multiple isolates from an Alexandrium ostenfeldii bloom in The Netherlands

Alexandrium ostenfeldii is an emerging harmful algal bloom species forming a global threat to coastal marine ecosystems, with consequences for fisheries and shellfish production. The Oosterschelde estuary is a shallow, macrotidal and mesotrophic estuary in the southwest of The Netherlands with large stocks of mussels, oysters, and cockles. These shellfish stocks were threatened by a recent A. ostenfeldii bloom in the Ouwerkerkse Kreek, which is a brackish water creek discharging water into the Oosterschelde. Little is yet known about the characteristics of the A. ostenfeldii population in this creek. We therefore isolated 20 clones during an A. ostenfeldii bloom in 2013, and characterized these clones on their growth and toxin profile in their exponential growth phase. The cyclic imines were identified by comparison of A. ostenfeldii extracts with the retention time and CID spectra of standard solutions, or with published CID spectra. We furthermore assessed the allelochemical potency and phylogeny of a selection of 10–12 clones. Morphology and molecular phylogeny showed that all clones belong to Group 1 of A. ostenfeldii. All clones showed comparable growth rates of on average 0.22 ± 0.03 d⁻¹. During exponential growth, they all produced a unique combination of paralytic shellfish poisoning toxins, spirolides and gymnodimines, of which particularly the latter showed a high intra-specific variability, with a 25-fold difference between clones with the lowest and highest cell quota. Furthermore, the selected 12 clones showed high allelopathic potencies with EC₅₀ values based on lysis assays against the cryptophyte Rhodomonas salina between 212 and 525 A. ostenfeldii cells mL⁻¹. Lytic activities were lower for cell extracts, indicating an important extracellular role of these compounds. A high intra-specific variability may add to the success of genotypically diverse A. ostenfeldii blooms, and make populations resilient to changes in environmental and climatic conditions.     

稻、麦秸秆对球形棕囊藻(Phaeocystis globosa)生长的抑制作用

考查了秸秆及其浸出液对球形棕囊藻(Phaeocystis globosa)生长的影响,分析了秸秆附着微生物及秸秆物理吸附作用对秸秆抑藻活性的影响,观察了稻杆浸出液对藻细胞形态的影响。结果显示,一定量的物理破碎稻杆、麦杆及其浸出液均可有效抑制棕囊藻的生长,灭菌与未灭菌秸杆之间抑藻效果相差不大。不同条件处理的秸杆浸出液总酚含量与抑藻作用间均存在很大差异,但两者之间并无明显相关性。原子力显微镜观察发现,秸秆处理组细胞膜破裂、细胞塌陷,结构不完整,细胞粗糙度明显增加。这些结果提示微生物和物理吸附对秸杆抑藻作用的贡献很小,秸秆中存在的或降解产生的抑藻活性成分是秸秆抑藻的主要原因;除了酚酸类物质外,秸杆中可能存在其他可以显著抑制藻类生长的物质;秸秆通过破坏藻细胞的膜结构,导致藻细胞出现空洞,细胞内容物流出,从而抑制或杀灭藻细胞。     

Use of two-dimensional gel electrophoresis to differentiate morphospecies of Alexandrium minutum, a paralytic shellfish poisoning toxin-producing dinoflagellate of harmful algal blooms

Contamination of shellfish with paralytic shellfish poisoning toxins (PST) produced by toxic harmful algal blooms (HABs) have been negatively affecting the shellfish and aquaculture industries worldwide. Therefore, accurate and early identification of toxic phytoplankton species is crucial in HABs surveillance programs that allow fish-farmers to take appropriate preventive measures in shellfish harvesting and other aquaculture activities to overcome the negative impacts of HABs on human health. The identification of toxic dinoflagellates present in the water is currently a time-consuming operation since it requires skillful taxonomists and toxicologists equipped with optical and scanning electron microscopes as well as sophisticated equipment, for example, high-performance liquid chromotography-fluorescence detection. In this paper, a two-dimensional gel electrophoresis (2-DE)-based proteomic approach was applied to discriminate between toxic and nontoxic strains of Alexandrium minutum. Variation in morphological features between toxic and nontoxic strains was minimal and not significant. Also, variation in 2-DE protein patterns within either toxic or nontoxic strains was low, but pronounced differences were detected between toxic and nontoxic strains. The most notable differences between these strains were several abundant proteins with pIs ranging from 4.8 to 5.3 and apparent molecular masses between 17.5 and 21.5 kDa. Groups of proteins, namely NT1, NT2, NT3, and NT4, were consistently found in all nontoxic strains, while T1 and T2 were prominent in the toxic strains. These specific protein spots characteristic for toxic and nontoxic strains remained clearly distinguishable irrespective of the various growth conditions tested. Therefore, they have the potential to serve as "taxonomic markers" to distinguish toxic and nontoxic strains within A. minutum. Initial studies revealed that the expression pattern of T1 was tightly correlated to toxin biosynthesis in the examined alga and may be used to serve as a potential toxin indicator.