The value of toxin profiles in the chemotaxonomic analysis of paralytic shellfish toxins in determining the relationship between British Alexandrium spp. and experimentally contaminated Mytilus sp.

Although phytoplankton is ubiquitous in the world's oceans some species can produce compounds that cause damaging effects in other organisms. These include the toxins responsible for paralytic shellfish poisoning, which, in UK waters, are produced by dinoflagellates from the Alexandrium genus. Within Great Britain (GB) a monitoring programme exists to detect this harmful genus as well as the Paralytic Shellfish Poisoning (PSP) toxins in the flesh of shellfish from classified production areas. The techniques used for toxin analysis allow for detailed analysis of the toxin profiles present in contaminated shellfish. It is possible to compare the toxin profiles of contaminated shellfish with the profiles from toxin producing algae and use this information to infer the causative microalgal species responsible for the contamination. This study sought to evaluate the potential for this process within the GB monitoring framework. Two species of toxic Alexandrium, A. catenella from Scotland and A. minutum from Southern England, were fed to mussels (Mytilus sp.) under controlled conditions. The toxin profile in mussels derived from feeding on each species independently, when mixed and when introduced sequentially was analysed and compared to the source algal cultures using K means cluster analysis. Toxin profiles in contaminated shellfish clustered with those of the causative algae and separately from one another during toxin accumulation and, where A. catenella was the sole toxin source, during depuration. During depuration after feeding with A. minutum and where mixed or sequential feeding was undertaken deviant toxin profiles were observed. Finally, data generated within this experimental study were compared to monitoring data from the GB official control programme. These data indicated that the causative algal species in sole source contaminations could be inferred from toxin profile analysis. This technique will be of benefit within monitoring programmes to enhance the value of data with minimal additional expense, where the toxin profiles of causative microalgae have been well described.

A review of the global distribution of Alexandrium minutum (Dinophyceae) and comments on ecology and associated paralytic shellfish toxin profiles, with a focus on Northern Europe.

Alexandrium minutum is a globally distributed harmful algal bloom species with many strains that are known to produce paralytic shellfish toxins (PSTs) and consequently represent a concern to human and ecosystem health. This review highlights that A. minutum typically occurs in sheltered locations, with cell growth occurring during periods of stable water conditions. Sediment characteristics are important in the persistence of this species within a location, with fine sediments providing cyst deposits for ongoing inoculation to the water column. Toxic strains of A. minutum do not produce a consistent toxin profile, different populations produce a range of PSTs in differing quantities. Novel cluster analysis of published A. minutum toxin profiles indicates five PST profile clusters globally. Some clusters are grouped geographically (Northern Europe) while others are widely spread. Isolates from Taiwan have a range of toxin profile clusters and this area appears to have the most diverse set of PST producing A. minutum populations. These toxin profiles indicate that within the United Kingdom there are two populations of A. minutum grouping with strains from Northern France and Southern Ireland. There is a degree of interconnectivity in this region due to oceanic circulation and a high level of shipping and recreational boating. Further research into the interrelationships between the A. minutum populations in this global region would be of value.

Evaluation of the MIDTAL microarray chip for monitoring toxic microalgae in the Orkney Islands, U.K.

Harmful or nuisance algal blooms can cause economic damage to fisheries and tourism. Additionally, toxins produced by harmful algae and ingested via contaminated shellfish can be potentially fatal to humans. The seas around the Orkney Islands, UK currently hold a number of toxic algal species which cause shellfishery closures in most years. Extensive and costly monitoring programs are carried out to detect harmful microalgae before they reach action levels. However, the ability to distinguish between toxic and non-toxic strains of some algae is not possible using these methods. The microarrays for the detection of toxic algae (MIDTAL) microarray contains rRNA probes for toxic algal species/strains which have been adapted and optimized for microarray use. In order to investigate the use of the chip for monitoring in the Orkney Islands, samples were collected between 2009 and 2011 from Brings Deep, Scapa Flow, Orkney Islands, UK; RNA was extracted and hybridized with generation 2 and 3.1 of the chip. The data were then compared to cell counts performed under light microscopy and in the case of Alexandrium tamarense to qPCR data targeting the saxitoxin gene and the LSU-rRNA gene. A good agreement between cell numbers and microarray signal was found for A. tamarense, Pseudo-nitzschia sp., Dinophysis sp. (r<0.5, for all) in addition to this there the chip successfully detected a large bloom of Karenia mikimotoi (r<0.70) in August and September 2011. Overall, there was good improvement in probe signal between generation 2 and generation 3.1 of the chip with much less variability and more consistent results and better correlation between the probes. The chip performed well for A. tamarense group I signal to cell numbers in calibrations (r>0.9). However, in field samples, this correlation was slightly lower suggesting interactions between all species in the sample may affect signal. Overall, the chip showed it could identify the presence of target species in field samples although some work is needed to improve the quantitative nature of the chip before it would be suitable for monitoring in the Orkney Islands.

Outbreeding lethality between toxic Group I and nontoxic Group III Alexandrium tamarense spp. isolates: Predominance of heterotypic encystment and implications for mating interactions and biogeography.

We report the zygotic encystment of geographically dispersed isolates in the dinoflagellate species complex Alexandrium tamarense, in particular, successful mating of toxic Group I and nontoxic Group III isolates. However, hypnozygotes produced in Group I/III co-cultures complete no more than three divisions after germinating. Previous reports have suggested a mate recognition mechanism whereby hypnozygotes produced in co-cultures could arise from either homotypic (inbred) or heterotypic (outbred) gamete pairs. To determine the extent to which each occurs, a nested PCR assay was developed to determine parentage of individual hypnozygotes. The vast majority of hypnozygotes from pairwise Group I/III co-cultures were outbred, so that inviability was a result of hybridization, not inbreeding. These findings support the assertion that complete speciation underlies the phylogenetic structure of the Alexandrium tamarense species complex. Additionally, the ribosomal DNA (rDNA) copy numbers of both hybrid and single ribotype hypnozygotes were reduced substantially from those of haploid motile cells. The destruction of rDNA loci may be crucial for the successful mating of genetically distant conjugants and appears integral to the process of encystment. The inviability of Group I/III hybrids is important for public health because the presence of hybrid cysts may indicate ongoing displacement of a nontoxic population by a toxic one (or vice versa). Hybrid inviability also suggests a bloom control strategy whereby persistent, toxic Group I blooms could be mitigated by introduction of nontoxic Group III cells. The potential for hybridization in nature was investigated by applying the nested PCR assay to hypnozygotes from Belfast Lough, Northern Ireland, a region where Group I and III populations co-occur. Two hybrid cysts were identified in 14 successful assays, demonstrating that Group I and III populations do interbreed in that region. However, an analysis of mating data collected over an 18-year period indicated a leaky pre-mating barrier between ribosomal species (including Groups I and III). Whether the observed selectivity inhibits hybridization in nature is dependent on its mechanism. If the point of selectivity is the induction of gametogenesis, dissimilar ribotypes could interbreed freely, promoting displacement in cases where hybridization is lethal. If instead, selectivity occurs during the adhesion of gamete pairs, it could enable stable coexistence of A. tamarense species. In either case, hybrid inviability may impose a significant obstacle to range expansion. The nested PCR assay developed here is a valuable tool for investigation of interspecies hybridization and its consequences for the global biogeography of these important organisms.