New real-time PCR assay for toxigenic Amphidoma languida

Azaspiracids (AZA) are a group of lipophilic toxins, which are produced by a few species of the marine nanoplanktonic dinoflagellate genera Azadinium and Amphidoma (Amphidomataceae). Amphidomataceae were found to be globally distributed in coastal waters and new areas of occurrence are regularly discovered. The AZA toxins accumulate mainly in shellfish and - when consumed by humans - can lead to the so-called azaspiracid shellfish poisoning syndrome (AZP). Given this serious threat to seafood production and to deepen knowledge about the distribution and risk potential of AZA-producing algae, an appropriate detection method enabling a fast identification and quantification for these toxigenic species is needed. Traditional light microscopy is time-consuming, requires expertise and is getting rather difficult when it comes to the detection, identification and quantification of small-sized plankton. To overcome this challenges, quantitative real-time PCR (qPCR) assays are increasingly used as a molecular additive. Basically, when amplifying the extracted DNA and using DNA standards, the amplification threshold (CT) gives information about the number of target species in the sample. For two AZA-producing species, Azadinium spinosum and Azadinium poporum, quantitative PCR assays have already been developed and successfully applied in the field. Another AZA-producing species, Amphidoma languida, was discovered in 2012 in Irish coastal waters and discovered as a new species within the group of Amphidomataceae - in close relationship with Azadinium spp. All available strains from Ireland, Iceland, Norway, Denmark and Spain produce azaspiracids. Moreover, Am. languida from the Atlantic coast of southern Spain was responsible for AZA amounts in shellfish above the EU regulatory limit, emphasizing the need for further investigations. We thus developed a quantitative TaqMan PCR assay, amplifying 60bp of the D2 region (located on the LSU/28S) of the ribosomal DNA (rDNA) to detect toxic Am. languida. To confirm assay specificity in vitro, cross-reactivity tests with DNA of a variety of related organisms were performed. This included 12 different Am. languida strains as positive controls, Amphidoma parvula, 10 Azadinium species (each including different strains), as well as 10 further related dinoflagellate species (Alexandrium spp., Gymnodinium spp., Heterocapsa spp., Karlodinium sp., Prorocentrum spp. & Scripsiella sp.). The developed probe and primer set successfully detected only A. languida strains. Currently, we perform tests of the newly-designed assay on spiked field samples to test and optimize the quantification ability of the assay. With this assay, we provide a tool for the rapid and distinctive quantification of the toxic dinoflagellate Amphidoma languida to be used in monitoring programs and bio-geographic studies

Molecular detection and quantification of the azaspiracid-producing dinoflagellate Amphidoma languida (Amphidomataceae, Dinophyceae)

Species of the planktonic dinoflagellates Azadinium and Amphidoma are small, inconspicuous and difficult, if not impossible to be identified and differentiated by light microscopy. Within this group, there are some species that produce the marine biotoxin azaspiracid (AZA) while others are non-toxigenic, therefore a requirement exists for precise species identification. A quantitative polymerase chain reaction (qPCR) assay for molecular detection and quantification of one of the toxigenic species, Amphidoma languida, was designed and extensively tested. The assay was highly specific and sensitive to detect and quantify down to 10 target gene copies (corresponding to ca. 0.05 cells) per reaction. DNA cell quota and copy number cell−1 were constant for four different Am. languida strains, and for one strain they were shown to be stable at various time points throughout the growth cycle. Recovery of known cell numbers of Am. languida spiked into natural samples was 95–103%, and the assay was successfully tested on field samples collected from Irish coastal waters. This new qPCR assay is a valuable tool for routine monitoring for the prevention of AZA-shellfish-poisoning caused by the consumption of contaminated shellfish and is a supportive tool for studies on the biogeography of this AZA-producing species

Morphological, molecular, and toxin analysis of field populations of Alexandrium genus from the Argentine Sea

In the Argentine Sea, blooms of toxigenic dinoflagellates of the Alexandrium tamarense species complex have led to fish and bird mortalities and human deaths as a consequence of paralytic shellfish poisoning (PSP). Yet little is known about the occurrence of other toxigenic species of the genus Alexandrium, or of their toxin composition beyond coastal waters. The distribution of Alexandrium species and related toxins in the Argentine Sea was determined by sampling surface waters on an oceanographic expedition during austral spring from ~39°S to 48°S. Light microscope and SEM analysis for species identification and enumeration was supplemented by confirmatory PCR analysis from field samples. The most frequent Alexandrium taxon identified by microscopy corresponded to the classical description of A. tamarense. Only weak signals of Group I from the A. tamarense species complex were detected by PCR of bulk field samples, but phylogenetic reconstruction of rDNA sequences from single cells from one station assigned them to ribotype Group I (Alexandrium catenella). PCR probes for Alexandrium minutum and Alexandrium ostenfeldii yielded a positive signal, although A. minutum morphology did not completely match the classical description. Analysis of PSP toxin composition of plankton samples revealed toxin profiles dominated by gonyautoxins (GTX1/4). The main toxic cyclic imine detected was 13-desMe-spirolide C and this supported the association with A. ostenfeldii in the field. This study represents the first integrated molecular, morphological and toxinological analysis of field populations of the genus Alexandrium in the Argentine Sea

Amphidomataceae in the Labrador Sea and western Greenland waters

Amphidomataceae in the Labrador Sea and western Greenland waters Azaspiracids (AZA) are the most recently discovered group of lipophilic marine biotoxins of microalgal origin associated with human incidents of shellfish poisoning. Over the last couple of years, four out of 27 described species of Amphidomataceae have been identified as primary source of AZA. Diversity and global biogeography of species of Amphidomataceae, however, still are poorly known. In summer 2017 we sampled the central Labrador Sea and the western Greenland coast from Gothaab Fjord (64° N) to 75° N for the presence of Amphidomataceae and AZA. In the central Labrador Sea, light microscopy revealed the presence of small Azadinium-like cells at fairly high densities of 9,200 cells L-1. Single cell isolation from that station yielded 14 clonal strains representing four different species, Azadinium obesum, Az. trinitatum, Az. dexteroporum, and a new species which is currently described taxonomically. For all cultured strains, no known AZA at measureable amounts were detected. From all stations along the cruise, filtered seawater samples were negative for AZA. Specific qPCR assays for DNA samples, targeting ribosomal genes of Azadinium and Amphidoma, revealed positive signals from the majority of stations along the Greenland west coast indicating a not yet recorded widespread occurrence, albeit at low densities, of Amphidomataceen species in that area. Solid phase adsorption toxin tracking (SPATT) samplers were long-term deployed during the expedition in a continuous water sampling system (FerryBox) and were negative for known AZA. The results highlight the presence of Amphidomataceae in the area but the lack of toxins in the field samples indicate a currently low risk of toxic Amphidomataceae blooms in arctic coastal waters

Molecular discrimination of toxic and non-toxic Alexandrium species (Dinophyta) in natural phytoplankton assemblages from the Scottish coast of the North Sea

Molecular methods provide promising tools for routine detection and quantification of toxic microalgae in plankton samples. To this end, novel TaqMan minor groove binding probes and primers targeting the small (SSU) or large (LSU) ribosomal subunit (rRNA) were developed for two species of the marine dinoflagellate genus Alexandrium (A. minutum, A. tamutum) and for three groups/ribotypes of the A. tamarense species complex: Group I/North American (NA), Group II/Mediterranean (ME) and Group III/Western European (WE). Primers and probes for real-time quantitative PCR (qPCR) were speciesspecific and highly efficient when tested in qPCR assays for cross-validation with pure DNA from cultured Alexandrium strains. Suitability of the qPCR assays as molecular tools for the detection and estimation of relative cell abundances of Alexandrium species and groups was evaluated from samples of natural plankton assemblages along the Scottish east coast. The results were compared with inverted microscope cell counts (Utermo¨ hl technique) of Alexandrium spp. and associated paralytic shellfish poisoning (PSP) toxin concentrations. The qPCR assays indicated that A. tamarense (Group I) and A. tamutum were the most abundant Alexandrium taxa and both were highly positively correlated with PSP toxin content of plankton samples. Cells of A. tamarense (Group III) were present at nearly all stations but in low abundance. Alexandrium minutum and A. tamarense (Group II) cells were not detected in any of the samples, thereby arguing for their absence from the specific North Sea region, at least at the time of the survey. The sympatric occurrence of A. tamarense Group I and Group III gives further support to the hypothesis that the groups/ribotypes of the A. tamarense species complex are cryptic species rather than variants belonging to the same species

Molecular discrimination of taxa within the dinoflagellate genus Azadinium, the source of azaspiracid toxins

Molecular probes were developed for the dinoflagellate genus Azadinium to discriminate among three taxa difficult to differentiate by light microscopy. This genus contains azaspiracid toxin-producing Azadinium spinosum, but also non-toxigenic species. Quantitative polymerase chain reaction (qPCR) and fluorescence in situ hybridization (FISH) assays were applied to cultured isolates and Azadiniumspiked field plankton. Molecular methods were highly specific and sensitive in the unambiguous detection of Azadinium, and thus are valuable for routine plankton, biogeographic and phylogenetic investigations

State of the art and new challenges for qPCR assaya for Amphidomataceae

Planktonic dinophyte species of the familiy Amphidomataceae attract attention as producers of azaspiracids, lipophilic phycotoxins that accumulate in shellfish and cause human health problem for shellfish consumers. About 30 species are describe, which are small and difficult to differentiate with routine light microscopy. These group of species is thus an obvious case where molecular methods for detection and quantification are needed. An overview is given on the state of the art and current use of qPCR assays for Amphidomataceae (Dinophyceae). New challanges related to quantification and assay specificity due to the continuously increasing diversity of species and strains are highlighted and discussed

Exploration of microbial biodiversity in polar glacial ice

The polar ice caps play a key part in providing an understanding of climate variability over the last eight glacial cycles and may give information about paleoenvironmental features and changes of microbial diversity in the past. Microbiological studies on polar ice cores are rare and focused so far on silty or accreted ice. Aim of our studies is to detect, characterize, and compare the prokaryotic diversity in different Arctic and Antarctic ice cores at different depths in order to learn more about relations of past and recent communities and about alterations of ancient communities in relation to climatic changes. Critical points in ice core analytics are the strong contaminations of the outside of ice cores as well as the limited availability of sample material. Hence, preparation/decontamination protocols were tested and adapted with inoculated and artificially contaminated ice cores. Our work on real older glacial ice started with material from the uppermost 200 m of the ice shield of Dronning Maud Land, Antarctica. Some pollen grains and a low abundance of bacteria cells were detected by Sybr Green staining in combination with solid phase cytometry. From 3 other ice core samples from about 130-179 m depth, approximately 2150 years old, DNA could be extracted and amplified. DGGE-analyses of amplified DNA revealed a low bacterial diversity, but one strong DGGE band-only present in the inner part of two of the three ice cores analyzed- could be affiliated to the alpha-proteobacteria with a high similarity to Bradyrhizobium japonicum. Clone libraries and metagenomic studies supported the dominance of this bacterial type that might jointly be responsible for N2O abnormalities in glacial ice cores