Azadinium Poporum Research Articles

Azaspiracid-59 accumulation and transformation in mussels (Mytilus edulis) after feeding with Azadinium poporum (Dinophyceae)

Azaspiracid-59 (AZA-59) was detected in plankton in coastal waters of the Pacific Northwest USA. Given that bivalves metabolize and transform accumulated phycotoxins, a strain of Azadinium poporum isolated from the coast of Washington State that is a known producer of AZA-59 was used in a controlled feeding experiment with mussels (Mytilus edulis) to assess AZA-59 accumulation rates and transformation into shellfish metabolites. Mussels started feeding immediately after the addition of A. poporum. Mussels were generally healthy during the entire experimental exposure of 18 days with prevailingly high rates of clearance (approx. 100 mL per mussel per hour) and ingestion. Mussels were extracted after different exposure times and were analyzed by liquid chromatography coupled with low- and high-resolution mass spectrometry. In the course of the experiment a number of putative AZA-59 metabolites were detected including hydroxyl and carboxy analogues that corresponded with previously reported mussel metabolites of AZA-1. A significant formation of 3-OH fatty acid acyl esters relative to free AZAs was observed through the time course of the study, with numerous fatty acid ester variants of AZA-59 confirmed. These results illustrate the potential for metabolism of AZA-59 in shellfish and provide important information for local AZA monitoring and toxicity testing along the Northern Pacific US coast.

Harmful algae diversity from a coastal upwelling system detected by high-throughput sequencing

IntroductionIn recent years, environmental DNA (eDNA) amplicon sequencing has been used to unveil plankton diversity in the field. Nevertheless, molecular methods, such as this, are rarely used in harmful algal bloom (HAB) monitoring programs, which mainly rely on morphological identification by conventional light microscopy.MethodsThe present study focused on a shallow marine environment (Ría de Vigo, Northwest Spain), where sediment and plankton samples were collected from 2016 to 2018.ResultsThe application of eDNA amplicon sequencing allowed us to simultaneously detect 25 potential harmful species (mainly diatoms and dinoflagellates) included in the IOC-UNESCO Taxonomic Reference List of Harmful Microalgae. Among these, causative agents of amnesic shellfish poisoning (Pseudo-nitzschia spp.) paralytic shellfish poisoning (Gymnodinium catenatum and Alexandrium minutum), azaspiracid producers (Azadinium poporum) and ichthyotoxic haptophytes (Chrysochromulina leadbeateri), were identified. Some toxic microalgae were better represented in sediment (e.g., Pseudo-nitzschia pungens, Gymnodinium catenatum) or planktonic fractions (e.g., Pseudo-nitzschia, Gymnodinium smaydae), confirming the importance of including both sediment and plankton fractions in eDNA monitoring studies. Despite the limitations of sequencing short amplicons, it was possible to discern in this study six Pseudo-nitzschia species and associate each of them with each seasonal peak produced in summer periods. Furthermore, several species previously unreported in Ría de Vigo (Pseudo-nitzschia turgidula, Chrysochromulina leadbeateri, Azadinium poporum) could be detected.DiscusionThese results point out the application of eDNA amplicon sequencing to expand our knowledge about harmful species in HAB monitoring programs and early warning systems for low abundant and rare taxa.

Azaspiracid accumulation in Japanese coastal bivalves and ascidians fed with Azadinium poporum producing azaspiracid-2 as the dominant toxin component.

The filter-feeding bivalves often accumulate marine toxins by feeding on toxic dinoflagellates that produce marine toxins. Azaspiracids (AZAs) are a group of lipophilic polyether toxins which have been detected in a variety of organisms in many countries. In our present study, accumulation kinetics and toxin distributions in the tissues of seven bivalve species and ascidians relevant to Japanese coastal waters were investigated by experimentally feeding a toxic dinoflagellate Azadinium poporum, which produces azaspiracid-2 (AZA2) as the dominant toxin component. All bivalve species and ascidians investigated in this study had the capability to accumulate AZA2 and no metabolites of AZA2 were detected in the bivalves and the ascidians. Japanese short-neck clams, Japanese oysters, Pacific oysters and ascidians accumulated AZA2 with the highest concentrations on the hepatopancreas, whereas the highest concentrations of AZA2 were found on the gills in surf clams and horse clams. Hard clams and cockles accumulated high levels of AZA2 in both the hepatopancreas and the gills. As far as we know, this is the first report describing detailed tissue distribution of AZAs in several bivalve species other than mussels (M. edulis) and scallops (P. maximus). Variation of accumulation rates of AZA2 in Japanese short-neck clams on different cell densities or temperatures were observed.

Structure and toxicity of AZA-59, an azaspiracid shellfish poisoning toxin produced by Azadinium poporum (Dinophyceae)

To date, the putative shellfish toxin azaspiracid 59 (AZA-59) produced by Azadinium poporum (Dinophyceae) has been the only AZA found in isolates from the Pacific Northwest coast of the USA (Northeast Pacific Ocean). Anecdotal reports of sporadic diarrhetic shellfish poisoning-like illness, with the absence of DSP toxin or Vibrio contamination, led to efforts to look for other potential toxins, such as AZAs, in water and shellfish from the region. A. poporum was found in Puget Sound and the outer coast of Washington State, USA, and a novel AZA (putative AZA-59) was detected in low quantities in SPATT resins and shellfish. Here, an A. poporum strain from Puget Sound was mass-cultured and AZA-59 was subsequently purified and structurally characterized. In vitro cytotoxicity of AZA-59 towards Jurkat T lymphocytes and acute intraperitoneal toxicity in mice in comparison to AZA-1 allowed the derivation of a provisional toxicity equivalency factor of 0.8 for AZA-59. Quantification of AZA-59 using ELISA and LC-MS/MS yielded reasonable quantitative results when AZA-1 was used as an external reference standard. This study assesses the toxic potency of AZA-59 and will inform guidelines for its potential monitoring in case of increasing toxin levels in edible shellfish.

Complex profiles of azaspiracid analogues in two culture strains of Azadinium poporum (Amphidomataceae, Dinophyceae) isolated from Japanese coastal waters determined by LC-MS/MS

Lipophilic marine biotoxins azaspiracids (AZAs) are produced by dinoflagellates Azadinium and Amphidoma. Recently, several strains of Azadinium poporum were isolated from Japanese coastal waters. In our present study, AZA analogues in two strains (mdd421 and HM536) of A. poporum were analyzed by several detection techniques on the liquid chromatography-tandem mass spectrometry (LC-MS/MS) and liquid chromatography-quadrupole time of flight mass spectrometry (LC-QTOFMS). The dominant AZA analogue in the Japanese A. poporum strains was AZA2. Other known AZA analogues were AZA11, AZA35, AZA2 methyl ester and AZA2 phosphate ester. Besides these AZAs, thirteen new AZA analogues were discovered in the two strains. A putative AZA analogue (Compound 1) with the smallest molecular weight ever found in nature was also discovered in the two strains. This is the first report describing detailed AZA profiles in Japanese isolates of A. poporum.

Diversity and distribution of harmful microalgae in the Gulf of Thailand assessed by DNA metabarcoding

Information on the diversity and distribution of harmful microalgae in the Gulf of Thailand is very limited and mainly based on microscopic observations. Here, we collected 44 water samples from the Gulf of Thailand and its adjacent water (Perhentian Island, Malaysia) for comparison in 2018. DNA metabarcoding was performed targeting the partial large subunit ribosomal RNA gene (LSU rDNA D1–D3) and the internal transcribed spacers (ITS1 and ITS2). A total of 50 dinoflagellate genera (made up of 72 species) were identified based on the LSU rDNA dataset, while the results of ITS1 and ITS2 datasets revealed 33 and 32 dinoflagellate genera comprising 69 and 64 species, respectively. Five potentially toxic Pseudo-nitzschia (Bacillariophyceae) species were detected, with four as newly recorded species in the water (Pseudo-nitzschia americana/brasilliana, Pseudo-nitzschia simulans/delicatissima, P. galaxiae and P. multistriata). The highest relative abundances of P. galaxiae and P. multistriata were found in Trat Bay and Chumphon (accounting for 0.20% and 0.06% of total ASVs abundance, respectively). Three paralytic shellfish toxin producing dinoflagellate species were detected: Alexandrium tamiyavanichii, Alexandrium fragae, and Gymnodinium catenatum. The highest abundance of A. tamiyavanichii was found in the surface sample of Chumphon (CHO7 station), accounting for 1.95% of total ASVs abundance. Two azaspiracid producing dinoflagellate species, Azadinium poporum ribotype B, Azadinium spinosum ribotype A, and a pinnatoxin producing dinoflagellate species Vulcanodinium rugosum, with two ribotypes B and C, were revealed from the datasets although with very low abundances. Six fish killing dinoflagellate species, including Margalefidinium polykrikoides group IV, Margalefidinium fulvescens, Karenia mikimotoi, Karenia selliformis ribotype B, Karlodinium australe, and Karlodinium digitatum were detected and all representing new records in this area. The findings of numerous harmful microalgal species in the Gulf of Thailand highlight the potential risk of human intoxication and fish killing events.

Toxigenic strains of Azadinium poporum (Amphidomataceae, Dinophyceae) from Japan and Vietnam, with first reports of A. poporum (ribotype A) and A. trinitatum in Asian Pacific

SUMMARYMorphology and phylogeny of small armored dinoflagellate Azadinium species isolated from Japanese and Vietnamese coasts were examined by light microscopy, scanning electron microscopy and molecular phylogeny inferred from ITS and LSU rDNA (D1–D3) sequences. Dominant azaspiracids (AZAs) were analyzed by using liquid chromatography/triple quadrupole mass spectrometer (LC/MS/MS). Sixteen and four culture strains were established from Japan and Vietnam, respectively. A phylogenetic tree confirmed our species identifications of Japanese cultures as Azadinium poporum (ribotypes A, B and C), A. trinitatum and A. zhuanum, and Vietnamese cultures as A. poporum (ribotype B). Typical cells of A. poporum had the thecal tabulation of Po, cp, X, 4′, 3a, 6″, 6C, 5S, 6″′, 2″″, A. trinitatum had Po, cp, X, 4–5′, 3a, 6–7″, 6C, 5S, 5–6″′, 2″″ and A. zhuanum had Po, cp, X, 3–4′, 2a, 6″, 6C, 5S, 6″′, 2″″. Cellular morphology of the three species were congruent with those in the original reports, in terms of the presence/absence of antapical spine and the position of the ventral pore, nucleus and pyrenoids, although small differences were also found in the nucleus position of A. trinitatum and A. zhuanum. AZA‐2 was the dominant component in Japanese A. poporum (ribotypes A, B and C), and AZA‐40 in Vietnamese A. poporum (ribotype B). AZA‐2 and AZA‐40 were not detected in Vietnamese and Japanese A. poporum, respectively. AZA analogs were not detected from A. trinitatum and A. zhuanum. This is the first report of the toxigenic A. poporum from Japan and Vietnam, with identifications of the major AZAs. The ribotype A of A. poporum and A. trinitatum were the first records in Asia.

Temporal and spatial distribution of Azadinium species in the inland and coastal waters of the Pacific northwest in 2014–2018

Azaspiracids, produced by some species of the dinoflagellate genera Azadinium and Amphidoma, can cause a syndrome in humans called azaspiracid shellfish poisoning (AZP). In 1995, mussels from the Irish west coast contaminated with azaspiracids were, for the first time, linked to this human illness that has symptoms of nausea, vomiting, severe diarrhea, and stomach cramps. The only confirmed cases of AZP to date in the United States occurred in Washington State in 2008 from mussels imported from Ireland. Shortly after this case, several others involving similar gastrointestinal symptoms were reported by shellfish consumers from Washington State. However, no detectable diarrhetic shellfish toxins or Vibrio contamination were found. Cursory analysis of Solid Phase Adsorption Toxin Tracking (SPATT) samplers suggested the presence of azaspiracids in Washington State waters and motivated a study to evaluate the presence and distribution of Azadinium species in the region. During the spring and summer months of 2014–2015, quantitative polymerase chain reaction (qPCR) analyses detected the presence of the toxigenic species Azadinium poporum and A. spinosum on the outer coast and throughout the inland waters of Washington State. In 2016–2018, standard curves developed using A. poporum isolated from Puget Sound and A. spinosum isolated from the North Sea were used to quantify abundances of up to 10,525 cells L−1 of A. poporum and 156 cells L−1 of A. spinosum at shore-based sites. Abundances up to 1,206 cells L−1 of A. poporum and 30 cells L−1 of A. spinosum were measured in the coastal waters of the Pacific Northwest in 2017. Other harmful genera, including Alexandrium, Dinophysis, and Pseudo-nitzschia, were observed using light microscopy at coastal sites where A. poporum was also observed. In some samples where both A. poporum and A. spinosum were absent, an Amphidomataceae-specific qPCR assay indicated that other species of Azadinium or Amphidoma were present. The identification of Azadinium species in the PNW demonstrates the need to assess their toxicity and to incorporate their routine detection in monitoring programs to aid resource managers in mitigating risks to azaspiracid shellfish poisoning in this region.

Distribution and abundance of azaspiracid-producing dinophyte species and their toxins in North Atlantic and North Sea waters in summer 2018.

Representatives of the marine dinophyte family Amphidomataceae produce lipophilic phycotoxins called azaspiracids (AZA) which may cause azaspiracid shellfish poisoning (AZP) in humans after consumption of contaminated seafood. Three of the four known toxigenic species are observed frequently in the eastern North Atlantic. In 2018, a research survey was performed to strengthen knowledge on the distribution and abundance of toxigenic Amphidomataceae and their respective toxins in Irish coastal waters and in the North Sea. Species-specific quantification of the three toxigenic species (Azadinium spinosum, Azadinium poporum and Amphidoma languida) was based on recently developed qPCR assays, whose performance was successfully validated and tested with specificity tests and spike experiments. The multi-method approach of on-board live microscopy, qPCR assays and chemical AZA-analysis revealed the presence of Amphidomataceae in the North Atlantic including the three targeted toxigenic species and their respective AZA analogues (AZA-1, -2, -33, -38, -39). Azadinium spinosum was detected at the majority of Irish stations with a peak density of 8.3 x 104 cells L-1 and AZA (AZA-1, -2, -33) abundances up to 1,274 pg L-1. Amphidoma languida was also present at most Irish stations but appeared in highest abundance in a bloom at a central North Sea station with a density of 1.2 x 105 cells L-1 and an AZA (AZA-38, -39) abundances of 618 pg L-1. Azadinium poporum was detected sporadically at the Irish south coast and North Sea and was rather low in abundance during this study. The results confirmed the wide distribution and frequent occurrence of the target species in the North Atlantic area and revealed, for the first time, bloom abundances of toxigenic Amphidomataceae in this area. This emphasizes the importance of future studies and monitoring of amphidomatacean species and their respective AZA analogues in the North Atlantic.

Diversity and geographical distribution of resting stages of eukaryotic algae in the surface sediments from the southern Chinese coastline based on metabarcoding partial 18S rDNA sequences

AbstractSurface sediment samples were collected from sixteen sites of the southern Chinese coastline in this study. Diversity of eukaryotic microalgae was examined using high‐throughput sequencing of 18S rDNA to understand the community structure and geological distribution of microalgal resting stages. A total of 218 operational taxonomic units (OTUs) were detected for eukaryotic algae, and only 75 OTUs (34.40%) were detected into species level. The algal communities were composed of sixty‐four genera belonged to twelve classes of four phyla. Dinoflagellates were the most abundant and diversified group, which accounted for the average of 56.26% and 53.70% DNA reads and OTUs, respectively. Dinoflagellates were dominated by Protoperidinium, Euduboscquella, Amylax, and Paragymnodinium. Chaetoceros, Thalassiosira, and Skeletonema were abundant in diatoms, while Pedospumella and Paraphysomonas in Chrysophyceae were the most abundant genera due to their massive occurrence in some particular sites. The high abundance of Protoperidinium sequences suggested the high nutrient levels in the southern Chinese coastline. Nineteen of the detected OTUs were assigned to harmful and/or bloom‐forming microalgae. The wide occurrence of the toxic dinoflagellate Azadinium poporum and high abundance of Alexandrium pacificum in some particular sites suggested the potential risk of human poisoning.

The effect of temperature and salinity on growth rate and azaspiracid cell quotas in two strains of Azadinium poporum (Dinophyceae) from Puget Sound, Washington State

Azaspiracids (AZA) are novel lipophilic polyether marine biotoxins associated with azaspiracid shellfish poisoning (AZP). Azaspiracid-59 (AZA-59) is a new AZA that was recently detected in strains of Azadinium poporum from Puget Sound, Washington State. In order to understand how environmental factors affect AZA abundances in Puget Sound, a laboratory experiment was conducted with two local strains of A. poporum to estimate the growth rate and AZA-59 (both intra- and extracellular) cell quotas along temperature and salinity gradients. Both strains of A. poporum grew across a wide range of temperatures (6.7 °C to 25.0 °C), and salinities (15 to 35). Growth rates increased with increasing temperature up to 20.0 °C, with a range from 0.10 d−1 to 0.42 d−1. Both strains of A. poporum showed variable growth rates from 0.26 d−1 to 0.38 d−1 at salinities from 15 to 35. The percentage of intracellular AZA-59 in both strains was generally higher in exponential than in stationary phase along temperature and salinity gradients, indicating higher retention of toxin in actively growing cells. Cellular toxin quotas varied by strain in both the temperature and salinity treatments but were highest at the lowest growth rates, especially for the faster growing strain, NWFSC1011.Consistent with laboratory experiments, field investigations in Sequim Bay, WA, during 2016–2018 showed that A. poporum was detected when salinity and temperature became favorable to higher growth rates in June and July. Although current field data of A. poporum in Puget Sound indicate a generally low abundance, the potential of local A. poporum to adapt to and grow in a wide range of temperature and salinity may open future windows for blooms. Although increased temperatures, anticipated for the Puget Sound region over the next decades, will enhance the growth of A. poporum, these higher temperatures will not necessarily support higher toxin cell quotas. Additional sampling and assessment of the total toxicity of AZA-59 will provide the basis for a more accurate estimation of risk for azaspiracid poisoning in Puget Sound shellfish.

Accumulation and transformation of azaspiracids in scallops (Chlamys farreri) and mussels (Mytilus galloprovincialis) fed with Azadinium poporum, and response of antioxidant enzymes

Accumulation and transformation of azaspiracids in scallops (Chlamys farreri) and mussels (Mytilus galloprovincialis) fed with Azadinium poporum, and response of antioxidant enzymes

Two novel azaspiracids from Azadinium poporum, and a comprehensive compilation of azaspiracids produced by Amphidomataceae, (Dinophyceae)

Two novel azaspiracids (AZA) with a molecular mass of 869 Da were found in Pacific strains of Azadinium poporum and characterized by tandem mass spectrometry and high resolution mass spectrometry (HRMS). One compound, AZA-42, was found in Az. poporum strains AZFC25 and AZFC26, both isolated from the South China Sea. AZA-42 belongs to the 360-type AZA that in comparison to AZA-1 has an additional double bond in the F–I ring system of AZA comprising C28-C40. The other compound, AZA-62, was detected in Az. poporum strain 1D5 isolated off Chañaral, Northern Chile. Mass spectral data indicate that AZA-62 is a variant of AZA-11 with an additional double bond in the C1-C9 region of AZA. In addition to the description of the two novel AZA, a comprehensive list of all AZA known to be produced by species of the genera Azadinium and Amphidoma comprising 26 AZA variants is presented.

Phycotoxins in scallops (Patinopecten yessoensis) in relation to source, composition and temporal variation of phytoplankton and cysts in North Yellow Sea, China

The North Yellow Sea is a major aquaculture production area for the scallop Patinopecten yessoensis. In this study, the temporal and spatial variation of phycotoxins in scallops, phytoplankton, and their cysts were analyzed during a survey conducted from June 2011 to April 2012 around Zhangzi Island. The study area is a semi-enclosed epicontinental sea surrounded by the Shandong Peninsula, the Liaodong Peninsula and the Korean Peninsula. The three main results of the study were as follows: (1) The saxitoxin-group toxins, okadaic acid and analogues, and pectenotoxins were the major phycotoxin residues found in scallops; (2) Six kinds of toxic microalgae were identified, Protoperidinium spp., Gonyaulax spp., and Alexandrium spp. were the dominant taxa; Seven types of potential marine toxin-producing dinoflagellates, A. tamarense, A. catenella, Dinophysis fortii, G. catenatum, Gambierdiscus toxicus, Azadinium poporum, and Pseudo-nitzschia pungen were identified as the primary source of phycotoxins and were present at relatively high density from June to October; and (3) azaspiracids and domoic acid might be new potential sources of toxin pollution. This study represents the first assessment to phycotoxins around Zhangzi Island in the North Yellow Sea.

Sympatric occurrence of two Azadinium poporum ribotypes in the Eastern Mediterranean Sea

The marine dinoflagellate Azadinium poporum produce azaspiracids (AZA) and has been recorded widely in the world. However, information on its biogeography is still limited, especially in view of the fact that A. poporum comprises several genetically differentiated groups. A total of 18 strains of A. poporum were obtained from the Eastern Mediterranean area by incubating surface sediment collected from Ionian Sea of Greece. The morphology of these strains was examined with light microscopy and scanning electron microscopy. Small subunit ribosomal DNA (SSU rDNA), large subunit ribosomal DNA (LSU rDNA) and internal transcribed spacer (ITS) sequences were obtained from all cultured strains. Molecular phylogeny based on concatenated SSU, LSU and ITS sequences confirmed three ribotypes within A. poporum and revealed two subclades within ribotypes A and C. Greek strains of A. poporum ribotype A were nested within ribotype A2 together with strains from Western Mediterranean Sea and French Atlantic, and Greek strains of A. poporum ribotype C were nested within ribotype C2 together with a strain from the Gulf of Mexico. Growth experiments on four selected strains revealed that ribotypes A and C from Greece differed in their growth at higher temperatures, indicating that they are physiologically differentiated. Azaspiracid profiles were analyzed for 15 cultured A. poporum strains using LCMS/MS and demonstrate that the A. poporum ribotype A from Greece produce low level or no AZA and A. poporum ribotype C from Greece produces predominantly AZA-40 (9.6–30.2 fg cell−1) followed by AZA-2 (2.1–2.6 fg cell−1). The first record of AZA-40 producing A. poporum from the Mediterranean suggests that this species is a potential source for azaspiracid contaminations in shellfish from the Eastern Mediterranean Sea.

Accumulation and transformation of azaspiracids in scallops (Chlamys farreri) and mussels (Mytilus galloprovincialis) fed with Azadinium poporum, and response of antioxidant enzymes

Azaspiracid (AZA) producing microalgae have been reported internationally and could potentially impact a variety of seafood. Scallops (Chlamys farreri) and mussels (Mytilus galloprovincialis) from China were fed with the AZA2 producer, Azadinium poporum, to study uptake, metabolism and oxidative stress in the shellfish. LC-MS/MS showed significant accumulation and differential metabolism of AZA2 in the scallops and mussels. In mussels AZA2 was metabolized to AZA19, with subsequent decarboxylation to AZA6. In scallops no AZA19 or AZA6 was observed, however, a novel AZA metabolite was formed that is isobaric with AZA19 ([M+H]+, m/z 886), but elutes at a different retention time. In addition it was noted that the scallop metabolite was stable during heating, while AZA19 has been shown to decarboxylate. Concentrations of reactive oxygen species (ROS) and activities of antioxidant enzymes were monitored. ROS levels increased slightly in the meat of scallops and mussels due to starvation in the acclimation and depuration periods, but reduced in the feeding periods with non-toxic Isochrysis galbana or toxic A. poporum. No obvious variations were found in activities for a range of antioxidant enzymes. These results provide new insights on the potential for accumulation and metabolism of AZAs in bivalve species relevant to this area of China, which is of importance considering the recent finding of AZA producing microalgae in the region.

Identification of Azadinium species and a new azaspiracid from Azadinium poporum in Puget Sound, Washington State, USA

The identification of a new suite of toxins, called azaspiracids (AZA), as the cause of human illnesses after the consumption of shellfish from the Irish west coast in 1995, resulted in interest in understanding the global distribution of these toxins and of species of the small dinoflagellate genus Azadinium, known to produce them. Clonal isolates of four species of Azadinium, A. poporum, A. cuneatum, A. obesum and A. dalianense were obtained from incubated sediment samples collected from Puget Sound, Washington State in 2016. These Azadinium species were identified using morphological characteristics confirmed by molecular phylogeny. Whereas AZA could not be detected in any strains of A. obesum, A. cuneatum and A. dalianense, all four strains of A. poporum produced a new azaspiracid toxin, based on LC–MS analysis, named AZA-59. The presence of AZA-59 was confirmed at low levels in situ using a solid phase resin deployed at several stations along the coastlines of Puget Sound. Using a combination of molecular methods for species detection and solid phase resin deployment to target shellfish monitoring of toxin at high-risk sites, the risk of azaspiracid shellfish poisoning can be minimized.

Adding new pieces to the Azadinium (Dinophyceae) diversity and biogeography puzzle: Non-toxigenic Azadinium zhuanum sp. nov. from China, toxigenic A. poporum from the Mediterranean, and a non-toxigenic A. dalianense from the French Atlantic

The marine planktonic dinophyceaen genus Azadinium is a primary source of azaspiracids, but due to their small size its diversity may be underestimated and information on its biogeography is still limited. A new Azadinium species, A. zhuanum was obtained from the East China Sea and Yellow Sea of China by incubating surface sediments. Five strains were established by isolating single germinated cells and their morphology was examined with light microscopy and scanning electron microscopy. Azadinium zhuanum was characterized by a plate pattern of Po, cp, X, 4′, 2a, 6′′, 6C, 5S, 6′′′, 2′′′′, by a distinct ventral pore at the junction of Po, the first and fourth apical plates, and a conspicuous antapical spine. Moreover, Azadinium poporum was obtained for the first time from the Mediterranean by incubating surface sediment collected from Diana Lagoon (Corsica) and a new strain of Azadinium dalianense was isolated from the French Atlantic. The morphology of both strains was examined. Small subunit ribosomal DNA (SSU rDNA), large subunit ribosomal DNA (LSU rDNA) and internal transcribed spacer (ITS) sequences were obtained from cultured strains. In addition, LSU sequences were obtained by single cell sequencing of two presumable A. poporum cells collected from the French Atlantic. Molecular phylogeny based on concatenated SSU, LSU and ITS sequences revealed that A. zhuanum was closest to A. polongum. French A. poporum from Corsica (Mediterranean) and from the Atlantic showed some genetic differences but were nested within one of the A. poporum ribotypes together with other European strains. Azadinium dalianense from France together with the type strain of the species from China comprised a well resolved clade now consisting of two ribotypes. Azaspiracid profiles were analyzed for the cultured Azadinium strains using LC–MS/MS and demonstrate that the Mediterranean A. poporum strain produced AZA-2 and AZA-2 phosphate with an amount of 0.44fgcell−1. Azadinium zhuanum and A. dalianense did not produce detectable AZA. Results of the present study support the view of a high diversity and wide distribution of species belonging to Azadinium. The first record of AZA-2 producing A. poporum from the Mediterranean suggests that this species may be responsible for azaspiracid contaminations in shellfish from the Mediterranean Sea.

Identification ofAzadinium poporum(Dinophyceae) in the Southeast Pacific: morphology, molecular phylogeny, and azaspiracid profile characterization

Azaspiracids (AZA), a group of lipophilic phycotoxins, are produced by some species of the marine dinophycean genus Azadinium. AZA have recently been detected in shellfish from the Southeast Pacific, however, AZA-producing species have not been recorded yet from the area. This study is the first record of the genus Azadinium and of the species Azadinium poporum from the Pacific side of South America. Three strains of A. poporum from Chanaral (Northern Chile) comply to the type description of A. poporum by the presence of multiple pyrenoids, in thecal plate details, and in the position of the ventral pore located on the left side of the pore plate. Molecular phylogeny, based on internal transcribed spacer and large subunit ribosomal DNA sequences, revealed that Chilean strains fall in the same ribotype clade as European and strains from New Zealand. Analyses of AZA profiles using LC–MS/MS showed an identical profile for all three strains with the presence of AZA-11 and two phosphorylated AZA. This is the first confirmation of the presence of AZA producing Azadinium in the Chilean coastal area and underlines the risk of AZA shellfish and concomitant human contamination episodes in the Southeast Pacific region.

Growth and toxin production of Azadinium poporum strains in batch cultures under different nutrient conditions

Azaspiracid-2 (AZA2) is the dominant toxin produced by Azadinium poporum strains AZDY06 and AZFC22 isolated from the South China Sea. Biomass and AZA2-production were examined within batch cultures with variation in experimental concentrations of nitrate (0, 88, 882, and 2647µM) or phosphate (0, 3.6, 36, and 109µM), different nitrogen sources (nitrate and urea) and media (f/2-Si, L1-Si, and K-Si) in the present study. Growth of both strains positively responded to nitrate or phosphate nutrients, but the growth status was significantly repressed by the highest additional level of phosphate (109µM). Both AZDY06 and AZFC22 grew well with higher specific growth rates, but with shorter growth periods, within f/2-Si medium spiked with urea than that within media spiked with nitrate. L1-Si medium with relatively high concentrations of trace metals was relatively favorable to both strains of A. poporum tested here. No obvious change within the toxin profile occurred in all cultures of both strains under the various nutrient conditions, although trace amounts of some suspicious derivatives of AZA2 occurred in some cultures. AZA2 cell quotas within both strains significantly (p<0.05) increased at the stationary phase under lower additional phosphate (0 and 3.6µM). Significant differences were not found within AZA2 cell quotas in cultures with additional nitrate ranging from 0 to 2647µM. The highest AZA2 cell quota and maximum AZA2 quantity per culture volume occurred in batch culture at the stationary phase under phosphate concentrations at 3.6µM. Neither A. poporum strain exhibited significant changes in AZA2 cell quotas within f/2-Si media spiked with urea or nitrate as nitrogen sources. The AZA2 cell quota of strain AZDY06 also did not change remarkably within f/2-Si, L1-Si, and K-Si media, however the AZA2 cell quota of strain AZFC22 within L1-Si medium was significantly (p<0.05) higher than that within f/2-Si medium.