Allan Cembella

Azadinium spinosum gen. et sp. nov. (Dinophyceae) identified as a primary producer of azaspiracid toxins.

Azaspiracids (AZAs) are a group of lipophilic marine biotoxins associated with human incidents of shellfish poisoning. During a research cruise to the North Sea, we analysed size-fractionated plankton for AZA by mass spectrometry and successfully isolated an AZA-producing dinoflagellate from the east coast of Scotland. As shown previously, an axenic culture of this dinoflagellate produces AZA 1, AZA 2 and an isomer of AZA 2. Here we give a taxonomic description of this new taxon Azadinium spinosum gen. et sp. nov., as a de novo producer of AZAs. Azadinium spinosum is a small (12–16 µm length and 7–11 µm width) peridinin-containing photosynthetic dinoflagellate with a superficial resemblance under light microscopy to gymnodinioids, but with a thin theca. The large nucleus is spherical and located posteriorly, whereas the single chloroplast is parietal, lobed, and typically extends into both the epi- and hyposome. The Kofoidian thecal tabulation is APC, 4′, 3a, 6″, 6C, 5?S, 6′″, 2″″. This plate pattern has an epithecal affinity to the Peridiniales and a hypothecal affinity to the Gonyaulacales, but is distinctly different from described dinoflagellate genera. The assignment of A. spinosum to the dinoflagellates is supported by molecular phylogenetic analysis of four genes, SSU rDNA, LSU rDNA (D1/D2 region), ITS and cytochrome oxidase (sub-unit 1) (COI). In agreement with the morphological description, phylogenetic analysis did not show any particularly close affiliation to the Peridiniales or Gonyaulacales, nor to any other dinoflagellate order represented in molecular databases. Consequently, we erected a new genus, Azadinium, for this taxon. However the ordinal affiliation of the genus is uncertain. This study represents the first description and confirmation of a new dinoflagellate species capable of producing AZA and is thus an important advance in surveillance programmes for toxigenic microalgae and toxins of human health significance.

The smallest known genomes of multicellular and toxic cyanobacteria: comparison, minimal gene sets for linked traits and the evolutionary implications.

Cyanobacterial morphology is diverse, ranging from unicellular spheres or rods to multicellular structures such as colonies and filaments. Multicellular species represent an evolutionary strategy to differentiate and compartmentalize certain metabolic functions for reproduction and nitrogen (N(2)) fixation into specialized cell types (e.g. akinetes, heterocysts and diazocytes). Only a few filamentous, differentiated cyanobacterial species, with genome sizes over 5 Mb, have been sequenced. We sequenced the genomes of two strains of closely related filamentous cyanobacterial species to yield further insights into the molecular basis of the traits of N(2) fixation, filament formation and cell differentiation. Cylindrospermopsis raciborskii CS-505 is a cylindrospermopsin-producing strain from Australia, whereas Raphidiopsis brookii D9 from Brazil synthesizes neurotoxins associated with paralytic shellfish poisoning (PSP). Despite their different morphology, toxin composition and disjunct geographical distribution, these strains form a monophyletic group. With genome sizes of approximately 3.9 (CS-505) and 3.2 (D9) Mb, these are the smallest genomes described for free-living filamentous cyanobacteria. We observed remarkable gene order conservation (synteny) between these genomes despite the difference in repetitive element content, which accounts for most of the genome size difference between them. We show here that the strains share a specific set of 2539 genes with >90% average nucleotide identity. The fact that the CS-505 and D9 genomes are small and streamlined compared to those of other filamentous cyanobacterial species and the lack of the ability for heterocyst formation in strain D9 allowed us to define a core set of genes responsible for each trait in filamentous species. We presume that in strain D9 the ability to form proper heterocysts was secondarily lost together with N(2) fixation capacity. Further comparisons to all available cyanobacterial genomes covering almost the entire evolutionary branch revealed a common minimal gene set for each of these cyanobacterial traits.

PHENOTYPIC VARIATION AND GENOTYPIC DIVERSITY IN A PLANKTONIC POPULATION OF THE TOXIGENIC MARINE DINOFLAGELLATE ALEXANDRIUM TAMARENSE (DINOPHYCEAE)1

Multiple clonal isolates from a geographic population of Alexandrium tamarense (M. Lebour) Balech from the North Sea exhibited high genotypic and phenotypic variation. Genetic heterogeneity was such that no clonal lineage was repeatedly sampled according to genotypic markers specified by amplified fragment length polymorphism (AFLP) and microsatellites. Subsampling of genotypic data from both markers showed that ordination of individuals by pair‐wise genetic dissimilarity indices was more reliable by AFLP (482 biallelic loci) than by microsatellites (18 loci). However, resulting patterns of pair‐wise genetic similarities from both markers were significantly correlated (Mantel test P < 0.005). The composition of neurotoxins associated with paralytic shellfish poisoning (PSP) was also highly diverse among these isolates and allowed clustering of toxin phenotypes based on prevalence of individual toxins. Correlation analysis of pair‐wise relatedness of individual clones according to PSP‐toxin profiles and both genotypic characters failed to yield close associations. The expression of allelochemical properties against the cryptophyte Rhodomonas salina (Wisłouch) D. R. A. Hill et Wetherbee and the predatory dinoflagellate Oxyrrhis marina Dujard. manifested population‐wide variation of responses in the target species, from no visible effect to complete lysis of target cells. Whereas the high genotypic variation indicates high potential for adaptability of the population, we interpret the wide phenotypic variation as evidence for lack of strong selective pressure on respective phenotypic traits at the time the population was sampled. Population markers as applied here may elucidate the ecological significance of respective traits when followed under variable environmental conditions, thereby revealing how variation is maintained within populations.

Implications of life‐history transitions on the population genetic structure of the toxigenic marine dinoflagellate Alexandrium tamarense

Genotypic or phenotypic markers for characterization of natural populations of marine microalgae have typically addressed questions regarding differentiation among populations, usually with reference to a single or few clonal isolates. Based upon a large number of contemporaneous isolates from the same geographical population of the toxigenic species Alexandrium tamarense from the North Sea, we uncovered significant genetic substructure and low but significant multilocus linkage disequilibrium (LD) within the planktonic population. Between the alternative molecular genotyping approaches, only amplified fragment length polymorphism (AFLP) revealed cryptic genetic population substructure by Bayesian clustering, whereas microsatellite markers failed to yield concordant patterns. Both markers, however, gave evidence for genetic differentiation of population subgroups as defined by AFLP. A considerable portion of multilocus LD could be attributed to population subdivision. The remaining LD within population subgroups is interpreted as an indicator of frequency shifts of clonal lineages during vegetative growth of planktonic populations. Phenotypic characters such as cellular content and composition of neurotoxins associated with paralytic shellfish poisoning (PSP) and allelochemical properties may contribute to intra‐ or inter‐annual differentiation of planktonic populations, if clonal lineages that express these characters are selectively favoured. Nevertheless, significant phenotypic differentiation for these characters among the genetically differentiated subgroups was only detected for PSP toxin content in two of the four population subgroups. By integrating the analysis of phenotypic and genotypic characteristics, we developed a conceptual population genetic model to explain the importance of life‐cycle dynamics and transitions in the evolutionary ecology of these dinoflagellates.

The Relevance of Marine Chemical Ecology to Plankton and Ecosystem Function: An Emerging Field

Marine chemical ecology comprises the study of the production and interaction of bioactive molecules affecting organism behavior and function. Here we focus on bioactive compounds and interactions associated with phytoplankton, particularly bloom-forming diatoms, prymnesiophytes and dinoflagellates. Planktonic bioactive metabolites are structurally and functionally diverse and some may have multiple simultaneous functions including roles in chemical defense (antipredator, allelopathic and antibacterial compounds), and/or cell-to-cell signaling (e.g., polyunsaturated aldehydes (PUAs) of diatoms). Among inducible chemical defenses in response to grazing, there is high species-specific variability in the effects on grazers, ranging from severe physical incapacitation and/or death to no apparent physiological response, depending on predator susceptibility and detoxification capability. Most bioactive compounds are present in very low concentrations, in both the producing organism and the surrounding aqueous medium. Furthermore, bioactivity may be subject to synergistic interactions with other natural and anthropogenic environmental toxicants. Most, if not all phycotoxins are classic secondary metabolites, but many other bioactive metabolites are simple molecules derived from primary metabolism (e.g., PUAs in diatoms, dimethylsulfoniopropionate (DMSP) in prymnesiophytes). Producing cells do not seem to suffer physiological impact due to their synthesis. Functional genome sequence data and gene expression analysis will provide insights into regulatory and metabolic pathways in producer organisms, as well as identification of mechanisms of action in target organisms. Understanding chemical ecological responses to environmental triggers and chemically-mediated species interactions will help define crucial chemical and molecular processes that help maintain biodiversity and ecosystem functionality.

Comparative gene expression in toxic versus non-toxic strains of the marine dinoflagellate Alexandrium minutum

BackgroundThe dinoflagellate Alexandrium minutum typically produces paralytic shellfish poisoning (PSP) toxins, which are known only from cyanobacteria and dinoflagellates. While a PSP toxin gene cluster has recently been characterized in cyanobacteria, the genetic background of PSP toxin production in dinoflagellates remains elusive.ResultsWe constructed and analysed an expressed sequence tag (EST) library of A. minutum, which contained 15,703 read sequences yielding a total of 4,320 unique expressed clusters. Of these clusters, 72% combined the forward-and reverse reads of at least one bacterial clone. This sequence resource was then used to construct an oligonucleotide microarray. We analysed the expression of all clusters in three different strains. While the cyanobacterial PSP toxin genes were not found among the A. minutum sequences, 192 genes were differentially expressed between toxic and non-toxic strains.ConclusionsBased on this study and on the lack of identified PSP synthesis genes in the two existent Alexandrium tamarense EST libraries, we propose that the PSP toxin genes in dinoflagellates might be more different from their cyanobacterial counterparts than would be expected in the case of a recent gene transfer. As a starting point to identify possible PSP toxin-associated genes in dinoflagellates without relying on a priori sequence information, the sequences only present in mRNA pools of the toxic strain can be seen as putative candidates involved in toxin synthesis and regulation, or acclimation to intracellular PSP toxins.

Azadinium obesum (Dinophyceae), a new nontoxic species in the genus that can produce azaspiracid toxins

Tillmann U., Elbrächter M., John U., Krock B. and Cembella A. 2010. Azadinium obesum (Dinophyceae), a new nontoxic species in the genus that can produce azaspiracid toxins. Phycologia 49: 169–182. DOI: 10.2216/09-35.1 The novel dinoflagellate taxon we describe here as Azadinium obesum sp. nov. was isolated as clone 2E10 from the North Sea along the Scottish east coast, the same locality as for Azadinium spinosum Elbrächter et Tillmann, the type and formerly only species for this genus. In contrast to A. spinosum, a known producer of azaspiracid (AZA) toxins, the isolate of A. obesum produces no known AZA analogues detectable by liquid chromatography coupled with tandem mass spectrometry. Azadinium obesum is a small (13–18 µm length; 10–14 µm width) photosynthetic dinoflagellate with a thin theca exhibiting the Kofoidean plate tabulation: Po, cp, X, 4′, 3a, 6″, 6C, 5?S, 6′″, 2″″. This species is morphologically distinguished from A. spinosum by slightly larger mean cell size, consistent absence of an antapical spine, the lack of a stalked pyrenoid and several details of the plate configuration. Among these thecal features, the first precingular (1″) plate of A. obesum does not touch the first epithecal intercalary plate and is four sided rather than five sided as in A. spinosum. Furthermore, in A. obesum the lower half of the first apical (1′) plate is very narrow and tongue-like, and precingular plates 1″ and 6″ are very close together, whereas these diverge in A. spinosum. DNA sequence and phylogenetic analysis elucidates and supports the separation (but close affinity) of A. obesum and A. spinosum, as well as the description of the former as a distinct species. Phylogenetic interpretation of the four genes analysed – internal transcribed spacer, 18S rDNA, 28S rDNA (D1/D2) and cytochrome oxidase I – further validates the recently erected genus Azadinium Elbrächer et Tillmann but does not clarify the position of the genus with respect to higher taxonomic levels within the subclass Peridiniphycidae.

Comparative Genomic and Transcriptomic Characterization of the Toxigenic Marine Dinoflagellate Alexandrium ostenfeldii

Many dinoflagellate species are notorious for the toxins they produce and ecological and human health consequences associated with harmful algal blooms (HABs). Dinoflagellates are particularly refractory to genomic analysis due to the enormous genome size, lack of knowledge about their DNA composition and structure, and peculiarities of gene regulation, such as spliced leader (SL) trans-splicing and mRNA transposition mechanisms. Alexandrium ostenfeldii is known to produce macrocyclic imine toxins, described as spirolides. We characterized the genome of A. ostenfeldii using a combination of transcriptomic data and random genomic clones for comparison with other dinoflagellates, particularly Alexandrium species. Examination of SL sequences revealed similar features as in other dinoflagellates, including Alexandrium species. SL sequences in decay indicate frequent retro-transposition of mRNA species. This probably contributes to overall genome complexity by generating additional gene copies. Sequencing of several thousand fosmid and bacterial artificial chromosome (BAC) ends yielded a wealth of simple repeats and tandemly repeated longer sequence stretches which we estimated to comprise more than half of the whole genome. Surprisingly, the repeats comprise a very limited set of 79–97 bp sequences; in part the genome is thus a relatively uniform sequence space interrupted by coding sequences. Our genomic sequence survey (GSS) represents the largest genomic data set of a dinoflagellate to date. Alexandrium ostenfeldii is a typical dinoflagellate with respect to its transcriptome and mRNA transposition but demonstrates Alexandrium-like stop codon usage. The large portion of repetitive sequences and the organization within the genome is in agreement with several other studies on dinoflagellates using different approaches. It remains to be determined whether this unusual composition is directly correlated to the exceptionally genome organization of dinoflagellates with a low amount of histones and histone-like proteins.

Preliminary Characterization of Extracellular Allelochemicals of the Toxic Marine Dinoflagellate Alexandrium tamarense Using a Rhodomonas salina Bioassay

Members of the marine dinoflagellate genus Alexandrium are known to exude allelochemicals, unrelated to well-known neurotoxins (PSP-toxins, spirolides), with negative effects on other phytoplankton and marine grazers. Physico/chemical characterization of extracellular lytic compounds of A. tamarense, quantified by Rhodomonas salina bioassay, showed that the lytic activity, and hence presumably the compounds were stable over wide ranges of temperatures and pH and were refractory to bacterial degradation. Two distinct lytic fractions were collected by reversed-phase solid-phase extraction. The more hydrophilic fraction accounted for about 2% of the whole lytic activity of the A. tamarense culture supernatant, while the less hydrophilic one accounted for about 98% of activity. Although temporal stability of the compounds is high, substantial losses were evident during purification. Lytic activity was best removed from aqueous phase with chloroform-methanol (3:1). A “pseudo-loss” of lytic activity in undisturbed and low-concentrated samples and high activity of an emulsion between aqueous and n-hexane phase after liquid-liquid partition are strong evidence for the presence of amphipathic compounds. Lytic activity in the early fraction of gel permeation chromatography and lack of activity after 5 kD ultrafiltration indicate that the lytic agents form large aggregates or macromolecular complexes.

Swimming speed of three species of Alexandrium (Dinophyceae) as determined by digital in-line holography

N.I. Lewis, W. Xu, S.K. Jericho, H.J. Kreuzer, M.H. Jericho and A.D. Cembella. 2006. Swimming speed of three species of Alexandrium (Dinophyceae) as determined by digital in-line holography. Phycologia 45: 61–70. DOI: 10.2216/04-59.1 Digital in-line holographic (DIH) microscopy was used to track motility in several related species of the marine dinoflagellate Alexandrium in response to temperature after acclimation at selected temperatures. Numerical reconstruction of DIH holograms yielded high-contrast three-dimensional images of the trajectories of many motile cells swimming simultaneously throughout the sample volume. Swimming speed and trajectory were determined for clonal isolates of A. ostenfeldii, A. minutum and A. tamarense within the temperature range from 8 to 24°C. The strains of these species revealed differences in temperature optima for growth and tolerance that were a function of both acclimation responses and genetic factors reflecting the origin of the isolates. The fastest swimming speeds were recorded at 24°C for cells of A. minutum. Acclimated strains of all three species swam significantly slower at lower temperatures, although fastest swimming speeds did not always occur at temperature optima for growth. Aged cells from stationary phase cultures swam more slowly than cells in exponential growth phase. Doublets from a rapidly dividing culture swam faster than singlets from the same culture, confirming the propulsive advantage of paired cells. Holographic microscopy is a powerful tool for the acquisition of detailed observations of swimming behaviour of microalgal cells in the form of three-dimensional trajectories over the appropriate temporal (sub-second) and spatial (micrometer) scales.