Malte Elbrächter

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.

Dinophyte chloroplasts and phylogeny - A review

Dinophytes acquired chloroplasts obviously early in evolution and later lost them multiple times. Most families and genera contain both photosynthetic and heterotrophic species. Chloroplasts enveloped by three membranes with thylakoids in stacks of three, containing peridinin as the main pigment, are regarded as the original dinophyte plastids. Pyrenoids are generally present. Stigmata, if present, are usually parts of the chloroplast or are modified original plastids. The form II type RUBISCO found in the dinophytes is unique for eukaryotes, otherwise known only in some anaerobic bacteria. It is disputed whether the original dinophyte chloroplasts are derived from a prokaryotic or an eukaryotic endosymbiosis. Various dinoflagellates contain aberrant chloroplasts. Glenodinium foliaceum and Peridinium balticum have a single complete endosymbiont, originally a pcnnate diatom. Podolampas bipes houses several dictyophycean symbiont cells. The “symbionts” of Lepidodiniurn viride and Gymnodinium chlorophorum ar...

Discrimination of the toxigenic dinoflagellates Alexandrium tamarense and A. ostenfeldii in co-occurring natural populations from Scottish coastal waters

Blooms of the toxic dinoflagellate Alexandrium tamarense (Lebour) Balech, a known producer of potent neurotoxins associated with paralytic shellfish poisoning (PSP), are common annual events along the Scottish east coast. The cooccurrence of a second Alexandrium species, A. ostenfeldii (Paulsen) Balech & Tangen is reported in this study from waters of the Scottish east coast. The latter species has been suspected to be an alternative source of PSP toxins in northern Europe. Recent identification of toxic macrocyclic imines known as spirolides in A. ostenfeldii indicates a potential new challenge for monitoring toxic Alexandrium species and their respective toxins in natural populations. In mixed Phytoplankton assemblages, Alexandrium species are difficult to discriminate accurately by conventional light microscopy. Species-specific rRNA probes based upon 18S and 28S ribosomal DNA sequences were developed for A. ostenfeldii and tested by dot-blot and fluorescence in situ hybridization (FISH) techniques. Hybridization patterns of A. ostenfeldii probes for cultured Alexandrium isolates, and cells from field populations from the Scottish east coast, were compared with those of rDNA probes for A. tamarense and a universal dinoflagellate probe. Alexandrium cell numbers in field samples determined by whole-cell in situ hybridization were much lower than those determined by optical microscopy with the Utermöhi method involving sedimentation chambers, but the results were highly correlated (e.g. r 2 = 0.94; n = 6 for A. tamarense). Determination of spirolides and PSP toxins by instrumental analysis on board ship demonstrated the presence of both toxin groups in plankton assemblages collected from surface waters near the Orkney Islands, and confirmed the association of A. ostenfeldii with spirolides in northern Europe. These results show that rRNA probes for A. tamarense and A. ostenfeldii are useful, albeit only semi-quantitative, tools to detect and discriminate these species in field studies.

Woloszynskia halophila (Biecheler) comb. nov.: a bloom-forming cold-water dinoflagellate co-occurring with Scrippsiella hangoei (Dinophyceae) in the Baltic Sea

Molecular analyses and subsequent morphological reinvestigation of clonal isolates germinated from cysts previously assigned to Scrippsiella hangoei (Schiller) Larsen revealed considerable differences to vegetative cell isolates of this cold‐water dinoflagellate from the northern Baltic Sea. The presence of hexagonal platelets on the cell surface and a characteristic acrobase on the episome agree with the description of Gymnodinium halophilum Biecheler. However, the arrangement of amphiesmal vesicles in more than nine latitudinal series indicates allocation of this dinoflagellate to Woloszynskia Thompson. We therefore reassign G. halophilum to Woloszynskia halophila. This species exhibits ultrastructural characteristics similar to Polarella glacialis Montresor et al. and symbiontic Gymnodinium Stein, such as stalked pyrenoids and a central eyespot consisting of multiple layers of crystal‐filled vacuoles. A close relationship between these dinoflagellates is also supported by 28s rRNA sequence data. The preference for high salinities identifies W. halophila as a marine species. The spiny resting cysts of W. halophila are identical to the cysts formed during the massive encystment events previously attributed to S. hangoei in the Baltic Sea. This suggests that W. halophila is a significant contributor to the dinoflagellate spring blooms in the Baltic Sea. Scrippsiella hangoei clones, in turn, produce noncalcareous and smooth‐walled cysts when crossed with a complementary mating type.

Sequence comparisons link toxic European isolates of Alexandrium tamarense from the Orkney Islands to toxic North American stocks

Summary Twenty-one cultures of the Alexandrium tamarense/catenella/fundyense species-complex, isolated from a bloom at the Orkney Islands, north of Scotland, were examined morphologically and tested for toxicity using HPLC analyses. All cultures belong to the morpho-species of A. tamarense (Labour) Balech rather than A. fundyense Balech and are as toxic as the most toxic A. fundyense isolates from North America. A subset was genetically analysed using sequence data from the D1/D2 region of the LSU rRNA gene (656 nt). Genetic analyses indicated that these A. tamarense populations were related to North American isolates. At least three classes of the D1/D2 region were found in the cloned material. Sufficient base substitutions in the D1/D2 region preclude introduction into the Orkneys by human mechanisms (i.e., in ballast water or in shellfish stocks). The sequence analysis supports a dispersal hypothesis in recent evolutionary time of North American stocks of A. tamarense into Northern European waters via currents, possibly as part of the hypothesised original dispersal in the North Atlantic from the Pacific. In contrast, other Western European isolates of A. tamarense , whose D1/D2 region has been sequenced, are non-toxic and belong to a different gene pool.

A new non-toxic species in the dinoflagellate genus Azadinium: A. poporum sp. nov.

A new dinoflagellate species, Azadinium poporum sp. nov., was isolated as three clones from the southern North Sea off the Danish coast. In contrast to the type species A. spinosum, a known producer of azaspiracid (AZA) toxins, the isolates of A. poporum produce no known AZA analogues detectable by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Azadinium poporum is a small (11–16 µm length; 8–12 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 the type A. spinosum by a slightly lower mean cell length/width ratio, consistent absence of an antapical spine, the presence of more than one stalked pyrenoid, and the conspicuous arrangement of the ventral pore, located at the junction of the pore plate and the first two apical plates. This latter feature also distinguishes A. poporum from A. obesum. As in A. spinosum, but differing from A. obesum, the first precingular (1″) plate of A. poporum touches the first epithecal intercalary plate 1a. DNA sequences and phylogenetic analysis of four nuclear-encoded genes, namely the internal transcribed spacer (ITS), 18S rDNA, 28S rDNA (D1/D2) and cytochrome oxidase I (COI), support the separation of A. poporum from (but with a close affinity to) other Azadinium species (A. obesum and A. spinosum), as well as its description as a distinct species within the recently erected genus Azadinium Elbrächter & Tillmann. Nevertheless, in spite of the phylogenetic consistency at the generic level, the position of the genus with respect to higher taxonomic levels within the subclass Peridiniphycidae could not be further clarified.

Amphidoma languida sp. nov. (Dinophyceae) reveals a close relationship between Amphidoma and Azadinium

Toxic algae such as Alexandrium and Azadinium have an important ecological impact and have originated several times independently within the dinophytes. Their closest relatives, however, are mostly unknown at present. A new dinophyte species, Amphidoma languida sp. nov., was isolated from Bantry Bay (Ireland) during a period of elevated azaspiracid toxicity in mussels. The new species was described in detail, and its phylogenetic position was analysed, by using a combination of light and electron microscopy, chemical detection methods, and sequence comparison of concatenated ribosomal RNA sequence data. Morphological similarities, such as cingular and hypothecal plates, the number and arrangement of sulcal plates, and the characteristic apical pore complex with a small X-plate centrally invading the first apical plate, indicated a close relationship between Amphidoma and Azadinium. However, no known azaspiracid analogues were detected in A. languida by liquid chromatography coupled with tandem mass-spectrometry. In a molecular phylogeny, the Amphidomataceae including Amphidoma and Azadinium were an independent lineage among other monophyletic major groups of the dinophytes such as the Suessiales, Prorocentrales, Gonyaulacales, and Peridiniales. Thus, the taxonomic affiliation of Azadinium is clarified, and our data may prove helpful in the development of specific and reliable molecular detection methods of toxic Azadinium.

Bacteria of the genus Roseobacter associated with the toxic dinoflagellate Prorocentrum lima

The dinoflagellate Prorocentrum lima is known to produce diarrhetic shellfish poisons. However, it is yet unclear if the dinoflagellates themselves or the bacteria associated with them produce the toxins. Here we analyze the toxicity as well as the spectrum of bacteria in two cultures of P. lima, namely P. lima-SY and P. lima-ST, which initially derived from the same P. lima strain PL2V. Toxicity tests, applying the Artemia bioassay revealed in both cultures high levels of toxins. The bacteria, associated with the two cultures, were identified by PCR/nucleotide sequence analysis of the 16S rRNA gene. From cultures of P. lima-SY the dominant sequence was found to share a 93.7% similarity with the sequence of Roseobacter algocolus [R. algicola]; the relative abundance was determined to be 83%. In addition three further sequences of bacteria, grouped to the α-Protobacteria have been identified: Paracoccus denitrificans [90.8%], R. algocolus [94.4%] and Rhizobium huakuii [92.6%]. The identification of bacteria in P. lima-ST revealed that most share highest similarity with Bartonella taylorii but with a relatively low score of 87%. In addition to this sequence, two sequences with high similarity to the genus Roseobacter were obtained. The other sequences identified have not been detected in P. lima-SY. Studies with pure bacterial strains, previously isolated from a culture of P. lima-ST and subsequently cultured on agar plates, revealed that none of them was identical to those identified in the dinoflagellate culture itself. An explanation for the change of the spectrum of bacteria in the different cultures can only be expected when axenic cultures from P. lima are available.

Exotic flagellates of coastal North Sea waters

Flagellate species have been shown to survive transocean passage by ballast water and the large dinoflagellateGymnodinium catenatum was introduced from Japanese to Tasmanian waters in this way.Gymnodinium mikimotoi—better known asGyrodinium aureolum—andFibrocapsa japonica as well asAlexandrium leeii are good candidates to have been introduced recently. Species which seem to have been introduced recently into the North Sea but apparently are transported from adjacent seas by currents into the region areGymnodinium chlorophorum andAlexandrium minutum. Species reported as introduced due to misidentifications areGymnodinium catenatum andLepidodinium viride. Under other names the speciesProrocentrum minimum, Prorocentrum redfieldii, andHeterosigma akashiwo have been known for a long time in the North Sea. The recent reports of threeChattonella species may be either due to introduction or they have been overlooked. The reasons why the introduction of flagellates into coastal North Sea waters is difficult to prove will be discussed.

Delimitation of the Thoracosphaeraceae (Dinophyceae), Including the Calcareous Dinoflagellates, Based on Large Amounts of Ribosomal RNA Sequence Data

The phylogenetic relationships of the Dinophyceae (Alveolata) are not sufficiently resolved at present. The Thoracosphaeraceae (Peridiniales) are the only group of the Alveolata that include members with calcareous coccoid stages; this trait is considered apomorphic. Although the coccoid stage apparently is not calcareous, Bysmatrum has been assigned to the Thoracosphaeraceae based on thecal morphology. We tested the monophyly of the Thoracosphaeraceae using large sets of ribosomal RNA sequence data of the Alveolata including the Dinophyceae. Phylogenetic analyses were performed using Maximum Likelihood and Bayesian approaches. The Thoracosphaeraceae were monophyletic, but included also a number of non-calcareous dinophytes (such as Pentapharsodinium and Pfiesteria) and even parasites (such as Duboscquodinium and Tintinnophagus). Bysmatrum had an isolated and uncertain phylogenetic position outside the Thoracosphaeraceae. The phylogenetic relationships among calcareous dinophytes appear complex, and the assumption of the single origin of the potential to produce calcareous structures is challenged. The application of concatenated ribosomal RNA sequence data may prove promising for phylogenetic reconstructions of the Dinophyceae in future.