Azadinium Poporum Research Articles

Azadinium poporum from the Argentine Continental Shelf, Southwestern Atlantic, produces azaspiracid-2 and azaspiracid-2 phosphate

The marine dinophycean genus Azadinium has been identified as the primary source of azaspiracids (AZA), a group of lipophilic phycotoxins known to accumulate in shellfish. Blooms of Azadinium in the southern Atlantic off Argentina have been described from the 1990s, but due to a lack of cultures, the diversity of South-Atlantic Azadinium has not yet been fully explored and their toxin production potential is completely unknown. During a spring 2010 research cruise covering the El Rincón (ER) estuarine system (North Patagonian coast, Argentina, Southwestern Atlantic) a search was conducted for the presence of Azadinium. Although neither Azadinium cells nor AZA in field plankton samples were detected, 10 clonal strains of Azadinium poporum were successfuly established by incubation of sediment samples. Argentinean A. poporum were more variable in size and shape than the type description but conformed to it by the presence of multiple pyrenoids with starch sheath, in plate pattern and arrangement, and in the position of the ventral pore located on the left side of the pore plate. In contrast to all previous description of A. poporum, isolates of the Argentinean A. poporum possessed a distinct field of pores on the second antapical plate. Conspecificity of the Argentinean isolates with A. poporum was confirmed by molecular phylogeny of concatenated ITS and LSU rDNA sequences, where all Argentinean isolates together with some Chinese A. poporum strains formed a well-supported ribotype clade within A. poporum. All isolates produced AZA with the same profile, consisting of AZA-2 as the major compound and, to a lesser extent, its phosphated form. This is the first report of a phosphated marine algal toxin. This first confirmation of the presence of AZA producing Azadinium in the Argentinean coastal area underlines the risk of AZA shellfish contamination episodes in the Southwestern Atlantic region.

Detection of Azadinium poporum in New Zealand: the use of molecular tools to assist with species isolations

A real-time PCR assay for the detection of species from the genera Azadinium and Amphidoma (family Amphidomataceae) was developed in order to screen field samples and to aid in the isolation of azaspiracid (AZA)-producing dinoflagellates. The assay was highly specific and sensitive and allowed the rapid detection of target species. Samples collected as part of the New Zealand Marine Phytoplankton Monitoring Programme were analysed using the Amphidomataceae real-time PCR assay. Azadinium poporum was detected in New Zealand for the first time, and a culture was successfully established. Extracts of this isolate proved to be of low toxicity to mice and did not contain AZA-1, -2 or -3. Field samples will continue to be screened with the aim of identifying AZA-producing species. The Amphidomataceae real-time PCR assay will be a useful tool for monitoring programmes and taxonomic surveys worldwide.

Azaspiracid variability of Azadinium poporum (Dinophyceae) from the China Sea

Azadinium poporum is a small dinoflagellate from the family Amphidomataceae which is known for the potential production of azaspiracids (AZAs) causative of azaspiracid shellfish poisoning (AZP). A. poporum has been recorded from European and western Pacific waters. Here we report on the high variability of toxin profiles within this species in Chinese coastal waters. Out of 16 analyzed strains of A. poporum from different geographic locations along the Chinese coastline, three strains proved not to contain AZAs, whereas 13 strains contained different combinations of AZA-2, AZA-11, AZA-36, a yet unknown isomer of AZA-1 (named AZA-40) and new AZA with yet unreported molecular mass of 853Da (named AZA-41). The new AZA-40, other than AZA-1 itself, belongs to the recently discovered “348-type” group, which in tandem mass spectrometry displays a group 4 fragment with m/z 348 instead of the group 4 fragment of the classic AZAs with m/z 362, indicating a shift of a methyl group from the C24–C40 part of the molecule (rings F–I) to the C2–C9 part (carboxylic side chain and ring A). AZA-41 apparently is a dehydro variant of AZA-2. In addition, a previously reported AZA with a molecular mass 871DA could be unambiguously assigned to AZA-11, which is known to be a shellfish metabolite of AZA-2. This is the first report of AZA-11 being also de novo synthetized by dinoflagellates.

Morphology, phylogeny and azaspiracid profile of Azadinium poporum (Dinophyceae) from the China Sea

Azadinium poporum is a small dinoflagellate from the family Amphidomataceae which is known for the production potential of azaspiracid toxins. A. poporum has been recorded from European and Korean waters. Here we present the first report of its occurrence along the coast of China. Morphology of Chinese A. poporum is similar to those from Europe and Korea. Several stalked pyrenoids surrounded by a starch sheath were revealed with light microscopy and confirmed by transmission electron microscopy. Among 25 strains from the China Sea we identified two distinct ribotypes (referred to as ribotypes B and C). ITS sequences of strains within the same ribotype are identical, whereas ribotype B and C differ from each other at 11 positions (98.3% similarity). A. poporum ribotypes B and C type differ from European strains (referred to as ribotype A) at 16 and 15 positions (97.5% and 97.7% similarity). The ITS region pairwise distance within A. poporum ranged from 0.017 to 0.022. Among all three ribotypes, no hemi-compensatory based changes were found within helix III of ITS indicating that they are conspecific. Azaspiracid profiles were analyzed for six strains and turned out to be unexpectedly diverse. Whereas no AZAs could be detected for one strain, another strain was found to contain a m/z 348 fragment type AZA previously found in a Korean Isolate and traces of two other unknown AZAs of higher masses. A third strain produced a novel AZA with a molecular mass of 871Da. Three strains were found to contain considerable amounts of toxic AZA-2 as the sole AZA, a finding that might elegantly explain the detection of AZA-2 in sponges in the Sea of Japan and which underline the risk potential of A. poporum blooms with subsequent shellfish intoxication episodes for the Asian Pacific.

First record of the genus Azadinium (Dinophyceae) from the Shetland Islands, including the description of Azadinium polongum sp. nov.

Azadinium is a dinophycean genus capable of producing azaspiracids (AZAs), a recently discovered group of lipophilic phycotoxins causing human intoxication via mussel consumption. Although initially described from the North Sea, the genus currently consisting of four described species is probably distributed worldwide. Here we report on Azadinium from the Shetland Islands, which are located in the northernmost part of the North Sea and are largely influenced by the Atlantic Ocean. Two strains of Azadinium were isolated from a single water sample. One strain was identified as Azadinium spinosum based on morphology and sequence data and had an AZA cell quota of about 20fg per cell, similar to all other described strains of the species. The toxin profile consisted of AZA-1 and AZA-2 in a 2.3:1 ratio and a yet undescribed AZA of 715Da. The other strain represents a new species and is here described as Azadinium polongum sp. nov. Like A. spinosum, but different to Azadinium obesum and Azadinium poporum, A. polongum has an antapical spine. A. polongum differs from A. spinosum by an elongated shape of the pore plate (Po), and X-plate, the location of the ventral pore, and the absence of a distinct pyrenoid with starch sheath. Molecular analysis based on SSU, LSU, and ITS sequencing supported separation of A. polongum at the species level. Detailed LC–MS analysis showed that A. polongum does not produce any known AZAs in measureable amounts.

New azaspiracids in Amphidomataceae (Dinophyceae)

Azaspiracids (AZAs) are a group of lipophilic polyether toxins implicated in incidents of shellfish poisoning in humans, particularly in northern Europe, which are produced by the small marine dinoflagellate Azadinium spinosum. Other related species/strains of the Amphidomataceae have not been proven to date to contain any of the known azaspiracids. Closer analyses of these species/strains by triple quadrupole mass spectrometry in the precursor and product ion mode now revealed four new compounds with high similarity to azaspiracids, all of them with a characteristic m/z 348 fragment but with absence of the m/z 362 fragment. These compounds were detected in three species/strains, i.e. in North Sea isolates of Azadinium poporum (molecular mass: 845.5 Da), in a Korean isolate which has been designated as A. cf. poporum (molecular mass: 857.5 Da) and in Amphidoma languida isolated from Bantry Bay, Ireland (molecular masses: 815.5 and 829.5 Da). Cell quotas of roughly 2–20 fg per cell were in the same range as found for AZA-1/-2 in A. spinosum. Structures for all compounds were proposed by interpretation of fragmentation patterns and high resolution mass measurements using Fourier transform ion cyclotron resonance-mass spectrometry (FTICR-MS).

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.

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.