The paralytic shellfish toxin profiles and global distribution of the dinoflagellate Alexandrium minutum Halim

A comprehensive review was undertaken of the distribution of the toxic dinoflagellate Alexandrium minutum Halim. The primary threat to humans caused by A. minutum is because of the production of paralytic shellfish toxins (PSTs) which can accumulate in seafood vectors, which if consumed lead to the human condition known as paralytic shellfish poisoning. Within this work it became apparent that the known global spread of this important species has increased, either due to range expansion, as a consequence of improved or novel investigation, or as a combination of these factors. Those populations of A. minutum capable of producing PSTs display a range of different toxin profiles with differing saxitoxin derivatives or differing proportions of common toxins being present. As yet no genetic basis for these differences in toxin productivity has been isolated although there are currently 4 recognised clades within the A. minutum species the two principle clades being the Pacific and the Global. A novel analysis of A. minutum toxin profiles was undertaken by applying K means clustering to PST profiles extracted from the literature. This analysis generated 5 distinctive clusters, each relating to a statistically separable toxin profile. Interestingly examples of each of the two major clades had representatives from different toxin profile clusters. This suggests further, currently undetermined genetic separation or relatedness between clades, as the differences in the ITS region of the ribosome upon which the clades are based, does not seem to be coupled to the specifics of the toxin profile. It also raises the possibility that there is an environmental driver for the differing toxin profiles, although profiles clustered geographically in some cases in others they were either cosmopolitan or spatially isolated. This new approach offers an additional tool with which to examine the relationships between populations and target further genetic research

An evaluation of Paralytic Shellfish Toxin occurrence and magnitude around the UK coast since 2008; using chemotaxonomy to maximise routine monitoring data

A dedicated monitoring programme exists within the UK for the analysis of marine biotoxins of microalgal origin, in shellfish from classified production areas. For England, Wales and Scotland this is currently delivered by Cefas on behalf of the Food Standards Agency (FSA) and Food Standards Scotland (FSS). This monitoring programme tests for three groups of toxins, including the paralytic shellfish toxins (PSTs), using chemical analytical techniques, as well as identifying their causative organisms, using light microscopy. Although toxicity and toxin profile within shellfish from around the UK is well documented, since the implementation of chemical analytical testing, the underlying causative microalgal species remain undetermined in most areas. This is due to a lack of resource within the monitoring programme to perform definitive identification following light microscopy and a lack of specific research work confirming the toxin producers in all areas. Presented here is an analysis of PST shellfish toxicity data from the UK monitoring programme displaying trends in distribution and magnitude of PSTs in shellfish over the last 15 years of monitoring. Further to this, toxin profile data generated following the implementation of HPLC-FLD for monitoring PSTs is analysed. This PST toxin profile analysis is used to offer a possible proxy method for the identification of microalgal species by their differing PST toxin profile as presented in the literature. The possible distribution of causative organisms is evaluated using this chemotaxonomic technique to allow for targeted future work

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) whilst 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 UK 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

Two differentially expressed ommochrome-binding protein-like genes (obp1 and obp2) in larval fat body of the European corn borer, Ostrinia nubilalis

Ommochrome-binding proteins function in coloration and detoxification pathways by transporting tryptophan metabolites, and increase in hemolymph concentration prior to diapause. Two ommochrome-binding protein genes from the European corn borer Ostrinia nubilalis (Hübner) (Onobp1 and Onobp2; GenBank accession nos. AY819651 to AY819655 and AY862870) were isolated. Relatedness to OBP-encoding genes was suggested by peptide similarity, phylogenetic reconstruction, and expression data. 21 single nucleotide polymorphisms between obp1 and 23 polymorphisms between obp2 alleles were identified, and resultant genomic markers were inherited in a Mendelian fashion. RT-PCR showed fat body specific Onobp1 and Onobp2 transcription. The Onobp1 transcript was RT-PCR amplified from fat body of 5th instars, whereas Onobp2 was expressed in fat body of 4th and 5th instars, and peaked in 5th instar wandering and 1 week old diapausing larvae. Expression suggests gene duplicates are maintained by change in temporal expression. The significance of Onobp1 and 2 gene products to O. nubilalis diapause physiology requires additional investigation

Polymorphic CA/GT and GA/CT microsatellite loci for Ostrinia nubilalis (Lepidoptera: Crambidae)

Ten polymorphic dinucleotide (CA/GT and GA/CT) microsatellite loci suitable for population genetic screening were characterized from enriched partial Ostrinia nubilalis genomic libraries. Sequence from 126 enriched small insert genomic library clones identified 25 CA/GT and 58 GA/CT loci that were unique. Perfect repeats tended to be short (n = 10–12). Ten microsatellites, PCR amplified from a Crawfordsville Iowa population showed a mean of 10 alleles per locus (range six to 20), and six of 10 loci showed heterozygote deficiency. Amplification of eight loci was observed in the sister species O. furnicalis

Nuclear small subunit rRNA group I intron variation among Beauveria spp provide tools for strain identification and evidence of horizontal transfer

An optional group I intron was characterized at a single insertion point in nuclear small subunit rRNA (nuSSU rRNA) genes of the imperfect entomopathogenic fungi, Beauveria bassiana and B. brongniartii. Insertion points were conserved among nuSSU rRNA genes from 35 Beauveria isolates. PCR-RFLP and DNA sequencing identified 12 group I intron variants and were applied to the identification of strains isolated from insect hosts. Alignment of 383– 404-nt subgroup IB3 group I introns indicated that four insertion/deletion (indel) mutations were the main basis of fragment length variation. Phylogeny reconstruction using parsimony and neighbor-joining methods suggested six lineages may be present among nuSSU rRNA group I intron sequences from Beauveria and related ascomycete fungi. Terminal node placement of Beauveria introns conflicted with previously published phylogenies constructed from gene sequences, suggesting horizontal transfer of group I introns. PCRRFLP among introns provided a means for the differentiation of Beauveria isolates

Beauveria bassiana haplotype determination based on nuclear rDNA internal transcribed spacer PCR±RFLP

DNA sequence alignment of the nuclear 5.8S ribosomal RNA (rRNA) gene and internal transcribed spacers (ITS) from Beauveria bassiana demonstrated that 6.62% sequence variation existed between nine isolates. A higher level of mutation was observed within the ITS regions, where 8.39% divergence occurred. Polymerase chain reaction restriction fragment length polymorphism, PCR-RFLP, and DNA sequence alignment of the ITS1 and ITS2 regions identified seven polymorphic restriction endonuclease sites, Alu I,Hha I, Hinf I, Sin I, Tru 9AI and two Tha I restriction sites. The allelic frequency of each genetic marker was determined from 96 isolates. PCR-RFLP defined 24 B. bassiana genotypes within the sample set, from which eight phylogenetic clusters were predicted to exist. AMOVA and Fst (θ) indicated that no significant correlation existed between B. bassiana haplotype and insect host range as defined by insect order from which each isolate was derived

Cassini CAPS-ELS observations of negative ions in Titan's ionosphere: trends of density with altitude

Observations with the Electron Spectrometer sensor of the Cassini Plasma Spectrometer (CAPS-ELS) have revealed the existence of negative ions in Titan's ionosphere. Negative ions are observed during encounters whenever the instrument points in the ram direction at altitudes 950–1400 km. Complex hydrocarbon and nitrile chemical processes are believed to take place which play a role in haze formation. The heaviest ions observed so far have masses up to 13,800 amu/q. Using data from 34 Titan encounters, we show for the first time negative ion density trends of different mass groups, including total densities, with altitude. We determine peak densities and the associated altitudes at which they are observed and the highest altitudes at which individual mass groups are found

Mining an Ostrinia nubilalis midgut expressed sequence tag (EST) library for candidate genes and single nucleotide polymorphisms (SNPs)

Genes expressed in lepidopteran midgut tissues are involved in digestion and Bacillus thuringiensis (Bt) toxin resistance traits. Five hundred and thirty five unique transcripts were annotated from 1745 high quality O. nubilalis larval midgut expressed sequence tags (ESTs). Full-length cDNA sequence of 12 putative serine proteinase genes and 3 partial O. nubilalis aminopeptidase N protein genes, apn1, apn3, and apn4, were obtained, and genes may have roles in plant feeding and Bt toxin resistance traits of Ostrinia larvae. The EST library was not normalized and insert frequencies reflect transcript levels under the initial treatment conditions and redundancy of inserts from highly expressed transcripts allowed prediction of putative single nucleotide polymorphisms (SNPs). Ten di-, tri- or tetranucleotide repeat unit microsatellite loci were identified, and minisatellite repeats were observed within the C-termini of two encoded serine proteinases. Molecular markers showed polymorphism at 28 SNP loci and one microsatellite locus, and Mendelian inheritance indicated that markers were applicable to genome mapping applications. This O. nubilalis larval midgut EST collection is a resource for gene discovery, expression information, and allelic variation for use in genetic marker development

A beta-1,3-galactosyltransferase and brainiac/bre5 homolog expressed in the midgut did not contribute to a Cry1Ab toxin resistance trait in Ostrinia nubilalis

Post-translational glycosylation of midgut epithelial protein and lipid receptors may be required prior to binding of activated Bacillus thuringiensis (Bt) Cry toxins. A 931bp cDNA encoding a putative 297-residue beta-1,3-galactosyltransferase (beta3GalT5) was cloned from larval Ostrinia nubilalis midgut tissue, and showed homology to Drosophila brainiac (brn) and Caenorhabditis elegans bre5 proteins. Single nucleotide polymorphisms (SNPs) were detected in coding and promoter regions of O. nubilalis beta3GalT5 (Onb3GalT5), of which 3 of 31 CDS SNPs were non-synonymous. SNPs within HaeIII and MspI recognition sites were confirmed by PCR-RFLP, and are Mendelian inherited. Analysis of F(2) pedigrees suggested an Onb3GalT5 SNP C660 fixed within a Cry1Ab-resistant colony was not correlated with Cry1Ab resistance traits, as measured by higher larval O. nubilalis weights when fed toxin-containing diet